Immunoglobulins of the Lizard, Tiliqua Rugosa

I M MUIL.OSI.OBULIN€I OF EH E LIZÀRÐ,

TILIQUA RUGOSA.

A thesls submitted to the Unlversity oi Adelaide

for the degree of L\octor of Philosophy.

by

tohn David Wetherall, B-Sc' (Sydney)

Department of Microbiology, University of J\delaide - fune, 1969. (f i)

TABLE OF CONTENTS

(iii) Summary Statement ... (vii) (viü) Abbreviations useci in text

CHAPTER l. INTRODUCTION : PI{YLOGEIfY OF ANTIBOÐY PRODUCTION. I

CHAPTER 2. INTROÐUCTION : PEIYLOGEITY OF IMMUNOGLOBULIN STRUCTURE. . 39

66 CHAPTER 3. IVTATERI,ALS AND fuIETHODS

91 CHAPTER 4. KINETICS OF AÀITIBODY PRODUCTION .. '

CHAPTERS.PREPARAIIoNANDCHARÀCTERISATIoN OF Î.I7.ARD IMMUNOGLOBULINS. . lIU

CHAPTER 6. STUDIES ON REDUCEÐ TEARD IMMUNOGTOBULINS 141

CHAPTER 7. SOME BIOLOGICAL PROPERTIES OF LUARD IMMUNOGT, OBUTINS t54

17I CHAPTER B. DISCUSSIOIV AND CONCLUDING REMAR¡GS

Appendix I. Computer program for calculaLing molecular weights from high speeci sedimentalion equili:-.rrium data . . k)

(xii) Acknowledgements (xiii) BibliographY (iii) SUMMARY

The work described in this thesis consists of two parts.

The first part is corìcerned with the immune response of the lizard'

Til-iqua rugosa. to the antigens, Salmonella tvphimurium' rat

erythrocytes and bovine serum albumi¡ (BSA). The second paft

pertains to the isolation and subsequent characterlsation of

Ilzard immunoglobulins which contained antibody activity'

The antigens mentioned above were found to be immunogenic

in &liqua and the formation of humoral antibody was observed'

The klnetics of humoral antibody production by this lizard were

less vigorous than the correspondtng mammallan responses and

were lnfluenced by the envlronmental temperature at which the

llzards were maintained.

Both the rate of production, and the quantity of anLibody produced fn lizards injected with -Úiþ!rylum or BSA and

maintalned at 20o were decreased relative to the response to tltese

immunogens at 30o. Lizards injected witþ BS.\ and maintained

at either 250 or 30o prociuced the sarne ultimate antibody titre

although this took longer to attain at ihe lower temperature ' The

catabolic humoral half }ives of purified lizard immunoglobullns

were measured and lt was found that the thermodynamic effect (iv) of temperature on protein biosynthesis could not account for the observed immune response of lizards maintained at 20o to either

S. tvphimurium or BSA.

The lizard synthesised two types of antibody which were readlly

dÍstinguishable by their size, (rapÍdly and slowly sedimenting),

and susceptibility to reduction with thiols. Immunoglobulins

containing antibodles against the immunogens mentioned above

were isolated from lizard serum. These purified immunoglobulins

were then characterised-

It was found that the two puriffed }izard immunoglobulins, whilst

antigenically related. were not antigenically identical. The

existence of at least tipo classes of lfzard immunoglobulin was

thus indicated. Thls hypothesls was substantlated by physico-

chemÍcal characterlsation of the immunoglobullns and their constituent

Iight and heavY chains.

The larger immunoglobulin was a macroglobulin, tlile properties

of which were very simllar to those of mammalian 7M ¡ it was

designated tizarci 'yM'. Light and hear,ry polypeptide chains could

be detecteci in a reduce,l and alkylateci preparation of lizard '7M"

Starch gel electrophoresis and a molecular weight cietermination

indlcated that the heavy chain of lizard 'yIvI' was similar to the (") mammallan p chain : the lizarci heavy chain was designated a 'Þ' chain on this basis.

The smaller iromunoglobulin was a 7S gloiculin which ln many respeÖts was anAlogous to rnammalian yG. It was designated lizarcÍ '?G'. Although lizard '.yM' And mammalian yM were very similar, slgnÍficant differences between lizard '7G' and mam¡nalian yG were observed.

When examined by electrophoresis llzard 'yG' had the mobilÍty of a fast y or p globulin. Human yG has a slower mobility and is

termed aT2T}lmmunoglobulin. In this respect lizard'7G' pig mouse resembleci the ?I 7G immunoglobulins found in guinea and

serum and more recently observed in rabbit serum'

Lizard,yG,insalinesolutlonwasfoundtoformaggregates.This

reaction was pH dependent and a homogeneous preparation wi'cir respect

to size could be cbtainecÍ by decreaslng the pH of the solvent to 6 ' i)

or less, or also by aading zllvl urea or 4M guanld-ine hydrochloride

to the solverrt. The s|. of lÍzarci 'yG' Ín the presence of 4M urea ¿tJ rw- - '¡¡as 6 .95 .

AnLibo<1y, specific for BSA, 'ui¡as isolated from i:he serum of

hyperimmunized llzards uslng an insoluble immunoadsorbent' Thls

anlibody was shown to possess identical propertles to the purified (vi) lizard'yGt immunoglobulin preparatlon.

Lizard immunoglobulins were found to ext¡ibtt opsonic acdvity and promoted the phagocytosis of several anLigens '

In conclusÍon, it has been shown that:- antlbodies; (i) the lizard, -Illlggg-Igggs.A, is capable of synthesising

(i¡) antibody production in this species was dependent on the ambient

temPerature;

0fi) at least two classes of lizard lmmunoglobr'tltn exist and each

may manifest anttbodY activitY; and

(iv) the physicochemlcal properties of the purified llzard immuno-

globutlns suggest that they are analogous to marimalian yM

and YG immunoglobullns '

--o0o-- (vif)

This thesis contains no materlal which has been accepted for the award of any other degree or diploma in any UniversiËy and to the best of my knor,vledge and bellef it contalns no materlal

previously pubtisheci or written by another person,

except when due refererrce is made in the text of

the thesis.

(I. D. \Metherall)

]une, 1969. (vlii)

ABBREVATIONS USED IN TEXI.

where posstble abbrevfations, symbols and conventlons recommended by the Editorial Board of the Biochemlstry Journal have been used.

Temperatures referrecl tc are in ciegrees Celsius '

The Vforlci i]ealth Organisatlon nomenclature has been used for nrarnmalian imrnunoglobulins, ancl an analcgous nomenclature has been adopted fcr lcwer vertebrate immunc-¡glcbulins since thelr synthesls ls ccntrclled by related genes '

AdcltË.onal abbrevlaticns usect have been clefineci where they first occur ln the text. The following list of abbrevfaLions has been included for convenience.

Ab antibodY. AR analYtical reagent ' BCG Bacille calmette - Guerin strain of Mvcobacterium tuberculosls . BGG i:ovine gämma globulln' BSA bovine serum albumin ' CFA comPlete Freund's adjuvant '

CM - cellulose carbox¡¡methyl cellulose ' c'S0complementunitbase

EÐtrA ethylened.iamlnotetraacetate ' (ix) El-"/' extincLlon coefficient of ¿ !"/" solution measured r cm' in a I cm. cuvette. H healry chain of an immunoglobulin' ILS fmmunlzed lizard serum' i'm' I trroamuscular or intramuscularly' IM) or intraperitoneally' i:p'IP) I trroaperitoneal KLH keYbole ltmPet hEemocYanin' L light chain of an immunoglobulin' Ioq^T anttbody titres have een reported either as the dilution 'z used(".s.I:l28lorasthelogarithm(base2)ofthe clilution (e . g. Loø rT = 7') ' M molar concentration ' z-ME 2, mercaPtoethanol ' p micron (rÛ-6 meffe)' OD oPtical densitY ' PCA pyrrolicì-2-one-5-carbo>rylic acld' ' PVC PolY,zinYlchloride ' RALantisenrmraisedinrabbitstowholelizardserum. S Svedberg unÍts (I0-I3 seconds)' ,2G.* standard sedimentatlon coefficient' coefficient at infÍnite dilution' "zo,* ^tandardÞ sedimentatlon SDG sucrose density gradlent ultracentrifugation' TCA trichloracetic acld' tris tris (hydro:

CHAPTER I

Paqe

A. PREAI\4BLE 2

B.PHH.oGENETICoRIGINSoFAI{TIBoDYPRoDUcTIoN 3 (i) ÐefinÍtÍon of antibodY -.. . '. 3 (fi) PhylelÍc capacity for antibody production " ' 4

(iii) Andbody production within subphylum vertebrata 6

(iv) Evolution of the immune response " ' l5

C. DISSECTION OF AIVTIBODY PRODUCTION IN LOWER VERTEBRÀTES ...... ". 2A

(i) The influence of environnrental temperature on antibodY Prociuction 2T

(ii) Evidence for immunological anamnesis ' " 32

(iii) Immuncgenicity in lower vertebrates 35 -2-

A. PREAÀ4BTE

A phylogenetic approach to -rriological problenis has yielded a great deal of information which is not restrlcted solely to comparatlve biology.

A comparative exanrination of certaln bÍological systems has demonstrateci that there is a sLroi;g selective pressure to retaln functional integrily. This is well illusirateci by a Çornparison of Èhe prlmary structures of various polypeptlde chains. An often quoted example is the primarf structure of the haemoglobin polypeptide chalns; ceriain sequences of amino aclds a.re preserved in all specles desplte varfabiliry in o¡her portlons of the molecule.

Further, if representaLives of various livlng species are examined, less highly evolved structure- function relatlonships can often be found in lower vertebrates and invertebrates. these have provided moclels which are more amenable to experimentation than the systems found in the rnore higtùy evolved species. The efficacy of this approach is well demonstrated by the extensive studles of bacteria and thelr vlruses which have led to great advances in our understanding of molecular biology in general. The overall complexiry of the antlbody response in mammals has led to a more fundamental expertmental approach to this subJect.

It is the hope of finding less complex experir¡rental mociels anci of gaining a better apprecÍatlon of the selective advantages of the immune response that has prompted the revival of interest by immuirologists ln the phytogeny of immunity. Should phylogenetlc stuciies of the fmmune -3- response provÍde clues on the relative importance of its various in vivo manlfestaLions, then the essence of the ability to synthesise antibodÍes may be more clearly delineated.

B.

B(i) DeËnition of antibodv

Before further consideration of this topic, it is necessary to deflne the term "antibody". Implicit in the term antibody is lhe concept of protection, and antibody is generatly defined on a functional basis. A Committee of the World Health Organisation however has provided an alternative definition based on recentlY acquired knowledge of the molecular architecture of mammalian antlbodles (Ceppellini et al. I964).

The W.H.O. deflnition classffÍes antibodies as immunoglobulins and cites three essentlal criterÍa for a substance to be an immunoglobulin'

These are:- (a) that it be a protein of animal origin;

(b) that it have some Structural features in common with other

immunoglobullns, and

(c) Urat although it need not necessarily have a biological function,

when it cioes that function must be antibody activity.

The scope of the W.H.O. ciefinition is sufficiently croad to be

appllcable to the antibodies of animals other than those of human

Figure l.l : EVOLgTION OFJITE \/ERTEBRATES

À chart cf geologic time, and a stmplífied famlty tree

of the classes of vertebrates, shourlng thelr rqnge and

relative abunc.ance through time .( Fron: Colbert, t 95 5 , ) EVOLUTION OF THE VERTEBRATES.

aO at g,t E c Duration ö .g o I E€ o >o IE åp CE P o E ERAS of PERIODS -9 -ã 8 'E .t O(a E at periods () 6¡; CI CL t, E 〠g ÈE E o o a9 À o Ê, iñ .

cENOZOtC 1 Quaternary 70 million years duration 69 Tertiary I 60 Cretaceous. MESOZOTC l t .7 13O million - I years duration. 35 Jurassic. I I ll-z 35 Triassic. I I I 30 Permian. I I I T U 25 Pennsylvanian i lI I 25 Mississippian. -l I lrr\ 40 Devonian.

PALEOZOIC 30 Silurian 3OO million years duration. 70 Ordovician

80 Cambrian TABLE }. I

PHT.Í,UM CHORDATA.

SUBPHIÍ,UM VERTEBRATA.

CLÄSS T AGI{¿\TITA (the jawless fishes)

ctAss 2 PLACODERIVII (early jawed fish, mostly armourec, g!gl-9xtinc.!) PISCES ctÀss 3 CHONDRICHTHYES (the cartilaginous fishes) POIKILOTHERMS CLASS 4 OSTEICHTTTYES (lower (the bony fishes) vertei¡rates )

CI.ASS 4 AMPHTBI..\ (the amPhlbians - Èhe earlies t tetraPods)

CL.\SS 6 REPTILA (reptiles -'colci--bloocÌed' scalY skin) TETRAPODA tetrapods with

CLASS 7 AVES (birds:'warm-blooded' ietrapods with feathers) HOMEOÎHERMS (hisher CL.ASS B M/\MMALIA (mammals -'vuarm-bloooeci' vertebrates) Lelrapccis with hair) -7 - of the vertebrates of considerable importance is the division generally into poikf lotherms and homeotherms. The homeotherms ' to maintain a relatively referred to as the higher mammals, are able

constantbodytemperatureÍndepencjentofthatofthesurrounciing Poikllotherms' environment by means of a homeostatic mechanism' and their body that is the lower vertebrates, lack this mechanism temperature Some tenperature varies with the environmental ' of iheir bociy poikilotherms however exercise a oegree of control (e'g' see Bogert' 1959)' temperature by behavioural mechanisms TemperaturecontrolbythesemechanismsÍsverylimited'andgenerally with that of their environment' speaking their body temperature fluctuates

Thepoikilothermicnatureofthelowervertebratesisanimportant factortobetakenintoaccountwhenassessinEtheircapabilÍtyto that antibody synt}resis produce an|ibody, since lt is to be expected (togetherwiththeirothermetabolicfunctions)wouldjremarkedly

influenced by the ambient temperature ' of serum factcrs in Metchnikoff was the first to study the role

acquiredimmunityirrpoiki}otherms(reviewedbylvietchnikoff,lgÛ5). temperature was a parameter Although Metchnikoff realised that ambieni experiments, it was in determining the outcome of his protection the temperature dependence widat ancl sicard (1897) who first described ofagglutininpro

û¿er the ensuing 60 years isolated reports were published whfch indicated thaÈ various poikilotherms were capable of antibody production and that these responses were very dependent on the ambient ternperature (more antibody was produced aË high anrbient temperatr"res, whereas often the response could be completely inhibited at low temperatures). IL was also Íound that the in vitro manifestalions of the interaction of mammallan antibodies with anligen could be demonstrateci with lower veriebrate antibodies. These data, together wlth historical perspectlve, have been extensively reviewed by Good

and Papermaster (1964). Only sali,:nt cie tails anci co¡rclusions vrill

be mentioned here.

Papermaster, Cond.ie and Good (1962) were the first to systematically

investigate the capaci|y of lolver verte]¡rates to synthesise antlbody'

As it was already known that teleost fish, amphil:ians and reptiles

were capable of antibody production (for references See review by

Good and Papermaster, 1964) their studies were directed towards the

lowest vertebrate classes frcr which living representatives were available

As may be seen from Figure l.I, these are the agnathans and

cartilaginous fish.

The fossil record indicates that five orders of the class Agnatha

at one time existed (Romer, 1962). Today, however, only one order

- the Çyclostomaia - has living representallves. The cyclostomes

ccntain trnro families, the hagfish anci the lampreys. The cyclostomes -9- are consiciered to be the living represenLatives of the most primitive vertebrates (Romer !962, f 962a) and for numerous reasons, cited by Good and Papernraster (r864), the hagfish is thought to be the more primitive cf the two. Lampreys are found in i¡oth fresh and salt water, thre marine form spawning in fresh vuater. flagfish, however, are enlirelY mari.tre.

Papermaster, Cond.ie and Gooa- (i962) commenced ¡;heir investi- gaLion on Ehe phylogenetic origins of antioody prociucticn with the

Californian hagfish, Eptatretus stoulii. Despite inLensive efforts at immunizali.on with a variefy: of antigens the hagfish aid not synthesise detectable antibody, and attempts to elicit a delayeci hypersensi tÍvity reaction'¡¡ith cornplete Freunci' s adjuvant mixed with BCG were also unsuccessful. Complete Freuncl's ad'juvant, containing 30 mg ./mI. of MvcobacterÍum butvricum ônd indian iril<,

failed to produce an inflammatory response when injected intra- muscularly. the homograft response was also stutj-ied, but failure

of both autografts and homografts to take prevented evaluation of results. Persistence of antigen Ín the circulation of the hagfish

was aIsO observeci with several anligens. ÀIlcwat:ce was made in

these Stuc,ies for the poikilciherntic nature of the hagfish as some

of the aÞove experimenËs lvere carrieci out at ciifferent temperatures. - 10 -

On the basis of these results Papermaster et al. (op' cit'-) concluded that the hagfish was devoid of adaptive in:munological * responsiveness at:d suggested tha'¿ ttris finding correlated with the a]:ser-rce of lymphoid cells commonly associated v'¡ith the immune response of higher vertebrates.

* Papermaster et al. (1962)

Irr this anii subsequent papers Paperir,aster, Gocc;i arrd their c,:.llaborators use the foltov¿iag criteri-a :o defír¡e 'airapiive iiiiriiunity'.

(a) ti-re ability to reject skin honrografts. (1¡) The aþility to qemonstrate oelayeci aLi::rgic responses. (c) fne abitity to rnai

Both cellular and hunioral aspects of immunity are thus incluoeci' The latter three criteria ((c), (d) and (e)) consritute the humoral aspect 'this of adaptive irnmunify anci it is this aspect with v¡hich review" is prlmarily concerned. - ll -

In a succeeding paper, Papermaster Condie, Finstad and Good

(1964) made a detailecl comparaLtve examlnation of a variety of lower vertebrates for aciaptive immune responses. RepresentaLÍves of both families of Cyclostome, as well as elasmobranchs ani holostean and teleostean fish vvere includeci in this study. 'fhe elasmobranchs and bony fishes were found to be capable of antibody prociucÈion, although i:he respOnses Obtained were IesS vigorous ano. more erratiC than those Íound i¡ higher vertebretes. The ¡nost interestirrg resulis reported in this paper relrte to lhe cyclos[onies. Despiti; Íurther extensive investigations on the hagfish, E. stouiii, no evidence for arry of the elements of adaptive irrrärunity as cefined by these workers was forthcoming. The oÈher cyclostome investigated was the sea

Iampre5', _PeÈronrvzon rnarinus. Studies on the lamprey were limited to the response to haemOcyanin ancl bacteriophage TZ in small groups of spawnirrg animals, and. inqicated to the authors - " tl-rat the lamprey may have ð lor¡ degree of irnmr.lnologic responsir¡eness - "

The equivocal nature of these resulis or¡ the lamprey and the highly

significant posftion of this anirnal in phylogeny led Finstad and Good

(1964) to a further nore complete str:cty of its adeplÍrre imrnunoloEical responsi-.reness .

Finstqd_ ancj Good (i9b4) four¿o that 'rhe lamprey could proouce

speciiic antibooy to kiileci Bruceila cells, rejecl hornoçirafÈs .rnc1 -12- develop a delayed allergic reactÍon to old tuberculin. The adaptive responses of the lamprey were very feeble Ín comparison with mammalian responses and antibody procÍuction to most of the anligens used was not observed. Soluble BSA and bovine y gloirulln as well as bacteriophage T2 were not cleared from the lamprey circulatlon.

Although haemocyanin was cleared from the circulation, haemagglu- tinating or precipitaling antibody to this anttgen was not found.

In contrast to the hagfish, a family of lymphoid cells in the peripheral blOod, a primiiive thymus, airci a primitive spleen with srnall foci were found in lhe lamprey. A responsive proliferation of mononuclear cells in the primitive bone ntârror^/ of the protovertebral arch íollowlng antigenic stimulation was also observed.

It is possibte that part of the difficulty in eliciting antibody

synthesfs in the lamprey (and other primitive vertebrates) is caused

by a lack of appreclation of the correct immunizing procedures. For

instance, Marchalonis and Edelman, (1968) subsequently eÌlciteci

anttbody production by the lamprey to bacteriophage f2, theteby

independently confirming the work of Flnstad and Gooci, (l96rt¡ ¿¡¿

suggesting that further experimentation may reveal a broader spectrum

of antl.gens to whtch the lamprey may respond.

The precedtng discusslon has Indicated thðt the hagfish, Eptatretus

gtoulii. the lowest known vertebrate, ls devcid of adaptlve fmmuni$ - 13 - andôlSo,thatthelamprey,@,anothercyclostome, is capable cf a cornplex form of adaptive immunity involvlng cellular andr humoral responses .

On the basis of these finclings, ancÍ the knowledge that the other vertenrate classc.s exhibii adaptive imrcunlty, Papermaster, Good and their colleagues conclucied. that "adaptive imnnunity and its ce]lular anci humoral correlates cÍevetoped in the lovuest ','ertei:rates, anci

that a rising level of Ímmunologic reactÍvity and an increasingly ciifferertiiateci ancl complex imm.ui:ologic rnechariism are oicserved going up the phylogenetic scale" (Papermaster et al. 1964). Fror:l the

viewpoint of ihis thesis, which is crientated tor¡'ards aclaptive humoral

responses, their iuiportani corrclusiOiì was ihat 'che hagfish o,vas

incapable of procÍucing circr:iatiiig e,.ti.)ücií iit raspoiìse to ailtigenic

stimulation, v.ihereas the lairprey r:ould, a1:reil v'leait-ly'

In the ProceeclÍr¡gs of a rn¡orkshop on irevelopntenial ImrrrunoloEy

(Smith, Miescher anC Gooct, lå66) much ol the recently availa¿le data

on the phyLoEeneEic origins of adaptivc- immune responses has been

summarisecì. Finstai and Goocl (1966) have contributed a chapter

io ti:is volurno- in whfch they reviev,'ed their ovu'n and i:heir collaborators'

.¡/ork on tjre phylogenetic devcloprnent of aeaptive irnrnurlity and included

also so¡t're previously unp,rblished d.etails. Their conclusÍons were

unaltereC however. The hagfish rernaineci Lhe one vertebrate which -t4- could not be induced to synthesise humoral arrtibody, whereas its fellow cyclostome '¡-he lamprey did synthesise antibody, allhough weakly,andtoonIylantÍgen@)outofthe10with which it was stimulatecì'

As a result of their shrdies on immunoloEic responses in the (1967) have axololl, $fredon mexicanum, ching and wedg,rrood questloned whether hagfish are truly devol d of immunological responses ' they found axolotl a¡tibodies were almost entirely inactivated by heating inimunized axolotl serurn to 560 for 30 minutes, and mention that this procedure was carriecl out by Papernraster et al' (1962) on hagfish serum prior to assaying it for antiirody activity. They also suggest that the influence of the ãnvironmental temperature at which the hagfish were mair:tained (t 0o anci occasionally 20o) may have masked any irnmur¡e resporrses elicitect.

Their first point, i.e. heating of hagfish serum to 560/30 minutes would seem to be a valid criticism and shoulci be settled by further experimentation. However, when the failure to detect hagfish antibody

is consÍdered in conjunction with the absence of ry other sign of

aciaptive immunity, the probabilíty that hagfish do not synthesise

antibody is enhanced. Their second point relating to the masking

lnfluenCe Of envlronmental temperature on adapLive immune responsive- nessinthehagfishisprobablyirrelevant,sincehagfishcommonly - 15 -

Iive at about 40, ancü ln Keeping with the informatton cLerived from stuciies on other poikitotherms (reviewed in section C(i)) one would expect immune respcnses in hagfish to be easily oetectable at I0o.

It woul.i be unwise however to assume that further Study of the hagfish has nothing more to offer. Its phylogenetic position withÍn the vertebrates and its singular lack cf detectable adap"'ir,'e immunity render it one of the most interesiinE species availai:le for study in the search for biological rnechanisrns ancestral to the ad-aprJve immuniiy evicieirt in other vertebrates .

Anti.lcody prociuction lry many other lower vertejJrales in additÍon to those discussed airove has also beer¡ ciernoirstrated-. The jììore significanÈ examples of thes= si;cìies have been su-i:-jr:risriueci in Taoles

1.2 ar:,d I.B. Ta-ole 1.2 shows a subürivisior, cf thr: lcwer verieirr¿ies into classes and ortlers and inclicates those orders r:f irrhich at least one representative has been shown capable of anticccty prociuction'

Table 1 .3 details the more recent studles on antibody produciion in

poikilotherms ancl also the antigens used to elicit these anliboclies.

B(iv) lr¡tion of the Ímmune resÞcnse

In the relatively short space of 7 years sufficient data has

become available to allow soÍie speculaiions cn the evolution of

the irnmune response. TABIE I.2

SUBDIVISION OF THE MATOR CIÀ,SSES OF THE LO\MER \ÆRTEBRAIES *

CLASS SUBCIA,SS ORDER

Agnatha Cephalaspldomorpha Cyclostomata**

Chondrichthyes Elasmobranchii Selachli ** Batoidea**

Holocephali Bradydontl Chimaerae

Osteichthyes Actinopterygii Chondrostei ** Holostel ** Teleos tei **

Choanichthyes Dipnoi (Sarcopterygii) Crossopterygii

Amphibia Apsidospondyli Anura **

Leposponoyli Urociela ** ,4,poda

Rept"ilia Anapsioa Chelonia **

Ðiapsida Lepidosauria RhynchocephalÍa ** Squamata (Lacertilia**) (Ophidia **)

Archosauria CrocicüIia **

* Extinct subclasses and orders have been omitted. Ccmpiled from Romer,1962: Colbert, 1955: Young, 195J.

** Indicates that at least one representatÍve of this order has been shown to be capable of antlbody producrion. ÎABLE T.3 SOME LOV\/ER VERTEBRATES SHOWN TO PRODUCE ANTIBODY, AIID THE * ANTIGENS ETICITING PRODUCTION

* SPECIES OF A¡IIMAI A}]ITIGENS REFERENCES

I AGNATHA Cvclostomata

Sea lamprey (Petromyzon Br. abortus I t 2 3 marlnus) Hu. EH TÃ phage f2 5

II CHONDRICHTTryES Selachii Nurse shark BSA 6 (Ginglymostoma cirratum) PR8 7 Lemon sirark (Negaprion BSA 7, I breviros tris ) PRB; Chicken erythrocytes 7 Smooth dogfish (IVlus telus Lj¡quluq-haemocyanin 9 canis) Horned shark KLH I (Heterodontus tr-ancisci) phage T? t, 3 Leopard shark (Triahis phage T2 L2 ,13 semifasciata) KLH I3 Batoidea Guftarfish (Rhinobatos Br. a_bortus 3 productus) KLH; phage T2 l, 3

III OSTEICHTHYES ChondrosteÍ Paddlefish (Polyodon BT. a-bortus; BS¿ç BGG/CPN spathula) phage T2; KLH 3 Sahnonella É[ antigens; d Various erythrocytes t I

* Abbreviations for antigens are described at the end of the TaþIe. TABTE I .3 (cntd)

SPECIES OF AIiTIMAL À}ITIGE REFERENCES

I{olqstet eorvftn (¡mta calva) Phage T2.; KLH t Br. abortus 3 Shortnosed gar (LepÍsosteus BSII,; PRB 14, l5 platostomus)

Teleostei Margate (flaemulon album) BSA, PR8 14, 15, 16 S. oaratvpht B l6 Goldflsh (Carasslus phage øxr74 t7 auratus) sea urchin sperm t8 BSA 19 Bullhead (Amei¡¡rus melas) phage T2 I Garp (Cyprinus carpio) sea ¡rrchin sPerrn 18 Rainbow trout (Salmo B. salmonicida 20 gairdneri) B8A KLH 2t Black bass (Micropterus KtH I salmoldes)

MtvIPHIBIA Anura Bullfrog (Rana catesbiana) phage f2 22 phage øxL74 l7 Frog (Rana plpiens) S. tvohosa a 23 Frog (Rana temporaria) Br. abortus; S. Pullorum 24 South African clawed toad S. tvphl flagella 25 z6 frenopus laevis) T. ovriformls Marine toad (Bufo marÍnus) S. adelaide flagella 27 S. tvphosa H; BSA RGG z8 Sheep erythrocYtes 36 Urodela A¡

SPECIES OF A}IIMAI. JTNTIGENS REFERENCES

V REFTILIA Chelonla Painted turtle (ChrysemYs KLIf 30, 3l ptcta) Tortolse (Testudo ibera) Br. abortus 32 Pond tortoise (Emys Sheep serum protelns; BGG 33 orbicularls)

Rhvnchocephalla Tuatara (Sphenodon S. adelaide flagellin 34 punctatum)

Squamata Desert iguana S, tvphosa H; BSA; RGG 28 (Dfpsosaurus dorsalis) Sleepy lizard (Tiliqua S. tvphlmurium O antigens; rugosa) Rat erythrocytes; BSA 37 Cunningham's skink Sheep erythrocYtes 36 (Egernia cunninghami)

Grocidilla Alligators (Alligator KLH 35 mis sissippiensis)

ABBREVISIIONS FOR ATIITIçENS gr. a.boitus Brucella abortus ktlled cells Hu. EH Human erythrocyte H antlgens phage f2 bacterloPhage f2 BÉ¡A bovlne serum albumln PRg influenza A virus strain PR8 KtH keYhole ltmPet haemocYanin phage T2 E. coli bacterioPhage T2 BGG/CÍA, bovine y globultn + complete Fleurd's adjuvant 8., salmonfcida Bacterium salmonlcida T=lcwifo*tis antigens of the clllate@ RGG rabblt gamma globulin HGG human gamma globulin BGG bovine gamma globulin TABLE r .3 (cntd)

REFERENCES FOR TABLE I.3 l. papermáster, Condle, Fínstad and Good (1964) 2, Finstad and Good (1964) 3. Finstad and Good (1966) ¿1. Boffa, Fine, Drilhon and Amouch (1967) 5. Marchalonis and Edelman (1968) 6. Clem, DeBoutaud ar¡d Slgel (i967) 7 . Sigel and CIem (1966) 8. Clem and Small (1967) 9. Marchalonis and Edelman (1965) 10. Good and Finstad (196a) 11. Pollara, Suran, Finstad and Good (t968) 12. Papermaster (1966) 13. Sr¡ran, Tarail and Papermaster (I967) 14. Clem and Sigel (1966) 15. Sigel anq Clem (1965) 16. CIem and Sigel (1963) yi . Uhr, Finkelsteiir ancl Franklin (i962) IB. Cushins (1942) 19. Everhart and Shefner (1966) 20. Ridgway, Hodgins and Klontz (1966) 2L, HodEins, ll/eiser and Ridglvay (1967) 22, Marchalonis and Edelman (1966b) 23. Iftueger and Twedt (1963) 24. Alcock (196s) 25. EIek, Rees and Gowing (1962) 26. Lykakis and Cox (1968) 27 . Diener and Nossal (1966) 28. Evans (1963) 29. Chtng and Wedgwood (1967) 30 . Grey (1963) 31. GreY (1966) 32 . Mauns (1963) 33. Lykakis (1968) 34. Marchalonis. Ealey and Diener (1969) 35. Lerch, Huggins and Bartel (1967) 36. Talt (1967) 37. Thfs thesis. - 16 -

The available data suggest that the first appearance of the lmmune response occurs fn the lower vertebrates, ancj. is paralleled by ihe development of lymphoid elements (Finstad, Papermaster and Good,

1964; Good, Finstad, Pollara and Gabrielsen, I966). The studies on the cyclostomes and other lower vertebrates reviewed above suggest that the capacity for aclaptÍve immune responsiveness evolved in the flshes of the Silurian and Devonian periods of the Paleozoic erê'

The sequential development of this attribute is not understoocl.

The hagfish, the lorn¡est known vertebrate, seems to be cievold of

any facet of adaptlve lmmunify whereas its fellow cyclostome, the

Iamprey, displays a complex pattern of adaptive immune responses ' It is pertÍnent then to quesÈion v.rhether the evolution of this attribute

OcCurred aS a Single 'all Or none event', or aS wOuld Seem more

reasonable, aS the result of a gradual series of oevelopments' It is not yet possÍble tc answer this question. Clues may corne frorrr

further studies of the cyclostomes and other primitive fish' It is

an unfortunate possibility however that even if a graduated cievelopment

did occur, its origins may be lost in the extinct progenítors of the

lower ffshes; e.g. the ostracoderms and placoderms of the Paleozoic

era.

One of the values of a phylogenetic study of a complex biological

phenomenon is that a dissociation of constltulive elements will often

become apparent. PhylogenetiC analysis of the immune response Of -t7- lower vertebrates however has not revealeci any true dissoclation of its cellular and humoral correlates, althoughr (as stated by Finstad

and Good (I964)) this might have been expected. The possibility

stÍlI extsts that this may be achieved in future studies.

Àlthough representatlves of many invertebrate phyla and of the

Subphylum Vertebrata have been examined for adaptive lmmune responses,

the other 3 subphyla which together with the vertebrates comprise

Phylum Chordata seem to have been overlooked. These subphyla

are the Hemlchordata (marlne worm like creatures), the Cephalochordata (Iancelets etc. lncluding Amphioxus), and the Urochordata (tunlcates andthelralties).ByvirtueoftheirphylogeneLl'crelatlonshlpwith

the vertebrates, representatfves of these subphyla deserve a close

scrutiny for any of the ancestral elements of the adaptíve lmmune

responslveness.

One of the most significant questions relating to the evolullon

of the immune response is why such a response should have evolved

at all. This question has been discussed by Papermaster et aI' (f964),

Finstad and Good (1964), Finstad, Paperrrast€t and Good (f9641 and

more recently by Burnet 0968).

These authors have cogently argued that the defence mechênlsms

of fnvertebrates are efficlent, and ln relatfon to protectlve capaclty

agalnst lnvaslve saprophytlc and pathogenic mlcro-organfsms there -t8- ls no obvlous reason why the vertebrate system of adaptfve immunlty should have evolved. Indeed Papermaster et al. (1964) have concluded that the defence mechanlsms of the invertebrates (phagocytosls, cellular prollferatton, non speciflc and non tnducable humoral substances, etc.i see revlew by Good and PaPermaster, 1964) are retalned ln a recogntzable form ln the vertebrates. The attribute cOnferred on the vertebrates by vi¡tue of their adaptlve immune responsiveness, è¡td whtch seq[ns to be lacking in the invertebrates, may be the abitity to recognÍze as foreign subtle an'.igenic differences such as may be associated with the histocompatioility antigens within a species.

As a result of consideratlons of this nature it is generally thought that the aclapttve immune response may have evolved prirnartly as a means of deallng with aberrant cells whether they are the result of somatic mutatlons flhomas, 1959; Finstad, Papermaster and Good'

I964; Flnstad and Good, I964) or of neoplastic origin (Burnet, f 968) and that the assoçlaÈion of thls response wlth protective capabllitjes was fortuÍtous.

The above conslderations have emphasised when and why the

vertebrate adaptlve fmmune response evolved. It is also of lnterest

tO speçulate how, 1.e. from what evOludOnary "raw material" tl¡is

response evolved. - 19 -

In a thought provoking paper on the molecular basis of the immune response, Dreyer, Gray and Hood (1957) have pointed out that by the tlme the first immune response was recognizable a complex system of proteins must have extsted. they have hypothesised that these molecules (i.e. antibodies) arose as a late evolutionary offshoot of a basic type of membrane-bound chemoreceptor system which evolved as an essential part of the differentialion of multicellular organisms. Further, taking into account the relatively late evolu'Lionary appearance of these molecules, they also suggesi that this mechanism evolved for functions lnitially unrelaËed to the imrnurie respcnse as we know it.

The idea of specific receptor sites on ceII surfaces being ihe ancestral form of antioodies is an attractive one. It emphasises the importance of cell-cell interactions during the evolution of immunity whÍch is in accord with the ideas of Burnet (1968) that specific cellular responses of the type characterislic of homograft immunity and delayed hypersensitivity came earlier than humoral antibody production during

evolution. Finstad and Good (1965) have also cornrnented on the

increase in the influence of cellular immunity relative lo humoral

immunity in primitive fishes v"'here this disparity seen'rs to render

these species unusually susceptible to ihe clestrucËive and necrotizing

effects of adjuvant emulsions - -2û-

It 1s interesting to note that Cantacuzene (I919) described a strong tendency for bacteria to "stick" to cell surfaces in ascidians

(tunicates) in which a bacterial septicaemia had been induced. This is one of the few lnstances where a protochordate has been invesligated and it may support the concept of cell receptor sites functionlng as ancestral antibodies. It would be lnteresÈing to repeat such studies in protochordates utilizing modern critical methodology'

c. DISSECTION OF /\AITIPODY PRODUCTION IN TOWER VERÎEBRAIES

Many factors determine the ulttmate result obtained when an anligen

is introduced into the tissues of an animal. These include the nature ,

dose and route of administration of the antigen; the previous

experience of the recipient ln relation to the antigen or other antigens

that cross react r¿ith iU the d-egree of lyrrrphoid inaturation of ihe

antmal and its genetic enciowment. If the anirnal is a poikilotherm

the environrnental temperatr.rre at whlch it is maintained will also

affect the outcome '

It is not too surprisÍng then that wide variations are often observed

in the immune response o f animals, even within a given species,

and that the means of eliciting antibody production in most animals is stÍll largely determined empiricalty. Much of the published data

on anlibody production in lower vertebrates is based on experiments

in whtch only a small number of animals were used. Çonsequently -2t-

It is very ciifficulË to describe the humoral response of lower verteirrates in a precise manner. Furthermore, the influence of environmental temperature on poikltothermic anlfbody production renders a comparison of ihese responses wiÈh mammalian antibody responses less meaningful.

Keepirig ln mind the llmitations fmposed by ihe natr.¡¡e of the system, tlree aspects of anlibody productlon fn poikilotherrns have been chosen for further discussion. These are :-

(a) The influence of environmental iemperature on antibody production.

(b) EvÍdence for immunological anamnesis, ani

(c) Immuriogenicity in lower verle¡f,rates. c (i) Tha inflrrêncê ofe ¡rrzi rnnrnonf al k¿m frrrê ôn antiìrorlv ororluction

It has long been known that there is an associatlon between antibody produc'.ion and ambient temperature in poikilothermlc vertebrates (e . g.

Widal and Sicard, 1897), In general it has been found that an increase in the environmental temperature of an immunized poikllothermic vertebrate results |n enhanced antibody production, whereas a decrease in the environmental temperature causes complete or partÍal inhibition of anLibod.y production. Some of the early data has been reviewed by

Btsset , (1947). Despite intermittent attention this phenomenon ls still poorly defined and understood.

Since much of the published data cn the temperature dependence of antibody formation is confusing the following frruo hypotheses are -22- suggested in order to provide a basls for assessment of the data. Tlre hypotheses are :-

I. The reSponsive temperature range, over whích antÍbOdy formatiOn

may be observed, reflects the temperature of the animal's normal

habitat. il. The rate of appearance of circ'¡lating antlbody in a pofkilotherm, following sultable immunogenic stÍmulation within its responslve

temperatr:re range, is depencient on the rate of synthesls of

antibody at that temperature aLcÍ at least one other temperature

dependent step.

The following discussion has been considered Írorn the viewpoint

of establlshing the feasibility of these two hypotheses ' The revÍew

is Íntended to be selective and critical and no attempt has been made

to be comprehensÍve'

In a particularly lnteresting study, whlch bears on several

lmportant aspeets of the imrnunology of polkilotherms, Talt (1967)

investlgated the temperatqfe dependence of antÍbody production in

bhe toad, Bufo madnuF , and the lizard, Eqernia cunningh+ml, to sheep erythrocytes. Talt found that the toad was capable of respondlng

to this antigen at 2CIo, 25o, or 3Üo to approximately the same extent,

althought}reresponse!\TasmuchsloweratthelowerÈemperatures.

The lizard however responcied very differently at these three temperatures -23-

Less antibody was produced at the lower temperatures than at the higher temperatures anê this dffference was not overcome by time, i.e. the maximum titres obiained v\'ere citfferent for each temperature and took longer to attain at the lov¿er temperatures '

Evans and Cowles (IgS9) and Evans (1363) investigated the antibody response of the Caltfornian desert lguana, Dipsosaurus dorsalis,

to the lI antigens of -S,:lXPþEg at varying amblent temperÊtures ' The

antibody response was very weak at 250, Eood at 35o, and fair at 40o,

i.e. an optimal temperature for antibociy production between 25o and

4ûo seemed to exlst for thls animal. The desert iguana is well adapted (Cowles to hfgh temperatures, its lethal temperature being weII above 4i¡o

and Bogert, 1944).

several publlcations have indicated that antibody production ls (1940) also possible at low temperatures in some specles' Srnith was

able to de'cect aggluttnlns to Bacterium salmonicida in carp, Gvprinus qardneri' carpio, brown trout, salmo trutii, anci rainbow trout, salmo

maintained at It¡o. Ridgway (1962) siudied ¡he imniune response of

the cold water sable fish, Anaplopoma fimoria, maintained at 5-Bo

to red cells of the silver salmon and tbe steel-head. He reported

that after many injections of antíget'i and after a period' of'78 days an

agglutinating titre of l:246 was obtained' -24-

Thus it is clear that the normal enr¡irontnent of the species of animal being invesLigatecl musl be takerr into account ln studies of the effect of temperature on anLibody production. In many instances, thereiore, comparisons of response among different species mAy not be possible.

The discovery by Evans (1963) of an optimal temperature for antibody production in the desert iguana is very interesting. This was the first time such a finding haci been reported ancl the generalÍty of thfs phenomenon deserves further investigation.

The simplest hypothesis to explainthe effect of temperature on antibody production oy poikilotherms would be ihat temperature affected the rate of antiboCy production, but not the ullimate titre of antibody produced in the serum. Several investigators have pui:Iished reports supporting this hypothesis. I{owever, there are alsO some

data available which inciicates that a more complex mechanism is

operative, at least in some instances.

Cushing (1942) was the first investigator to exanfne the rate of

antibody production in a poikilothermic vertebrate maintained at

variouS temperatures. He used two SpeÇies of fish, the carp,

Cyprinus carnio, and the Eoldfi.sh, ,Carassius auratus. The fish

v/ere mainÈained at either l50 or 28o and lmmunized with a single

intraperitoneal injection of sea urchin spermatozoa. Cushing found -25- that both species produced agglutf nating antibodies, although antibodÍes were not detectable in the fish nraintained at 150 until

B days after they had become detectable in the fish mafntained at 28o .

After 25 days the antibody titres Ín both species of fish were as high in the "cold immunizeci" fish as in the "warm immunized" flSh. A similar experiment was performed by Tait (1967) and has already been referredtoabove.Hefoundthattoads,-@,immunized intraperitoneally at 20o , 25o, and 30o with sheep erythrocytes produced the same ultimate antibody titre but that the rate of antibody production was proportional to the temperature. Taitrs resultg, including those for the lizard Eqernia cunn-inqhami which will be ciÍscussed later, êre shor^tn in Table 1.4 which was adapted from his ihesis. TABLE I.4 marinus) nd lizards âc ) maíntained at verwi no frrres. l¿.dao tecl from Tait, 19671.

No. Day anlibodY Day antibocly Maximum titre first detected reached rnax. Loo-, animals Temp. in srand. Stand. Stand. 9tt"'Mean IvIean group Mean dev. cev. cíev.

TOADS 5 30 4 1. 3 1I 3.1 8 I,O q 5 25 7 3. T4 1.3 rt 1.4 tç, 5 20 I 3 2. 3 3.5 l0 1.0 LU}\RDS C 0 \J 5 30 I L9 I8 6.0 I 0. 6 5 25 t7 1.3 2s t.3 I 0. tl * 2U 54 -* 66 -* = * Thu results at 20o are given for only one animal ' -26-

It seems, therefore, that at IeaSt in certain species ancÍ under some circumstances, temperature affects only the overall rate of antibody production and not the ultÍmate titre attained.

Bisset (1948) was the first to claim that, in addition to the

temperature dependence of the rate of anlÍbody synthesis. there was

a further temperature dependent step which influenced the overall

rate of appearance of humoral antibody and was capable of fnfluencing

the ultimate humoral anttbody titre attained. Unfortunately Bisset's

conclusions were Often not experimegtally justified. The major

criticism of Bisset's worl'; lies in Lhe use of small grroups and ln

sacrificing one animal for each experimental observalion, thus making

no allowance for indlvidual variation.

In these experlments Bisset (1948) injected groups of frogs,

Rana tempqraria, maintained at 20o and 80 with Pseudomonas punctata,

after which two of the groups of frogs were transferred to the reciprocal temperature. Bisset claimeci ihat no airtibody prociucÍion coulii be

detected in frogs roaintaineci at Bo, while rapid antibociy procìuction

occurred in frogs kept at 20o. Furthermore, frogs immunized at the

lovr temperature began to produce antÍbody when the temperature was

increased. From these experirnents Bisset postulated that two

mechanisms operated in the development of immunity in cold-blooded

vertebrates. The first was the potenlial to produce anlÍbody, whlch _27- could occur at low temperatures. The secOAd was the synthesls and release of antibody into the clrculatlon. It was this second step that Blsset claimed was temperatu¡e dependent. Blsset also clalmed that a decrease ln the temperature of frogs wtth good anttbody titres resulted ln a rapld decrease in the tltre of these clrculatlng antibodtes. This result implies that frog anttbody has a very short half life at low temperatures. Thls result fs consldered further in

Chapter 7 where the turnover of immunoglobultns as a functlon of temperature ls discussed.

Bisset later modified the above concluslons on the effect of temperature on antlbody productlon. In a subsequent paper (Btsset, t949) he reported that ffogs immur¡ized-and held at low temperatures produced antlbody on admlnistratlon of cortlcosterolds. Bisset then suggested that whlle temperature did not affect the synthesis of antlbody, lt d,id affect the release of thts antibody lnto the cfrculation.

Several reports have claimed that Bisset's work was not reproducable and slnce hls expertmental methodologry was suspect, lt ls perhaps not surprfsfng that hls views have tended to fall into dlsrepute and a good deat of confusion has arisen' Thus Maung (1963) and Alcock (r965) were unable to obtain antibody productlon when either adrenal cortical hormone or cortisone was injected into immunized animals maintalned at

Iow temperatures. Also Evans (1963) argued that ff , as Blsset suggested, -28- only the release of antibody was temperatr.re depencient, then animals immunlzed and. maintaineo at a low temperature for some time should., when transferred to a wermer errvironment, exhibit a rapld appearance of ani:ibody tn the circulation. He did not observe this fn either the ciesert lguana, Dj@ or the toad, Bufq marinus.

Elek, Rees and GowinE (1962),|n a carefully planned and executed study found that African clawed toads, Açlqgpus laevis,, immunized at

80 with S. tvphosa H antigen and then transferre d to 27o , between the 45th and 79th day post immunlzalion gave rise to hlgh circuladng antibody tltres in less than 60 hours. fmmunized toads maintalned at 80 did not produce circulating antibdy during the 95 day period over whlch they were examlned. Injectton of adrenocorLicotroplc hormone (ACTH) or cortlsone into the cold fmmunlzed toads was also follor¡rzed by the rapld appearance of a moderate Litre of circulating antibody. As 60 hor¡rs is too short a perfod for de novo synthesls of the antibody detected in the cold immunized toads, they concluded that the release of preformed antibody accounted for their data.

Using a single cell technique for studylng in vitro antÍbody production l(rueger and I'\'vedt (f 963) set out to demonstrate antibody production and fts relationshlp to temperature using cells isolated from the red pulp of the spleen of the fuog, Rana oipiens. Formallnlzed

S-. tvphosa was injected lnto frogs maintalned at etthe r 26o or 40 . _29_

FOllowing immunization, serum v¡as collected and the spleen removed'

Microdrops of mixtures of splenic cells and bacteria were placeci in each square of a divideci cover slip (I spleen cell: l0 bacterla per drop) and after tncubation were assayed for antibody. Antlbody productton vras scorecl by immobilization of more than 25% of tfie bacteria in each orop. The serum agglutinin iiÚes of frogs kept at

260 reached a maximum of I : 64û and antibocìy production by spleen cells isolated frorn these animals v¿as clctected when they were incubated at 260. I{on-immunized cont¡ols did not show serum

agglutinins, nor did a preparalion of their spleen cells produce

fmmobilizôtion of any bacteria. The sen¡m of animals Ímmunized

at 40 dicl not contain detectable agglutinins and their spleen cells

dld not produce immobilization when incubated with bacteria at 40'

However, when the incubation of these cells was caried out at 260

antibody production \^,?as observed. More i.rnportantly, the supernatant

frOm splenic cell homogenates from " colct-immunized" fi.ogs showecí

a moderate ÈÍtre of 1 : l6Û. Spleen cell homogenates fronr non

immunÍzed frogs dicl not corrtain agglutinins '

Icueger and Tv,,ecit interpreted these resulis as inciicating that

Low ternperature inhibi.ted gecreLicn of intracelh-llar antlbody and lhat

this antibody vras released on subsequent iilcrease of temperature '

lhe rapid appearance of a relatively large amount of circulatinE

antibody has also been observeo i¡i sonie tromeotherms ' Thus FTiII -30- and Rowley (1967) showed thatlnJecH,on of S..tvpþimurlum endotoxln lnto gulnea plgs caused a l6-fold increase ln serum agglutlnln to rat erythrocytes" Incorporation of S35 label fnto rat erythrocyte absorbable material suggested that thls materfal was not syntheslsed de novo and was presumably a release of anlibody from extravascular storaEe sltes either intra or extracellular. Thls example ls quoted to emphasise the tenability of a release process being at least partially involved in the temperature dependence phenomenon.

Tait's results for the llzard, E, qunntnqllaml (see page 25), and also data relating to T. ruoosa presented ln Chaptet 4 of thls thesfs show that at low temperahrres antlbody production ls largely tnhtbtted ln these t$ro specles of llzards, f .e. in both instances lt has been found that temperature affected the maxlmum titre of circulatlng antlbody attained, as well as the rate at whlch this antlbody was produced,

On the other hand, Cushlng's data on the temperature dependence of antlbody production in carp and goldfish (described on page 24), and

Tait's data pertaining to the toad (page 25), indicate that tn these species, and wlthln the temperature ranges studied, although the overall rate of antlbody productlon varied with temperature, the ultlmate maxlmum antibody titre reached was lndependent of the temperature at whlch the anlmals were malntained. - 31 -

Tait (1g67 p. 146) has hypothesised lhat the differences ln the effect of temperature described above may be accounted for by the fact that

Iizards are not completely poiktlothermic. He presents some evidence which fndicates that llzards have a genetlcally controlled preferred optimum temperature which they attempt to maintain by characteristic daily behavÍoural pattenrs (e.$. sêê Bogert, I949 and

195g). Tait then argues that with the development of a relatively high preferred body temperatwe there is an associated rest¡iction in the range of temperatures over which phystological and biochemical activity can be carried out. Amphibfans and fish, however, have retained their ability to operate over the whole range of temperatures atwhich they can survive and, that only theraþ at which their various physiological processes occur is affected by teiriperature '

The extention of this situation is seen in the homeotherms, which have evolved physiological mechanísms for maintaining a constant

body temperature within nalTow limits and have therefore become

independent of the envlronmental Ëemperature conditions.

It woulcl seem reasonable to conclude that the effect of temperature

on antil¡ody production in poikilotherms ls a complicated phenomenon

and further experimentation to clarify the data of Krueger and fi¡¿edt

(1963) and Elek, Rees and Gowlng (1962) should be insHgated. It

seems that this phenomenon offers a ltkely example where the study

of the immunology of lower vertebrates may allow an expanded -32- experimental dissection of situatlons found in mammalian immunology.

It may prove an excellent experimental model for studies on the fate of antigen and the secreLÍon of antlbody from cells '

C (ii) Evidence for immunoloolcal anamnesis

lMtrettrer lower vertebrates have developed the capacity for immunological anamnesls (memory or secondary responslveness) has been generally regarded as a controversial question.

In mammalian species immunological memory is usually demonstrated in two ways which reflect the blfurcation of the immune response inio its cellular and hurnoral elements. Cellular lmmunological memory is typlfied by the accelerateci rejection of second set homogrrafts and humoral immunological memory by an accelerated and enhanced antibody production over and above that which v¡ould have been lnduced by the secondary antigenic stimulus in an unprimed anfmal. Since the distlnction between prlmary and secondary responses is quantitative rather than qualitative, the only precise way of differentiating these responses is by statistical analysis.

Nossal, Austin and Ada (1965) uslng statistlcally deftned parameters characterised the secondary humoral response of rats to S. adelaide flagella anÈigens.

This paper by Nossal et al. (op. cit:-) emphasised the complexlty of the parameters which mutually interact to elicit a statistically -33- definable secondary humoral response and which restrict the number of experimental sÍtuations follorrutng a primary antiEenic stimulus when a secondary response is demonStrable. Failure to appreciate

that secondary responses are only ellciiable under certain condillons,

and inexperience with the general requirements for producing

antibody responses in lower vertebrates has almost certainly caused

the present controversy.

Finstad and Good (1966, p. lB9) are firmly of the opÍnion that

a humOral anamnestic response caa j¡e ciemonstrated in certain

primitive fish. They consider ihe best evidence of memory was

obtained when the horned shark, Heterodontus francisci, anci the

guitarfish, Rl¡inobatos productus-, produced no demonstrable antibody

after primary antigenic stimulation, but after secondary stimulation

with the same dose of homologous antigen a vigorous respqnse was obtained. In this same paper (p.t78) Finstad and Good claimed to

have demonstrated a secondary antibody response to Brucella antlgens

in the lamprey, Petromvzon marinus'

There have also been a number of instances where failure to

demonstrate secondary antibody responses have been reported'

Grey (1963, 1966) claimed that there was no anamnestic response

in the painted turtle, C-hrysemvs picta, following a secondary injection of haemocyanin. In another species of tortoise,T@ iþ,¡e, Maung (t963) was also unable to obtain an increase in -34- antlbody titre following a secondary injection of Br. abortus.

Slget and Clem (1966) claimed that lemon sharks, Nggg[on brevirostlis, and nurse sharks, Ginglvlnos-toma clrratum, extribited only "weak immunological memory" to a variety of antlgens.

Evans, Kent, Attleberger, Siebert, Bryant and Booth (1965) were unable to eliclt a secondary response to formalinized -s:-EPbi in the toad BÉ marinus. However, when the antigen used was BSA they obtained a 2-3 fotd higher antibody titre after a third injecllon of antigen than after a single injection. Diener and Nossal (1966) studied the response of the same species of toad to S. adelaide flagella and using their statistical assess¡irenl roethoos v,'ere unable to demonstrate a secondary response, They also douoted v¡hether the results of Evans et al. (1965) with BSA would meet thefr criteria for

memory. Diener and Nossal hypothesiseC that the absence of

immunoloEical memory in the toad was due to the absence of a follicular

antigen trapping web and germinal centres ir¡ the lymphoid follicles of

thie species.

ft is possible that the above attempts to demonstrate a secondafy

response faited because of an unfavourable combination of experlntental

parameters. Finstad and Good's work clearly indicates that a

secondary response Ís possible, and an extensive effort to quantitall,vely

demonstrate this should be attempted, bearlng in mind that the -3s- parameters which govern this response in lower vertebrates may dÍffer from those operAtive in mammals and other higher vertebrates '

Evidence indicatfng that an anamnestic response may be eltcited in the lizard, !!liqua ruqgsa, is presented in Chapter 4'

there does not seem to be any dOubt that lower vertebrates can

exhiblt immunological memory when this is assessed by the accelerated

rejection of homografts in which it is believed that humoral antibody

does not pIaY an essential role.

Finstad and. Good (1966, p. 197) were able to ciemonstrate

accelerated second set skin homograft rejection in the larnprey. This

work was extended by Perey, FÍnstad, Pollara and Good (I968) who

clearly established that a variety of prlmitive fish extribited "memory"

fn rhis way. Hildeman and Cooper (1963) håve also reported that

goldfish, carassius auratus, rejected second set homografts in an

accelerated manner that was temperature dependent '

C (iii) Immunooenicitv in lower vertebrates

A survey of the literature pertaining to antlbody production in

Iower vertebrates (e.g. see Table I.3 following page t5) suggests

that it has often proven difficuli to induce antibody produclion in

these animals. Most success has been obtained using particulate antigens, particularly bacteria and viruses ' -36-

A number of reports have cornmented on the apparent relatlve lack of responslveness of some lower vertebrates, partlcularly to soluble antlgens. For example, GooC and Papermaster (1964, p'84) have commented on the lack of Success by ihemselves and others

at eliciting BSA antibocltes in fish, frogs and reptiles. Finstad

and Gooci (f 966) also reported that Br. aj;ortus was the only antigen

out of 1Û trieci that sLimulated antibociy production in ;he lamprey:

the other anuigens used were diphtheria toxoid, BSA, bovine y glol:ulin,

sheep and rabblt erythrocytes, keyhole limpet haemocyanin, typhoid

O and H antigens and bacteriophage T2.

In view of these reports it is pertinent to question why lower

vertebrates appear to be less responsive than higher vertebrates '

A number of reasons exist which either singly or in conjunction may

influence the Outcome of an attempt to immunize a lower vertebrate '

These include:

(a) Ease of induction of immunological tolerance or paralysis particularly with soluble antigens '

(b) An inherent lack of immunogenicity* ,o ,orn" of the antigens used. (c) A tack of knowledge cf the correct immunizi.ng schedules for lower vertebrates, whlch may differ significantly from those successfully used in higher vertebrates.

* -r-r!--¡t The following nomenclature has beer:r aciopteO. "Ar¡tigenicity' is considered an inherent property of the substance in question, whereas nimmunogenigity" depends also on the responsiveness of the recipient. Thus immunogenicity = antigenicity * responsiveness (after Davis et al. 1968). -37 -

lait (1967 , p.166) studied the influence of immunizing dose of soluble human serum albumtn (HSÐ on antibody production in the toad, Bufo marinus. He reported that doses of 50 mg.or greater of in HSA dissolved in saline when injectec! Íntraperitoneally resulted a relaijvely diminished antibody response. Better antibody productlon was obtained usÍng doses of less than 10 mg HSA' Tait

suggested that his data supported the hypothesis stated by Good and

Papermaster (I964), and mentioned on page 36 that immunological

tolerance or paralysis might be easlly inducar¡Ie in poikilotherms

with soluble protein antigens. In Chapter 4, however, some data

questioning this hypothesis is presented'

since Good and Papermaster's comment on the difficulty of

immunizing some lower vertebrates with BSA, I instances of successful (reference anlibody production to this anligen have been reported (1966) in Table I.3). Also, slnce the work by Finstad and Good

which only I antigen out of 10 evaluated was Íound to be immunogenic producLion in this in the lamprey, severel other instances of antlbody (1968) species have been reported. Marchalonis and Edelman

successfully immunlzed lampreys with bacteriophage f2, and Boffa, lamprey Fine, Drilhon and Amouch (1967) induced the synthesis of indicate antlbody to human erythrocyte H antigens. These resi¡lts that part of the difftculty in eliciting antibody produclion in lower 38- vertebrates has probably been due to ignorance of the sorrect immunizing procedures . It ls tmpossible at present, therefore, to cifaw any conclusions about the diverstty of the r6nge of immunogens recognized by lower vertebrates or to speculate whether this diverslty varies with thefr

phylogenetic classifica."lon. ExperÍence gained from further studies with these anímals should help overcome this problem' The

possibility that some lower vertebrates have an inherent restricted

abtlity to respond to some antiEens cannot at present be excluded' - 39 -

CHAPTER 2

Pacre

A. PERSPECTI\Æ 40

B. LOV\¡ER VERTEBRATE IMMUNOGLOBULINS 44

(i) Physlcochemical properties and isotypic heterogeneity of lower vertebrate immunoglobulins 45

(it) The specificity of lower vertebrate anlLbodies. . 58

(1if ) N-terminal amino acid sequences of lower vertebraie lmmunoglobulins ... j.. 6l

C. CONCLUDING REMARKS 64 -40'

A. PERSPECTIVE

In the prevlous chapter the diversity of the poientlal for the lmmunoglobuLin production was reviewed. In this chapter nature of the immunoglobulins synthesised by members of the

Subphylum Vertebrata will be dlscussed' until recentl¿ evolutionary studies vüere organically orienLated order into the and r.lerlved from rhe efforts of biologÍsts to introduce of dfversÍty of llving things. This was done by uslng a system parameters Due taxonomy which took into account morphologÍcal ' however' to recent accomplishments in blocheri:istry and genetics, evoluLlonary a new perspective of evoluÈion has emerged in whÍch structure of biological changes are reflecteci as changes in the chemical evolution" molecules. The sLirnulus leading to studies on "molecular and functional has been ernergence,. in remarkable detail, of strucÈural

features of some proteins and nucleic acios ' by the The principles of molecular evoluiion are well itlustrated intensively haemoglobin molecute which has been one of the most investigatedproteins.Amirroacidsequencesoftheconstituent polypeptidechainsofhaemoglobinfromavarietyofsourceshave intra and been establÍshed. Examination of these has revealed polypeptide chains interspecies sequence homologies between the whichsuggestevolutionaryrelationships'Asaresultofthese the haemoglobin studies a mociel has emerged for the evoluiion of -41 - molecule from the single polypeptide chain of myoglobin' A fatrly

comprehensive survey of studies on protein evolution may be found (1968) in the reviews by Dixon (1966), Nolan and Margolfash and

also in the symposium edited by Bryson and vogel (1965)' The Doolittle evolution of immunoglobulins has been reviewed by Singer and

(Ig67), Lennox and cohn (I967) and Hood, Gray, sanders and Dreyer (le6i). from the Two general prlnciples have become clearly established as accumulated mass of data derived from studies on evolu'tion

reflected in changes in the structure proteins. These may be stated

as follows : (i) closely relatecì organisms generally have proteÍns whose amino related acici sequenees êfe lirof€ similar than those of cistanlly organisms.Itfollolvsthatthemorecloselythetwodifferent polypeptidechaÍnsresembleeachotherinanÍndividual,the resulted in their rnore recent has been the gene duplicatton *'hich

ciivergence, and

(ii) parrial or complete duplication of cistrons has been a successful

mechanismfortheintroducLÍonofnevrproteins. success These prlncfples have already been applied with conspicuous

to the as yet llmited amino acid sequence data on immunoglobullns ' (e.g.seerevielvsbySingerandDoolittle,}966;LennoxandCohn,

1967; and Putnam, TitanÍ, Wikler and Shinoda' f 967)' -42- The extraordinary heterogenelty of i:he immunoglobulins of higher vertebrates and their unique property of having many dlfferent antlbody specificlLies superimposecl upon a constant baslc structure poses an exclting challenge to exponents of molecular evolutlonary studies on immunoglobulfns. tn additlon to thls structure-functton problem the constitutive treterogeneity of the immunoglobullns has poseci a paradox to the current concepts of the genetic control of protein bfosynthesis. It has proven impossible to reconcile the hypothesis that one cistron codes for one polypepticie chain with the findlng that each immunoglobulin light (L) and heavy (H) polypeptiae chaÍn is a single "apparently" encodeci by two cistrons which are expressed as contlnuous polypepti<ìe chain comprisÍng a constant and a variable segnient. Indeeci, the heterogeneity of the ir¡rmunoglobulins is perhaps the most distinctive at:ci confusing ospec'É of this protein systenn' It would seem reasonabte to expec; that Liris multiplicity of form is ciue to their funclion as antii:odies. but 'ùrere Ís no evidence ihat this is

so and some of the complexity fs known to be quite unrelated to

antibody sPecificitY.

There are three main classes of Ímniunoglobulln present in all

mammalian Sera that have been studie

vary considerably from one species to another. A number of minor

classes are also known to exist; however, because of thelr small

relaLive concentretion they u'lll noL ìce consiclered further here ' _43-

The three major classes are ciesignated yG, yM and 7A (VüHO nomenclature; ceppetlini et aI. 1964) and in normal human serum thetr concentratÍons are approximately I200 mg., 125 mg. and 280 mg' per IilC rnl. Of serum respectively. Their common characterislics are

that they may all manifest antibociy activity, and- that they all have a

similar quaternary* structure. They are related antigenically but are

not iderrtical. Hence they atl contaln common and distinctive elements;

the common parts are the light (L) polypeptide chains whlle the disLinctlve

parts are the heavY (H) chains,

Immunoglobulins have been founci to consist of a unimolar ratio

of L and H polypeptide chalns. some carirohydrate mcieties are

usually attached to the H chains. The L chains are common to all the the immunoglobullns within a species and ere resporrsible for Ï{ intraspecies anligenic cross reactivity between classes ' The

chains vyithin a species differ from class to class and exhlbit the distinctlve ai:Ligeriic specificities (calleci iscrypic) upon which the

ciivision into classes is based'

* of euaternary stmcLure is definect as the rnode of arrangement qua- the subunits comprislng a protein molecule ' Proteins wÍth definite ternary structure but whose subunits are linked solely by non covalent

interactions are called "multimers" (after Crick and Orget' I964)' - 44- B. LO\MER VERTEBRATE IMMUNOGLOBITLINS immunoglobulin One of the most distincLi.ve features of the mammalian constitulive system of proteins is the contrast be|ween their extensive heterogeneity heterogeneity anci the uniformity of their structufe ' The in the of the intact molecules is ultimately determined by cüfferences

primary structure cf their component polypepÈide chains. Yet these protein chains, that is the L and H chains, are as characteristic of this enzyme systenr as are the cornponent polypepticie chaír,s of a complex

which is homogeneous ir-¡ botil st¡uctural anO functjonal senses '

centereo Interest in lov,rer vertebrate immunogloi.rulfns has been from two earlier on their heterogeneiry anci quaternary structure. Apart I963) publications, (uhr, Finkelstein anci FranklÍn, 1962; ancl Grey, has only useful informallon on the nature of these irnmunoglobulins information' which become available durlng the last flve years. This firmly establishecÍ is described in detail ln the fol}owÍng pages, has proteins composed that tower vertebrate lmmunoglobulins are multichain counterparts of L and H polypepLide chains analoEous to their mammalian ' It ls clear also that both higher and lower vertebrate immunoglobulins data have evolved from a common ancestral protein. The available exhibft support the contenLion that lower vertebrate immunoglobulins

at least some of Lhe tntermediate stages of this evoludonary

development. -45- B(i)

utu, Ptnkelsteln and Franklin (1962) demonstrated that chickens,

frogs and goldfish injected wlth bacterlophage øX 17 4 produced

anËboilies whlch could be separated by suclþse denslty gradient

ultrasentrlfugation (SDG) into rapldly and slowly sedlmenting types '

They suggested that these anttbodies were the eqUivalent of mammalian were 7M anfl yG antlbodies respectlvely. Two further obsenratlons also made. It was found that the antlbodies produced by the frogs respect and goldfish had 7 to p electrophoretlc mobilitfes and in thls

resemþled mammalian antibodies. However they also found that the

slowly sedimentlng (approxlmately zs) frog and goldfish antibodles

were partlally susceplible to mfld reductiorr* with 2, mercapto-ethanol

* The extent of the reaclion between immunoglobulins and thiol reducing agents is dependent on the experimental parameters employed' ls When susceptibility tà m1ld 2, mercapto-ethanol (z-ME) reduction used as a crlterlon for discriminating between yG and yM type antibodies pH the experimental copditions usually * mployed are 0.lM z-ME at 7.4 for I hor¡r at 37o. Unless explicitly stated otherwise, these are the experlmental parameters , Or their approximate equtvalents, connoted when the expre.rior, 'mild z-ME reductlon' is used in the above text' The reductlon of S-S groups to S H groups may be reversed by oxfdlsing The reverse reactlon is usually prevented by alkylalion of agents. or the reduced proteln with a sultable reagent such as iodoacetamlde lodoacetlc acid. Apart from its convenience, there ls little to recommend this technlque since its applicabllÍty ts limlted to well deflned antibody best method systems. Denslty-U"¡"""tt giádi"nt ultracentrifugatlon remalns the for dlsttngutshlng llght (7S) and macroglobulln antlbodles ' -46-

(2-ME: û.IM T-ME/I frour/370). In thls respect these immunoglobullns differ from mammallan yG, the haemagglutinating and precipitatlng properLies of whtch are resistant to mild reduction with thiols. This finciing indicates that at least minor structural dffferences exist between mammalian yG immunoglobulins and the slowly seciÍmenting frog and gotdfish immunoglobulins. The rapidly sedimenling antibodies of all three animals were whclty susceptible to mild Z-ME reductlon as a¡e mammalian yM immunoglobulins.

Grey (1963, 1966) studied the antÍbodies synthesised when the pafnted turtle, c4rysemvs plcta, was inJected wfth keyhole limpet haemocyanin (KLH). His findÍngs were in general agreement with those of Uhr, .æ1. (1962). The turtle antibodies were found to be of low electrophoretic mobility and to be of two sizes, approxfmately

I8S and 75 as determined by sDG. Both types of antlbody were

susceptlble to mild 2-ME reduction.

The ffrst successful attempt to elucidate the nature of a lower

vertebrate immunoglobulin was made by Marchalonis and Edelman

(I965). these authors injected dogfish, (NþglgLggsg¡þ - an

elasmobranch) with Limulus haemocyanin. About a month later the

dogfish were bled, and they were able to isolate from the serum a

l7s immunoglobulln possesslng antibody activity. They did not

find a slowly sedimenting (i.e. approxlmately 75) fmmunoglobulin -47- wlth antibody actlvlty. They were successful, however, in lsolating a 75 protein whlch possessed structural similarities and was antigenically ldentl.cal to the l7s immunoglobulin, thus fulty juslifying classification as an immunoglobulin also'

Further lnvestigation of these immunoglobulins revealed that they were composed of polypeptide chalns physicochemically similar to mammalian immunoglobulin L and H chains. By immunodiffusÍon techniques utilizing specific antisera it was found that the antigenÍc specificities of the L and H chains of both the 17S and 7s immunoglcbulins were icientical '

Two dimenslonal high voltage electrophoresis of tryptlc hydrolysates further of the polypeptide chains isolateci fronr clogfish irnmunoglobullns

indicated that the H chains of iroth types of immunoglobulin had for similar primary structures, and this was also found to be true had the L chains. It was clear however that the L and H chains

different primary structures. The authors suggested therefore that

the dogfish had only one predominant class of immunoglobulin' pH when examlned by urea starch gel electrophoresis at acld it

was observed that the H chains of both types of dogfish immunoglobulin chaln of had an electrophoretic mobility sÍmilar to the p polypeptide class of dogfish human 7M. It appeared therefore that the predominant vertebrates immunoglobulln resemblecl the 7M tnrmunoglobulins of higher '

A subsequent publÍcation, (Marchalonls and Edelman' 1966a)' -48- presented data on the molecular weight, amino acld composition and carbohydrate content of the L and H chatns of each of the dogfish immunoglobulins. These data confi¡med that the H chains of the

75 and l7S dogfish immunoglobulins were very similar to each other ancl also that they physicochemically resembled the mammalfan

¡r H chain.

The nature of the imrnunoglobulins of the bullfrog, Rana catesbiana. has also been lnvesrlgated by Marchalonis and Edelman (1966b)'

They found that when this anuran amphibian v¡as immunized with bacteriophage fZ both rapidly anci slowly sedimenting antibooies were synthesiseci. Serological analysis c-rf the immunoglobulins of which these bullfrcg antibc¡dies were representative indicated that they were antigenically related but not identical. Starch gel electrophoresis, in the presence of urea and at acid pH, of the reduced and alkylated bullfrog immunoElobulins revealed that their

L chalns were slmllar but that their H chairsheiddifferent electrophoretic mobllities. Furthermore, the bullfrog 75 immunoglobulin H chain had

an electrophoretic mobiliþ- in this sytem similar to the mammalian

y chain and the l8S immunoglobulin H chaÍn was found to be similar

to the mammalian p chafn. These findings suggested that bullfrog

75 and 18S immunoglobulins were analogous Èo mammalian yG and

yM immuncglobulfn classes respectively. Carbohydrate content

determinations on the intact mOlecules and molecular welght -49- determinations of the bullfrog L and H chains confirmed thfs contenti'on'

Thus Marchalonls and Eclelman showecl ihat at least two classes of bullfrog immunoglobulfn existed, and that when judged by thetr physicochemical propertles these two classes appeared to be analogous to mammaltan yG and 7M lmmunoglobulín classes'

On the basls of the above dlscoveries Marchalonls and Edelman

(f 966b) hypothesised that - "the 7G immunoglobulins emerged at some polnt 1n evclutton between the elasmobranchs and the anuran amphibians. "

Unfortunately, only the t75 dogfish immunoglobulln described by Marchalonis and Ecielman (1965) exhibited andbody activlty.

Nelther the naturally occurring 7S 'r1t4'*nor the 75 subunits of re{uceci I7S '7M' antiboiies displayerl antibody activity' In vlew

of the antibociy activfty exhibited by other elasmobranch 7S 'yM'

* the following nomenclature has been adopted throughcut this thesis for lower vertebrate immunoglobullns ' ,yM' refers to those lower vertebrate lmmunoglobulins, the H p chain of whlch is physicochemlcally stmllar to the mammalian chafn' These tmmunoglobulins are further designated 75 or l9S, depenciing on their slze. ,yG' referS to those lower vertebrate immunoglobulins whose H chain physicochemically resembles the mammalian 7 chaln. ThÍs nomenclah¡re has been used since it {s believed that related genes control the synthesfs of lower vertebrate 'yM' ancl mammalian '?M' and lower vertebrate 'yG' and mammallan'yG' -50- immunoglobullns, which is described below, it seems probable that the absence of detectable antlbody activlty in the dogftsh 7s '7M' immunoglobulin was due to the relatÍvely short lnterval between immunlzation and collectlon of serum. However, it is possible that thls represents a peculiarity of this species (cf ' the response of the paddlefish, P. spclbl¿l-ê, Pollara, suran, Flnstad and Good,

(1968) Table 2 .2) .

During 1967 data on three more elasniobranch species were published. clem and srrall (1967) lnvestigatec lemon shark,

Neoaprion brevlrcstris. immunoglobulins; Clem, De Boutaud and sigel (1967) nurse shark, Glnqlvmostorna cirrat]rm immunoglobulin; ancl suran, Tarail and Papermaster (I96i) leopard shark, .Triakis.

s emlfasciata fmmunoglobulir¡s'

These investigations showecÌ that all three species of shark

synthesised macroglobulin antibody fairly shorlly after immunization (in of with an antlgen. and that after a much longer period excess

6 months) a slowly sedimentlng - approximately 7S - antibody was

also synthesised. Isolatlon and characterisation of these fmmuno-

globulins and their constltuent polypeptice chains confirmed the

prevtous work of lvlarchalonls and Eclelman on the dogfish' The

three specÍes were founcl to have one major isotypically defined class of immunoglobulin, the physicochemical propertles of the

H chain of which resembleC the p chain of mammaltan yM ' -51 - The sea lamprey, PeBomvzon marinus, |s the most primltive vertebrate reported capable of synthestslng humoral antibody and the nature of its immunoglobulins fs therefore of consfderable interest'

PoIIara, Pinstad and Good (1966) were the flrst to descrlbe a lamprey lmmunogrlobultn. They found lamprey Brucella antlbodles to be associated with a protein whose sedimentatlon coefficient was intermediate behveen 75 ancl l9s. The unusual size of lamprey anü'bodies (1967) was also demonstrated by Boffa, Fine, Drllhon and Amouch who reported that lamprey antibocly to human erythrocyte H antigens was associated with 10.9S and 6'65 proteins observed during analytical ultracentrifugation of whole lamprey serum '

yet The most extensive examinatior-r of lamprey immunoglobulins They reported has been that of Marchalonls and Edeiman (1968)'

successfully immunized lampreys with a single iniecti'on of bacteriophage the responses f2. The response obtained was very much weaker than

of hlgher vertebrates to the same antigen, and the bacterlophage

neutrallzing abllity of the lmmune lamprey serum was poor in comparison Despite wlth the serum of other vertebrates lsrmunized wtth thls antlgen.

thls they were able to localize the lamprey antibody activity throughout klnetics alt the ftactionation steps by virtue of the sensitivity of the of phage neutralization antibody assay used' wlth 6'6S They found the lamprey antÍbody activiry to be assoclated

and I45 fractlons of lamprey senrn and to possess p' electrophoretic -52- mobltity. serological analysls of the 6. 6S lamprey immunoglobulln proteÍns and also to be showed it to be free of other lamprey serum antigenicallyeitherveryslmilarto,oridentÍcalwith,thel4S ln quantl'ty immunoglobulin. The 145 proteiU cOuld not be isolated butwasthoughttobeanaggregateofthe6,6sprotein,sincethese twoprotelnsappearedtobeantigenÍcallyidenticat.Theseimmuno- properties not usually globulins were found to have some interesting

observeci in lmmunoglobulins '

were assocfated by non- Each consisted of L and H chains which the L ar¡d H chalns of covalent interations. The molecular weight Of ''' 25'000 and 70'000 the 6.65 'yM' immunoglobulin wðs approxlmately respectively.Becauseofttsdefinitecompositionandlackof is clearly a multlmer" interchatn covalent bonds the 6.65 lmmunoglobulln (afterCrickandOrgel,1964)'The6.6simmunoglobullnwasfoundto in aqueous solutlons: undergo concentration dependent cüssociatton facilltated this dÍssoclation' dissoclatlng solvents such as acetic actd TheimmunologlcalrelationshipofthelsolatedLandl{chainstothe fntactmoleculewasfoundtodiffermarkedlyftomthatobservedwlth immunoglobulinsfromotherspecfes.TheisolatedHchaindldnot

6 65 molecule; the isolated react with rabbit antiserum to the intact ' unrelated to the intact L chain was also found to be antigenically molecule,althoughtheLchainsfromreducedandalky}atedand untreated6.ssimmunoglobulinwereantigenicallyidenttcal.To -s3- explaln thls MarchalonÍs and Edelman suggested that the antigenlc propertles of the L and H chains depended strongly upon the conformation of the polypepttde chains, and that thls was different ln the dissociated siate ancÍ in the intact molecule. Lamprey 6.65 immunoglobulin, ln contrast to mammalian 7G, yM and yA, was unaffected by treatment wtth cys teine-activated Papain.

Although Marchalonis and Edelman suggest cautlon ln interpreling the alngular properties of the lmmunoglobullns descrlbed above, it seems clear that lamprey lmmunoglobullns are multichain molecules consistlng of L and H chalns analogous to the L and p chalns of mammallan lmmunoglobulfns. The dlstlnctive properties of the lamprey lmmuno- globullns are not lncompattble with current ideas concerning the evolution

of protein systems. Further analysfs of the multtmerÍc nature of lamprey

Ímmunoglobullns should yleld new informaü.on relatlng to varlous aspects of polypepttde chain interactlon in antÍbody molecules.

prevlous The data discussed so far would seem to substanüate

speculation that the evolutionary development of the immunoglobullns was "trans-specific" in character (cited by Diener and Nossal' I966)' (1968) Using data published up to I968, Marchalonis and Edelman re-stated thelr hypothesis, first proposed in 1966, that the gene

spectfylng y type polypeptide chalns emerged at or prior to the phylogenetic

level of the anuran amPhlbians. -54- Marchalonls has pursued this topic fi¡rther and has examined the immunoglobulins of a reptlle and the Australian lungflsh'

In his investlgatiol of lungfish lmmunoglobulins (Marchalonis,

1969) Marchalonis found that serum from the Aus¡.allan lungfísh,

Neocerôtodus forsterl contained a 19S immunoglobulin with an H chain comparable to the p chain by standard crlterla' A llght lmmuno- globulin , = 5.gs, was also found whlch the H chain dlffered "|,o,* 'n of from both mammalian p and y chains by having a molecular welght

about 38,000. Marqhalonis suggested that thls low molecular

welght tmmunoglobulin may represent an immunoglobulin class unfque

to the DÍpnot. An ¿typical immunoglobulin of similar size has also possible been reported ln duck senrm by Grey (1967). It fs, of course,

that this 5. gs lungfish immunoglobulln may be a 7 type immunoglobultn the Dipnol modlfied by specialization over the long perfod dr.fing which ftom have been distinct from the ancestors of the Grossopterygians'

whlch the ancestral amphibians were derlved'

unfortunately, due to the rarÍty of the species. Marchalonis did

not have an opportunity to immunLze a lungfish and consequently from vuas only able to isolate biologically inactlve immunoglobullns

lungfish serum. These findings relating to lungfish immunoglobullns as are therefore condifional on their future positlve identificaLlon

immunoglobulins by exhibÍtinq antibody activlty. The physicochemlcal dlstinctive' attrfbutes of the immunoglobulins as a protein system are so -55- however, as to rencter it very procable that the lungfish proteins isolated, by Viarchalonis vìi€re inceed ir,rnultoglcbulins ' A vslde variety possess of lov¿er vertebrate immunoglobulins have ncw been shown io ihe distinctive properties which characterise the mammalian immuno- globulins and it fs unlikely that ihe lungfish irnmunoglobulins are so different as to defy f solation.

The Dipnot comprise an order of the fleshy, finned, bony fishes via wffich are removed from the direct line of ascent of the tetrapoda

the ancestral amphibia. Consequently, the demonstraiion of isotypic

heteroEeneity of lungfish immunoglobulins requires rhat the earlíer

hypothesis of Marchalonis and Ecielrnan (1966b and Iii58) be mociified' of i mmuno- A suitable alternative woulci be that isotypic heterogeneity the globulins evolved subsequent to the phylogenetic appearance of of tetrapoda. Further investigaÈions of the immunoglobulin classes further various o-rher representatives cf tl^re bony iishes will allow

refinemerrt of this hYPothesis ' imrouno- several reports have recently been puclished 'jescribing the

globulins of rePtiles.

NiarchalonÍs, Ealey and Diener (]969) reported that the New Zealand

Luatara, sphenodon punctatum, possessed al least tv;o Ímmunoglobulin

classes, ciesignated 75 'yG' and 19S 'yM" analogous to the (1 also corTesponding mamrßalian classes. Lykakis 968) -s6- reported that the European pond tortoise, Emvs orbicularis, possessed both 75 ,yG' and l95 '7M' classes of immunoglobulin. In thÍs instance however Lykakis only demonstratecl that two types of antibody distingulshable by thelr size were synthesised. Although he refers to them as 75'7G' and l95 'yM'immunoglobulins, no evidence for isotypic heterogeneify nias reported.

The avaÍlable data relatlng to the nature of lower vertebrate immunoglobulins has been summarfzed in Table 2.2. Thts table

summarizes the number ônd type of immunoglobulln classes detected

in varlous lower vertebrates, together wlth some of their physicochemlcal

parameters.

The work of Clem and Sma|l (]967) on lemon shark immunoglobulins

has uncovered an interesiing anomaly. They found that the natr'rrally

synthesfsed 75 'yM' BSA antibodies were able to agglutlnate antigen

coated erythrocytes and that this activlty was resistant to mtld thlol

reduction. That is, these antibodies appeared to be polyvalent' In contrast, the 75 subunfts ob'tained by reciuction of lemon shark 19S

,yM' BSA antibodies were not able to agglultnate antigen coated

erythrocytes. Hence the I9S '7M', lemon shark antibody has simllar

properties to mammalian yM antibody which also loses its agglutinating

properties upon reducLlon to 75 subunits. The lemon shark 75 '7M'

anttbody, however, extribits the agglutinating and thlol reductlon

resistant properlles usuälly associated wtth mammallan 7G antlbodles ' TABLE 2.2 Àl'I'D soME or tnun IMMUNoctoBItLTN clÀssEs DETECTED IN vARIous LoïvER vERTEBRATE€*, PH:lt¡lC OC H EMICÀL PAR Atvf Ef ER'S of -3 Immunoglobulln Motecular Specles of Anlmal Carbohydrate Glasses Content (%) H Chalns (and reference) detected L Chatns

(g) t?M' 0 140 2.o I.AMPRÉY 6 6S NM 4.6 t 2.0 i 69.G f . marlnus) t 4S '?Mt and

(a) t* 20 .5 + 0.3 i 73.4 + 2.0 MOCITH DOGEISH 7S '?M' ! 1.8 198 !6 . canls) :7.6 tyM' hl 982 + 25.5 0.1 + 0.5 I zl.6 ! 2.8 and 17S i8 -7 + 0.9 (r96s & ls

i22 23 7l ! 3 SHARK 7S '?M' 3.51 t 0.11 16r!s - . brevlrostrls) 3 (g) t7M' 0.19 869 + 6l 22-23 7l ! and Smalt (1967) I 9S 3.67 !

22 (approx.) 22 (approx.) SH¡TRK ¡0.2S 'TM' .e.o(b) NM clrratum) 22 (approx.) 70 (approx.) , DeBouta¡d irn.rs(n) '?M' ir.rnþ) (c) t7M' 22 76.5 ! 1.8 SHARK 7S NM semlfasctata) 9. NM (c) . 22 77.0 ! 2.0 uran and PaPermaster t7 a 4S 'rM' (1967), Suran, larall TABLE 2.2 (cntd.)

" Immunoglobultn Carbohydrate iMolecular L0 Specles of Anlmal Classes Content (%) H Chalns (and referencb) detected ' L Chalns

PADDLEFISH 7.3 23 .50 0.56 75.30 + 0 .75 (P. spathula) 9S 'yM' NM 870 + ! Pollara, Suran, Flnstad (h) (f)** i 0. 50 o ? ie3 . ot ! 0.92 37.9s t AUSIRJ{IUIN LUNGFISH ,(e) NM NM (h **: (h) 70.28+ l.l0 (N, forstert) 19. nr(s) 'rM' i23.r4 ! 0.93 Marchalonfs (1969)

(a) 53 6 + r.6 BT'LL FROG 6.zs(9) 'yG' i.z.r o.l 22 0 ! 0-s (R. t NM catesbiana) (a) 20.0 + 1.0 72.1 + r,6 Marchalonls and tg.os(e) 'yM' i10.8 ! 2.6 ** TUATARA t7G' 7S NM NM NM NM (S. punctatum) tyM' Marchalonts, EaleY r8s and Dtener (f 969) (s) (d¡ 22.4 51 .0 LUARD ; 6.95 'yG' r.8 t 0.3 ls8 (g) (a) i (T. rugosa) l9.rs 'yM' 6.7 + 1.0 9 53 22.4 77 ,O Thfs thesls

mammallan and yG immunoglobulin ,7Mr and ,7G, are to be read as analogous to the lsotyplcalty defined 7M classes resPectlvelY. TABLE 2.2 (cntd.) Table Immunoglobulln * antigens used to tdentify these immunoglobulins are detailed in 1.3. The recognlzed by thei¡ antigenic classes not yet assoclated wlth antÍbody actÍvlty but which have been wtth (**) superscrfpt and phystcoóhemtcal propertles have been designated a ' NM Not measured. (chapter (a) Garbohydrate determtned uslng the anttuone reactlon 3) ' method of winzler (1955) uslng (b) carbohydrate determined as proteln bound hexose ustng the orclnol mannose as a standard' (c) gsUmated value onlY. aqueous solutions and lts L and (d) immunoglobulln undergoes concentration dependent dlssoclatlon ln Thls hence the varlability in the molecular H chatns appeared to be held together by noncovalent lnteractlons; wefght determlnatlons. elatlvely hlgh ambtent (el Desplte repeated antlgenlc stlmulatlon over a perlo temperature,nodemonstrableanttbodyoftheughtwasfound.Itlsnotclearthe paddleflsh ltself whether thls flndtng ls a reflectlon of the antlgens ' was found whlch may represent a class of (f) An atypical slowly sedlmenung (.10,* = 5.95) immunoglobulln of this tmmunoglobulin was i¡nlque to the tungflsh. The molecular weight of the H chatn lmmunoglobulln or p chaln' approxlmately 38,000, hence it ti dfsslmtlar to either the mammallan 7 of the protelns' (g) Values quoted are the "lO,r gel method of rhorun and Mehl (1968)' (h) Molecular welghts determlned by a modiflcation of the acrylamlde (w) Weight average mblecular weights ' -57- It must be concluded therefore that structural differences exist between the naturally synthesised lemon shark 75 'yM' molecules and the 75 subunits obtained by reduction of lemon shark l9S 'yM' molecules. The natue of these dlfferences remains to be elucidated.

Perhaps the naturally occurring 75 '7M' Ímmunoglobulin Ís a prototype of the 75 'yG'immunoglobulin which fs evldent in the tetrapoda?

Polyvalent 75 'yM' an tibody has also been reported for the nurse shark (cited in Clem and Srnalt, 1967) but was not reported for the dogflsh (Marchalonis and Edelman, I965). As mentioned previously,

this may be due to the short immunization period employed in thf s investigatlon, but may also represent a specfes difference.

1.wo more polnts on the physicochemical nature of lower vertebrate immunoglobulins remaln to be nrentioned.

Firstly, despite the rest¡icteci, lsotypic heterogeneity of lower

vertebrate immunoglobulins, constituitive heterogeneity similar to that

posSeSSed by mammalian immunoglobulin has been demonstrated' The

studies of Clem and Small (1967), Suran and Papermaster (I967),

Marchalonis ancl Edelman (I965, 1966b, 1968), and also Pollara,

sr.¡¡an, Finstad and Good (1968) lnclude data on the electrophoresis

of reduced and alkylated lower vertebrate immunoglobulins ' These

data show that when assessed by this crlterion the constituent L and

H chains of these immunoglobulins are as heterogeneous as their

mammalian counterparts. CIem and Smalt (I967) consider thls to -58- be lnd'icalive of the presence of common (constant) and ciistinctlve

(variable) reglons as in rnammalian L and tr{ chains.

secondty, the possibtlity thai o'cher classes of immunoglobulÍn

i:re completely exist in Lhe more primiti.,ze lOi¡¡er vertebraLcs cannot dismissed. If , for some reâson, a certain class of irnniunoglobulin pari:icularly dici not manifest antibody activity Ít coulcl be overlooked' if present in relaLively low concentrations. In the lower fishes so far studiecl, however, this does not appear likely. For example, lemon clem and small (I967) failed to detect any other protein in shark L shark serum which could cross-react with their rabbii anti have chain sÊ*lnir. Since within a species differeriÈ imtirunoglobu]Íns ai¡sence similar L chafns, this lack of reaclion incij'cates the çrrobable ofappreciableamountsofotherhuiloralimrnunoElo]:ulins.

te D(ii) S ofl ver Thehallr¡arkofanyhurnoralitl.munologicalresponseisits specificity. This specificiiy is neciiated iry antibociy rrrolecules of similar v¡hicir have tlie capacity to cllscrimirrate betv;een ligancs extents: struct.,:re i:y conrbining with iirern to detectal¡ty ciiffereni structures the grreater the difference in affÍnity lorh¡¡o closely relatecÍ -þefore of antibociies the more specific the antibooy. Even the affirrity couldbemeasured,Landsteinerrecognizedtheimporianceof regarciingspecificityasacontinuousspectJumofaffinitiesby

defining specificity as the dÍsproporlÍonal actlOn of an anLii:ody -59 - (f .e., populatlon of anttbody molecules) on a population of related antigens (Landsteiner, 1945). Sirotinin (1959) clalmed to have shown that goldfÍsh antibody to horse Serum proteins was much less specific than the corresponding mammalian andbodyl That this does not appear to be the case was shown by the work of Everhart and Shefner (1966) who were prompted þ Sirottninrs statement to examine the speclflcity of fieh entlbody'

Even lf Sirotinin's data were correct, and this ls doubtful because

Everhard and shefner could not reproduce it, sirotinln's conclusion that flsh antibody was less specific than mammallan antlbody was not justifÍed. Because of the relationship between specificity and affinity of antibodies the degree of cross reaction between two antigens

does not provide a tnie index of specifici$; high-affinity antibodies

tend to have broader specificity than low-affinity anltbodies and

sirotinin mentions no data relating to the avidity of his fish antlserum'

Everhart and shefner (1966) compared the specificity of goldfish and the antt BSA with rabbit anti BSA by studying both cross reactlons avtdity of the antlbodies concerned. They were unable to detect any

cross reactfons between goldfish anti BSA and sheep semm albumin

(SSA), human sen¡m albumin (HSÐ and horse serum albumin (HoSA)'

* Strotlnin clalmed that " .. . when injecting goldfish (Grassius au¡atus) wlth horse serum nearly the same titre of precipltln against horse gerum as agalnst rabbit serum ls obtafned.'' -60- Rabbit antl BSA serum however cross reacted with the SSA 78%, HSA t4% and HoSA 12% when rneasured at the equlvalence polnt of the homologous reaclion. Uslng an lmmunoabsorbent Column - carbo¡y methyl cellulose to whlch BSA had been covalently llnked - they found thar the goldfish antlbody was much less avld than the rabbit

BSA antibody.

The guesÈ1on of the relative avldlty of lor¡ver vertebrate antlbody fs an lmportant one deserving of more investigatlon. Grey, (1963 and 1966), reported that relative to rabbit antl haemocyanln, turlle antibodies to the same antiEen were of low avidtty and frrther, that whereas the avidity of the rabbit antibody increased markedly durlng

the course of immunization, ttre avidity of the turtle antlbody remained

constant durtng a 1t3 day period following Ímmunlzatlon.

There are no other publfcations directed exclusively towards an

examination of the specificity anci,/or avidity of lower vertebrate anËbody. often, however, a passing reference to speclficlty has

been made fn papers dealing with other aspects of comparatlve immuno-

chemiss. Por example, Marchalonls and Edelman (1968) in their

investlgatlons of lamprey antibody to bacteriophage f2 found that the

serologlcally unrelated bacterioPhage fI was unaffected by the lamprey

antibodY. In general then, there is no evidence that lower vertebrate

antlbodies are less speclflc than their mammalian counterparts ' -6t- However, slgnfflcant dlfferences in the quality, i.e., avidtty of lower vertebrate antlbdy relatlve to mammalian antibody have been demonstrated. llltrether this difference wltl be found to be a general one, and lf so, whetherlt is important in terms of the evolution of the lmmune response remains to be determlned.

Ð(iil) N-terminal amino acid sequences of lower vertebrate immunoqlobulins

The rationale, either direct or indirect, of many studies on the nature of immunoglobulins, parücularly those on lower vertebrate tmmunoglobulins, is to count the genes involved in their productÍon and also to assess their evolutlonary relatedness. These aims are best achieved from a conslderation of the primary structures of the protelns involved.

The amtno acid Sequence of a proteln Ís in a sense the phenotyplc expresslon Of the information carried in the genome. The prlmary structure of a protetn is directly related by the genetic code to the

structure of fts colTesponding gene or cistron, and knowledge of a given genetic mechanism or the structure of a gene may be gained

by knowlng the primary stn¡cture of the proteln co¡esponding to thls

gene. A simple extension of these princtples in effect permits one

to determine the number of genes or clstrons involved in the synthesls

of a given protein system by studying either directly or indÍrectly

the structures of the proteins involved, In so far as the physico-

chemical propertles of a polypeptide are a reflection of i ts primary -62- Structure, these properiÍes provide a less satisfactory alternate means of investiÇating these ProþIems. only two studfes involving aminc acid sequencing of lower vertebrate immunoglobulin polypepticìe chains have T¡een pubtlshecl to date and both of these must be regard.eci as the vanguard of furfher more extenslve investigatÍons. Both reports clescribe the N-terniinal arnino acid sequence of certain primltfve ftsh H and L chains. A summary of

this data, together with the sequences of some mammalian L and H

chains for comparative purposes is presented ln Table 2.3.

Suran and Papermaster (1967) Cetermlneci the N-terminal hexapeptide

amÍno acid sequence of leoparo shark, T, sernifasciatg, H and L

chains derived from both 195 'y[tI' and 7ß '7lVI' immunoglObulins.

PoIIara, Suran, Finstaci and Gaod (I96S) exarnined the sequence of

the Ì.I-terminal penlapeptide of the L and H chains of paddleflsh,

P. spathula , 19S 'ylvÏ' immunoglobulin,

A nunrber of points of evolutionary significance have become

evicrent from ihe daia preSetr'ceci. in '¿hese two papers. Thse are AS follows :-

(a) The H chains of botþ leopard shark and paddlefish irnmunoglobulins

were found to contain free terrninal NH, moieties. ThÍs flnding

contrasts w-ith the presence of blocked N-termtnal resldues in

higher vertebrate H chains anci lambda type L chalns. The blocked

terminals of higher vertebrate polypeptlde chains are due to the l.tBLE 2 .3 N.TERMINAL AMINO ACID SEQUENCES OF THE t AI{D H POLYPEPTIDE CHAINS OF SOVIE IMMUNOGLOBITLINS

N-terminal amino acid residue No Species of animal Polypeptide Chafns (and reference) t23456 * LEOPARD SHARK l7S '7M' H chaln Glu IIe Val Leu Thr GIU (T. seml- fasciata) 7S '?M' H chain GIu Ile Val Leu Thr Glu Suran and Papermaster L chatn Asp Ile Val Leu Thr (r s6 7) 't*' ,l3t (25"/.1 Val pooled) iG.". Glu Glu * PADDLE FISH :19S'yM' H chain Asp IIe VaI lle Thr (P. spathula) Pollara et al. Pooled L chains Asp lle Val IIe Thr (1s68) 0

MOUSE Kappa ¡Asp lle Val Leu Thr Gln Hood, Gray and L chains Gln Met Dreyer (1966) VaI

HUIVÍAN Kappa Asp lle Val Leu Thr Gln Niall ano chaln GIu Gln Met Edman (1967) and 75 myeloma Glu lle Val Leu thr Gln Hood, Gray and L chains Dreyer (1966) + Hood ancl :Lamocia PCA Ser Ala Leu Thr Glu Ein (r 968) :L chalns Pro Ala * - Mu¡o, residues iclentified. Values fn parentheses are yields cal- culatecl as moles phenylthiohydantoÍn (FTH) amino acids per mole of L chain (22,QOg g.) or H chain (7Û,000 g.) uncqrrected for procedural losses. + The N terminal amino acid residues of most mammallan lambrla L chalns and H chatns are usually blocked by the cyclization of a gluta- mlne residue to form pyrrolid-2-one-5-carbo>ry1ic acid, abbneviated PCA. -63- cycltzôtion of a terminal glutamine residue to form pyrrolldone

carboxylic acfd (abbreviated PCA) -

(b) Leopard shark l9S '7M' and 7S 'yM' H chalns were found to have

almost ldentlcal N terminal hexapeptide amino acid sequences '

Thls data substantiates that descrlbed earlter where the ldentity

of l9S 'yM' and 7S 'yM' H chains was flrst postulated'

(c) An lmpressive degree of homology was found between leopard

shark and paddlefÍsh L and H chains. In addition, a slmilar

degree of homology exlsted between these chalns and kappa L

chatns of human and mouse origin. Such evÌdence adds credence

to the hypotheses that the immunoglobulin polypeptide chalns

had a common ancestor and that this primcrdtal gene was of the

kappa tYPe.

(d) Neither Suran and Papermaster (1967), nor PoIIara et al' (t968)

have discounted the possibility that chains with blocked N-

termlnal residues may exist and have Stated their intention to

Investigate this aspect further. Pollara gl-at. (196S) reported

a maximum of.25"/" recovery $ncluding estimates of procedural

losses) of N-terminal aspartic acld from thelr paddlefish L chain

preparation. They suggest that this low recovery may be due to

the presence of another population of L chalns with blocked

N-terminal resldues, presumably of the lambda type' -64- (e) Pollara et al. (1968) have made a further lnteresting speculatlon

that the homology of the N-termlnal sequences of immunoglobulln

chatns of species as dlvergent as sharks, mouse and mau

tndlcated that a relatlvely htghlY evolved situation was being

examlned, and consequently that the immunoglobulin ancestral

genes must have arisen ln prevertebrate forms. In addltion,

Suran and Papermaster (1967) have suggested that the conseryation

during evolution of structural homologles fn the N-terminal

seguences of the L chains they examined Índicates that these

structures may be essential for antibody function.

C. CONCLUDING REMARKS

From the survey preselìted four main points can be made. These

are

(i) The production of immunoglobullns appears to be restricted to the

vertebrates.

(Íi) Alt the immunoglobulins which have been lnvestigated have been

shown to consist of mul|ichain structr¡res composed of polypeptide

chains whlch are clearly analogous to mammalian L and H chafns ' (iii) ln certaln specles fewer classes of immunoglobullns have been

observed than ln mammals. However, electrophoretic analysls of

these immunoglobullns has shor¡vn that a constitutive heterogenelty

slmflar to that found ln mammallan lmmunoglobuling does occur' -65-

(iv) It ls possible that the ancestral immunoglobultn gene was of the

kappa L chaln type since amfno acid sequence data, although

llmlted, indlcates an impresslve homology of elasmobranch

lmmunoglobulin L and H chalns with mammalian kappa type L

chains.

It seems that lower vertebrate immunoglobulins represent a trigttly evolved situatlon and that recognlzable lmmunoglobulin genes must have arlsen fn prevertebrate forms . It nray weII be, that as Dreyer

Gray and Hood (1967) have stated, the basic genetic mechanisms which now control the blosynthesfs of immunoglobullns inltially evolved " for functions having little or no relatlonship to the vertebrate lmmune response. "

The work presented in this thesis was commenced to gain more information about the synthesis and structure of llzard immunoglobullns ' -66-

CIãAPTER 3

Paqe

A. COLLECTION, MATNTENANCE A¡TD MA}TIPULATION OFLEARDS ...... 68

B. ANTIGENS USED ...... 70 C.ANTIBODYÀSSAYS...... 7l 75 D. ANALYSIS OF ANTIBODY IiV LEARD SERUM ' . ' (i) Analytical reduction with Z-mercaptoethanol' ' 75

(ii) Sucrose density gradlent ultracentrífugacion ' ' 76 E.PREPARA'TIONOFAIVTISERÀ...... "' 76 77 F.CHENIICALSAI\IDGENERÄLCTIEMICAI'N{ETHODS" (i) Chemicals .. - ' " 77 (ii) Buffers ... 77 (iii) petermination of proiein concentration .. ' 7B

(iv) CarUyhycirate cietermination ' ¡ ' 79

(v) Concentration of protein solulions ' t ' ' 79 (vi) Dialysis ... ." "' "' BT (vii)Ultracentrifugation ". "' "' BÚ (viii) Amino acld analYsls ' ' ' 83 6 7

Paqe G. FRACÎIONATION OF T.TZARD SERUM 83

ANAInICAI PROCEDURES

(i) Bectrophoresls ... 83

(ii) Ouchterlony analysis . -. - B3

PREPARATI\Æ PROC EDURES

(tü) Sodium sulphate precipltation 84

(i*r) Cet flltration . . . 84

(v) Ion exchange chromatography 85

(vi) Zone electrophoresis . . -, 86

(viÍ) Removal of lipoproteins. . 87

TT. INCORPORATIOI$ OF IìAÜIOACTIVE ISOTOPES INTO Í]ACTERIA AND PROTEINS¡ ...... 88 ? (i) Incorporatlon of P3 irrto S. tvphimurium . . . 88 131 (ii) I¿belling of proteins with I- . - . ... 89

I. REDUCTION OF LøARD IMMUNOGLOBULINS 90 68- In thts chapter the materfals and methods useci in the experiments Minor ciescribed ln the following chapters are set out in detail ' below have techniques and modificalions to tlre techniques described been occasionally been used in individual experiments and these have noted in the approprlate places in the text'

A. * known in The ltzards, (Figure 3.1) Xl!!guaÆ' commonly were collected south Australia as the 'sleepy' or 'stumpy tail' lizard,

from the Port Pirie and lVlurray BrÍdge disiricts of south Àustralia'

Lizards were used as soon as practicable after capture ' thermostatlcally In all biologÍcal experiments the llzards were kept in battery of controlled boxes. I-Teat vúas supplied co Lhe boxes using a The tops of tl:e ¿l x 15Û waÈt lamp oulbS ccntrolleci by therri,ostat. on wooden frames boxes were coverecl with plastic sheeting niounteci Hessfan bags to provtde insulatlon ancl to permÍt suntight to enter' of the were provtded for the lizards to lie under. Rectal temperatr'res the experiments Iizards were recorded at various intervals throughout from the temperature and were never found to vary by more than ! lo

stipulated in the text.

* (I950) used in thls thesf s The revÍsed nomenclature of Mitchell is ' lizard was prevtously called Trachvsaurus-ruqosus ' Mitchell This a found that the only feature separating rusosus from llllggg'was to be an modlffcation (shoriening) of the tail ' I{e considered this phylogenlc cha¡acter' example of speciallzalion rather than a basic AccordlnglyheproposedsuppressionofTrachYsaurusandreferredgeneral ruqgsus (as ruoosa) to X'itiqua. fnts proposat is now finding acceptance. Flgure 3.1 Photograph showfng the lizard, .Ilflgg-lggg.eEl, used throughout thÍs study.

It is commonly called ifie " sleepy" or

"stumpy tail" Iizard.

(Length of speclmen about 10 inches)

-59-

The llzards were fed ad [ib. On bananas, mfnced meat, tomatoes, applès and eggs. Vt/ater was conlinuously proviti'ed'

BLeedinq of Lizards using Lizarcis were blei by cardiac puncture, wlthout anaesthetic, was inserted a hypodermfc syringe fitÈed with a 2? gauge rreecile wirich under the foreleg of the lizarcì. Immediately after the needle was procedure, removed the area v¡as swabbed with 707c alcohol. This ensured together with the precaution of using only very sharp needles ' that a lizard could be bled many times ' that Lizard blood was found to olot rapidly and it v,'as also found particularly if Seconclafy clots often formed in the separated serurii, had clotted' it had been rernoved from i:he red cells shortly after these blood overnlght ThÍs cilfficulty was overcome by leavlng the clotted was at 40 before removing the serum. The usual procedure adopted Itwas io allov¿ the blood to stanci at room temperature Íor I hour' was removed' then 'ringed', and refrigeraied overnight before the seruÍrÌ

The iniection of Lizarcts -were routes, Throughout rhis project lizarcis lnjeciec. iiy three

intramu s cul ar, i ntraperi toneal atrci intravenous' part the leg Intramuscular lniections were made iirto the fleshy of ' only one All foru IeEs were used. as the occasion demanded, although injection was glven in each leg during muliipte injection schedules ' -70-

Intraperitofreal lniectiogs were performed by injectlng beneath a scale on the ventral surface into the perf toneal cavl,ty.

Int¡avenous f{¡lectlons could not be made directly, due to the scaly exterior of the llzard. A small lncision was made on the front ventral surface of the llzard's foreleg while lt was under ether anaesthetlc. The tissues were gently separated and a veln exposed, whlch was then injected with a 30 gauge needle. After lnjectlon the skin was pulled together, sPrayed wlth plastic skln, and bound with adheslve plaster, Llzards injected intravenously by thls method remained ln good health for at least slx weeks.

B. AÀIÎIGENS USED

Sallqonella tvohlmurlum the A smooth str.aln of -s:_u¡himurfgm, which had been typed by Instltute of Medlcal and Veterinary Science, Adelafde, and found to have Kauffman whlte o-somatlc anugens l, 4 , 5 , L2 and flagella antigens I and 2 present was used. Two subst¡alns (M206 and CS) were avallable (after Furness and Rowley, 1956).

A vacclne was prepared from an overnight shaken broth culture '

The organlsms were washed once ln saline and then resuspended in alcohol at a concentration of about 109 permlandleft to stand overnlght at 40. they were then washed twlce in saline and resuspended in sallne at 2'¿ x l0l0 organlsms per mLand stored in two ml.allquots at -2Oo until requlred. Numbers of organlsms -7L- were determined by viable counts prior to the alcohol killing and also by turbtdfty measurements of the killed suspension at 650 mp in a o Shlmadzu spectrophotometer, Al/30 irilution of liJ" organisms/ml. was founcl to have a turbidity at 650 m¡r of C.16. Bovine senrm albumln Bovine serum albumln (Commonwealth Serum Laboratories, Parkville,

Melbourne) Cohn Ftactlon V was clissolved in saline at the appropriate concentratlon. Rat ervthrocvtes Rat erythrocytes were obtalned by cardiac puncture of chocolate

brown rats. They were collectecl into Alsevers solution and washed

three Limes ln saline before use.

C. AI\TTIBODY ÀSSAYS Llzarci antlbody to rat erythrocytes was assayed by dlrect haemag- glulination. Antibocty to S. typhimurlur? was determined by a passlve haemagglutination assay. AntÍbody to BSA was determineci by Ouchterlony clouble ctiffuslon, quantitative precipitation or by a passlve haemagglutination aSSay. These rnethods are Set out below'

Dlluent, for haemaqolutinatÍon as sgvs * Lizard serum was founci to lyse sheep erythrocytes, particularly *- It should be mentJonecl that the backgrounct tltre of lizard sen¡m to sheep erythrocytes dld not lnterfere w!Ëh the passlve haemaggluti- naLlon assays descrlbed above. Provtclecl the lytfc effect of llzard serum was ellmlnated by the use of d'iluent containlnø NaTEDTA the backgrouncl agglutinatÍon litre was never greater than 1:2'- Hence it was not necessary to absorb sen¡m with normal sheep cells prlor to tltratlon. 72- lf coated with BSA or bacterial lipopolysaccharicìe, io a dllution of l:64 or I:t28. Thts lysis could be prevented by prior heaLtng of the lizard serum at 560 for l0-20 mlnutes, or by lncorporating d,isodlum e thylenediamine te (NaTEDIA . ztl AR) in the dilutl'ng fl uid traacetate Zt. used. The latter method v,¡as chosen for preventing lysis and in all the haemaggluiination assays used NaTEDTA (C.003M) was added to the diluent.

The compositlon of ihe diluent was as follows :-

Saline (ü . 15M) I0Û ml. Aqueous KIIAPO4 (lM) 4.4 ml. Equivalent to 22.0 rnl of 1M phosphate buffer, pH 7 .4 Agueous Ksi{OP4(IM) 17.6 mI.

NaTEDTA ZHZO (ÄR) I.I2 s - Distillect .¡¿ater to nrake 1.0 litre ü,t2% sodium azicie vt'as added to prevent contêminatÍon'

LizarcÍ anii rat ervthrqgytes

Dfrect haemagglutinations were carrieci out using a fir¡al suspension of û.5% 3 x washed rat eryihrocytes tltratee agains¿ serlal lvvofold oilutÍons of serum,

LizarC qntÍ Salmonellq tvphimurium

A modÍfication of the passive haemagglutlnation technlque of

Crumpton, Davies and Hutchison (1958) was used.

A 2,5"/" suspension of 3 x washed sheep erythrocytes was mlxed with a solution of 5Û vg./rnl.of alkali'ueated S. tvphimurium

Iipopolysaccharide. The lipopolysaccharide was prepared using 73- the phenol extractlon iechnlgue of Westphal, Luderitz and Bister

(1952) and the alkali sensitizalion was carrieci out as described by

Crumpton et al, (I958). ilhe ceils were incubateci with the lipopoly- saccharid.e for I hour at 370 anci then washed three Ënles with cold saline and suspended at I% v/v in saline.

To establish optimal conditions for sensitizalir¡n of the erythrocytes, various concentrations of alkali treateci lipopolysacchêride were tested for production of the htghest titre against a hyperimmune rabbit dntf -[!yphl4!$g!Ê serum. In thls way 5Û Fg. of lipopoly- saccharide per mI. was found to be a salisfactory sensillzing concentratlon.

Lizard anti BSA

Tluee assays for BSA were used. In those instances where a qualiÉattve indicatfon of B8Ä antiJcoCy was desired the Ouchterlonl'

method' descrfbed by Campbell, Garvey' Gremer and sussdorf

(1964, page 145) was used. Absolute amounts of BSA" antibody were

determined using the quanlitaÈfve precipiLin technique (Campbell

qtêI!, !96+, page f 31ì). Usually quantitative estjnraiions of BSA

antibodywere carried. out using a possive haemagglutinalion technÍque.

This particular assay is ¡iot wiciely ulilized a¡rd is described in

detail below.

Passive haemaqqlulination assav for anti BSA

A modlfication of the method of Gold anci Funcienberg (1967) was -74_ used in whtch BSA is coated onto sheep erythrocytes through the agency of chromium ions, C.3*.

Three times washed sheep erythrocytes were sensitized as follows :

Tv.renty volumes of a 5% v'/v'suspension of erythrocytes were sedimented tn a wide bore centrifuge tuoe and the supernatant fluÍC renroveci. One volume of a 17á sallne solution of BSAwas added anci rnixeo by careful shaking. Immeciiatcly follov,,Íng thls, I volume of a freshlv,prepared A.l% salir¡e solution of chroriiic cì:lorlcÍe (CrClrAR) was aclded and the mlxture ir.r{nediate}v mixed Ly gentle shaking.

The cells were then left to stand on the bench for 6Ü rninutes. The sensiiized cells were then diluted wlth 50 volumes of saline and washed 3 x with cold sallne. A I% v,/v saline suspenslon was prepared for use.

The sensÍtivity of this assay for BSA aniibody r¡;as deterrnined by tttrating dilutlons of a ltzard antl BSA serum contalning amounts of

BSA antibody determineci by the quantitative precÍiritirr method. The

titration was carried out sirnilarly to the other haernagglutination

assays descrfbed above. The experimentally determined correlatÍon

'between haemagglutination tiire of a sample of lizarci serum and its

BSA antibody content (pg . ) is siiown in Figure 3 ,2 .

Antibodies to BSA v{cre è}sc ..,ecected by passÍve l-raemagglutlnation

assays using sheep eryihrocytes to lvhich BSA iraci bc,en attached by

the i:is ciiazobenzidine ancl tannic acici methods (loth described by

-:;_ Fis. 3 .2 Lizard anti BSA serum haemagglutination tÍtre as a function of the concentration of i8 BSA antibody (pg ./mI. serum).

15

I q) ir ! -d H 10 c o -d +¡ rd É -d ! J E't o o' o É o 0) rõ E o C\¡ Þ o 5 tsl

o

1.0 2.0 3"0 4.0 Iog dilution of serum 10

53 00 s30 53 5.3 0. s3 BSA antibody Ín serum (vg./mI.) 75-

Danials and Stavltsky, f 964). The chromlum method, however, was found to be the most reproducable and stratghtfortrrard to perform.

D. A¡TALYSIS 9F AI{TIBODIES IIit LIZARD SERUM prelimlnary data on the nature of lizard antlbodies was obtalned by measurlng thelr suscep|tbllity to reductlon wtth thiols, and by

analytlcal sucrose density gradlent ultracentrlfugatlOn. ß) Ar¡alvtical reductlgn with-Z: mercaptoethanol

The susceptlbility of llzard antlbody to reduction wlth thÍols was

determined with 2-mercaptoe thanol (abbrevlated 2 - ME; Chemische

Pabrik, Switzerland)' The following rnethoci was used :

I volume of serum was acided to I volume of a [Ì ' 20 M solutÍon of 2-ME in phosphate buffered saline pH7 '2' The mlxture was then incubated at 37o for I hour' Re- oxldation of the sH groups formed durfng this reduction was prevented by alkylatlon uslng a 2 fold molar excess of iodoacetamlde. The alkylation was carrled out in an lce bath for 5 minutes. Thls mlxture was then tltrated in the usual manner.

It was found by experlence thôt ff the tltration of reduced ltzard

serum was çarrled out ln a haemagglutinallon assay diluent containlng

0.02M z-ME, recovery of reduced lizard andbody actlvlty did not

occur. Consequently many titratlons of reduced lizard serum samples

which had not been alkylated were carried out in thls modlfled dlluent. - lrõ -

(11) Sucros e densttv oradiqr¡t ul tracen trl fu qatlon

Preltmfnary analyses of the slze of llzard antibodles were determlned using sucrose denslty gradient ultracent¡ifugatlon'

A gradient was formed by layering in cenHfuge h¡bes I mI. each of l0% , 25"/o, 35% and 40% solutlons of sucrose (¡R) fn phosphate buffered IM sallne, pH 7.0, Approxfmately l0 ng. of proteln (0.5 ml. of a l:3 dilutlon of llzard sen¡m) was layered on top of the gfradlent. The loaded gradtent was then placed Ín a

Splnco SW39 rotor and centrlfuged at 35,000 r.prn. for 18 hours in a splnco Model L ultracentrlfuge. The gradlent was fractionated

by puncturlng the bottom of the tube and collecting the effluent with

the ald of a drop countlng fractlon collector '

Mammalian l9S and 7S antlbodies were used aS markers in

estimatlng the slze of the llzard antlbodles. Estlmates of the

seömentatlon coefftclents of the sedimentinE llzard antlbodles

were also obtalned uslng the method and tables of McÉþen (1967).

E. PREPARATION OF AI''ITISERÀ

Antlsera to llzard serum and to lizard imrnunoglobulfns were prepared tn rabbtts by two lmmunlzfng procedures '

Bebbit anti lizard serum Rabblts were given t mt, of normal llzard serur[ emulsifled in

complete Freund's adjuvant,intramuscularly followed by four doses

of 0.S ml. of lfzard serum int¡avenously at weekly intervals thereafter. -77-

About two weeks after the flnal inoculation the rabbits were bled by cardlac puncture.

Rabblt anti llzard lmmpnoqlobulln

Since only small quantitjes of llzard immunoglobullns were avaÍIable, a different lmmunizing schedule was used. The protein

(approx. 3-5 mg.) was mixed with complete Freund's adjuvant and

emulsified wtth a sonic probe untll a stable emulsion was formed.

This mlxture was then injecteci subcutaneously at many sltes along

the back of the rabbit and also into the footpads. Tvro to three

weeks later, after satisfactory test bleeds, the rabbit was bled by

cardiac puncture.

F. CHEMTCAI.S AI{p GFNERÀI CH-EMTCAI M,EIHODS

(i) Chemicals

'Analytical Reagent' (An) grade chemicals were used ln preference

to laboratory reagent grade whenever possible. Where signlflcant, the

use of an analytical grrade chemical is deslgnated in the text by AR'

(ii) Buffers

The compositions of buffers are stated in the appropriate places

in the text and were taken from Documenta GeiEf (Dfem edttor) 6th

edition (f 962), orWllllams and Chase (f 968, Pâ9ê 365).

A phosphate buffer system, (KTLZPO4/KZHPOa), was used for

cellulose ion exchange chromatography. The relationship between

the molar ratio of KTHPO* and KH,PO4 and pH for this system was 78- determined empirically and found to be

pH = 6.71 + 1o910

sod.ium azide (ts.ü2"/.'l was usuatly added as a preservative to all buffers, with the excepiion of those buffers intencied for use with cellulose ior¡ exchangers.

Trls(hydro¡rymethyl)aminomethanehasbeenabbrevlated to tris.

(iii)Uetermination ot Pr

Relative pfotein concentrallons were determirred using either the

Lowry rnodificatlon of the Folin-Clocalteu rneËhod Éowry, Rosebrough,

Farr and Randall, 1951) or by measuring opttcal densities at 280 mp

with a shlmadzu spectrophotometer. The Folin niethod was particularly

useful in those fnstances in u'hich the proiein v;as cüssolved in a

buffer such as veronal which absorbs at 28C m¡r'

Absolute estimates of protein concentratiorr v';ere determlned ln

huo ways. rv\Ihere thu sample was plentiful, exhaus'tÍve dialysis

against a volatile buffer, ê.Sr ôInßonium carborrate, followed by the lyophillsation anct weighing w'as utilized. Grenerally, liowever,

exrincricn coefficie"t Gl;; 2':rJ mp )was useo- to coiivert opïical

clensÍty readings at 28t mu into mass units '

For these purposes the following extinctior¡ coefficients were

used : 79-

1o1 7 + (measr.¡red) E:'"lcm BSÀ = .0 0.2 _r% normal lizard serum + 0.5 (measured) Ë-Icm = l0.B

_7o/. lmmune lÍzard serum 10.8 + 0.5 (measured) !;-lcm = _L% E: " lizard 13.8 1.0 (measured) Icm 'yG' = ! tif- lizard'yM' = I3.0 (assumed)

(iv) Carbohvdrate determf nation

hoteln bound carbohycirate was deterniineii as hexose usingr the anchrone method and a glucose standard. Anthrone determinations were carried out uslhg the technique described ln Kabat and Mayer (f 96f ) with the following minor nrodification which gave nrore reproducable results : tÌre reagent rnlxture containing anthrone solution anci sample was placeci in a t¡olling rlvarer oath for 3 niinuÈes insteaci of a !0o water bath for 16 minutes.

(v) ConcenÍraLion of protein solulions

The following methods were used for the concer¡traliorr of protein soluiions :

(a) Oialysis against solid sucrose followed by dlalysis against an

appropriate buffer. The high concentratlon of sucrose obtained

inside the dtalysis bag prevented clenaturation of the protein.

(-þ) DÍalysis against 'Aquacide II' þroduct No. 17851 of Calbiochem,

Los Àngeles, U.S.A.), followed by dtalysis against an approprlate '80-

buffer. Thls method was particularly useful where large amounts

of sucrose were undesirable and was used extensively during the

la¡-ter part of the work.

(c) Îüegative pressure oialysis. Sartcrius mentbrane filters (Cat.

itto. 132û,J) surrour¡cled by oilute buffer urrder negatirre pressure

were used for the soncentratiorr of purifieci or partially purified

immunoglobulins .

(o) Occasionally samples were concentrated by the acidi'LÍon of solid

or a satlirated solution of ammonÍum sulphate (4P,,) at 40 to a

Íinal ÇoncenÈratfon of 5Û% saturatior¡. The precipitaies formed

were centrifuged and re-dissolved in a suitable buffer.

(e) tyophilization after exhaustive dialysis against volatlle buffer

was somellmes used. Lyophilization causes loss of antibody

activity and was only used where biological aclivity was no

longer required.

(ø) Piaivsts

viskinE dialysis tubirig (unicn carbide cor¡:, cl:icago, u.g.A.) was llseci tlrroughout. sizes 8/92, lJ/sZ, 23/?i and c55 were

avaÍIabte . Síze IS/32 was used for the dialysis of reduced immr¡no-

globulins because of its smaller pore size.

(vii) Ultracentrifuqaticn

Preparattve ult¡acentrifugation, (for exanrple during the purf-

ficatlon of s. tvphlmr¡rium lipopolysaccharide) anrj sucrose ciensity -81 - gradient ultracentrifugation (describeci in section l-(ii), Chapter 3), were carried out in a Beckman Spinco MoCel L ultracentrlfuge.

A Beckman Spinco lvÍodel Ë analytical ultracerrtrifuge was used io determlne size heterogeneity of serum and serum fracH.ons, to measure sedimentatlon coefficients and to make direct esÉmates of the molecular weights of purified lmmunoglobullns.

(a) Measurement of sedlmentatlon coefflcients

Sedlmentatlon coefficients were estimated from temperature

controlled sedlmentation velocity experiments using schlieren

oplics under conditions described ln the appropriate places

in the text,

Apparent sedimentation coefffcients ( s ) ciefined rry the eguatlon,

s (ln svedberss) = *2. * ^:dt- I (where x !s the distance fronr the rniripoint of rhe seolmenli,ng pea!< io ¿he axls of rotation, | |s the Liriie it: secoricis and r'¡ is the angular velocity of ro¿a¿ion)

\,vere calculated qsinE the iritegraËeci forrn of the above equatlon,

or by the rapid graphical neihod describeci by lvlorkham (1960).

Standard sedlmentation coefficients, that is sedimentation

coefflcients corrected So as to colrespond to a reference solvent

having the vlsCoslty and denslty of wôter at 20o, were esttmated

by the usual equaLion (Schachman, 1959, page 82)-

Denslty and viscosity data for the various solvents useci were

taken from the Tables published by SvedberE and Pedêrson (f940) 82-

and by Kawahara and Tanford (1966). Values for the partial

specific volumes of lÍzarcf immunoglobullns were calculated from

the amlno acid compositlon data presentecl in Chapter 5; the

procedure for doing this is described in cohn anci Edsall

(r 943 , pege 37 5) .

(þ) Size heieroqeneitv

The sample in question rvas subjected to a sedimentaliorr velocity

experiment using schlieren optics and the relaiive proportion of

each constituent was esH.rnated by measurement of the area urrder

each peak expressed as a percentage of the total ôre¿. Since

secÈor-Shaped cells were usecl, these fÍgures \/ere corrected for

radial dilution (Schachman, 1959, page 7t)'

(c) Direct estimation of mclecular weiqhf

Direct measurements of the molecular weights of lizard lmmuno-

globulins were lr¡ad€, usirrg the high speed equilibrium (menlscus

depletion) method described by YphanÈis (1S64). Equilibrtum

was attained at ZSo usirrg 3 mrn column heÍghts in a double

sector cell anci interferertce optics ' Photographs vdere taken on

Kodak II G plates and measurements froni the photographs were

carried out wlth the aia of a microcornparator '

The calculation of weíght average molecular weights from the

experimental data was facflitated using a computer programme -83-

(see Appendix l) written by 1fr, D.f . Fennell* and the Unlversity

of Àdelaide's CDC-64Û0 digital computer. Viscosify density

and partial speclfic volume data were obtafned. from the sources

mentioned irr sectlon F (vif) (a).

(viii) Amfno acid gnalvsis

All amino acÍd analyses were carrÍed out in ihe Department of

Biachemlstry, Unlversity of Adelafde, using a Technicon automatic amino acid analyser.

G. .Fry\CTIONATIOhT OF LUARD SEIUM An,alvlical procedures

(i) 4lectrophoresis

Immunoelectrophoresis in agar EeL and elect¡ophoresis on paper

and cellulose acetate strips were lrsed to analyse lizarci serum

and preparations of lizar

were used whlch ariÊ summarlzed in Table 3 ' l '

(il) Ouchterlonv analvsis

This technique was used to investlgate ant'igenic cross reactlons

between lizarci immunoglobuli,ns, and to qualitatively detect BSA

antibody. The principles of thls technique have beerr revlewed

by ouchterlony (wefr, 1967 , page 655). The technlque used

* Departrnent of Physical and Inorganlc Chernistry, University of Adelaide. TABLE 3.I

Supportlng medlum Name Purpose Reference and buffer system.

Immuno- 0 .B% Ion agar (O>

Strlp Cellulose Acetate Analysis of Cellulose electrophoresls (Oxoi¿, U.K.) whole Acetate: Paper: lMhatman serum \Â/llltams & 3MM veronal Chase (1968) buffers pH 8.6 Paper: Beckman Splnco Manual No. RIM-S with minor modiflcaLions.

Starch Gel Hydrolysed starch Analysis of Method of electrophoresfs (Connaugttt M.R. reduced and Edelman and Lab. Canada) in alkylated Poulf k (1961) . 8M Urea and immuno- formate buffer globulins

PVC zone Polyvinylchloride Purificatfon Method used electrophoresf s (Pvc) I. c.I. Corvic and isolatlon described D55,/9. Veronal of serum page 86. buffers pH 8.4 protein fracllons ' 84- was adapted with minor modifications from campbell, Garve¡¡,

Cremer and Sussdorf (1964). heparalive procedures

(iii)Sodfum sulphate p

This procedure was used to prepare lizard serum fracLions of

enhanced immunoglobulin content.

AZ4o/" w/v solution of anhydrous sodium sulphate (ÀR) in Û.05M

.k tris- HGI buffer pH 8.0 wes added to the protein solution untll the

desired concentretlon, usually 14% ot 16%, was obtained. The

sodium sulphate solutlon v¡as added slowly with slirring an<Í at

room temperat]¡re. Aiter 10 r¡iirrutes the precipitate was collected

by centrifugation and n'ashecl lwice in a sodium sulphate solution

of 'lhe ciesireci final cor¡centJaiion. The washeci precipitate was

then dissolved in e minimel volume of the colci l:uffer reguired for

the next fractionaLion step; this was usuaily 0.05M tris I{CI pH 8'Û'

The dissolved precipitate was lhen dialysed agalnst ltt volumes

of the buffer in which it was dissolved.

The concentratlons of sodtum sulphate used for precipitating

Iizard immunoglobulins are described in the text of Chapter 5.

(iv) Gel filtration

sephadex (Pharmacia Fine chemtcals, uppsala) was used for

tris - tris ftrydro:

The columns were made from good quality glass tubing of the deslred dimenslons and lvere consbtlcted so that mixing of the effluent volume was minimal. The irase of the column was made had using a rubber bung through which a 17 gauge syringe needle mesh been pushed and over wiúch ccarse and fine layers of nylon

had been placed.

(v) Io+ e4ghanqe ChromatoqraPhv

Ðiethylaminoethyl cellulose (ÐEAE) anci carboxyrnethyl cellulose -vV.B.lì' Balston Ltd', (CIVTC) C\\Ihatman lon-exchanEê Celluloses,

Kent, U.K.) were used. Eachfonexchangerwaspreparedforusebywashingwith0.5M with sodium hydroxide and 0.5M hydrochlorlc acld and equilihration

the appropriate buffer as descrlbed in the experlmental notes

supplted by the manufacturer (whatman Acivanced lon-Exchange

Celluloses - Technical Bulletin IE2) '

Before application to the ion exchange cellulose the sample batch lvas equllibrated wlth Èhe starting buffer by cialysis ' Both

and gradient elutton technique were employed ' 86 -

Linear or concave gradients were formed with the aid of perspex gradÍent devices scheniatically shown |n Figure 3.3, which also shows the equation describing the gradients so formed. By varying the volumes of solutions useci and their initial concentrations the desired gradient elution profÍIe coulci be olctained (adapted from

Peterson and Soþer (i962).

(vi) Zor¡e elec troPhoresis

Zone elecirophoresis was carrled out usipg polyvinyl chloride powder (PVC - Imperial che¡i:ical Industries of Auslralia and New

Zealand - Corvic D. 55,/9) as 'ihe supporting mecìiuni' This technique was primarily used for rernoving iract+ in;purities, particula fly armacroglobulias, from preparatioi¡5 oÍ immunoglobulin '

occasionally whole lizard serurÍi v,¡as subjecteci to zone electro-

phoresis as the first step in ffactionalion of serum, but the relatively small sample capacity llmited the practlcability of this

method.

The following method lvas used :- (ionic PVC powder was fined and wast¡ecl v'rith veronal buffer

strength 0.05, pH 8.4). After renrovlng surplus buffer, the PVC (50 cm) suspension was poured irrto a rectangular perspex tray cm x tl

lined with thin polyethylene plastlc sheeting. The tray had one

removable side edge whictr facilltated applícaiion of sample and

subsequent cutting of the PVC i:lock into strips for elution' Figure 3.3

Dlagrammatlcal representation of the apparatus used for produclng linear and concave concentratlon gradients for DEAE and CMC fon-exchange chromatography.

The gradients produced by the apparatus are described by the

equad.on : Az/At cT = c2- (cz-cLl (Ì-v)

A linear grradient ls formed if A1 = A, (fiøure (a))

A concave gradlent Ís forrned if Al > a, (fleure (b))

M Vlagnetic stirrer

Al, A2 Cross sectional area of the perspex cylinders c Initlal concentrations of the solutions t' 2 placed in each perspex cylinder: the gradient formed extends from C, to Cr'

v The fraction of the total volume of the gradient eluted, i.ê., o 1 v < L.

c The concenl¡ation of the gradient at time T T wherr a fraclion v- of the total volume has been eluteci, l.re ., c14 Çr1Cz. Ar Az I Az Ct Cz I Cz

_F outlet outlet M It t ///tt///t/ /tt tl, /rt t ttrrtlrlt// ,/ tt t ,/t/

c2 c2 Co c E o o c o o C) =o € 3n ct E"t o.25 0.5 l€ 0425 0á Ù75 to Y Y Linear Gradient. Concave Gradient. -87-

The PVC block was drled with absorbent paper. A, I cm. wide

segment wàs then removed from the block and cirieci s$.ll further with absorbent paper. The sample was added to this segment whlch

was then replaced ln the block, care brging taken to ensure adeguate

contact between the sample segment and the rest of the block.

Electrophoresis was then carried out ln a cold room at 6 volts per cm.

for 36 hours. When electrophoresls was completed the block was

cut into I cm. wlde segments ani each segment placed Ín a 50 ml.

ceng.fuge tube containing 5 ml. of cold veronal buffer. After rnixing,

the slurry fcrmed was filtered through a sintered glass funnel under

slight negallve Pressure . the protein content cf eacþ fraction was then estimateC, uslng

the Folin Lowry tecþn1que (Lor,vry et aI. f 951). Protein concentrðtion

and antibody activity profiles \rere

fractior's pooled and concentratect.

(vll) Removal of llooProteins

Contaminating lipoproteins were often present in preparatfons of

lizarci ryM' immunoglobullns. These were removed by the ultraCen-

trifugal flotatlon method of Havel, Eder and Bragdon (1955).

In thls method the denstty of the proteln solution is increased

from approximately l.0t {i.e. 0.15M sallne) to 1.20'oV the additlon

of a concentrated salt solution of denslty f .346. The resultant

mixture is then centrifuged (in a Spinco Model L ultracentrifuge) at 88-

35,000 rev/min. for 6 hours irr an SW39 rotor. Any lipoprotefns present float on the surface of the protein solution and may then be easlly removed wfth a fine pipette or by ustng a cent¡lfuge tube sUcer.

The concentrated salt solutjon contains 153 .ii g. of sodium chlofide and 354.0 g. of potassium bromide per litre' In general' the volume (B) of this stock solutlon of density 1.346 necessary to raise the densfty of a volume (A) of a protein solutlon wlth an fnitial densfty of I .005 to a density x, is given by the equation :- l.005xA + l.346xB = (A+g)xX

When the destred resultant ciensity of the mixture is I.20 the volume

(B) of the coücentrated salt solution to be acfded io a given volume

(Ð of protein solution is giverr bY : B = 1.345 A

subseguent to the removal of lipoprotelns the hlgh salt concen-

tration of the proteln solulion may be reduced by dialysis.

H. AI\TD PROTEINS

2 (i)

The bacterla were labelled with P32 using the technique

described by Benacerraf , Bíozzl, Halpern anci stiffel (1957).

The organisms were labelled by allowlng them to multlply In a

medium to whlch p32 fruA been added as orthophosphate- -89- t3l (r1) T.abelllno of oroteins with I

the fotlowing adaptlon of the chloramlne T method of Hunter and

Gneenwood (I962) was used.

Materials required : (a) tl2t o, II3r, carrier free iodide in aqueous solution, free ffom

reduclng agrent. þ) Protein solution 1n phosphate buffer ui.15M, pH7.4 atÛ.I - lo/. concentration.

(c) So¿tum iodicle solulton (l mg./rr,!.) in phosphaie buffer 0'l5M

pH 7.4.

(ci) Chloramine T (BDH) solution J.u mg./mL'in phosphate i;uffer

ü. SIvf pH 7 .4.

(e) Soeium metùtsulphite solu'rion (IÛ rng -/ml.) in phosphate

buffer 0.15M PH 7.4.

The radioactive iodide, the amount of which depends on the

desired speciflc activity (usually about Û.5 mc. per mg. proteín)

was added to the proteln solutlon and the mixture continuously

stirre.d. sodium todide (0.I m1.) may now be added, although this

step was not obligatory. The chloramine T soludon (c.1 mI.) was

then added, and after 5 mlnutes the reaction was termlnated by

the addition of the sodÍum meiabisulphite soluiion (t.I mI')'

unbound radioactive iocline vvas removed fronr the reaclion mlxture

by exkraustive d.ialysis against phosphate buffered- saline or by -90- passage of the reactlon mlxture through sephadex G-25. Usually an efficiency of labelling gireater than 50% was obtalned.

I. REDUCTION OF LIZARD IMMUNOGLOBT'LINS Lizard lmmunoglobulins, and also mammalian immunoglobullns used as controls,were separated lnto thetr constltutlve L and H chains by reductlon with Cleland's reagent (¿ittrio*rreitol - DTT, product number 233155 of CalbÍochem, Los Angeles, californla, U.S.A.) tn a dtssociating solvent. The experimental detalls of these reacflons are descrÍbed in the text of the thesis (Chapter 6) ' -91 -

CHAPTER 4

Paqe

A. NATURAL ANTIBODIES IN NORMAI, LIZARD SERUM 92

B. KINETICS OF A}JTIBODY PRODUCTION . . . 93 (i) Lizard resp'onse to S. Wphimurium C5 " ' 93 (ii) Lizard response to rat erythrocytes ' ' ' 96 (iii) Lizard response to BSA ... 98

(iv) Brief summary of data , " ' ' ¡ t0t

c. DISCUSSION ...... '.. 102 (i) Dose of immunogen ' .. t02

(ii) Types of lizard antibody produced ' " 104

(iil) The effect of environmental temperature on antibody productlon by.the lizard' 105

(1v) The speciflcity of lizard antibody ' ' ' 107

(v) Evldence for a secondary humoral response r08 -s2-

A.NATURALANTIBoDIESIN}{ORIVL\LtuARljSERu1V,I

Lizard serum, in cgnimOn v¿ith the serum oÍ Other vertebrates, wAs of founcl Ëo contain'natural'antibociies. Tapie 4't lists the titree these nat,.ual antibod,ies to erythrocytes from seven vertebrate species; the titres presented are the geometric means of the titres of ten lndividual lizard sêrð.

Atl the natural antlbodies were found to be wholly susceptible to

reduction with 2-ME (0'LMß7o/1 hour) and in the case of rat eryth-

rocytes vtere shown by sucrose density gradient ultracentrifugation

(SDG) to be macroglobulin in nature. The specificity of the reaction from the between natural antibodies in lizard serum and erythrocytes of 'sera' variety of vertebrates studied was examfned by the absor¡'';tion were as speclÍic These stuciies indicated that lizard natural antibodies of I : 32 as those of mammals. Thus when Ll.zard serun v¿ith a titre vras to rat erythrocytes, and a titre of I : 16 to ra-obit erythrocytes, was absorbed with rat erythrocyies all antlbody 'co the rat erythrocytes was not renroved but the antibody titre to uhe rabbit erythrocyies

affected. The increase in volume of the serum sample during absorption

was taken into account in these experiments ' Lysis of the erythrocytes used in these haemagglutination assays lysis indicated the presence of complement in }izard serum' This

could be prevented by using NarEDfA (0.0Û3M) in the haemagglutinating minui:es' diluent, or by heating the lizard serum in a 560 lvater bath for l0 TABLE 4.I

\ÆRTEBRATES IN NORMAL LIZARD SERUVI

Reclprocal of Source of Range No. lizards/tO * less Erythrocyte Titre of with tltres Tested Titres thanl:2

Sheep Less than 2 0-4 s/ro Rat 16 0-64 t/n

Guinea Pig 0 Lo/Lo Rabbtt 16 2-r6 0i1.0

Human Rh+ve 0 Less than 2 0- 16 7 /ro load 0 Lo/ro (8. marinus) Blue Tongue 0 toA0 Llzard

* Geometric mean titre of I0 tndividual titres ' -93-

A complement tttration uslng rabbtt haemolysln and sheep erythrocytes as describedln Kabat and Mayer (t961, Pô9ê I49) showed that fresh normal llzard sen¡m contalned approximately 60 C'50 complement unlts. Thus it was found that normal lizard serum contained natural antibodies to erythrocytes from other vertebrate species ' The actfvity of these antibodies was manlfest in haemolytic and haem¡gglutlnatlon reactlons .

Because llzard serum did not contain agglutinins to sheep

erythrocytes these were chosen for use in passive haemagglutination

assays.

B. KINETICS OF AI{TIBODY PROD.UCTION was The atms of this lnvestlgaLion were twofold' Firstly' it by intended to gather data on the kinetics of anttbody production thellzard,Tlliggg4g@secondly,itwasintendedtoimmunlze lizards in order that a supply of immunized lizard serum was available'

thereby permltling the characterisation of the immunoglobullns of

this species. llzards The humoral anLibody responses obtained by immunizÍng

with S. typhimr¡rium C5, chocolate brown rat erythrocytes and BSA are

described tn the following Pages '

B(i) L{zard lesponse to S. tvphimwium C5'

The first attempts to immunize lizards were wlth salmonq.lla '

salmonella tvphlmurium c5 was chosen and an alcohol killed 94- vacclne was prepared (see Chapter 3). Both intramuscular and intraperitoneal routes of antigen adminlstration were employed.

Intravenous lmmunization was technlcally impractical and thls method of immunizatfon was not investÍgated. Flgures 4.1 to 4.3 detail the results of the experiments with this antigen.

Flgure 4.1 shows the result of intramuscular injecdorts of anligen mixed with complete Freund's acijuvant (CFA). Three i.m. injections of antlgen, of which only the first ccntainecì CFA, were injected over a I0 ciay períod and resulted in a strong antibody response in lizards maintained at 30o, Antibody was only detectable after day 10 and then proceeded to rise rapidly until a peak Litre was reached at approx- imately day 40. After this time the level of circulatlng antibody decreased very slowly and was still appreciable at day 160' In other experiments an appreciable titre was still present 210 days after the initial immunizing fnjection. I lilhen a single i.m. injection of antigen wes glven (1.e. 10

organisms + CFA) the response obtainecl in lizards maintained at 30o wôs not as vigorous as when 3 antlgen lnjections were made. The

response obtained f s shown in Figure 4.1 . It can be seen that the * maximum titre obtained (logrT = 9) was lower than in the mul.ti injection case 0og2T = 11) ancl took 16 ciays longer to be reached. ' Hu"*agglutination assay titres r*'i}l be repcrÉed eÍther as the dilution used, or as the logarithm (base 2l of. the dilution. For example, the litre of an antibody titration which is positive for 5 serial twofold ciilutions wiII be reported as I : 32 ot as logoT = 5. -95-

The lmportant lnfluence of environmental temperature on antlbody production f s demonst¡ated by the third curve in Figure 4.1 whlch

shows the response of lizards maintained at 20o to a single i.m' lnjection of t08 organlsms * CFA. This response was followed for

ll0 days when 3 of the 5llzards died. A similar antlbody response

was obtalned when lizards malntalned at 20o were glven 3 lnJectlons

of anHgen over a I0 day Perlod.

The response obtalned followlng a single l.p. inJectlon of antlgen

(108 orgar¡lsms + CFA) into llzards malntained at 30o ts shown in

Figure 4.2. In thts experiment anttbody was first detected on day I

and thereafter rose to a maximum tltre of losrT = 6 at about day 30 '

The clrculating antlbody remalned at this level unlll day 60, when a

second injection of 108 organ-lsms wlthout CFAwas made. This

caused a very rapid increase in the antibody titre, and a new hlgher the maximum antibody titre of hosrr = 10 was reached 16 days after

second antigen injecLion. This response was simllar to a secondary

response.

AII the antibody produced by the lizard in response to antigenlc

stimulation with S. typhimurium was found to be susceptible to mild

z-Irif| reducÈlon. Sucrose density gradient ultracentrlfugatlon of

fmmunized ltzard serum showed that only macroglobulin antlbody

was syntheslsed throughout the entlre response perlod lnvestlgated'

Representative SDG proflles of serum taken ffom immunfzed lizards Figure 4. I ANTIBODY PRopVPTIoN BY LIZARÐS TO-S. ÎYPHJ¡4URIUM INTECTED I. M.

Antigen : Alcohol killed S. lvphimu¡ium C5 Ab. assay : Passive haemagglulination

Legend : o'{ Regponse of.2t) lfzards tnaintained at 3,3o to 10o opganisms + CFA i.m. at day 0, followed by 10o organisms, without CPA. at days 7 andlo(+) o HRe to IO )

tr--{ Re to t0 )

Each point represents the arithmetÍc mean of the üo9rl' s of each group of lizards.

Fisure 4,2 ANTIBOÐY PRODUCTION BY IIZAJìD.S AT 30o T.O S. TY?HIMURIUM INTECTED I.P.

Antlgen : Alcohol killed S. lvohimurium C5.

Ab. assay : Passive haemagglutination

Legenci : O'--o ReçPonse of l0 lizarcis co an 1.p' injection of 10" organl,sms + CFA at day 0, followe d bya further l0o organissts i.p. at day 60 ( 1 )

Each point represents the arithmetÍc mean of the logrTis of the

I0 lizards . * ¡ooo mr¡rroolunr¡rpr¡ rrrnr{ ' rOC. HAIIAOOLUTII{AYIO¡{ TlÌIË-t f- E- t + I :'o I I B I t t \ t I I t t I I 9 t II t óo+ t t I lç I i ã I !- I I I t t ¡ l* I I t I I E I I I Í 6 I i{I a.o

ü

Ë t o É É Fisure 4.3. SDG PROFILES OF TIZARD ANTI SJEHIMURIUM SERA

Profile A SDG of lizard anti -L_lUp@, serum taken from llzards 30 days post 1.m. admlnistration of antigen.

Profile B: SDG of lizard antl.S.:-W,phimr.uium serum taken from IÍzards 50 days post i.p. admÍnistration of antigen.

Profile C: SDG of lizard anli S. tvphimurium serum taken from lizards I60 days post i.m. admir¡lstration of antlgen.

Profile D: SDG of lizard antl S. lvphirnurÍum serum taken from lizards 80 days post i.p. administratlon of antigen.

Legend : H Optical denslty at 280 m¡r. -l H Haemagglutination titre OPTICAL DENSITY. (28O mul.

cl

o

oN

lt t D I Io o2

C- 3 E m P

ô

o @ I

o al

t-3UIlr NOtrVNrtnlOOVlrfVH j96 I at various stages throughout thefr response Ëc -g-:-lyphimurium are shown in Figure 4.3 .

B(ii) Lizard response to rat ervthrocvtes

several attempts to stlmulate lizarcls to produce antibody to

sheep erythrocytes were unsuccessful. Both i.m. and 1.p. routes

of immunlzatton were used, as was also a wlde range of doses, with

ancl without CPA. Only margfnal responses resulted, however'

Since there was an apprecfable background titre to chocolate

brown rat erythrocytes. these cells were tried next and an antlbody

response was obtainecl. RepreSentative response curves are shown

in Flgr:re 4.4. This figure shows t}e responses of llzards maintained

at 30o to i.m. and i.p. immunlzation wlth this antigen. The first observation, whlch has been founcl in every instance, was a decrease ín in the backgrouncl titre tc the rat erythrocytes foltowed by a rise tltre, In the case of the i.p. route, the tiÚe returned to its back-

ground level and remaineC there. If a seccncl injection of anligen these (1 mI. 50% suspension of erythrocytes Í.p.) was then given to

lizards, a response belleved to be similar to a secondary response

was obtained. The antibody titre dici not drop as before, but rapidly

lncreased to a maximum tit¡e of loerT = 9.5. This titre persisted

for about 45 days and then started to subside '

similar responses were obtained using the i.m. immunlzatlon route wlth this anllgen and a typicat i.m. response curve is shown in -97-

Flgure 4.4. In the example shown, l0 lizards were lmmunized at day 0 wlth rat erythrocyte stromata + CFA. The titre of the natural antibody which had been tresent now decreased. At day I0 a fr'¡¡ther i.m, lnjection of I mI. of a 50% saline susiJensÍon of rat erythrocytes

(without CfA) was macie. The antibocly titre now increased rapidly

(anc! exponentially) un¿il a maximum titre of logrT = 9 was reachecl at about day 50. The anttbody level then commenced to decline slowly and by clay 195 was approachlng the background titre again.

At this time another dose of I ml. of a 50% saline suspension of rat erythrocytes was inJected intramuscularly. The antibody tltre lmmediately lncreased qulckly : a maximum titre of losrT = 11 was obtained 35 days after this last injectlon of anËgen. Thls response was belleved to be a secondary humoral antibody response.

The anttbody synthesised during the early phases of the lizard response to rat erythrocytes was wholly susceptible to mild 2-ME reductlon, and by sDG ultracentrifugation it was shonrn to be approx- imately l9S (rabbÍt macroglobulin antiborJy used as a size marker)'

Later |n the response, ê.9. at clay 35 followlng 2 x 1.m' injections,

75 anttbody coulcl also be demonstrated by SDG ultracentrlfugation

uslng rabbtt 75 antiboOy aå a slze marker. Tt¡.is 7S lizard anti

rat erythrocyte anü.body was resistant to mlld Z-ME reductlon' Figure 4.4, 3

Antigen : Chocolate brown rat erythrocytes.

Ab. asgay : Di¡ect haemagglutination.

Legend EH I ml. of a 50% v,/v suspension + CFA injected i.P. at days 0 and 58 ( I ) Each point represents the arithmelic mean of the logrT's of 5 lizards .

H 10 mg. rat erythrocyte stromata + CFA i.m. at day 0, followed,:y I nrl. of a 50% v,/v suspenslo^n of rat erythrocytes at days lC and fSS t | ). Each point represents the arithmetic'nrean of the LogrT' s of l0 lizards.

* indlcates that serum taken from llzards at these times throughout

the response were examined by SDG and the profiles are shown in

Figure 4.5. LOO¡ HAttlßlrflNrlflol rflnE-l

+

Ë

ù I +

t tã l* ä Ir

ã l* L- l8 lì/t! E

ú

-Þ It¡

It ot¡ tt¡ Figrure 4.5.

Proflle .Ac This proftle is of lizard serum taken from llzards

35 days post Ímmunization as shown tn the t'tll'

response curve portrayed Ín Figure 4.4.

Profile B: This profÍIe is representative of that obtafned for

Ifzard serum taken from polnts on the 1.m' immune

response curve shown in Figure 4.4whLch are

Índicated bv an asterf sk (*).

Legend: H Protein concentratlon: opllcal density at 280 mp.

r--{ Rat erythrocyte haemagglutination titre '

t-l Rabbit anti sheep erythrocyte haemaggluttnation ¡i¡¡s (19S size marker). OPTICAL DENSITY. (zaOm¡ ) Ot o

.tt ..1 Ð --D-- Þ -___:___á c) -a------::---'¡ I -*-'-" oz o

- z C --"t--":--- r 3 Ir' -t @ r' m Ð Ito

Þ @

(,¡ o o o o.5 ']Hllr NOtrvNlrnlÐÐvnlvH 1- 98-

In Ftgure 4.5 two representative SDG profiles of lizard anti rat erythrocyte sera are shown. Lizard anti rat erythrocyte sera, even from animals 240 days post immunization, contalned signiflcant proportions of both macroglobulin and 75 antlbody as shown by SDG ultracentrlfugation .

B(fti) Llzard responsg to BSA

At the time when this investigation was commenced the published literature was not encouraging about ihe ability of lower vertebrates to synthesise BSA anLibociy (see for example Good and Papermaster,

1964, page 84). In view of this, and particularly as the fÍrst attempts to elicit llzard BSA antibody appeared to be unsuccessful, the author was disposed to believe that the lizard was unable to syntheslse BSA antibodies.

It was thought however that the dtfficulty môy possibly lte in t}te means of detection of these antibodies and the Farr ôssay using Il31 -

BSA was tried since it had the advantage of detectlng 'incornplete' or non preclpitatlng antibody as well as normal antibody. Use of this assay clearly demonstrated BSA antibodies in the serum of lizards which had been immunized i.m. with BSA ernulsified with CFA.

Further studies, in which a variety of passive haemagglutination assays were triecí, showed that these antlbodies were not 'incompleter, and resulted in the adoption of a haemagglutination assay for

BSA anttbody in which BSA was coateci onto sheep erythrocytes -99- uElng chromlum chlortde (see Matertals and Methods).

Purther preliminary experiments suggested that the i.p. route was

more effective than the i.m. route for aclminlst¡ation of the BSA, and

also that positive Ouchterlony doubte diffuslon tests could be

obtainetJ uslng serum from llzards which had been lmmunized intra-

perttoneally with BSA + CFA for about 2 months.

An experiment to determine the dose dependence of the lizard

response to BSA was undertaken-

Solutlons of BSAIn sallne rangtng from 20 lrg. per ml. to 400 mg.

per ml. were prepared, mixed with an equal volume of cFA, and

emutslfled. One ml. of each of these preparations was then injected i.p. lnto ltzards malntalned at 30o. The haemagglutlnation tltres

of the sen¡m of these anlmals were then measured at 0, I0, 20, 40

and 60 days post immunfzation. The results have been summarized

tn Table 4.2. the surprislng aspect of these results was the wide

range of doses of BSA that elicited gooci antlbody responses.

Dlminishecì responses were obtalned Íf CFA was not used wlth the

BSA; thus a close of 30 mg. of BSA alone ellclted a maximum litre I logrT's lower than the maxlmum tftre obtalned when CFAwas usecl'

Three lizarcls were each lnjected with 20C mg. of BSAIn sallne wlthoutCFAtoseelf immune paralysls could be lnduced' However, ôll the llzards synthesf sed antf body TABLE 4.2

RESPONSE OF IIZAIIDS A1 3OL' TO VAIìTTNG AIWOUNTS OF B8A*

means of Log Tts Mass BSA Number Post lmmunÍzalion 2 Injected Ilzards at ï)av : î1 0 I 0 ?.n 4 0 60

200 mg. 2 c 4 t2 15 l5

I00 mg. 4 0 4 ll r5 l5

50 mg. 2 0 2 l0 r3 l5

30 mg. 4 fì 4 8.5 13 r5

l0 mg. 2 û 2 7.5 1I 14.5

5 mg' 2 n 2 7 .5 13 15

r mg. 6 0 2 4 r0.5 14

9 100 ¡rg . 4 0 2 6 I

lo Fg. 4 0 t 2 I I

* Each injection of BSA was in a total volume of I mI. of an

emulsÍon composed of I volume of BSVsalfne and 1 volume of CFA

and was Ínjected intraperitoneally. - lui¡ - which, 60 days after lmmunization, was present in sufficient quantities to produce a posltive Ouchterlony test vrhen a I mg./mI' solution of BSA was used in the aniigen well.

Figure 4.6 shows the response of lizards tc 3u mg. BSA + CFA at 3iio , zso anci 2,Jo, and also the response to 3c i¡g. BsAwithout

ÇFi\ at 30o. The enhancement in response ci.ue to ihe use of CF.A' can easily be seen.

It can also be seen that variation in tlle temperature at which the lizards were maintained influenced the response obtaineci' It was interesting tc note that the only ciÍfference between response of the llzard to BSA at 25o and 3Ûo was that there was a delay in

f-\ the appearance of the maxirnurn titre (1.e' lo9rT = 15) at 25-;

however, the ultimate maximum tltre reached in both cases was was the saroe. At 2 0o the maximum titre attained 0ogZT = 4)

much lower and the overall rate of appearance of humcnal antlbody

was signif:icantlY diminished.

The following flgure (Flgure 4.7) illustrates the response of

lizards maintainecl at 30c io i.p. injections cf 3t mg' BSA + CFA

anq I mg. BSA + CFA. The 2-ME reduclion resistar't'iitres are " also shown in this figure. It was clear ihat z"NiE reduction "

reslsùant lizarci BSA antfboc.ly was synthesisecÌ early f n the respcnse,

although lt took longer to appear.whqn a I mg'' tiose cf BSAwas

lnjectecl (25 iJays) than v¿hen a 30 mg. Lrose of BSA was lnjectecì (12 days). .{ j-J-t a

- lot - d) (.

Op \ ADË SDG ultracentrifugaüon confirmed that thls z-Iiv,.IE reduction resistant lizard antlbody was slo\,\7ly sedfmenLing, i.ê. 75.

Detectable macroglobulin anttbody was not present in the serum of lizards 65 days after they had been immunized wfth 30 mg.

BSA + CFA and rnaintained at 30o. All the antlbody by this time was 7S and wholly resistanto to z-ME reducÈion.

Some representative SDG profiles of lizard anti BSA serum from various stages in the response to thís antigen are shown in Figure 4.8.

Although all the data presented for the response to BSA relate to the í.p. immunization route it was found that i.m. immunization also resulted in anflbody production. The 1.p. immunizlng route is to be preferred however slnce lt elicits better responses to thÍs antlgen and is technlcally easler to perform.

B(iv) Brlef summarv of data

The data presentedin section B have been summarlzed in Table 4.3

and some of the findings båve been ölscusscid tn.,the followtng section.

* A difference of I dllution in the haemagglutlnatlon assay was not consldered to be a signfficant drop in tltre. Figrure 4,6 . INIECTED I',P.

Antlge4 : Sallne solutlon of BSA either wlth or wlthout CFA.

Ab. assay I Passlve haemagglutlnatton.

Legend H 30 mg. BSA + Cqå i.p. lnto llzards ûnaintatned at 30Ion day 0. Each polnt represents the arith¡netlc mean of the logrl's of l0 lizards.

H 3ü mg. BSA + CFÄ1.p. fnto llzards fuiatntatned at 25Ion day 0. Each pofnt represents the arlthmetic mean of the logrl's of 5 liza¡ds.

.H 30 mg. BSA + CFÀ l.p. into llzards þarnblned at 20")on day 0. Each potnt repre,sents the arithneÈlc mean of the logrT's of 5 llza¡ds.

H 30 mg. BSA o¡ìlyÄ.p. into llzards tnatntained at 30Jon day 0. Each polnt repres€nts the arith¡netlc mean of the. logrT's of 7 llzards. úal.Ã.

30'cæF.

I¡ G t-_ t- - Þ9 -Þ =J .c'o - l¡¡- ñ I 3()'c. ) 2()'c+cF.

g) t¿10 20 ao æ BSA I.P DlrRArloll OF BEsPor{sE. (DaY4. o Figure 4.7. BSA INTECTEÐ I. P.

Antlgen : Sallne solutlon of BSA emulsified with an equal volume of CFA.

Ab. assay : Passive haemagglutinatlon-

Legend: As on diagram.

Each point.on the response curve represents the arithmetic mean of the logrT,s of S lizards for the I mg. BsAresponse and of l0 lizards for the 30 mg. BSA response. T Et¡¡ t- z I k

=Ð 0J 0 . H Total ant¡body t¡trc. þo' c) ¡l¡¡ H 2tE resistãrt t¡trc. þo' c) g

I x, &608() 1@ 120 lmgt BSA+CE DuRATþrl oF REsPoilsE. fo"p}

5 l¡¡ Ê E

9- I æTotal antibody titre. (SO'C) f= H znE resistant t¡tre. c, (!, t t¡¡ I Ia{

x, .lO 6() 80 1()0 120 D nlg' ñ¡ +CE DrrRATKtlt oF REsPoilsE- (Dtrd. Flgr¡e4.8.SDGPJÌoFILESoFtIzARDÄNTIBSASERA

Proflle A : SDG profile of lizard anti BSA serum taken frorn Iizards 18 days post i-p. lmmunization wfth 30 mg. BSA + CFA.

from Profile B : SDG profÍIe of lizard anti BSA serum taken lizards 30 days post i.p. immunlzation wlth 30 m9' BSÀ + CPA ]

taken from Proflle C : SDG proftle of llzard anti BSA serum llzards 65 days post i.p. immunization wlth 30 mg' BSA + CPA.

280 mp' Legend : H Proteln proflle : optical denslty at

H Lizard anti BSA haemagglultnation tiEe profile '

Mtld z-M¡E reduction reslstant llzard anti BSA haemagglutination titre profile ' c I'l t.5 A 64

:r lltìlr Êl It 8l lÞ NI t8 ;l IE FI lzll 3Ll ,12 -l t2 lr Ël l9

Days after lvlax. Types of AT,ITIGEN injection S econdary TEMP. No. of Day Ab. titre Ab. and of antlgen response Dose (degrees) Lizards Íirs t loVrT. detected fmmunizlng route detected to reach

BSA 15 60 Both NA 30 mg. BSA + CFA IP 30 I 0 I 7 26 t9s NA 30 mg. BSA IP 30 7 I t0 l5 77 and NA I mg. BSA + CFA IP 30 5 15 98 7S NA 30 mg. BSA + CFA IP 25 5 I 4 60 antlbody NA 30 mg, BSA + CFA IP 20 5 t6

S. ^TYPHIMURIUM I0' organisms + CFA (3 in I0 daYs) tnjections 38 NA IM 30 20 I t I Only 56 NA 30 l0 I 9 l9s 80 NA 20 5 22 2 an'cibody 2s NA 30 t0 I 6

t0 l6 (+) organisms at daY 60 IP 30 IO NA Table 4 .3 (cntd ) Days after Day Ab. Max. Types of Secondary ATITIGEN TEMP. No. of fnjection (degrees) Llzards first tltre Ab. response Dose and logrT. of antlgen detected immunlzlng route detected to reach

l0 mg. stromata + CFA (daY Û) + I ml. 50% susp. + NA I 48 (day 10) IM 30 l0 Both As for day 0 & daY l0 r9s 50% susp. + I ml. 11 35 and + (day IM 30 IO NA I95) 7S antibodY ml. 50% susP. I L' 3Z NA rJ NA 4 + CFA (daY 0) IP 30

As for daY 0 + mI. 50% susp. + I NA 9.5 52 (day sB) IP 30 5

NA Not applicable ln thÍs lnstance ' .102-

C. DISCUSSION

These experiments have indlcated that the llzard, Tllisua rusosa' is capable of producing large quanttties of antibody to the three antÍgens studled. Nthough Tait (t967) reported god antlbody productfontosheeperythrocytesinanother1izard,@ lt was found that thls anligen ellciteci only a very feeble response in Tiltqua, even after a variety of immunizing schedules was tried' In general it was found that poor anLibody responses resulted unless cFA was included with the first injection of antigen.

This was found to apply to each of the 3 antigens used, and was parLicularly evident with small doses of BSA. For example, doses of I mg. or less of BsAinjected i.p. without CFAdld not produce a measurable response by 40 days; when the BSA was emulstfied with CFA however, and then injected, measr¡rable responses could

be obtained with even 10 Pg doses.

Some of the other characterlstics of the immune response ln

Tilloua are descrlbed below.

C(i) Dose of immunoqen

Generally it was found that relatively large doses of immunogen

were necessary to eliclt good antlbody responses '

A substantial response by the lizard to S. lvphtmurlum was ellclted

by three i.m. lnjections of 108 alcohol killed organisms, only the first of whlch included. CFA. A less vlgorous response was obtained -103- when only I injection (l0B organisms + CFA) was given. It was found that a good antibody respolìse to rat erythrocytes wôs ellcited following several i.m, or i.p. injeclions of I mI. of a 50% suspension of rat erythrocytes (again only the first lnjection contained cFA).

Greatly diminlshed responses were obtained if I mLof a l% suspension of rat erythrocytes was substituted for the 1 mLof 50% suspenslon'

A most surprising findlng was the extreme dose range of BSA

(+ CFA) which ellcited good antlbody responses. For example, both

30 mg. and I mg. doses of BSA when injected with CFA ellcfted the same maximum titre 0ogZT = 15)¡ however thls took 17 days longer

to a6aln with the lower dose. The dose response to BSA + CPA for

the lizard has been summarized ln Table 4.2 ' Tait (1967) reported that antibody production in the toad, E-:-l4gElEE,

was lr¡hlbited by large or multtple smaller doses of human serum

albumin and suggested that hls data supported a previous speculatlon

by Good and Papermaster (1964 page 84) that immunological paralysis

might be easily inducable ln lower vertebrates with soluble and'gens.

The data presented in Table 4.2 Ls not st¡ictly apPlicable to this

hypothesis since the antigen, when emulsified with CFA, can no

Ionger be regarded as soluble. However, when each of 3 lizards

was injected i.p. with 200 mg.of BSA dissolved in sallne (wlthout

CFA), each synthesised appreclable amounts of antlbody' -lQA- C(ii) Tvpes of.llzard antibodv svnthesfsed It was found that the lizard coulcl syntheslse two types of antlbody inttially disLinguishable by their sÍze and susceptibtlity to 2-ME'

The macroglobulin antiooCy had a seclimentation coeffictent of apprord,mately I9S. This was estimated by sDG ulracentrifugation using a rabblt 7M antibociy as a size marker. Itwas also calculated from the tables relatlng to sDG ultracentrÍfugatlon pu.bllshed by

McEwen (1967). Llzard macroglobultn anÈlbody was wholly susceÞtible to mtld recluction with 2-ME. The other type of lizard anttbody had from a secllmentatton coefflclent of approxlmately 75 (also estimated reduction' sDG ultracentrlfugatiOn data) and was reslstant to Z-ME types of antibody The immunoglobulln classes to whlch each of these

belong ls considered fn Chapters 5 and 6 ' only macroglobulin antibody was produced by the lizarcl in response produced to immunizatlon with -E-:.lylhiñ. If any 7S anttbody was detected it must have been present in concentrations too low to be bythepasslvehaemagglutinationassay.Althoughthedataisnot 24Ú lays afier includeci ln section B(i), the antibociy founcì in llzarqs An immunization wlth this immunogen was entirely macrogloi:ulin' analogoussituationhasbeencltedbyDavis'Dulbecco'Elsen' immunized with Ginsberg anrl Wood (I968 page 463) for rabblts pneumococci. Marchalonis salmonella ancJ for horses lmmunlzecl wlth response et aI. (I969) have aISo described a prolongecl macroglobulin -105- ln the tuatarô, @ immunized with S. a4elaide flagella. Grey (1963, 1366) reported that another reptile, the painted turtle, ChrvseJnvs-picta, synthesised only macroglobulin antibody over a I00 daY Period.

Both t95 and 7S antíbodies were elicited during the response of the lizard to rat erythrocytes. However even in lizards wt¡lch had been lmmunized for 240 days,both l95 and 75 antibody were present in apprecÍable quantitles .

The response of the lizard to BSA was surprlsingly vigorous, and resembled the mammalÍan response to this antlgen. Both 195 and

75 antlbodies were elÍcited, however in contrast to the responses

to the other 2 immunogens,the macroglobulin response was transitory

and by 65 days post 1.m. immunizationwith 30 mg' BSA + CFAonly

75 antibody was evident as shown by SDG ultracentrlfugalion.

Al though the llzard response was slower than the response of rabbÍts

to this immunogen, comparable quantities of antibody were synihesised:

the serum of hyperimmune lizards contained in excess of 5 mg '

specific antibodY Per mI. C(iii) The effect of envlronmental temperature on antibgdv Þroduction bv the lizard.

The data presented on the responses of the lizard to BSA and

S. tvphimurlum (see table 4.3) show that the environmental temperature affects the production of antlbody. -IÛ6-

When BSA was used as an immunOgen the responses obtained at

2So and 30o fndicated that the overall rate of anlibody production was slower at the lower temperature, but that the Same maxlmum titre was eventually reached 38 days later. The response of the lizard to BSA and S.. Ivphimurium at 20o however was entirely different' At this temperature the maxlmum titres reached throughout the responses were ôppreclôbly lower than those reached at 30f and the rate of antlbody production, partlcularly in the response to S. tvohimurium' was much slower.

These data demonstrate that Ín thls species : (a) a factor,other than the thermodynamic effect of temperature on protein biosynthesisraffects the total amount of antibody

produced when llzards are immunized and maintalned at 20c'

This factor cioes not seem to be operative at 25o and 30o, and

(b) t he rate of antibociy production is temperature dependent, being slower at lower temperatures '

The effect of a sudden decrease in the environmental temperature

on the antibody tltre of lizards whose titres were rising exponentially

Ís shown ln Figure 4.9. It can be seen that the decrease in

temperature causes a cessatlon of antibody producllon and the

tttres remain steady for some time' Tait (I967) observed simllar findlngs in his study of the responses sheep of tJre lizard, E. cunninqhami, and the toad, B. marlnus, to Figure 4.9 THE EIFECT OF A DECREASE IN ENVIRONMENTAL

LEARD

Legend - rH Lizard response to BSA + CFA o'{ Lfzard response to S, tvphlmurlum + CFA

Theenvfronmentaltemperatureofthelizardswas decreased from 30o to 20o at the li.mes indicated by the arrows tî1r. BSA+CF -.1 I ilGI Þl

O--O--O_ l , i s. TYPHIMUn[-ilu+cF'\ \ 'd o l-l 3 H =lut -l

to 20 30 ¡lo 50 nre /orvs). -107- erythrocytes (see page 25). The drastic reductions in antibody tÍtres reported by Bisset (1948) and Elek, Rees and Gowing (1962) when animals which had been lmmunized at a wôrm temperature and were actively producing antibody were suddenly transferred to a

Iower temperature was not observed.

C(iv) The specificitv of lizard anlibody

Several experiments were performed which indicated that the antibodies ellcited by these three immunogens were speclfic'

It was found that lizard anti S. tvphimurium senrm contained antibody specific for the Salmonella O-somatic antigens l, 4, 5, & 12' s. adelaide did not cross react with lizard -$-3¡h!mur'i.um antibody'

Lizard anti rat erythrocyte sera did not agglutinate sheep or guinea pig erythrocYtes.

the specificity of lizard BSA antibody was assessed by

Ouchterlony double diffusion tests of this serum against the sera of

various other vertebrates. A hyperimrnune lizard anti BSA' sen¡m

containirrg 5 mg, BSA antibody per ml. was tested against rat, mouse, rabbit, pÍgeon, chicken, horse, calf , sheep, human and guinea pig sera. Precipitation lines were observed only against sheep and calf (1.e, bovine) serum which indicated that lizard BSA antibociy

cross reacted with sheep albumin. slnce both the sheep and the

calf belong to the mammalian order Artiodactyla, and as nelther is closely related in phylogeny to reptlles, this result ls not -108- sr¡rprislng. In Chapter 5 some evidence ls presented suggestl,ng that the avldity of 75 lizard BSA antibody was comparable with the avidlty of ZS rabbit BSA anttbody. The lack of cross reactions of llzarc antÍ

BSA with the albumins from a number of other vertebrates is therefore an lndication of a htgh degree of specificity and cannot be accounted for by a relallvely low avldfüy of llzard BSA antlbody'

There ls nc evidence then to suspect that lizard antlbody is less specific than mammalian antibody.

C(v) Evidence for a seco.nclarv humoral response'

On several occasions dr¡rlnE this study evidence suggesting that the lizard is capable of a secondary humoral response was obtainecl'

The occasions on which this is thought to have been demonstrated have been indfcated in Table 4.3 (facing page 10f )'

An acceleratecl production of antlbody was also observecl when llzarcls immunlzed with S. Wphlm!¡rium were subjected tc a second presentatlon of antlgen (see Figure 4.2). However, lnsufficient

clata were avallable to distinguish between the response obtalned

and the primary response which would have occurred with thls dose of antigen. Hence tt is not possible to clecide with certalnty lf

this response to the seconcl injection of antigen was a true seqondary

response. -109-

The best eviclence for a seconclary response was obtained ln the response of lizards to rat erythrocytes. Ia this example (see

Flgure 4.4) the response of the llzard to a second injectlon of rat erythrocytes differed completely from the response to the inttial inJection of this antigen : the serum antibody titre lncreased very rapidly wlth no ttme lag, and the ultimate antibody tttre obtained was much greater. thts type of response was obtained when llzards were lmmunlzed elther intramuscularly or intraperttoneally with rat erythrocytes. The response obtained to the second presentation

of antigen was acceleratecl and enhancecl and was belleved to be a

true secondary humoral response. - 110 -

CHAPTER 5

Paq_e

...... llI ^ INTROÐUCTION

B. PROPERTIES CF LIZI)RÜ SERUM '. ' 11r

C. PREP/\RATION OF LIZARÐ IMMUNOGLOBULINS

(i) Preparation of lizard 'ylvi' iuimunc-'gloilulin 115

(ii) Preparalton of lizarci 'yG' intmunoglobulin TT7

(iii) Preparation of lizarcl specÍfic B51L ani:ibcdy r23

D. REACTION OF BSA I/VITH LIZARD AltII BSA ' ' ' 127

E. CHÄRÄoTERISATIoN oF LIZARD IMMUI\ToGLoBuLINS

(i) .Antigenic cross reaclivitY t?8

(if) Amino acid comPosltion L29 I3I (iii ) Carbohydrate content . - .

(iv) (a) Measurement of sedimentation coefficients. . I3I r33 (b) Molecular weight measurements " '

134 F DISCUSSION ... .-. "' llt -

A. INTBO-DUCTTON

In the previous chapter it was demonstrated that the llzard,

Jiliqua rusosa. was capable of arrtibociy synthèsis. I\¡/o types of antibody were produced which were readily distinguishable by their

size and susceptibility to z-M.E reduction. In this chapter the nature of the immunoglobulins containlng

these two types of antibody was lnvestigated furtber. Methods

were developed for isolatlng these immunogrlobulins from the serum

of lmmunized llzards. Some of the physicochemical parameters of

the isolated immunoglobullns were then determlned. These studies

suggest that this species of lizard possesses at least two isotyplcally

dÍsLtnct immunoglobulin classes .

B. PROPERTIES OF LUARD SERUM

Prior to the fractionation of llzard sen¡m a survey of some of its

properties was canied out.

Lizard serum proteins were obtained by }ycphilizatlon of lizard

serum whlch had previously been exhaustively dialysed against a

solutton of ammoniurn carbonate. The optical density (OÐ) at 28Ü nrp

of a standard solutfon of ltzarci serum proteins dissolved in 0'IM

Sodium hydroxide + û.15M sodiurrr chloride was measured and the

extinctlon coefflcient aÈ 280 mp of a 1"/. w/v solutÍon of lizard

Sen¡m proteins in a I cm. quartz cuvette was found to be I0.8 + 0'5 tl? -

(i.e. tl*"*. 280 mp = lü.8 I [r.5). Thls value was used in subsequent determlnations of the protein content of both normal and immunized lfzard sêrô. The average proteln content of llzard s.erum was 48 mg. protein per ml. of sen¡m. This value varied between llzards and was usually lower in the serum of lizards whlch had been In captivity for a long period. Lizard serum comprlsed slightly in excess of 50% the volume of whole lizard blood.

The size heterogeneity of lizard serum proteins was investigated by analytical ult¡acentrifugalion and Sephaciex G20Ù gel filtralion chromatography usinE the serun¡ of lizards immunized with S ' tvohi- murlurn. A photograph of the analytical ultracentrffuge pattern obtained for normal lizard serum is shown in Figrrre 5.11 which faces page

The gel filtration elution pattern obtalned is shqnzn in Flgure 5.14.

Both these technlques indlcated that lizard serum contalned l9S, 7S

and 4S protein ftacLions and tn thls regard resembled mammalian sera.

In contrast to mammalfan sera hqwever, lizard serum contained relatively large proportions of macroglobulÍn protelns and correspon-

dingly relatively diminished proportions of the 45 proteln fractfon

whlch cogesponds to lÍzard albumin.* The relative proportion

*- Lizard albumin was isolated from lizard serum fractions and identlfled by lts immunoelectrophoresis precipitin arc. It was found to have a sedimentation coefficient of 45 ' - 113 - of each serum fraction was estimated using ciata obtalned from

Sephadex GzûU gel filtration and has been sumrnarized in lable 5 .1 .

The dlstrlbu'cion of lizard S. tvphimurium aniibody in the gel filtration

elution pattern ls also shorivn in FÍgrure 5.14 and confirms the previous

findtng reported ln Chapter 4 that this antibody was excluslvely

macroglobulin ln nature.

The electrophoretic properties of llzard serum protelns were

lnvestlgated using PVC block electrophoresis, paper strip electro-

phoresis and immunoelectrophoresis -

Whole llzard serum, which had been prevÍously dialysed against

the veronal buffer useo, was subjected to PVC block electrophoresis

(Chapter 3). The eleclrophoretogram oi¡tained |s ehown in Flgure

5.18. The profile shows a relaLive preponderance of p mlgrating

proteins and an ajfsence of a cìistinct y glol¡ulin fraction. The

clistribution of lizard S. tvphimurium antibociy activity is also shown ln this figure.

Paper strip electrophoretic analysis (Chapter 3) was carried out

on a number of individual lizard serum samples, and also on human

and rabbit sera for control purposes. Representative patterns obtained

are shown Ín Figure 5 .2 . It shoulci be menlioned that thÍs technique

was usecl only to eslimate the relative proporLion of each electro- phoretic fraction. It is clear from the patterns depicted fn Flgure -tr4- 5.2 that lizard serurn lacks a d,istinct electrophoretlcally defined y globulln fracLion. In lizard serum these proteins merge wlth the f, globulins and together make up about 52% ot the total lizard serum proteins. Representative de¡rsiometric tracings of the electro- phoretograms used to estimate the relative proportlons of each fractlon are shovrrn ln Figure 5 .3

The enhanced resolutlon obtainable by lmmunoelectrophoretic analysls of senrm protelns was utllized to further assess the compo- sltlon of llzard serum. Rabblt anti whole lizard serum GA'L), prepareci as described in Chapter 3, was usecl to cievelop the pattern of precfpitin arcs, For comparative purposes a sample of normal human serum developeci with goat antl human serum was also analysed under identical conditions. Representalive patterns are shown in

Figure 5.9 which faces page I28. These analyses shovved that lizarci Serum contained a large number of ciistinct proteins. \iVitLI this technique some y migrating proteins were detected' The preponderance of B proteins and the relatively low concentraLion of albumin fraction previously mentlonecr were also evldent.

The results obtained by these investigations have been summarlzed in Table 5.1 and have a direct bearing on the methods used to isolate lizard immunoglobulins. The absence of a dlstlnct electrophoretic y fraction, whlch would be expected to contain immunoglobullns, Fisure 5.14 GËL FILTRATION OF TIZARD SERUM

Gel fÍltraÈion chromai;ography of lizarci anti -Êd!Þ!ryig¡g

serum (4 mI.) on a column of sephadex G200 (2.5 cm. x 100 cm,)

elutecl wlth tris-ilcl buffer ({,.Û5M tds-Hcl + 0-2M Nacl),

pH B. u.

Legend :

El,ution profile of protein (OD 23i; mp).

ra Lizard S. tvphimurium antibody activity '

N.B. A slmilar pattern was obtained if normal lizard senrm was used in place of immunizeC lizard serum.

Ffsure 5.18 PVC ZONE ELECTROPHORESIS OF LIZARD SERUIVI

PVC zone electrophoresis in veronal buffer (ionic strength 0.Û5¿

pH 8 .41of. serum fro¡li lizards immunieed v¡ith S. tvohlmurium

Cathodd 1-ve) to the left of the origin.

Legend :

H Proteln concentratton profile : OÐ 75Û mp of the Folin reaction.

H LÍzard S, tvphi4rurium antiþodv activity'

N.B. A similar pattern was obtained if normal lizard serum was used in place of immunlzed lizard sen¡rn ' Optical Density (zsO mf¡) Optical Density þeO m¡) I oD

oa

\t o o +õ' 3 .ll .ll o o¡ o clÐ o o=o 5 w 3 2r z =A c 3 q3 or ir .qoo

lìto o

cr J o o Figure 5.2 : PAFER STRIP.ETECTROPHOREqIS qF VARIOUç SERA

Paper strtp electrophoresis cf human, rabblt, and fndivldual ltzard

serum samples. The variatlon in the lntenslty of the stained bands (stained wlth

Amido Black t0B) ts due to variable proteln concentratl';ns of the gerä ussd. FnPtß grltP ELECTROñ0nE$8 OF YAR¡OI'E 8CIA.

lftrmm. Llz¡td t. Llz¡lü 2. Llz¡rd 3. LErrú e. Lbblt. -f,

ü t d * 3 I - {D tp9 O¡,, - I tt ü l üttt; +n Fiqure 5,3 : DENSIOMEIIRIC TRACINGS OF PAPER STRI? ETECTROPT.IQREIOGRêMS SHOWN INJis. s . 2

A Human serum (normal)

B. Llzard L

LLzard 2.

I'he arrows indtcate the albumln peak and the

polnt of application of the sample.

The relative proportions of each proteln fraction

were determlned from the areas under each Peak. A

+ I

B

+ t

c

+ve. + | -ve- Albumin. Origin TABLE 5.I

PROPEFJIES OF LIZARD SERUM

A. Extinction coefficient :- E!% oÍ lizard serum = IÛ'8 ! Ü'4 I cm. ZUU mp

B. ProteÍn concentration :- Range 35 - 55 mg./ml'

Mean 48 mg ./ml'

C. Electrophoredc heterogeneity' Relative concentratlon of electrophoretically deflned facLlons :-

y+Þglobuttns 52%

e globullns 24"/' Albumln 24%

D. size heterogreneity, (from gel filtrati.on chromatography on Sephadex G200). Relative concentration of :- Macroglobulinftactlon 28%

"75" Protein fraction 47%

Albumin fractton (4S) 25% trs - almost certainly accounts for the difficulties encountered in the fsolatfon of llzard 75 immunoglobulln. c. PREPARAÎION OF TTZARD IMMLINOGLOBULINS

(Í) Preparatlon of lizard 'vM' immunoolobulin

In Chapter 4 Ít was reported that macroglobulin antioodles were produceci when lizards \¡/ere irnmunized with either S. typhimurium or rat erythrocytes. The immunoglol¡ultn containing antibodies against these two lmmunogens v¡as isolated. Prelirninary

the procedr¡re used to isolate lizard '7M' immunoglobulin has

been diagrammatically summarized In Figure 5 .4 and is ciescrlbed in detall below.

The lmmunized lizarci serum was preclpitated twice with sodium

sulphate at a final concerrtration of L4% w/v and then washed with a

14"/.w,/v solution of sodium sulphate. Approximately 2Ù% of the

total lizard serurn proteins were precipitated. The precipitated

protein rvas dissolved in a minimal volume of tris-HCl buffer* * ' tri"-Hcl buffer whÍch is û,ûSlvi in tris-HCl + û.20M in NaCI and is of pH 8.Û, will be abbreviated to tris-HCl' - tr6 - (0.05M tris-HCt + 0.20M NaCl), pH 8.0, anci cüalysed against

I00 volumes of this buffer. Eighty percent of the haemagglutinating antibody activity was recovered and the specific haemagglutinali'ng anltbody activity* increased fourfolci (20 to 80 haemagglutination unlts per mg. Protein) .

The dialysed protein solution was then fractionated on a Sephadex

G20û column equilibrated with tris-HQl buffer. Two protein peaks were eluted from the column, the antibody activity being associated w-ith the first peak which corTesponded with the void volume of the peak column (see Figure 5 .48) . The fractlons comprising this were concentrated and cüalysed against a large volume of U'10M phosphate buffer, pH 8.Û. and then applied to a D[AE-cellulose column and a

batch elutlon procedure fotlorred (see Fig. 5.4C). The antlbody

protein was eluted with 0.5M phosphate buffer, pH 7.fJ. This

fracLion was pooled and concentrated and had a specific antibody

aclivlty of about 400 haemagglutinating unlts per mg. of protein.

When this fractlon was examined by immunoelectrophoresis

(developed wlth RAL), two precipitin lines were observed which

were similar to the 7M and aZ macroglobulin precipitin lines which

occur in mammalian serum. Lipoprotein could also be detected ín

this fraction unless the lizarOs from whici: lhe serum had been * Reciprocal of the haernagglutination assay titre divided by the protefn conccntration in mg./mL. Figure 5.4 : DIAGRAMMATIC REPRESENTATION OF THE ,VM' PROCEDURE UÇED TO PREPARE TIZARD IMMUNOGI9BI'TTIN FROM .IMMUNEZED IIZÀRD SERUM

Step A. Sodlum sulphate precipitation (14% w/v).

Step B. GeI filtralion on Sephadex G2t C of the 14% sodium

sulphate precipitate dissolved in iris-HCI buffer.

Step C. DEAE-cellulose batch chromotcarraphy of the leading

peak from the Sephaciex GZ-'¿ gel filtration elution profile.

Step D. PVC zone electrophoresis, in veronal buffer, of the

DEAE-cellulose fracticn containing the antlbcdy

activity, (i.e. the protein eluted with Û.5M

phosphate buffer , pH 7.0, shown in Step Ç),

Legend:

H Proteln concentratton (OD 283 mp or FolÍn OD 75Û mp).

la Haemagglutinatlng antibody activity.

Detalls of the proceciures are includecÌ in ihe text and also !n

Materlals anci Methods. f 414 10 Used later E c rols o for prepn @ 24i¿ of N + IG. (tc ø. s. o o ns. IE at P-147" .9 2c.c. Dissolve CL Í14:¿l Tris buffer o o25M +l Gel filtration

B fr D EI â ol E @t NI olo -: 6l .= Êl at ol c ôl oo ol .g -tol (E .s) ol .9 o q oct -V9 pool -10 o1020 30 EFFLUENT VOLU FRACTION NUMBER. -TL7- obtained had been starved fon I week prior to being bled. Llpo- proteÍn, when present, was evident by its opalescence, and also by an Arnido Black IÛB stainable smear on the anodic side of the

sample well of the immunoelectrophoretic pattern. If thls protein was present it was rernoved by ultracentrifugal flotatlon before

proceeding with the next stage of fractionation (see 'Rernoval of

lipoprotelns' - Chapter 3, Page 87).

The llpoprotein free protein fraction was concentrated and dialysed

against 4 x L;¡ volumes of- veronal buffer in ;creparalion for the final

step - PVC zone electrophoresis. This technigue was calTieci out

as cìescribed in Chapter 3 anci successfully separateci the 'yIVI' and

û macroglobulin still present in the preparation. A typical electro-

phoretogram is shown in Figure 5.4D. The antibody protein was

present in the most cathodic fraction. The protein present in this

fraction had a specific antibody activity of about 800 haemagglutinating

units per mg. and was homogeneous when examined by immuno-

electrophoresis. An irnmunoelectrophoretic pattern of the isolated lizard'TM.immunoglobulinisshowninFigr.rreS.gwhichfaces

Page 128'

(1i) Preparatlon of liz,ard 'vG' immunoglobulin

Slowly sedimenting (7S) llzard antibociy was detected in the

serum of llzards which had been immunized with rôt erythrocytes

and BSA. The immurroglobulin fraction containing anlibodies tl8 - against these two immunogens was isolated.

The procedures usually er¡ployed for the preparation of 7G from mammalian Serum $/ere unsuccessful when applied to lizard Serum.

These methods usually depend on tt¡e lov¿ electrophoretlc mobility of 7G lnrmunoglobulins. Slnce it was shown in section B that lizard serum does not contain a dlstfnct 7 globulin fraction the lsolation of

'7G' from lizard serum mlght be expected to be relatively more difficult and somev¡hat anôIogous to the isolation of 7A from mammalian serum. Also, in contrast to mammalÍan serum, over 50?á of Lhe total lizard se4lm proteins comprise the electrophoreflc fracLion of lowest mobilily. Although lizard 'yM' immunoglobulln also occurs in this group of lizarci serum proieins, Íts size and ciensity distinguish it sufficiently to permii its purificailon in a similar manner to the

purificalion cf mammalian YÏVI.

D[AE

from mammalian serum, and in order to invesligate the chromato-

graphic behavior¡r of lizard '7G' a comparative examination was made of the DF.\E-celtulose chromatography of lizard ' mouse and rabìcit anti BSA Sera. The BSA anlibody present in these sera was shown

by SDG ultracentrifugation to lce exclusively 75.

A batch of DEAE-ceLLulose was prepared for use as describeci 1t9 - * in Chapter 3 and equilibrateci v,;ith tris phosphate buffer

(0.ClM, pH B.tl). Sar,-rples of each of the 3 anti BSA sera were dialysed against this buffer ( 4 x 2ûC volume changes over 24 hours).

One serum sample, containing approximately g0 mg. protein, was loaded onto a column (1 cm . x 26 cm. ) which had been packed under

8 lb. of nitrogen with 2.¿,9. (dry weight) of the prepared DEAE.

The serum was washed onto the column wiih the tris phosphate buffer and the col.umn eluted with a further 5,3 ml. of this buffer.

At thfs point a concave concentratlon and pH graclient was prepared using the perspex grad.ient device described in Chapter 3 (p.85) :

25û ml. of tris phosphate buffer (û.ÛltuT, pïl f .i')) was placed in the wide cyl.inoer (i.e. A 1) and 5i,¡ ml' of phosphate buffer (Û.5M, pH 4.5) in the narrow cylinder (Í.e. AZ). Formation of the gradient was corrlmenced by openir,E thetap joining,Al and.A.?and the gracüent was then pumpeci onto the DEAE-cellulose column ' Five mI. effluent fractions vrere collecteci. Thfs particuLar gradient rn¡as chosen since it was known to saiisfactorily resolve i:uman and rabbit immuno- globulins. The protein content of each fraction was detenrrined by

measuring its absorbancy at 28C mp, and the dlstribution of anLibody

throughout the fracLions determined by the haemaggluLination reacllon. * This buffer was prepareci by titrating IM tris with tM KHZPO4 such that a l:lûû dilution of the resultant mÍxture had a pH of 8.0. 12ü - This experinrent was repeated with the olher 2 samples of anti BSA sera using the same cclumn and batch of DEAE-cellulose and the same preparations of the buffers. Protein concentraticn and anLibody activÚy distrÍbr.rtion profiles were cirawn for each serum. The profiles obtained are shown ín Figure 5.5 and are strictly comparable wÍth each other, Differences ln the behaviour of each serum on the DEAE-cellulose were apparent : llzard serum proteins were bound most strongly.

In the case of rabbit anti BSA senim (Figure 5.5C) a peak of essentially pure yG was eluted with the U.ülM trls phosphate buffer.

A further small peak of antibody activity w-as eluted after the gradient had been commencecl.

The behaviour of the mouse anti BSA serum ls shown in Figure

5,58. It can be seer¡ that no protein was eluteci with the initial buffer and that about 6Û ml. of the gracient had been used before the first protein was eluteci. Ttt'o peaks of antibody activity were observecl which may be due io the resolution of the sevcral n¡ouse

75 yG inrmunogloPulin clesses.

The behaviour of lizard antÍ BSÀ is shown in Figure 5.5A'-

Apart from a very small protein peak eluted wlth the i¡itial buffer

the butk of the lizard serum proteins wes not eluted until more than

100 ml. of the gradient had been passed through the column. It -tzr- was interesting to note that the lizard BSA antibody was also resolved inÈo 2 peaks. Since all the BSA antibody ln this serum had been shorr¡n to ce 7S it seems possiicle that 2 types of lizard

7S antibocty were presenl,. These cr¡uld be analogous to the several subclasses of marrrmaliarr yG imnTunoglobulins, or perhaps distinct

75 r7G' and 75 'yM' lizard immunoglobulins rnay exist.

thls experiment was repeated several Limes and the protein and antibody profilee shown in FiEure 5.5 were found to be reproducable.

The method ultimately adopteci for the preparation of lizard ryG' was determlned emplrically and is shown cüagrammatlcally in

Figure 5.6. Four steps are involved. These are :-

(a) SocUum sulphate precipitation (Figure 5.6A').

It was found ihat although llzarci 75 antibody was not precipitated by 14"/" w/v sodium sulphate, a 16"/.w/v sodfum sulphate concen- tratlon dld precipitate aË least 8C% of the antibody acll.vity and this was used as the initial step in the Ísolatfon of lizard 7G.

To avoid wastage of lizarci serum, lhe soluble fractions remaininE after the sodium sulphate precipitation ancÍ gel filtration steps used ln isolating lizard ryN4r were also usecl for the preparation oi lizard

7S 'yG'.

(b) Sephadex G2ûe gel filtration chromaiography (Flgure 5.68).

The protelns from Step (a) were cllssolved in a minimal volume BSA SERA Figure 5.5 DEI\E:9EIJ.IIIOSE 9HROIvIATOGRAPHY OF A1ÙTI

Appro>dmately 90 mg. of proteln was added to a D[AE-cellulose column (l cm. x 26 cm.). The proteln was eluted wtth the followlng buffers :-

(t) 50 ml. 0.01M trls-phosphate pH 8.0i and

(tl) A ccncave gradlent (total volume 300 ml.) fiom 0.01M trls- pH phosphate, PH 9.0 (250 ml.) to 0.5M phosphate buffer, 4.5 (50 ml,).

Legend :

Protein (OD 2S0 mt¡)

r.< Haemagglutlnatlng antlbody actlvtty

A. Llzard antl BSA serum ) ) contatnlng 73 BSA B. Mouse antt BSA serum ) antlbod,y onlY ) C. Rabbft antl BSA serum )

The gradlent proftle (smooth lfne) ls shown in Flgure c, and was ( commenced ln each lnstance at the polnt lndlcated bV the ô¡ïo\nt + ). Ttre equatlon descrtbtng the gradlent used ls :- .207 A2 cr =r-0-vr) (slnce 0 2s7 ) 2 A1 where c, = concentratlon of the gradleQt at a umê 1l when a fractfon v, of the total volume of the gradlent has been wlthdrawn.

profiles To enable conrparlson of the elutlon and an$,body actlvfty deptcted !n thls flgrrre varla$on tn tt¡e er¡perl¡nental parameters was mlnlmtsed by use of the same column and batch of DE'AE and the same piepäfatlof¡s of buffers and eluttng procedure ' DEAE -CELLULOSE CHROMATOGRAPHY OF AI{TI-BSA SERA

A

I 0

B

t'0

0

c â E o I o Ò o. > o 3 ! ! ¡ G I Ë û =a a ¡ ã C Ê o I c¡ E ¡ o â ¡o I !C I ê ! o .9- (,E ¡

I 0 200 250 300 1020 0 50 100 150 EFFLIEXT YOLUIE (nl). Flgure 5.6 DIAGRAMSIATIC REPRESENTATION OF THE PROCEDURE USED TO PREPARE LEARD '7G' FROM LUARD SERUM

Step A. SodÍum sulphate precipitaLion 16% w/v.

Step B. GeI filtration on SepLradex Cr20Ú of the

l6% sodium sulphate precipitate dissolved in trÍs-HCI buffer.

Step C. DEAE-cellulose batch chromatography of the

aniibody containing fraction from Step B.

Step D. CM-cellulose batch chromatography of the

arrlibociy containing fraction from Step C.

Antibody activity profiles are shown as a broken line '

The continuous lines are protein peaks. A SoDril Sttfll¡E PFECfffATÛ'|. C DEAE-CELL()sc Clfrl¡AIOGRAPttY'

2 \rcLs 242 NEso¿ + 16Z FRECIPfTATE 1 \ðL LIZARD SEFI¡,I l- 9 E d STEP B a 5 .-POOL4\ e0O3M P+ r-lolvl pl-l 8'0 -0.12MpH 7'0 pl-l 7.0 EFFLIJENT VOLlT.l€

(SEPIIADEX CM-CELLULOSE CI{ROIVtAÍOGRAPÈIY' B GEL FILTRATþN G2OOI D

o @ N I I I F E I U' g, z z I U U t o o I ) J a I F (It- re+OOL-r'. À o /\ --\ o .-ûOlM P'-ì pll 7'O pll 6l() EFFLU€NT FRACTþT{ EFFrlfNf \Otr.hÆ -t22- of trts-HCl buffer and fractÍonated on Sephadex G20Û . 7S antlbody activlty was four¡d in the seconci peak eluted ffom the column' These fractions were pooled, concentrated and dialysed against phosphate buffer (0.04M pH 8. íi).

(c) pnAn-cellulose chromatography (Figure 5. 6C).

'Ihe antibody protein sarnple Írom the previous step was adsorbed onto DfiAf-cellulose equillOratecÍ with C. t4IVi phc'sphate buffer pH 8.0 and eluteci witl'r t,.lzIVÌ phosphate buffer PII' 7 'ii ' The protein eluted contained nrost of the anLibociy aclivity and represented

60"/" of the total protein applied to the column. The conditlons for

thls batch process had been prevlously determined using a linear

concentratlon gradient of phosphate buffers.

The antibody protein fraction was concentrated and dialysed

against phosphate buffer (0.01M pH 7.0) in preparatlon for step (d)'

Immunoelectrophoretic analysis of the proteln fraction at this stage

shorn¡ed that 3 prqteins were present. One of these was shown to

be a transferrfn because of lts abllity to blnd raclfoactive iron ¡FuSg).

(d) CM-cellulose chromatography (Ftgure 5.6D).

The fraction resulting from step (c) was applied to a CM-cellulose

column equilibrated with phosphate buffer p.Lìtvi pH 7.0). The

lizarci 7S antibody bounci strongly to this cationic ion exchanger

ancl it was pgssible to elute it from the column ln pure form Oy -123- batch elutfon with phosphate buffer (0.15M, p¡l 6.0) after the other phosphate contamlnaüng proteins had first been removed by passage of previously buffer of lower molarity. These conditions had also been

determined using gradient eluLion techniques '

heary Although the method just descrlbed Ís lndlrect and subJect to

procedural losses, lt does permlt the lsolation of immunoelectro-

phoretically pure lfzard 7s immunoglobultn with antibody actlvity' of About 30 mg. of immunoglobulln could be obtafned from 50 ml'

lizard serum. An immunoelectrophoretic pattern of the isolated lizard

,yG, lmmunoglobulins is shown in Figure 5.9 which faces page 128'

(iii) Prgparatfon of llzard antibodv specific for BSA

A cellulose immunoadsorbent was used to isolate lizard antibody

specific for BSA.

The lmmunoadsorbent was prepared by covalently blnding BSA

to the carboxymethyl moieties of carboxymethylcellulose by the

carbodiimlde reaction. water was added to z g. of cM-cellulose

powder (\Mhatman cM70) unttl a slurry was formed which could be

continuously mlxed uslng a magnetic stlrrer. BSA (2S0 mg') was

added and the pH of the resultantmlxh¡re was adJusted to 5'0'

Carbodifmide reagent, (I-Cyclohe>q¡I-e- (2-morpholir¡o-ethyl) - -r24- * carbodllmlde metho-p-toluenesulphonate, Aldrich-No' c.10 , 64t-2. I g.) was then added and the mixture allowed to react at room temperature for 24 hours with continuous stirring. During this reaction the pÊI was maintained at 5.Û. The carboxymethyl cellulose peptidyl BSA (CM-cellulose-BSA) forrned was thoroughly washed to remove excess reagents and ciried '

CM-cellulose-BSA (1 .5 g. ) r,r'as equilibrated with 0.05M phcsphate

buffer pH 7.4 containing 0.10M sodiun: chloride and packecl into a ** column 1 cm. x 20 crrr. The sample of anli BSA sen¡m (2.5 mI.)

was ciiluted with 2.5 mI. of the equiliirration buffer, washed onto

the column and allowed to remain Írr ccnLact with the immuno-

acisorbent for 30 rninutes '

The loaded colurnn vyas eluted with phosphate buffer until the

optical density at 28t) mp of the eluent was less therr a.125. The

majority of the serum proteins were eluted from the column: these

were collected, pooled an,l subsequently recycled. The BSA

antibody whlch had acìsorbed to the cM-cellulose-BSA was eluted

from the column by passage of a 0.1M glycine-Hcl buffer pH 2.{i,

containing 0.05M Nacl. The eluent was collected in 2.5 ml. *-' A water soluble carbo

l/tlhen aII the protein had been eluted from the column and the optical density at 28Û mp of the glycine buffer eluent had reh¡rned to zero, the CM-cellulose-BSA in the column was washed with the inltial phosphate buffer until the pH of the eluate returned to7 '4'

The sen¡m proteins whlch had not been acisorbed when first applied were agatn passaged through the column and a second batch of BSA antfbody eluted as before. Thls process was repeated a third time'

After 3 passages nc more ant[body could be lsolated from the senrm protelns. The elutÍon profiles obtained bytfre 3 successive passages are shown in Ftgpre 5.7, The successlve passages extracted 55%,

27% and,70/" of the total BsA antibody content of the serum respectively.

The 3 batches of specÍf1c BSA antibociy were pooled and then

dialysed dgðtnst 2 xUi$ volumes of phosphate buffered saline, pH7'0.Whenthisprep,arationwasconcentratedandsubjected

to fmmunoelectrophoretic analysfs, a small quantlty of a contaminant

was detected. Passage of the preparation through sephadex G200

removed this contamlnant. It was obsenred that the specific

antlbody lsolated was easlly clenatured because storage at 40 Fisure 5.7 : EIUTIOIV OF LIZARD BS4 ANTIBOITY FROM Ai\ü IMVIUNOADSORBENÎ

The figure shows the elution profile of.lizarå specifíc BSA aniibody from a CM-cellulose-BSA immunoadsorbent column with glycine-HCl buffer (0.15M, pH 2.0). 2.5 mI. of lizard anti BSA was applied to a I cm. x 2t cm, column contalnÍng l-5 g. of immunoadsorbent.

Legend

H Elution of antibody which ¡our¡d to the immunoacisorbent on ffrst passage of the lizard serum through Lhe column. (55% of the total antibody &'as bound on this presentalion).

H Elution of antibody which bouncí to the immunoadsorbent on seccnd passage of the lizarci serum through the column. (27% of the total aniioody was bounc on this presencation).

H Elution of antibcdy which $ourrCì to the immunoaosorbent on third. pasÉage of the lizaro serum tfuough the column. (7% of the lcial antibociy v¿as bounc oi'l tirls presentation). WITH ELUTION OF SPECIFIC LIZARD ANTI BSA GLYCINE -HCl BUFFER PH 2'O.

1 E o Â| 03 t ts Øz u¡o J

IÞ o O'l

5 10 15 20 FRACTION NUMBER. - 126' in phosphate buffered sallne, pH 7.0,resulted in the fornration of an appreciable precipitate of denatured protein. This rn'as also observed wiih lizarcl ,7G' immunoglobulin preparaticns. The antibody prepared in this manner reacteci with BSA in an ouchterlony test.

An immunoelectrophcretic pattern of the purffied specific anLibody is shown in Figr:re 5.9 (facing page 128). The pattern obtained agar when whole lizard anti BSA serum was electrophoreslsed ln an gel (as fcr immunoelectrophoresis) and then "developed" with a (Figure 5'9)' 1 mg ./ml. solution of BSA 1s also shown in this figure fast electrophoretic The precipltin line formecl lllustrates the relatively the behaviour mobilfty of the lizarC and.body and is conslstent with

ofthisantibodyonDEAE-cellulosechromatographyreportedln

sectlon ç (lt) .

A sample of rabbit anti BSA serum taken from a hyperlmmunized was also passed rabbÍt containing anti-ocdy exclusivety of the 7G type that the behaviour through the immunoacsorbent column. It was found in Figure 5'7 of the rabbit antibociy v¡as almost ideni:-ical to that shown less for lizard antibody, although the rabbit antiserum containec was unchanged BSA antlbocly than the lizard serum. This result evenifthebufferlngcapacityoftheglycineelutingbufferwas

altered by dÍlutlon with salfne (I ; IÛ)' -L27- In contrast, a mouse anti BSA serum, which contained some macroglobulin antibody aud a low total antibody level displayed eluted different elution parameters. The mouse BS¿,1, antibody v¡as with less glycine-HCl buffer (the peal< of the eluLion profile occurred at fracticn lÛ) and only 35?ó of the total anlibod'y was retained on the immunoadsorbent clurlng the first passage of the senrm'

This finding indicates that the avicìity of the lizard BSA antibod'y greater than was comparable wlth that of rabblt BSA antlbody and the absence that of mouse BSA antibody. These results suggest that from a of cross reactions of lizard anti BSA senrm wit} the albumins 4) is an indication number of other vertebrates (mentioned in chapter ofahighdegreeofspeciflcity,andnoiduetolowavidify'

D. REACTION OF BSA WITH LIZARD A\TTI BSA

lhequantitiesofBsAantÍbodyinvarfcusbatchesoflizardanti quantitative precipitln reactfon BSA sera were determined using the dfssolveci (Chapter 3). Varying quantltles of BSA (10 pg' - 2'0 mg') in0.25mI.oÍboratebufferec]salÍnewereacldedtoc'25mI.of formed were removed lizard anti BSA serum. The immune precÍpitates bycentrlfugationarrcianalysecifortheirproteincontent.The antÍbody and supernatants were testecl for the presence of excess antigentoensurethatatnreequivalencezonehadbeenreached. A and B' are The results obtained with two ltzard serum batches, Fiqure 5.8 : OUANTITATIVE PRECIPITAI'IOhí OF BSA wïTH TIZARÐ ITI\TTI BSA

Varying amounts of BSA (in U.25 ml.) were addeci to Û'25 mI. immunized lizard serum.

Abscissa : Logarfthm, BSA added (+,g) to 0.25 ml. lizard

anti BSA serum.

Ordinate : Optical densÍty at 280 m¡r of a sodiurn hydro>dde

(û.lI{) solutÍon of the 4 x cold saline washed

immune precipitates ( H )

A and B refer to two batches of lizard anti BSA serum.

Batch A contains 5.zl mg. BSA antibociy per mI. of lizard serun:'

Batch B contains 2.7 rng, BtA antibod.y per nil. of lizard serum'

The molar antiþody to aniigerr ratio for ihis system was 3. l:'1. PRECIPITATION OF BSA WITH LIZARD ANTI BSA

+ Antigen Antibody+++ exoess.+ +++ excess. A

o.l a. € o slc ro 20 30

ú, C êo o .9 ÉL o 03 B

o.r

ro 20 30 (to¡$ 0oo¡ts) (tooopsù Logro BSA added (lrg) to O.25ml. lmmune Lizard Serum -t28- shown ln Figure 5.8.

at using the data shown in this figure and uttlizing the fact that precipltate, the equlvàlence polnt all ihe BSA was in the im¡nune was calculated' the antibody content of each batch of Iizard sefum used mg' BSA Itwas found that batches Aand B containeci 5'/i and 2'7

antibociy per ml. cf lizarci semm respecilvely' calculated' The ratio of anLiboLly to antigen at equivalence was also be 6'7 l' If The mass ratio of antiborjy to antigen rças founcl to : to be I50'OCC the molecular weight of lizard yG antibocì'y is taken be 3 l : I (see chapter 6), then the molar ratio was calculated to ' ' for'the These values are very similar to the colTesponding ratios (cited in BSVrabbit anti BSA system reported by singer and Campbell Day, 1966, Page 139)' E. l.i

(i) Antisenic cross reactiviw the antigenic cross reactivity of lizard 'yfui' anci '7G' immuno-

globulins was invesLigated by ouchterlony double dtffuslon tests whole llzard ln agar gel uslng rabbit antisera prepared agalnst shown in serum ancl llzard '7M' immunoglobulin. The results are

Flgure 5.10.

In Figure 5"104 the pattern obtalned rnzhen both immunoglobulins that were dtffused against RAL serum is shown' It can be seen occurred with a llne of ldentity was formed, however spu¡ring also Fiqure 5.9 : IMMUNOELECTROPHORHIIC PATIERNS

I. Human serum (both wells) developed with goat antl-human

serum in the centre trough.

2. Lizard sen¡m (both wells) developed with ralcbit antl-lizard

senrm.

3. Human yG (both wells) developed with goat antl-human

senrm tn the centre trough.

4. Lizard 'yG' d-eveloped with rabbÍt anË.{Ízard serum.

( Serum of lizards hyperimmunized wiih BSA electrophoresed

and the pattern cieveloped with a saline solutÍon of BSA

(l mg./mL.) in the centre trough.

6 Lizard 'yM' develcpeci with rabbÍt anti{izard serum.

7. Lizard ,yM, developed with rabbit anii-lizarci serum in the

left hand trough and rabbit anti-lizarii 'yM' in the right

hand trough. ro

i' F\ F. C-{ I w r €

(r) Fiqure 5.1Û : OUCHTERLOIVY ANALYSIS OF LEARD IMMUNCGLOBULINS

A. V/eIl I lizard ryG' Well 2 lÍzard'7M' Well 3 llzarci'7M'

lMe1l 4 rabblt anli-lizard '7ld' serurn.

B lMell I lizard 'yG'

WeIl 2 lizard anttbody specific for BSA Well 3 rabbit anti-lizard serurl.

C. (photo) TVell I llzard 'yM' WeII 2 llzard'7G'

Well 3 lizarci 'yM' WeIl 4 rabbit anti-lizard serum.

A diagrammatic representation of the lines of identÍty and the spurs formed in the ouchterlony pattern shown in Figure C. is also included. O

@a a o j

Ð -t29- each preclpltin line. This indicates that these lmmunoglobulins have common antlgenfc determinants but also have determinants with the unlque to each lrnmunoglobulin. This result ls consistent classes hypothesls that these immunoglobulins represent two dlstinct '

botlt This hypothesls was supported by the pattern obtalned when serum lmmunoglobultns were reacted wlth rabblt antl llza¡d '7M' ' line of this pattern ls shown in Ftgure 5.I08. In thts lnstance a identlty was formed, but spurrlng only occr.rred wtth the '7M'

precÍpitin line, indlcatlng that lizard 'yM' and '7G' share anligens

but that the 'yM' immunoglobulin contains antlgenic determlnants

not present ln the '7G' lmmunoglobulin'

that lizard lmmuno- The probable explanaLion of these results ls globullns, like higher vertebrate tmmunoglobulins, contaln common

L chains and cltslinctive H chalns '

(ii)@

Theamlnoacldcompositlonsoflizarcìimmunoglobullnswere steln determined accoring to the standard proceciure of spackman,

andMoore(r958)usingaTechniconautomaticaminoacfdanalyser. at II0o for 22 Samples were hydrolysecJ wlth 6M hydrochloric acid

a¡'l lntemal standard' hours and 48 hot¡rs. NOr-Ieucine was used as Thevaluesrepontedaretltemeansofthe22hourand43hour serlne, half-cystine, analyses, except for the values for threonine, TABLE 5.2 *

Human *+ Ltzard Immunoglobulins immunoglobullns Amino (Spec Acid 'yGt 'yG' 'yM' yG yM

Asp I I o I 8.6 8.2 8.6 7.6 8.7 Thr** I a 0 4. 7 8.7 Ser** 8 5 9. I 7.8 t0.3 7,8 Glu I 3 I 13. 8 L2.8 12.0 12.8 Pro 5 3 7.0 6.3 6.4 6.3 Glv 4.1 4 9 3 7 3 6 3.7 4 4.2 Ala 4.1 4 3 4 a 2 3 VaI. 5.8 ç, I 7 I I 6 7.8 Cys/2** 2.3 I 6 I 9 2 3 I.9 A Met I.6 0 G I = 0 9 1.4 2 3 3 2 Ilu 1, 2 4 0 3 3 Leu 7 I 6 I 7 6 7 7 7 6 TYr't* 6 2 5 3 5 0 6 4 5 t /, 4 0 4 2 T 7 4 6 4 I Phe E Lys ( I 5 7 5 7 ¿t I His 3.1 3. 3 2 2 2 4 2.2 Arg 5.8 6. rJ 6 I 4 3 6.r (N-NH^)** 2.3 2. 5 t 5 I 6 1.5 (1.e.. ¡ùo1o" N)

TOTATS 100.1 100.I 99.5 100.0 99.6

free * E:

_* ¡ à v) ¡ a, (n o 0) N > û Oi .1 È\ -> o rJ) .c -c u' ('' (,) (r) o o L É Ë g ¡{ 9 rd rú L. o¡{ f{ rt{ l+-l (ts{ l+{ r0 ró ro ur Þl ts N N N rn4 o o J ) a J -'{ ¡l{ Fl{ F¡ FI Þ¡ co o a co É llizard'yM' U 6.2:7.2 8.1 8 6 tt.2 7 a 1

;Lizard'yG' 6.5 6 .2:6.3 I I 2 9.6 6.4

:Lizard Spec. lì e: :BSA'ycl I û 7 r3.3 r3.9 lI.5 Je J: (t Dogfish 17S 'yM' 0 .J 2 I 10.1 ll.I 7.2 1 (r Dogfish 75 'yM' 0 I 9 0 10.9 6

(2 Bullfrog 'yG' 0 4 I 10.5 I I

(2 Bullfrog'7M' 0 ll.3 9 6

(3 0 6.5 Human yG

(g 0 Human TlvI

Amino acid compositions taken from followlng sources : (t) Taken from Marchalonis ancl Eclelman, (f 966a)

(2) Taken from Marchalonis and Eclelman, (1966b)

(S) Taken from Scht¡ltze and Heremans (1966) -130- tyrosine and amlde nitrogen which were obtained by extrapolation of the 22 and,48 hour hydrolysls values to zero time. The results have been summarizecl in Table 5.2, Lizard immunoglobullns resemble those of other vertebrates in their high content of the amino acids serine, threonÍne, aspartic acid and glutamic acld.

An assessment of the "compositfonal relatedness" between llza¡d immunoglobuttns and those of other vertebrates wôs canled out using the method of Metzger, shôp|ro, Mosimann and vlntor (1968). A maffix of dlfference indíces for various immunoglobulins was calculated and ls shown tn Table 5 .3 . (Calcutatl'ons were facÍIitated by use of the untversity of Adelalde's cDc6400 computer). The median DI obtained by comparing 630 palrs of protelns (Metzger-et aL-

I958) was 25. The values reported fn Table 5.3 are considerably less than 25 ancl inciicate that the proteins compared in this table comprise a related group.

In this method the amino acid composltlons of two proteins are compared and a measure of their relatedness, the difference index

(Dl),esttmated. The ÐI is defined as 50 L[mes the sum of the mole fractlon dffferences per 100,000 g. protein for each amino acld in the two protelns being compared : two proteins wlthout any amlno aclds ln commonwould have a DI of I00, and two idenü.cal protelns would have a DI of zero. -I31 - (iit) Carbohvdrate content of llzard immunoolobulins

The percentage of carbohydrate present tn llzard immunoglobulins was determlned as hexose using the anthrone reactlon and a glucoSe standarcl (Materials and lviethods page 79). The hexose ccntent of a sample of human yG was cietermined at the same time to Serve ôS a control.

Lizard '7G' immunoglobulln was found to contaln (r.8 + 0.3)%

hexose, and lizarcl '7N{' immunoglobulin was found to contaln (1.4 + (6.7 t I . O)Z hexose. The human yG was found to contaÍn 0.2)% hexose, in agreement with published values (Cohen and Porter,

I s64) .

(1y) furalvtical ultracent¡-ifuoatíon of lizard immunoqlobulins

(a) Se¿imentation coefficients.

Standarcl sedimentatlon coefficients (i'"' t20,* tult"") were

calculated for each inrmunoglobulin at several concentrations from

sedimentation velocity clata obtained using a Spinco Model E analytical ultracentrifuge with schlteren optics ' The schlleren

patterns obtained for these immunoglobulins are shown fn Figure 5'lI '

Lizarci'yM'was founcl to have u tTr,uu = I9'IS' The concen-

tration dependence of the standard sedimentation coeffÍcients of this

immunoglobulin is shown in Figure 5.llB. The sedÍmentation velocity pattern for thls lmmunoglobulin revealed small amounts Pioure 5.1I : ANALITICAT IILTRACENTRIFUGE PATTERNS OF TIZARÐ SERUM AND LIZARD TMMI'NOGLOBULINS A. Ulffacentrffugal pattern of not al ltzard serum ' Protein concen- traËon, l0 mg ./mI.; photograph taken 4Û minutes after reryhins mar

B. Ultracentrifugal pattern of lizard '7M' irnmunoglobulln ' Proteln concentration, 3 mg./ml.; photograph taken 16 mlnutes after reachlng maximum épeeO of 50 ,740 rev./m¡n.¡ phase plate angle 600; sãtvent tris-Hgl. Sedimentation proceeded from left to right.

ultracentrifugat pattern of lizard 'yG' immunoglobulin. Proteln concentraLion, 5 mg ./mI.; photograph taken l6 minutes after reachins maldmum speeO of 56,100 rev./min.l phase plate angle 60u, acetate buffer 0.15M, pH 5'l' Sedimentation proceeded"ol.tent, from left to rlght.

Protein D. ultracentrlfugal pattern of llzard '7G' immunoglobulln' concentration, 4 mg./mL.; photograph taken 24 mlnutes after plate angle reachins ma¡dmum ãpeea of 56,100 rev./m¡n.; phase ;0"; ;á.r"nt, trts-Hgl. Sedlmentation proceeded from left to rf ght.

Protein E ultracentrifugal pattern of lfzard '7G' immunoglobulin' 4 mg ./mL.; photograph taken 40 minutes after concentration, plate -6-o-1;ã""ir,reachlns maximum ãpee¿ of SO,l0ü rev./mln-l phase angle rrir-HCI + 4M urea. Sedimentation proceeded fronr left to rlght- t.\

I Figure 5 . I lA : Standard sedimentation coefficients of lizard '7G' as a function of concentration (solvent-tris-HCI + 4M urea) I 0

7 0 o

6 0 o

3 O 5 0 c! o 4 0

3.0

1 .0 2.0 3 .0 4.0 5.0 Concentration of lizard 'yG' (mg./ml.)

Figure 5.118 : Standard sedimentation coefficients of . Iizard 'yM' as a function of concentration (solvent-tris-HCI) 20

I9 o I8 o 1 L7 o C\t U) I6

I5

0.7 t.4 2.1 3.5 Concentration of lizard '7M' (mg ./ml.) 132 - of contaminants with sedimentation coefflclents of about 325 and 65.

HÍgh molecular welght contaminants have been found ln many purified macroglobulÍn preparaLions, (e.g. Marchalonis and Edelman (1965h

Turner and Rowley (1g63); Kunkel (1960))'

The schlleren sedimentation pattern for purified lizarci 'yG' immunoglobulln unexpectedly showed two peaks with sedlmentation coefficients of approximately 7S and I5S. Ftgther investigation showed that the high molecular weight component appeared to be an aggregate of the 75 component. It was founci that the proportion of

the high molecular weight eomponent decreased with decreaslng pH

of the solvent and it was absent when the lizard '7G' was dissolved

in solvents of pH 6 or less. Although hÍgh sAIt concentrations ' e.g. I.5M NaCl dicl not remove thls Component, it was absent when the solvent contalned 4M tuea or 4M guanfdine hydrochlortde. (See

Flgure 5.llC, D, and E.)

The standard sedimentatlon coefficients of this immunoglobulin

were therefore determlned in trls-HCl buffer containing 4M ur@ô' fhe s1.^ for lizard 'yG' immunoglobulin was found to be 6.95. The ¿u ,w concentretion depenclence of the sedimentation coefficient for this

protein is shown in Figure 5, ltA' -133-

(b) Motecular weight measurements .

The molecular welght of each lfzard immunoglobulin was measured using the high speeci sedtmentation equilibrlum method described by used YphanH.s (1964) (see also Chapter 3). A Spinco AN-J rotor was fn these experiments to minimise rotor precession at the relatlvely low speeds used.

The solvent used for lizard '7M' was iris-HCI buffer' Because of its polymerising properties, it was necessðry to determine the molecular weight of lizard '7G', in the presence of 5M guanidine hydrochlorÍde. Iodoacetamide (Û.005M) was also added to the solvents since Lamm and small (1967) have reported that this minimised size heterogenefty of the lmmunoglobulins caused by aggregation by through the formation of intermolecular disutphide bonds initiated

any free sulphydryI groups present'

The following results were obtained :-

(i) Llzard ryM' lmmunoglobulin; molecular weight 953,000.

Solvent : trls HCI buffer; equilibrium obtained at 943I rev '/min'

(ii) Llzard ,yG, immunoglobulin; molecular weight 158,000. solvent: 5M guanidine hydrochloride; equillbrium obtalned at

!7 ,25Û rev./min-

C¿aphs of the logarithm (base e) of the protein concentraticn,

measured in fringe numbers, versus the radial dfstance squared Figure 5.12 Yphantis sedimentation equilibrium molecular weight determinations. Plot of the logarithm of the concen- tration (in fringe numbers) versus radj.al distance squared. The line is a least-squares fit of the data. Lizard 'yM' tris-HCl + 0"005M

iodoacetamide "

I d 0 o .FtU rd l{ ! c C) o -2 o o ö' o È -4 49 49.s 50"0 2 Radial distance squared (cm. )

I o .HÐ rd Ðç 0 c o) Lizard 'yGn 5M O c guanJ.dine IlCL + 0"005M o o iodoacetarni.de " Ot o -1 Ë

-2 49.0 s0 " 0 51.0 2 R.adial distance squared (cm ) -134-

("*.2) for these determinations are shown in Figure 5.12; the straight lines shown are least squares fits of the data' The plot for departure from linearlty at the bottom of the ceÌl in the n'laterÍal ltzard ,7M' indicates that a quantity of high molecular welght alrsence was present fn the sample of this immunoglobulin used' The thÍs sample was of any curvature in the plot for lÍzard 'yG' shows that the 5M guanidine homogeneous with respect to size in the presence of

hydrochloride used as solvent'

immunoglobullns Since the partial specific volumes (î) of the lizard an error of were not determlned experimerrtally in ihe solvents usecl, for Thls alone about 2% coulO exist in the values calculated i' estimates I{ence would result in a 59é eITOr in the molecular welght ' and 'yM' the error in the molecular welghts reported Íor lizard 'yG'

would be between 5% and 8%'

F. DISCUSSION irnmunoglobulln' each A macroglobulln and a slowly sedimenting manifestingantlbodyactivity,havebeenisolatedfromlizarciserum' immunoglobullns These have been designated tizarc 'yM' and 'yG' properties which respectively on the basis of their physicochemical

have been summarlzed Ín Table 5 ' 4 '

to those of The properties of lizarcl 'yM' were very similar with thiols reduced mammalian yM lmmunoglobulins ' Mild reduction TABLE 5.4

PHYSIC OC HEMICAL PAIU\METERS OF LEAIID IMMUNOGTO BI'LINS

Parameters 'yM' 'yG'

Þ

mobilitY EIec EophoretÍc p TttoP (Paper : Veronal buffer PH 8.6) ('fast' 7)

Carbohydrate content (as hexose by anthrone (6.7 ll.cì)% (r.8 + a.3)"/" method)

PartÍal spectfic volume, Ç (calculated from amino acid 0 .7I0 0.722 compositions)

Sedime ntation coefflcient 19.I 6.9 (svedbergs) "?0,*

Molecular weight (weight average - YPhantis 953,0ûù I58,000 methoci)

--æÞ.*: -l3s- it to 7s subunits wÍthconcomitant loss cf antibody activlty' In contrast to the close resenrblance of lizarci 'yM' and mammallan 7M, the properties of lizard '7G' differed stgnificanily from mammalian yG in several aspects. These included its elect¡ophoretic mobillty

and ease of PolYmerisation.

In sectlon B it was reported that liza¡d serum did not contain however a a dlstinct electrophoretic proteln fraction. There was

5C% of the total serum 'fast'y to P fraction whlch comprised about proteins. Electrophoresis experlments (both PVC zone electrophoresis BSA antiirody and immunoelectrophoresis) showed' that 73 lizard anti

had the electrcphoretic mobility of a fast y'

TheabovefindÍngwasconfirmedbycomparingthebehaviourof

IlzarC 75 wlth that of rabbit and mouse yG antlbodles on DEAE

cellulose chromatography. This experirnent, vrhich was ciescribed' in section C(ii), showecr that lizarcl'yG'bounct to the ion exchanger Rabbit anti more firmly than either rabbit or mouse 7G antibodles ' for this experiment BSA serum and mOuse anti BSA serum were ChOsen of the since rabbtt antl BSA antibody has an electrophoretic mobility of a y2 type (i.e. a ,slo\ry, 7) and mouse anti BSA antlbody conslsts

mlxture of slow and fast y's called V, and V, 7G immunoglobulins'

DEÀE cellulose ls The relatively strong bÍnding of llzard antibody to provldes an a reflection of lts electrophoretic properties and -136- explanation for the diffÍculties encountered in purifying thts immunogloi¡ulln. It was interesting to observe that lizard and rabbit antibodies were also resolved inio two fracticns as was mouse antlbody. The antibody actlvity Ln each fraction was reslstant to

}-MfE reduclion. The partial resolutlon of mouse 75 yG anlibody into two fractions has been clocumented (e.g. Fahey, 1967) and more recently Faulk and Pondman (1969) have reported the existence of two types of rabbit 7G antibody which differed in electrophoretic mobillty. The elution profiles shown in Figure 5.5 are consistent with these reports, and it would seem that at least two types of

lfzard 75 antibcdY may also exist.

The physlcochemical parameters of lizard 'yM' and '7G' immuno- rvVtren globullns have been summarlzed in Table 5.4 ' these data were

considered, particularly in conjunction wÍth the studies on reduceci

immunoglobulÍns reported in Chapter 5, it was evident that at least description tr¡,,o classes of lizard immunoglobulln occurred ancì that their

as 'yMt anci 'yG' immunoglobulins wes a reasonah'Ie one'

The polymerÍsatlon of lizard 'yG' , demonstrated by analytlcal

ul.tracentrifugation, was an unexpected finding. \Mhen thls prOperty

is consldered together with the 'fast' y electrophoretic mobillty of this protein lt is reasonable to question whether thls lmmunoglobulÍn

could be analogous to the mammalian yA immuncglobulln class ' 137 -

There are four reasons which suggest that this hypothesis is not Justffied. These are :-

(r) The low hexose content of this immunoglobulin (1.8%) was

comparable with the hexose content of mammalian yG immuno-

globultns. Immunoglobullns of the 7A class have been found

to have about 4-5 times this amount'

(2) The molecular weight of the lizard 75 immunoglobulin seemed to

be more consistent with that of a yG type immunoglobulfn' This

point was substantiated by the data presented ln the following

chapter where it was shown that the molecular wefght of the

H chaln of lizard '7G' was close tO that of the human y chaln' yA The molecular weight of the H chain from mammallan immuno-

globulÍns is about 65,000 higher, (e'g' see Cebra and SmaII'

1967').

(3) The quantities of this antibody found ln the sen¡m of hyper-

immunized lizards were, by analogy with mammalian systems,

more in keeplng with it being of the yG immunoglobulin class'

The levels of humoral 7A antibody are usually characteristlcally low (Davis et aI.. 1968). Although this analogy need not of necessltyholdforlhisspeciesof}izardÍtdoesprovide

circumstantial evidence in favour of a yG immunoglobulln class

belng lnvolved. -138-

(4) Nthough lizarci 75 antiboCy was bound to ÐEAfrcellulqse more

ftrmly than either mouse or rabbit 7s antibody, it was eluted

well before llzard 7M antibody. In mammalian systems 7A

antibodles are uSually eluted from DEAE-cellulose wlth the 7N'I

immunoglobulins .

Although the evÍclence presently available lndlcates that this immunoglobulln is analogous to rnammalian yG its tutusual properties warrant further investigatlon.

The isolation, by means of an lmmunoac;.scrbent. of lizarci antibody was a specific for BS:\ tllustrateci several points. Firstly, that this better method for ihe isolation cf lizard 'yG' imrnunoglobulins ti:an

the relatively teditus 4 steç,r prcceciure:-'utlined in section C0i) -

parlicularly as the inimunoacsorbent cculcl be reused. secondly, antibcd'y comparison of the elutiurr characterisiics of rabbit and lnouse tc isolate speclfic for BSA (from the same immunoacisorbent column used

Iizard specific antibcciy) wlth the eluLion characterlsLics of the lizard tc that antfbody suggestecl that thls antibody was of sfmilar avidity from of the rabbit antibody. since the rabbit ant'tbody was lsolated to be of the serum of hyperimmunlzed rabbits anii cculd be expected

hfgh avidity Ít appears that this species cf lÍzard is capable of

synthesising high avidity antlbody. Grey (1963. t966) reportecì

that turtle antlbocly to KLH was of low avidity relatfve tc a rabbii

antibociy against the same immunogen, and Everhart and shefner -139- (f 966) reportecl that goldfish anti BSA antibcdy was of low avicüty relative to a rabbit anli BSA antibody. In the latter fnstance' however' it was not stated whether the goldfish antlbody wôs of the macroglobulÍn or 75 type and Everhart and Shefner may have cornpared dlfferent classes of antibody.

The reaclton of BSA wtth lizard antJ BSA anlibody was studied using the quantitative preclpltin methocl (secticn D) ' Three findings warrant mention.

(I) Comparison of the shape of the precipitln curves shown in

Figr.fe 5.8 with precipiLin curves obtained using rabbit anti

BSA serum from a hyperimmune rabbit substantiated tþe view,

presente,J abcve, that the lizarcl airiibocly had a similar avidity

to rabbit antibodY.

(2) The unusual precipitating properties of goldfish antiborJy reported

by Everhart and Shefner (1966) (i.e. 3 equivalence points ancl

a large antigen to anlibocly ratio) were not cbserved for this

poikilothermic sPecies .

(3) The molar antiborJy to antlgen ratio at equivalence was calculaied

to be 3.1:I, ln good agreement with previously reported values

(e.g. slnger and campbell, citeci ln Day, 1966 page 139) for

' colTespondfng marnmallan BSÀ antibody systems ' -140- The amlno acid compositions of the lizard immunoglobulins were determlned prlmarily to estimate the partlal specific volumes of these proteins. These data were also used however to assess the related- ness of ltzard immunoglobulÍns and those of some other vertebrates.

The method of Metzger et al. (1968) was used and a rnatrlx of difference indices (DI) has been shown in Table 5.3 . It was interesLing to note the fcllowing sequence of DI's'

(a) Dogfish l7S 'yM' DI = 3.2 antigenically ldentical Dogfish 75 '7M'

(o) BulHrog 'yNi' DI 4.1 ) tyG' Bullfrog ) ) tYMt Lizard ÐI = 6 ) each pair is Lizarci 'yG' ) antigenicallY ) riis tinct .DT Human yIVi âri ) Human ,YG )

(c) Interspecies yG compariscns DI 9.6 ic 1Û.5 InterspeciesYM DI = 7'ltoltl'l'

(d) The median DI for 630 pairs of proteins \rl'as 25 (Metzger-æl' rs68).

These values for the ÐI fa]| in the orcler expected from phylogenetic

considerations and ten¡l to support the view that the twc lizard

immunoglobulins investlgated represent different classes .

In conclusicn it may be stateci that two classes of lizard immunc- globulin were isclatect and characteriseci. It Ís suggested that they

are analogous to the rnammalian yM and 7G immunoglobulin classes ' -141 -

çHAPTER 6_

Paæ

A. INTRODUCTION r42

B. EXPERIMENTAT PROCEDURE 143

C. RESUTTS

(i) Starch geì electrophoresls. . . 146

0i) C.el fÍItration chromatography t47

D. CONCLUDING REMARKS .. aaa lsl -142- A-@.

Edelman (1.959) was the first to demonstrate that an immunoglob- ulfn could be spllt lnto smaller components by reductlon wlth a t̡lol.

He reduced human yG wtth z-ME fn the presence of 6M r¡rea and oÞ sen¡ed the formation of fragments wlth an average molecular weight of appro:d.mately 50,000. About the same tlme Porter (1959) publlshed his results on the cleavage of rabblt lmmr¡nogrlobultns with crystalllne papaln. These two dlscoverles lnstigated many extenslve lnvesÈlgat- lons on the quaternary structure of lmmunoglobullns, the net result of which was the characterisation of the L and H chalns common to all immunoglobullns .

Edelman,s technique fon dlssociattng hurnan 7G was unsatlsfactory slnce the resulting L and H chalns were insoluble, except ln concentrat- ed urea solutlons, and had lost thei¡ biologlcal activity. Flelschman,

pain and Porter (lOOZ¡ therefore re-examined the condiLions for reduclng immunoglobulins in the absence of denaturlng agents such as urea'

They found that lf , after reduction with a thiol and alkylation wlth

lodoacetamlde, the reductlon mixture was allowed to dialyse agalnst a

dlssoclating solvent such as IN proplonfc acld, t}en two components

resulted. These components were separable by zone electrophoresfs, 9f

more sfmply, by gel flltratlon ln the presence of IN propionic acld'

These two components were found to be the L and the H chafns of the

lmmunoglobulln and when prepared ln this manner were fou¡rd to retaln -143- their solubility ln the absence of urea, Thls method, or variations of it, remains the most satisfactory way to isolate soluble, btologically active immunoglobulln L and H chalns.

The dlfferentiation of immunoglobulins fnto classes ls based on antigenic speclflcttles unique to the H chalns. In keeplng wlth thelr an4genic distinctlveness the H chalns of each Immunoglobulln class dlffer in thelr physlcochemlcal propertles. Mammalian 7M and 7G

H chalns are easlly distinguishable when their slze and electro-

phoretic mobtlity dr.rring starch gel electrophoresls ln 8M urea and

at pH 3.0 are cornpared : the p and the y chalns bave molecular

weights of approxlmately 70,000 and 53,000 respectlvely and also

have different electrophoretic mobllities, the 7 chain movfng further

lnto t¡e gel than the p chaln. It has been shown that these

differences in H chalns appear to be a characteristlc of the immuno- globullns of all the specles so far examined Êee chapter 2).

In thts chapter the size and electrophoretic mobility of ltzard

lmmunoglobultn L and H chalns has been investlgated ln order to

establtsh whether the 7S and l7S immunoglobulins belong to the

one lmmunoglobulln class.

B. Þ(PERIM_EIIÎAL PROCEDgRE Lizard and human immunoglobutins were reduced in the followlng

manner:- -144- Each immunoglobulln was dissolved in a solvent containing

2M guanidine hydrochlorlde, 5M urea and 0.55M trts-HQl buffer pH 8.1 . The protein concentration was adjusted to approxlmately 5mg-/mI-soliddithlothreitot(Çleland'sreagent'abbrevfated

DTT) was added to a fÍnal concentration of 0.lM and the mixture allowed to react at roOm temperature for 2 hours. Alkylation was carried out in an ice bath by adding a l0% mola¡ excess of solid iodoacetamide to the reaction mixture and gently mixing for I0-15 minutes.

The reduced and alkylated immunoglobullns were used as follows :

(i) Starch gel elecrroPhoresis : Analytical, vertical, low voltage starch gel electrophoresis

was carried out in the presence of 8M urea and at pH 3 according

to the method of Edelman and Poulik, (I96f )'

(ii) GeI filtralion column chromatography :

A modÍfied version of the procedure described by small and

Lamm (1966) was followed.

A column (1.6 crr. x I00 cm.) was packed with sephadex ç2O0

which had prevlously been equilibrated with an aqueous solud'on of

2M guanldine hydrochloride and 5M urea. Polythene canula tubing

was used for the inlet and outlet flttings of the column since guanidfne solution extracts ultraviolet absorbing material from I45 - rubber. A relatlvely constant flow rate was obtained by adjusting

the column for upward fl.ow of the liquld phase. Care was also taken

to minlmlse the effluent mlxfng volume at the top of the column.

The packed column lrzas equillbrated by passing 600 ml. of solvent

through lt: a steady flow rate of 4-5 mI. perhourwas obtained.

Samples containfng a final concentraLl,on of l0% sucrose were applied

at the base of the column and 2.0 ml. fractions were collected ftom

the top of the column wfth the aid of a fraclion collector. The OD

at 28û mp for each fractÍon was measured with a Shlmadzu spectro-

photometer.

The main purpose of this column was to determlne tf the H chains

of lizard 19S 'yM' and 7S 'yG' lmmunoglobulins differed in molecular

welght. The column was therefore first calibrated with proteins of

knou¡n molecular weight, namely human yG - molecular welght

150.000; reduced and alkylated human L chains, molecular weight

22,000; reduced and alkytated human 7 chains, molecular weight

55,0ü0 (Fahey, 1965h and BSA, molecular weight 67,000 (Phelps

and Putnam, 1960). The vold volume of the column (Vo) was

determined by measuring the elution volume of the macroglobulin

peaks of rabblt anti BSA and llzard anli BSA sera. The characterlstic

elulion volume (Ve) of each of the proteins listed above was then measured and a calibration curve comprising the oarameter ffi as '146- a function of the logarithm of the molecular weight was drawn for the column and is shown in Figure 6.3.

C. REST'LTS

(i) Starch gel electroohoresis of reduced and alkvlated lizard and human immunoolobulins

Figure 6.1 shows the patterns obtained when reduced and alkylated

human yM and yG and reduced and alkylated lizard 'yM' and '?G'

lmmunoglobullns were sublected to starch gel electrophoresis .

It can be seen that the L chains of all the lmmunoglobulins have

a similar mobility and also that the lizard immunoglobulin L chalns

exhlbit similar heterogeneity to the human L chains. The human 7M

immunoglobulin used was a myeloma prgtefn and therefore more

discrete L and H chain bands were obtalned for it in the starch gel.

As was expected, tbe H chains of human yG and 7M were found

to be different, the 7 chaln movlng ahead of the P chain. The H

chains of lizard '7M' and '7G' were also distinguishable. The

Ltzard ,¡r' chain had slmilar moblllty to the human P chaln. The

llzard '7' chaln however appeared to be heterogeneous and to have a slightly different mobtlity from the human y chain, although Íts

mobillty was still greater than that of the '!r' chaÍn. The reasons

for the unusual appearance of the llzard 'yG' heavy chain band are

not apparent. It may be due to an artefact resulting from denaturation Fisure 6.1 : ST¡IRCEI-GEL ELECTROE¡ÍORESIS OF IMMUNOGTOBUf,IN-S

A photograph, anci sketch of the photograph, of a starch gel

electrophoretlc pattern of recÌuced and all

A Reduced and alkylated human ?G

B Reduceci and alkylated lizard 'yG'

c Reduced and alkylated lizarcl '7M'

D Reduceci and alkylated human 7M

The human yfui was a myeloma protein.

Electrophoresls was carried out in formate buffer pH 3 .0 in the

presence of 8M urea. Electrophoresfs was performed for 16 hours

at approxtmately 6 volts per cm. The gel was then sllced and

stalned with Amicio-Schwarz l0B. (Method of Edelman and Poullk,

1961). ABCD

+L CHAINS

+H CHAINS

o - êor€ln

ABCD

(- L CHAINS

<+ H CHAINS

orçrn

-æìÞ+> -I47- of the protein during preparation, or due to an lmpurity in the sample

- although the lizard '7G' was homogeneous when examined by immunoelectrophoresis using rabbit anti lizard serum to develop the

preclpitin lines. An alternative possibility, whlch cannot be

excluded, is that several related types of H chain were present

ln the origÍnal sample.

(iI) Gel filtratÍon of reduced and alkvlated lizard i¡Lmunoqlobt¡lins

The elution profiles of reduced and alkylated human yG and lizard ,7G, ,7M, and are shown Ín Figure 6.2. The callbration "rr*" ffi versus logarith¡n molecular weight) for cietermining the molecular

weights of the eluted peaks is shown in Figure 6 ' 3 '

with The expected elutton pattern from the column was obtalned proteln peaks reduced and alkylated human yG (Figure 6.24). TWo

were evÍdent, which represent the H, Í.ê. 7 chalns þeak 1) and patterns the L chalns (peak 2l of. the 7G molecule. More complex

were obtained for the reduced and alkylated lizard lmmunoglobulins.

In both lnstances, however, protefn peaks were obtained which

coffesponded to the L and H chafns of these Ímmunoglobulins '

The pattern obtained with reduced and alkylated lizard 'yGt

(Ftgure 6.28) was the most complicated and dlfficult to interpret' for peak I was such Four protein peaks were evident. tire ffi ratlo that the protein comprising this peak w-ould have a sÍnrilar molecular 148 - weight to unreduceci lizardlyc" The second peak (2) could aL-H represent (on a molecularweight trasis) a p type heavy chaln, chain complex, or a H-H chain' complex' Each of these entiÈies (3) ha¿ a v,roul.l be eluteci about this position. The ihirci peak to be molecular weight coïTesponding to a y chain and Ís believed

the H chain of lizard 'y?'. . The fourth peak (¿l) hao a molecular

weight correspond'ing to L chains '

The pattern cbtaineci for recuced and alkylatec lizard '7M' peaks corres- (Figrrre 6.}cl) v,Ias ûrore easily interpreteil. ProÈein (peak 3) ponciing io ihe ïj, i.e. 'p'chain (peak 2) antj the L chain a proteia vrere easily dlscernible. .Another peak (1) v¡hich containe'1 withamolecularweighcC|orrespondingtoaTssubunitoftheintact yM Iizarci 'yM'was observed. Incornplete reciuction of rabbit (l'L)66b)' immunoglobultn has also been reported by Lamm and small

was eluteci from A further peak of ultraviolet absorbable niaterial thecolumnaftertheLchainshadbeenelutec].ThisPeakisnct hov,rever was such that show-n in Figrlre 6.2. Its elutlon volume the reduicing lt would be composec of small molecules aud represents the immunogloiculin and alkylating compouncls added to the colu'rnn with

samples.

the column' The Lizarcl anti BSA serurn was alsc passecl through to remove fractions collectecÌ $rere ciialysed against saline in order l4g -

the u¡ea and gruanidine hydrochloride anc'l then assayed by the passive haemagglutination methccf for anlibody activify. Lizard BSÂ antlbody was eluted from the column in a single peaì< with a ffi ot I.41 - 1.53. By reference to the calibraticn curve (Figure 6.3) a molecular wei.ght

of about I50,000 is inclicated for this antibody.

The molecular weight calibration curye shown in Plgure 6.3 may

be written as :- Ve Log (molecular weiglii) 6.583 - x (1) IC ".953 Vo

.Determann (1:168) has reF,orted a sirnilar equation, also deternrineci

empirically, for molecr-llar v'reight estimations using Sephadex G-20ü.

This equalicn is :-

Lcgl (molecular weight) 6.698 - o.9BZ (2) O = " # The simflarity of these two equatlons substanliates the hypcthesls

that the parameter ffi f" essentially independent of the dlmensions of the column and experimental condilions useC.

The range of values of the elution parameter, ffi , for the L arrcl H chains of lizarcl 'yG' and 'yM' and also for lntact lizard 'yG' has

been indicated in Figure 6.3. From these cia'ca the following estimates

c¡f the n'¡olecular weights of these proteÍns were made : Flsure 6.2 t cEt HITTRATION OP REPUCED &ND AttffLAIEP IMMUNOqÉ9BuL.LNS

The elutlon profiles of reduced and alkylated human and lizard

Immunoglobulins from a column of Sephadex G-200 equillbrated wlth

2M guanidfne hydrochlorlde + 5M uÍêê.

6.24 reduced and alkylated human 7G

6.28 recluced and alkylated llzard '7G'

6.2Ç reducec an,j alkylated lizarci '7M'

Column citmensions: 1.6 cm. x lri'ù cm.

Flow rate : Approximately 4-5 ml .¡how, reversed flow.

A further peak of ultraviolet absorbable substances (the reducing and alkytating agents usecl) was eluted after the peak correspondlng to the L chafns. These peaks are not shown in this Flgure ' A

1.0

20 &

B ? ol€l Ârl

It-l .nl l¡¡lzl

o 0.1

10 20 30 40 50 FRACTIOÍ{ NUMBER. Ffsr¡re 6.3 : SEPIIAQH( G-200. MOIECULAR V\¡EIGHT CATIBRATION CURIIE

lþ u" a funclton of the logarithm of molecula¡ weight for a Vo Sephadex G-200 column equillbrated ln 2M guanidtne hydrochloríde

+ 5M urea.

Ordinate : {i.e.ffi)

(n¡olecular weight) Abscissa : Logarfthm 1t

The slope of the calibration }ine was found to be -0.953¿ ðrICi

Its intercepE on the loer' molecular weight axls, *ten# = 1,

5.630. The equation clescribing loqlO (molecular weight) as a function of # is of the formY = M.X + constant. The emplrlcal equation for the calibraÈton curve is therefore :-

Logl' (molecularweight) = 6'583 - Cl'953 " ffi' 2.5

HU]UAN L CHA[.IS. 2- L

,YG. LIZARD HUMAN YCHANS I H O{A|NS. VE I VO LTZARD 'úm' B SA. H CHAINS' I

UG' HUMAN YE

1

1ll

55 00 +0 45 t0 -150-

Lfzard'yG' : (i) Intact 'yG' Max. value 17 4,04c Min. value 130,000 Mean lSr,ooo

(ii) L chains Max. value 25, 000 g, Min, value 1 900 Mean 22 , t¡¡g

(iii) H chains ('T') Max. value 6û, iiÛc N,Iin. value 46,üùLr Meag 5I, ÛÙC

Lizard 'yM' : (i) L chains :1,s for llzard 'YG'

(ii) H chain ('p') Max. value 87,000 MÍn. value 66, 000 Mean 77,A00

The areas of the H and L chain protein peaks of lizard '7G' and

'yM, were estimated. the complexlty of the eluHon patterns,

particularly for lizard 'yG', , limlted the accuracy of these estlmates '

It was found, however, that the area enclqsed by the II chain peak

of both immunoglobulins (i'e' Figure 6'28 peak (3) and Figure 6'2c

peak (2)) was 7Û% - BI"/. afthe total area enclosed by the L and H

chain peaks, since the extinction coefficients of the H and L chains

were not known, nG aftempt was made tc calculate the relatlva raags

raLio of H anci L chains for lizard immuncglcirulins ' It ts clear ,

however, that on the basis of the areas uncier ihe oD 28íJ m{-t curve, - lsl - these immunoglobulins rn¡ill prove simflar to the already characterised mammalian fmmunoglcbulins ¡,r'hich have a unimolar ratio of H ancl L chains.

D. CONCLUDING RENTARKS

The aims, stated at Lhe beginning of thls chapter, have been

realtzed. The I{ chains of both lizarcl'7G' anci'yM' have been shown

to differ in their rnoi:ility durinE starch gel electrophoresis and' alsc

in their rnc¡Iecular weights. Further e'¡ldence for the existence of

at least two classes,'-¡t lizarC imrnuncglcbulirr has thus been provicecl'

hellminary estimates cf the molecular weights of ihe H and L chains

cf lizarcÌ immunoglobulins have beer¡ macle. .A value for the molecular

weight of intact lizarci '7G' in reasonable agreement lvith the value

oi¡tainecl in the prevÍous chapter has alsc been obtained, together with presumptive evldence for a unirnolar ratio of H to L chains '

Both lizard lmmu¡roglobulins differecì from humatr aud rabbit

immunoglobulÍns in their susceptlbility tc reduction. whereas used human yG wes cornpletely resclved by the reduction methoci were only lnto H ancl L chain ftactions, both lizard' 'yG', anci 'yM',

partially split. The behaviour cf lizar

gloilulinsprecluiedf-uri;herattemptscrtrec.l.ucLiorl. - IS2 - protein The behaviour of ltzarci 'yGl was unexpected. Although peaks correspünclirig i..r the !I and L cl¡alns cf this immunoglcirulin (i.e.peaks(3)anc1(¡¿).Figure6.2B)wereevi

ancl atkylated PreParation .

one of these components (peak 2) appeared tc have a molecular

welght oÍ about 70,OOo, i.e. similar tÐ a P chain' This cculcl be the Tlj; inÈerprete,l as incliceliaE Lire prese¡¡çE: of scrie 75 '7l,f in

,?G, preparatiori. Hov¿ever, tr;il:hlUt n-r'¡re clata thls explanation cf 7s'yG' cônnoÈ be accepted si¡ce L-ÏT ancl I{-T{ chain c"rmplexes preseilce rryoulC v¡c¡ulci also have slrnilar molecular v¡eights arrcì tl¡eir The rernaining peak be an<¡ther possiirle explanation for thiS peaìc' t"' fn the.lizard'yG'profile (i.e. peal; (I) Figure 6'28) corresponds lizard 'yG' â prctein with a rnolecular weight simÍla|ic ur¡rec'luced explain thls peal<; and the presence of thfs mclecular specles niay being present however, the p<¡sstblliüy of a contamlnating ¡:rotein prepcrrai:ion cannc.t be cornpletely exclrrueC even ihough the crigír:al witl¡ rabbit was immunoelecircphoretÍcally homogeneous lvlren testecl

anti whole lizarti serum'

Ttiroughoutthisstuciylizard,yG.hasbeenfoundtohavesome in a starch gel is u'r¡usual propertles. Its elc'ctrcphoretic rirs-nilirf shown to less than that oi¡serve¿i for mammalian yG' It has bcen - t53 - aggregate easily fn a manne¡which may be reversed by lowerlng the pH, or by the additton of urea. Althcugh homcgeneous by immuno- electrophoretic analysis, starch gel electrophoresis in the presence of 8M urea ancl at pH 3 resultecl in an unusual H chain pattern' It appears to be particularly suscepiible to cienaturation once remr¡vecl from the stabilizing envirc'nmenl of other Lizard serum proteins ' The data obtained in these expe riments, however, support the argument that ihe 7S lizarcl inirnunca¡lobulin recr¡ceci anci alkylated was a '7G' type as distinct fron¡ a'yA' type immunoglobulin. The need fcr lr'cre informaricn abcut this prolein is clearly iniicatei'

wlren the values cbiainetl fcr lhe niolecular v'reights of lizarc1 imnruiroglcl:ulin L and lI chains are used lc accounL for the molecular weight values of lizard. '7VI' and 'yG' re1:orteci in the previous chapl"er it seerns clear that ihe quatemary structl¡'e of lizard immunoglobulins resembles that of mammallan immunoglobulins . That is, lizarcl 'yM' and 'yG' may be represented by the fcrmula (L-H)n v,zhere fcr 'yG'n = 2andfor'7lv{'n = 10.

In conclusion, definite evldence for the existence of tw-o classes

of lizard immunoglcbulins has beerr presented. However the

possibility that more iharr 2 classes exist has not been precluCed' pnc-e again lizarcl 'yG' Ìras cltsplayed properties not usually

:êssoqiateci r¡¿ith the YG i:n'.1Ínllnogl'o¡3ulin cf namrnalian species ' - t54 -

CHAPTER 7

Paoç

15s A. OPSONIC PROPERTIES OF LIZARÐ /AÀTTIBCDIES 156 (i) Results t57 (ii) Discussion a

159 B.THETURNoVERRATEoFLIZARÐIMMUNoGIoBULINS. (i) Experlmentalprotocol ..' "' "Ú r60 (ii) Analyslsof data" "' "' "' r60 _r3t r63 (iii) Turnover of lizard '7M' Iabelled with I- ' " l3l 164 (iv) furnover of lizard '7G' labelled with I-'

o L64 (v) Ðenaturalion of lizard immunoglobulins ' ' ' ' (vi) Discusslon "' "' 'rr "' I66 155 -

Two biological properties of lizard immunoglobulins are consldered in this chapter, and the results obtaÍned compared with the corresponding propertles of mammallan antibodies. the properties investfgated were the opsonlc actÍvity of lizard antlbodies and the rate of catabolism of lizard immunoglobulins.

For these studies intravenous lnjection was required, which

involved minor surgery on one of the linrbs of each lÍzard used ' this, unfortunately, may have introduced an undesirable variable since the possibility exists that the injection site may have become

infected leading to alteration of the physiological state of the

animal, a shortening of iheir effective experimental life, or both '

However, in practice it was found that atl the lizards injected

intravenously remaineci healthy and obvious signs of local infecijon

were never observed. In fact these studtes demonstrated the feasibílify of pursuing biological experiments requiríng intravenous

fnjection using lizards as experimental animals '

A the The abflity of some mammallan antibod'ies to participate in

phagocyttc ellmlnalton of foreign antlgens from ihe mammalian (1962)) clrculation has been well documented (reviewed by Rowley '

These anttbodles are called opsonins' ts6 -

In this secHon data is presented on the abllity of lizard anttbodles to promote phagocytic clearance of several antigens.

A meast¡re of opsonfc activlry is the phagocytic index, K. Thls parameter is determined by the slope of the llne obtained when the logarlthm of the concentratlon of the lnjected antigen ls plotted as a function of ttme, Thus

K = lontO Cl lontO C2 Tz Tt where C, and C, are the concent¡ations of circulatlng antigen at tlmes T, and T, resPecLively (Benace¡raf , BLozzi, Halpern and

Sttffel , I957) .

(i) Results

In this investigation antigen concentrations were generally determined indirectly, uslng radiolsotopically labelled antlgens .

The only excepLion was colloldal carbon, whlch ls easily measured turbtdimetrlcally. The clearance curves were plotted over a tlme fnterval of flve minutes.

opsonlzed s. tvpEimurium, strain M206, (Furness and Rowley,

f gS6) was used in t¡is study. The bacterla were opsonized as follows:

One volume (109 bacterfa/mL ) was mixed with one volume of

serum diluted if necessary to prevent agglutination and placed ln

the reffigerator for 30 minutes. The bacterfal suspenslon was then Flogle 7.1 CTEARANCE OF P32 LEBH,I.ED S. ÎY?ÏÍIMURIUM M2Û6

FROM LIZARDS w 2t-22o),

o{ Clearance of bacterla opsonized with lmmunized

lizard serum. Each potnt represents the mean value

of results for 2 lizards *

o- - € Clearance of unopsonlzed bacterla. Each point represents the mean value of results for 4 llzards ' o lil

I 2 3 4 5 rme /urnures\ Figure 7.2 ; Correlation of the phagocytic index (K) with the haemagglutination titre of circulating S. tvphimurium antibody in the serum of immunized lizards.

0.2 oo ooo

o o M

X õc) -d O 0.1 -F{p o

O o Þl OO (ó -c O.

o o o

Lz3 4 5 6 ,7 B 9 i0 log haemagglutination titre -l 2 -157- washed 3 times with 20 volumes of cold saline and resuspended to a concentration of l0B bacteria per ml. in saline.

A summary of the results obtained is set out in Table 7 .l and a

typical clearance curve is shown in Figure 7 .1; the values set out ln Table 7.1 were computed from simllar plots.

In addition to the results shown in this Table the phagocytic

indices of. P32labelled S- tvphimurium M206 injected into a number

of immunized lizards were also determined, Vt¡ith llmiLing antibody

concentratlons there was a correlation between the phagocytic index

and the level of circulating antibody to S. tvphimurium as detected

by the haemagglutlnatlon assay. These results have been showp in

Figure 7.2.

(il) Df scussion

It was found that nelther colloidal carbon nor lizard erythrocy'tes

were cleared from tJre lizard circulation. The clearance of S. iyphi-

murium M205 in lizards was found to be very similar to the clearance

of this organism by mice. In both instances, clearance was enhanced

above the background rate (I( = 0.02) in immunlzed anlmals possessing

ratsed anttbody levels. Both lÍzard and rnouse antibody appeared

capable of promotlng phagocytosis in the lizard - an interesting

flndlng consldering the distant phylogeneLic relaLionship between

these two specles. The prlme role of antlbody in effecting the TABLE 7. I

PHÀGOCYTIC INDICES IN THE LIZÀRD

Antigen No. of ArithmeÈic mean (and modiflcations) lizards of phagocYtic used index, K t. Colloidal carbor¡ 2 0 (a) 2. Lizard erythrocYtes 2 u

3. P32 labelled s. ùvr¡hiinr$iuln M206þ) (i) normal iracteria + 0.02 (il) iracterfa opsonized wiih (a) ir,rnrune lizard serum 2 û.20 (b) normal mouse serum 2 O.II

+. P32 labellecl S. tvJohiinqriurrr M20ô opsonlzed with senrni of inrmunizeo

2 û.2û 2 ü .19 s. Il3l labelled BsA ln (U) (l) normal liza¡ds (ti'rre = ';¡ 2 0 (lt) lmmune llzards (titre = l:128) 2 0.11

Footnotes (a) LizardrqrythrocYÈe s were labellecì *ltù CrSl by lncubalion rn¡itÌr Nar6t" on '

(b) The bacteria \A,ere tabelled with P32as descrlbed ln Chapter 3 '

(c) These llzards were malntafned at the apprctprlate temperature for at least I wee j.: Prlor to use.

(d) Tltre refers to the haemagglutlnatlon titre obtained uslng the passlve haerragglutlnalion ðssay for BSA anttbody described ln Chapter 3 ' ts8 - the clearance of antigen is emphasised by the finding that within

Iimiting antibody concentration (titres of zero to I : l2B, i.e. lo9, T = 7), the phagocytic index increased directly with the Litre of antibody.

Another finding of Interest was the apparent lack of dependence of K on environmental temperature. Phagocytic indÍces of opsonized

S. tyohimurlum were not slgnlficantly di.fferent in lizards equllibrateci and maintained at 30o or 2Ao- 22o. This finding must be regardect as preliminary because of the statfstical limitations of the experiment.

However, it is consistentwith the finding of Tait (IS67, p.159) v¡ho reported that in vltro phagocytosis of sheep erythrocytes by lizard anci toad macrophages was temperature itidependent, relative to the phagocytosis of sheep erythrocytes by guinea pig macrophages. this topic deserves further study, parlicularly in relaËion to the temperaiure

dependence of the initial steps in the inouctio'rr of aniibody synlhesis in poikilotherrns.

The results obtaineci on the clearance of iodine 13l labelled BSA

again Serve to demonstrate the necessity of anlibody for clearance'

It was also found that passively transferred antibody from an immune

to a non-immune lizard servecl to enhance the rate of clearance of

BSA in the latter. I59 -

B. THE TURNO\IER RATE OF TIZARD IMMU.NOGLOBULINS

The concept of a dynamtc state, or turnover, of body protein was first suggested by Borsook and Kelghley (r935) and was confirmed experimentally fn the studies of Schoenheimer and hls colleagrues on the metabollsm of rabbtt plasma protetns (Schoenhetmer, 1942).

It has been established that ln homeot}erms a steady state exlsts where protein catabolism is balanced by synthesis of protein.

Thus, a measure of the rate of turnover of a protein gives a measure of the rôte of synthesis of this protein.

During an lmmune response specific antibociy of a particular iramunoglobulin class is cietected in the serum of a responding ¡nimal because the actual rate cf synthesis of this antibody is greater tJran

the net rate of synthesis of that class of immunoglobulÍn. Provided

that poikilotherms resemble homeotherms by manifestlng a steady

state with regard to serum protein levels then determinatÍon of the

turnover rates of fmmunoglobullns at two different temperatures would

give an estimate of any differences ln the rates of immunoglobr-rll'n

synthesis at the two temperatures, and indirectly, some indication

of the possible rates of synthesis of speclfic andbody at these

temperatures.

Data ls presented. on the turnover rates of lizard '7M' and '7G'

immunoglobullns at 20o anci 3oo. -160-

(i) Exoerlmental orotocol

The work described may be divided into three stages ' (l) Purified lizard ryM' and 'yG' immunoglobulins were prepared using the procedures described fn Chapter 5. These were then

labelted with iodine-I3I (Materials and Methods). Relatively

low speclfic aclivitles were used to minimlze denaturation of

the proteln during radio labelling.

(2) The labetled proteins were inJected intravenously lnto the rear

Ieg of the lizards whÍch had been kept at a specified temperature

for at least a week prior to injection. The fate of the labelled

proteins in the vascular system of the lizards malntained at the

specified temperature was then followed as a function of Lime.

The data obtaÍned was analysed to provicie estimates of the half-Iife ffr/rì of the lizard immunoglobulins.

(3) Expericrents on the Labelleci Ínrmunogl cbulins and on the senrrn

of lizards lnjecteci with these labellei immunoglobulins were

also performed in order to provlde some iciea of the extent of

cienaturalion of the labelled proteins.

(il) Analvsis of data

The data has been analysed using the open two compartment

model schematfcally depfcted ln Figgre 7.34 and flrst proposed by

Campbell, Cuthbertsoh, Matthews and McFarlane (1956). Flgure Z.3A

Dlagrammatical representation of an open two cornpartment model for the metabollsm of plasma proteins (after Campbell et al. 1956).

k rate of synthesis of proteÍn. s = kI kZ = rates of exchanges between plasma and extravascular lymph. kg = rate of loss of protein by catabolism.

Figure 7.38

Protein actÍvlty at time T Diagrrammatical plot of log I t Protein ac'iivity at time zero versus tlme showlng how the parameters ê, b, C, and C, maY be obtained. The curve obtained ( æ ) ls characteristic of-the opeã two compa4tment system portrayed in Figure A and Ís of the form X = C, e-at + CZ e-þr. The line B ( o'æ ) is obtained by subtraclion of the extrapolaieci reglon of the original ct¡rye from the orlginal curve, When a greater than two compariment system is encountered subtraction of the first extrapolated line ylelds another curve which after a time becomes linear and a further extrapolatlon and subtraction may be carried out ff desired. A SITES OF SYNTHESIS lnjection of labelled ks protein.

kr PLASITIA LYMPH lntravascular =k, Extravascular

ke

CATABOLISM

I B

l- o o o Czo .E .E o o o.5 \ o .== \ y(, E(, Cl o \ o) o slope = -a o \ \ slope=-b

TIME 'r6l -

Although this mocÍel is an oversimplification of the actual in vivc state, ft allows for a reosonable estimate of lhe turnover rate of ihe proteins lnvolveci. VÍore accurate clata, e.g. obtained by using larger groups of lizarcis and better control of the preparation anci cienaturatlon of the immunoglobulins, would be required'to lustify use of the more complex and lnformative multi-compartmental analyses available.

In the two compartment mociel aII the extravascular compartments are consldered jointly and are called tymph (symbol L). It is assumed that proteln moving from one compartment to the other mixes instan- taneously rn'ith the contents of the receiving compartment, and also

that metabolism of the protein takes place exclusively in the plasma compartment at the fractional rate of turnover kr '

If fP*) is the concentration of labetled protein molecules in the plasma, p the specÍfic activfty of P, and if (L*) is the concentra'rion

of labelled molecules in the lymph compartnrent, lhen the differential

equations describing the system r'vÍth respect to time are :-

d(p*) - k' (P*) + k"(L*) - k"(P*) ...... (1) -----:- = L ¿ \) dt d(L*)= kr(P*) kr(L*) ,...... r. (21 L . ---- dt D* and the solution to these equations for p (1.e. !) is:- p = + "' r" o" ..' f3I ", "-u' "r"'o' -162- where a + b k + kz + k^ (4) l ó t, (s) and ab kz ''3

Since at time t = zero, the specific activity of P i.e. p equals unity then

+ (6) c ta+ Çzb kI oe and

(71 c +C I taa I 2

SoluHon of equations 4 to 7 permlts calculation of the rate constants kl, kZ, k3.

(b-a) 2 k I cI Ut (8) aC +bc 2 I k aC +bC (s) 3 2 ] ** kg ai¡ (10) aC + bC 2 I

Since the catabolisn' of plasma proteins follows first order reaction kinetics (cited by Schultze anci Heremans, 1966 p. 454) the half life

E ,/rl of the protein catabolized is determined by:

ala Gt) T r/2

** In the computation of k the catabolic rate constant, equation (10) must be multiplied bY 2.30 3 slnce the values for a and b are obtained from a logl0 plot. -163-

Calculation of Èhe rate constants reguires values for C, , CZ, a and b and these may be determined graphically from a plot of the logarithm of the fraclion of administered radioaclivlty recovered as a function of time. Thls method, illustrated in Figure 7.3B, is loosely called "curve peellng" and has been describeci by Beeken, volwller, Goldsworthy, Garby, Reynolds, stogsdill and stemler

(1962) and also Dawes (1967 p. 286)'

1ii1¡ Tu¡nover of Il3I labelled lizard 'rrM' immunoolobulin

Two groups, each of five lÍzards, were acclimatised to environ-

mental temperatures of 20o and 30o respectively and then injected

intravenously witþ 0.5 mg. of labelled lizard'7M'. The rate of loss

of label from the serurïr was then determined by sequential cardiac

bleedings over a perioci of 20 days 0.5ml. btood was taken each

Lirne. The lizards were maintained at their respective temperatures

throughout the 20 daY Period.

The data is first presented as a graph of the percentage radio-

activity relative to that at time zero , versus time (Figure 7 ' 4) ' The latter part of these curves can be used to provide a first estlmate of flgure, (Flgure 7.5) shows the the half life, T ,¡r. A subsequent peeling" same data plotted on a semi-logarithmic scale with the "cun¡e

breakdown for determining the values of C, ' CZ' a and b' These have been measured and values for ka and T t/Z computed: the results are shown in Table 7 '2 ' -164-

(iv) ofI I3I belled llzard tv(ìr irnmunoqlobulln

The previously described experiment v'¡as repeated using lizard ,yG, in place of the 'yM'immunoglobulln, Two groups of five lizards were followed over a perÍod of 40 days.

Figure 7.4 shows graphs of the removal of radloactlvely labelled protein from the serum of llzards versus time, and Ffgure 7 '6 shows

the semi-log plot of this data also as a function cf time. Values taken from these for k, and T L/2*"t. coii:puted from measurements graphs and Ëhe res,-rlts are also summarizeci in Taole 7.2.

(v) Denaturation of the llzard immunoqlobulins

since clenaturation of lhe protein would give rise to spurious

values for the turnover of lizard immunoglobulins care was taken

whenever possible to avotd this. The immunoglobulins used ln

these experiments were freshly prepared. They were also preparecl kept as rapldly as possible anci Èhe various intermediate fractions chilled. Relatlvely low specific activities of radioactivity

(5u0 pc./mg. protein) v¡ere also used'

several experiments were carried out which gave some measure of slze heterogeneiiy, this being an inclex of cienaturatlon' these

experiments were as follows :-

(a) sucrose density graclient analyses (chapter 3) were performed TABLE 7 .2 o 20 TURNO\IER R.ATE DATA FOR LEAR^D IMMUNOGLOBULINS AT ÀI{D 300.

Lizard'yM' Llzard'yG' Parameter o o 2oo 3oo 2t 30

tional catabolic rate constant k, (davs -I) o.o5ü ü.1s4 a.023 o.osl3

Iife of protein (daYs) I3.5 ) Prom kr; 1å r3.8 4.6 3Û.5

From relative activitY versus lime Plot (i.e. 'a'value) T4 5 36 2L -165-

on samples of the labelled lizard '7M' and '7G' diluted wlth

lmmune lizard serum. The antibody actlvlties of the added serum

acted as size markers for the 195 and 7S protefn peaks respectively'

Lizard anti rat red blood ceII anÈibody was used as the l95

marker for '7M' and lizard antl BSA as the 7s marker for '7G',.

The results indJcate that wtrile the bulk of both immunoglobulins

were reasonably homogeneous wlth regard to size, a disLlnct

proportion of polymerised material was also present '

(b) Sucrose density gradient analyses were also performed on sarnples

of serum taken from the lizards injected with the labelled protein'

The profiles ob'.aineci, together with those of (a) above, are

presented in Figure s 7 .7 anci 7 .8 . It may be seen that both

immunoglobultns remained lntact within the circulatlon of the

llzard. As most of the grossly denatured protein would be rapidly phagocytosed, this is a fgrther indicaLion that undenatr'¡red

labelled immunoglobultns were present in the lizard circula|ion'

(c) Evidence that the labelled lizard lmmunoglobullns had similar

electrophoretlc propertles to the normal protelns was provÍded

by autoradiography of immunoelectrophoresls patterns of the

tnitial samples and of senrm samples taken from the test lizards ' .yG. Elgure 7 ,4: REMOVAI oF II31 I.ABELLED LIzARÐ AITID 'yM! FROM THE CIRCULATION OF TIZARÐS MAINTAINEÐ ÀT 2OO AI'TD 3OO.

Ordinate : Percentage radfOacttvlty remaining in the

serum at time I relative to its value at Bme zeto.

Absclssa ¡ Time in days, I

I I

I r-{ tG AT 2ob t H trG AT 3ob 50 I o\ \ \

I to n 30 ¿lo uE-@$€)

a >-{ rt AT 20'c

,o e

50

t0 t\-...

-_ -a-

123¿lt6' t0 toll1213raÉló ulE-rgâre) Flgu¡e 7.5: REMovÀL oF ¡r3r Iå,BEILEÐ LIZARÐ 'vM' FRoM THE CIRCUIÂTION OF TEARDS MAIN'TAINED AT zoo AI.TD 3oo.

Ordlnate : Logarlthm (base l0) radioactivity remaining ln

the sen¡m at tl¡ne T relative to lts value at

Èlme zero.

Absclssa : Time in days. \ o, ,I Æ 2&. \È Q+

2a óalo t2 1l ló la rre lorreì.

Ir AT 3dc.

I \ I

21ótp12Llóll rre lo¡rsl æFlar¡re 7.6 a REMovAL oF rl3r T.ABELLED LZARD 'TG' FRoM THE CIRCTJI.AIION OF TEARDS ÙIAI}IÎA;INBD AT

zCIo A¡üD goo.

Ordlr¡ate I Iogàffürsr (bese l0) radtoacgvfÈf'¡n the 6st¡m

at dme f selaËve to tts value At tls6 Eæ'

Absclssa ! Tltne ln dayS. I

!

I I

I

i

i

I

E 8

a5 e t t

6 ô .t a't r â h t k t c' E to A ñ át a{ A R E R il R

ñ a

9 o I I a 6 I -¿ (, E t) Figue 7.7 ¡ SUCROSE DENSITY GRADIENT ULTRACENTRI-

FUGATION OF LIZARÐ SERUM CONTAINING

r13I LagnlJED TEARD 'yM'

I3l A. A sample of I Iabelled llzard '7M'

ttiluteci with normal lizard sen¡m.

B. A sample of serum taken from lizards injected

4 days prevÍously with Il31 lun"lleci lizard t?Mn.

The arrow refers to the posftion of lizard antl rat erythrocytes

macroglobulln antibody used as a l9S marker. F EEli cPil/luBE.

c No ôt

¿ o r¡l ¿ râ f t I ó J I ¡ t 4 '' ---- ¿y d Ël o< -\ \ -.-+-_ \ Flqure 7-B SUCROSE DENSITY GRADIENTS ULTRACENTRI- FUGATIOIV OF LIZARIf SERUM COI\EAINING tl3l LegeLLED LIZARD 'yG', -

A. A sample of Il3l labetled lfzarcl '7G' dtluted

with llzard anti BSA serum r

B. A sample of serum taken from one of the

lizards lnjected 5 days prevlously wÍth r Il3 labelled lizard 'yG',

The arrow refers to the position of lizard BSA antlbody dsed as a 75 rna¡ker. \ \ t \ ê \ -\. \È- ¿o d -.è - --â-- êf\¡ ã I I tn

ó o a.t a¡ (Ð ïõi-rÏilñv.net ïolTfln-íw¿5 1?¡I Ëã Ißz I 1 -166-

(vÍ) DÍsgussion

The slope 'a' (reference Figure 7.38) is clearly a first approximaticn to the catabolic rate (kr) for a plasma prolein and Campbell et al.,

(1956) have suggested it be called the "ap¡:arent catabollc rate."

Earlier publÍcatlons (cited by Campbell et aI., 1956) have often reporteci thÍs value as the real catabolic rate " After equilibration between the lymph and plasma, however, the net flow of labelled protein between the exÛa- and intravascular spaces must reverse,

so that ihe plasma which is ccnstantly lcsing labelled material

through catabolism, is also constantly being replenished with fresh

amounts of labelled material from the lymph. Hence k will be greater ? than the apnarent k, rneasured by the slcpe 'a'-.

If k^ anci the slcpe'a'were equal. ther¡ (frcm equaLicns B anii l0) J woulci be zero, implying a ccmplete lack of transfer of material k.I from plasma to lymph. This coulcl only occur when :-

(a) there was no extravasatfon of protein;

(b) the time for equilibrium between lymph and plasma was zero, or (c) catabolism proceeds in both lymph anci plasma at identical rates'

The first of these three possibÍlities could be approximated to

tn the cêse of yM fmmunoglobulin which Ín the human system at

Ieast is almost entirely intravascular- -167'

The results in Table 7.2 substantiate the use of a two compartment moclel systern. In both instances k, is greater than 'a' (x 2.3Û3) and the half life obtained by calculation cf k, is sLrcrter than either the value obtained from the plots of percentage relative activity versus tÍme (Figure 7 .4) ar from the slope 'a' (Figures 7 .5 and 7 .6l-. It is u"U^ates for agree perhaps slgnificant that these two T L/Z '7M' more closely than cio those for '7G' . This woulci be a reasonable finclfng if a larger percentage of '7M' inlmunoglobulin than 'yG' immunogloT¡ulin remainec in the plasma.

The forntation of an¿ther exi:onenlial curve when the lÍne c, is drawn and. sublraci:eC frcm the original úecay curve indicates that the system is really nrore ccmplex and in a more extensive stucly a

three compartment model at least shoulci be consllered.

The most sÍgnificant aspect of the data in Table 7.2 is the with ternpera- varlation observed in the T t/Z of each immunoglobulin ture.

If the assumptions made at the beginning of this chapter prove

justified, and the half lives of lizard irnmuncglobulfns at dlfferent

temperatures reflect clifferences in the rates of synthesÍs cf specÍfic

antibody at these temperatures, then it is clear that the temperature

dependence of antibcciy production by this species cannot be

explalned solely by the thernrodynamic control cf temperature on -168- proteln blosynthesis.

It was found that the half !Ífe of lizard 'yM' at 20o was 3 times as long as lts half life at 30o. Hence lf lizard anlibody specific for S. tvphlmurium was synthesÍsed only as fast as the immuno- globulin class lt represents (i.e. '7M'), then the maxlmum circulatlng antibody tltre of lizarcls immunized ancl maintaineci at 20o vuoulC be * reacheC 60 x 3 -- lgrl days after injectÍon of the anLigen. This was not observed : the maximum titre attained by lizards immunized at

ZCo r,vas rnuch lower than for lizarcl-s irnmunizecl at 30o ancl even ihough the rate cf appearance cf this arrtibocly in Lhe serum was slower than at 30o, (inAicated by the slcpe of i:he antibody response curve shown in Figure 4.1), ihe nraximum titre was reached earlfer.

These results inclicate that in lizarcls immunized and maintained at 20o another mechanism was operative.oth.er than the decreased rate of antibocly synthesis at the lower temperature. Slmflar results were observed for the responses of lizards imrnunized with BSA and maintained at 20o ancl 3Uo.

It was noted that cluring the responses of lizards to BSA the rates

of appearance of cfrculating antlbocly, i.e. the maximum titre reached

divided by the tlme taken to reach this titre, were approximately * The number of day3 to reach the maximum circulating antibody rÍtre in lizards similarly immunized but mairrtained at 30" (reference Chapter 4). .169- * proportional to the ambient temperatures used and also that the ralio of these rates at 30o and 20o was 3.7 z !.

At 2So the maxiroum circulating antibody titre reached Ín lizards immunlzed with BSA was the same as that reached by similarly immunized lizarcls nraintained at 30o. This observation would be most easlly acc'¡ur:'ted for by hypothesising that only the rate of antibody procucilon was affected at this ternperature and that the other mechanism, operetÍve at 200, cloes lot Íunction at 250.

The nature of the mechanÍsm which gtverns the quantiLy of circulating antibociy synthesiseci at 2Ûo remains tc be elucldated'

Early stages in the inducticn of antibody prcducLion, e.g. anllgen processing, may be involveci. furother possibflity which should be investigatecl is that the physiological status of the animal may be altered at the lower teinperature. For example, a change tn the distribution of antibody between the vascular and extravascular

Compartments may occur, resulting in an apparent decrease in the quantity of clrculating antibody synthesised'

The values obtaineci fcr the half lives of lizard immunoglobulins

compare reasonably with the values repcrtec'l for mammalian immuno- * At 30o rate = 0.251 at 25o rate = û.15; ai 20 rate 0.06 . Data taken fronr Table 4.3, Chapter 4, page ÌÛ1' -170- globullns . For example, the half ltfe cf human 7G ln plasma is about 20 days. and this is 4 times as long as the reported value of 5 ctays for human yM (cited in schultze and Heremans, 1966, page 4TTl. The half life at 30o cf lizarci'7G'ln plasma is 13'5 days ancl thls ls 3 times the half life of 4.6 days found at 30o for

Iizard ,yM' ln plasma. The difference found in the half llfe,at a given temperature, of lizard 'yG' ancì'7M' is consistent with the p,revious contenLion that these immunoglobulÍns represent cilfferent immunoglobulln classes' -I7l-

CHAPTER 8

DISCUSSION A}TD CONCTUDING REMARKS. -t72-

The past five years have witnessed a dramatlc increase in the published data on the phylogeny of Èhe lmmune response and conseguently on the phylogeny of immunoglobulln structure. The commencement of thÍs proiect coincided with the beglnnÍng of this

period. The alms of the project were simply defined; lt was intended

to study the immune response of the lizard,Il[gge-q¿gosa, and

establlsh whether this species synthesised both yM and 7G

immunoglobulins with antibody activÍty.

When this project was commenceci there was much less data

on th.is subject than |s currently available. Many reports in the

literature had described the synthesis of only ¡¡asroglobulin antibody

during the immune responses of a variety of poikilotherms, at least

to the immunogens then used. the question of whether some

polkilotherms could synthesfse yG antibodies was unresolved.

The relevance of this questlon became more evident when it was

shown, initially by Marchalonis and Edelman in 1965 and later by

others, that although some poikitothermic vertebrates synthesised

both macroglobulin and 75 lmmunoglobulins, these belonged to one

isotypfcally defined lmmunoglobulin clas s'

The approach used in this study was to immunize lizards such

that both macroglobulin and 7s antlbooles were ellclted, and to _t73_ then use the antibody activtty to ldentify lmmunoglobulin containlng fractlons prepared from lizard serum. Once lsolated the purified

lmmunoglobulln cot¡}d be characterlsed, and the origlnal alm of

establishing whether two Immunoglobulin classes were synthesised

by this specles could be achieved. Two dÍstinct problems were

therefore encountered ln this project,'each of whlch proved more

demanding than was anticiPated.

the first phase of the study relateC to the induction of antibody

synthesis in Tillqua. Little was known at the start of the many

parameters which govern the inductlon of antlbody synthesis by

poikilothermic vertebrates and consequenlly the techniques eventually

found to be effeclive were largely determined empirically' The

antigens fnitially chosen were S. tvphimrrrfuE and rat erythrocytes

since these were readily available and the assays for antlbody to

these antigens were sensltive and easy to perform. Although these

two antigens were found to be immunogenlc in !l[igg., predomlnantly

macroglobulin antibody was eliclted. The a¡¡tibodies produced macroglobulln in nature agaÍnst s. -tvphlmurium were exclusively during the 240 days over whÍch the response was studied, and

although some 75 antibody was synthesÍsed in response to rat

erythrocytes. the predominant antibody synthesised agalnst this

antigen was macroglobulln. - t74 -

Attempts to obtafn a good 75 antfbod.y response led to the utillzation of other lmmunogens. It was subsequently found that

BSA elictted an excellent 7S antibody response, following a transient macroglobulin response. This is one of the few occaslons when such a response has been obtained in a poikilothermlc vertebrate.

Relative to mammallan responses to these immunogens, the responses of .Ii$gua were less vlgorous. This finding seems to be a general one for poikilotherms and ls undoubtedly a result of the dependence of the immune responses in these animals on the ambient temperature. It was also observed that an anamnestÍc humoral response could be elicited fn Tiliqua under certain conditlons; fn the case of the response to S. typhtmrr¡ium lt seems that a wholly macroglobulln secondary response was observed.

The successful ellcltation of humoral antibody production mentioned above led to the second phase of thls study whlch was the lsolalion and subsequent characteris atlon of lrnmunoglobulins, exhlbiting antibody activÍty, from lizard sen¡m.

A conslderable amount of difficulty was experienced ln tsolatlng purtfied preparations of the 75 immunoglobulln from lfzard Serum'

Modificatlons of the techniques useci for lsolating mammalian 7G

eventually proved successful; however this was at the expense of - L75 - good yields. fnvestlgation of the propertÍes of lizard serum protelns provlded reasons for this dlfficulty. Briefly, two factors were involved. These were, firstly the absence of a distlnct protein fraction of 7 electrophoretic mobility, and secondly, the fÍnding that the fractlon of llzard serum protelns with the lor¡¡est electrophoretic molcillty (a fast y or F fraction) comprised 50% of the total serum proteins.

Less dlfficulty was experienced lsolating lizard '7M' since its

sìze, electrophorelic mobility and ciensity were similar to mammalian yfut. The rnethods used for isolating mammalian yM were directly

applicable to the preparatlon of this immunoglobulfn from llzard serum '

The lor¡¡ yields of lizard '7G' obtaineci with the method used

prompted an lnvesllgaLion for a better method. This was achieved

tourards the end of the proiect, Lizard anttbody specffic for BSA,

belonging to the ,yGt immunoglobulin class, was prepaled from the

serum of hypertmmunized lfzards uslng an lnsoluble lmmunoadsorbent'

Physlcochemical and serological characterlsatlon of the lizard

lmmunoglobulins prepared, each of wh-ich manlfested antibody activlty, lndicateci that two immunoglobulin classes analogous

to mammalian yM and yG were present in the serum of immunized lizards. Although lizard 'yM' was very similar to mammalian 7M -176- ryGt some anomalles fn the properties of lizard were found whÍeh ryGl compounded the difficulty of working wiËh this protein. Lizard was found to denatufe very easily once lt was removed from the stabilizing environment of other llzard serum proteins. This was also fou¡rd to be the casê Tor lizard speclftc antibody of this inrmunoglobulin class. It appearecf in addition, that Several "types" of lizard 7S ünmunOglobr,rnin trray exlst slnce evidênce fcrr physico- chemical heterogeneity was observed. Unfortunately antisera speciflc for lfzard trnmunoglobrúin H chalns were not obtained durÍng the course of the proieoJ and thls aspect was not developed further'

several biological aspects of the immunology of this species were investigated in more detail.

The methods used to assay lfzard antibodies are artlfletal in the

sense that they do not rely on functional properties of antibodles '

It was of lnterest, tJrerefore, to examine a funcllonal aspect of lizard anlibody. The property chosen was the opsonic activity of IÍzard antlbody. Lizard antibodies were found to behave very

slmllarly to mam¡nallan antlbodies in promoting the phagocytosis

of several antlgens.

The temperature dependence of antibody formation in the lizard

was also investlgated further. It was Íounci that this specfes of -177-

Iizard d.fd not live well at 350. Experiments on their immunologlcal responslveness wele therefore carrfed out at 2Oo, 25o and 30o. Both the rate of production, and the quanLity of clrculating antibody produced in liza¡ds lnjected with S. typhimr¡rium or BSA and maíntalned at 2Of were decreased. relative to the response to these immunogens obtained at 3Ûo. Lizards injected with BSA and maintained at 25o and 30o produced the same ultimate antibody Litre, although this took longer to attain at the lower ternperature. The half llves of prrrified lizard lmmunoglobulins ln the circulation were measured and it was found that the thermodynamic effect of temperature on proteln blosyntheSis could not account for the observed immune response of lizards maintained at 20o to etther S. tvphimurium or BSA. At 20o at least two temperature dependent factors lnfluence the outcome of the lmmunization of lizards, Thls phenomenon may well prove to be

an lmportant one 1n the study of immunology. ff early steps in the tnductlon of antibody synthesis are lnvolved, for example antigen

processing, then a unique experlmental model for the study of this

facet of lmmunology will be avallable.

The data reported in this thesls are generally !n egreement with

current knowledge on the phylogeny of immunlty whtch was revleryried

in detail in Chapters I and 2. Vertebnate immunoglobullns comprise

a uniquely related complex group of proteins and are therefore of -178- much interest to both lctochemists and immunoloqists.

At thfs junclure it is perhaps of value to briefly reconsider the rationale of phylogenetic studfes on immunology and immunoglobulins.

This aspect was discussed at some length fn Chapters I and 2. The discussion presenteC ii-¡ these chapters has emphasised several concepts of fundamental importance.

Firstly, it Ís clear that the concept of the immunoglobulin mediated lmmune response as a mechanism devoted solely to protection from infectious elements is too narrow. A broader concept rnore cognizant of the immense biologlcal significance of the abilÍty to synthesise

"antibodies" is required. Some hypotheses have already been

proposed (Chapter I) and the concept that the adaptive immune

response may have evolved primarily ds a means of deallng with

aberrant cells is currently popular.

Secondly, lt fs no\^¡ apparent that even lower vertebrate lmmuno-

globulins represent highly evolved forms, and recognizable immuno-

globulin genes must have arisen in prevertebrate forms. Although

the data is scant, it appears reasonable to propose that the

conspectus of vertebrate immunoglobulin structure presently

available represents no more than the "top of the iceberg." The

origlns of L and H polypeptlde chain genes are as obscure as ever. _179_ Conceptu¿I hypotheses on possible functional propertles of unassoclated L and H chains are necessary for progress in this aspect. The problem is essentially one of not knowing what to look for.

Dreyer, Grey and Hood G967) have made a serious attempt to come to terms with these aspects which are conceptual rather than pracÈical fn nature. They have hypothesised that antibodies, i.e. lmmunoglobulins, arose as a late evolutlonary offshoot of a basic

type of membrane-bound chemoreceptor sysÈem which evolved aS an

essentlal part of the differentlaüon of multicellular organisms.

Hypotheses of this nature would seem to be gaining in popularity and

it is to be hoped that they wlll lead to the d.esign of fruitful

experÍments.

Of the vertebrates alive today the hagflsh occupies a unique

positfon. Papermaster and Good and their collaborators first directed

attention to this species when they reported that they were unable to -\M.H. induce it to synthesise antibody. Toenes, H. and Hildemann,

(personal communication) have recently reported, however, that they

successfully maintained this specfes at higher temperatures, and

observed antibody formation. Another group of animals almost

completely ignored to date and whtch warrant close lnvestigallon are

the protochordates. It is on these species that the author believes

future studies would be most profltably directed. APPENDD( I K) FORTRAN COMPUÎER PROGRAM USED FOR CALCUIÂTION OF MOLECUIÀR VITEIGHTS FROM HIGH SPEED SEDIMENTATION EQUITI¡3RIUM ÐATA (Yphanris, 1964).

PROGRAM JDVY (INPUT, OIITPIII) DIMENSION XSQ (rCü), Y00û), W(I{iC), CX(lrO), AVcY(r00) coMMoN A, C(21), O(eoc), TITLE (B) 99 READ 19, (TITIE(I), I=l , 8) rs FORlviAT (8A'10) IF (TrrtE (l).EQ.IoH )stop READ 20,M,FI, F2,F3, F4, P5 20 FORI\4AT (13 , F7 .3 ,P7 .4,84. I , F6.3, 810.3) MM=O .0 DO 22 f=I, M READ 2I,CX0), CYl, CYz, CS, Cy4, CYs, CV6 2t FORIvIAT (7F7.31 xsQ $)= (1çx(I)-Fl )r'Fz+ F3 )* *2 AVCY(J)= (Ct'l + CYz+ CY3 + CY4 + CYs + CY6'j. /6 . 0 FRED= (AvcY(I )-AVcYtûl /T 4 PRINT 24,! ,CXÛ), CYI , CyT, C1¡3 ,ÇY4, CYs, CY6, AVGYT), FRED *AVCY(])= 2 4 F ORMAT (I 0X'tJ=*13 *CX 0)=* El 0 . 3 *CYIS ARE* 6Et 0 . 3 I*El 0 . 3*FRED='IEI 0 . 3) rF FRED. G[.0 .0.123 ,22 23 MM=MM+1 Y(MM)-i\rOc(FRED) xsQ(MM)=xsQ0) W(MM)=1. g 22 CONTINUE M=MM DO 30 NORDER=I,2 cALt tsQPot (M,xsQ,Y,W, 0, 31, 4, l,NORDER, Û) PRINT 26 2 6 F ORtviAT v / / / /L zKr HrX3 HSQ r 4XI HYI zxs i{YCAJ,C I 4x6H MOL WT /') DO 29 I=1, M SZ=0.0 NUT=NORDER+ I DO 28 I=1, NUT Z= (I-t)*c $)*xSe (r) ** 0- z ) 28 SZ=ffi*Z SMW=SZ*F5 29 PRINT 27 ,t,XSQ0) ,Y0) , D(I), Slvf\A¡ 27 FORN4AT (r0)G3, 4815.4) 3 O CONTINUE GO TO 99 END (xi) APPENÐDí I (cntci.)

Factor I (FI) Arithmetic mean of the microcomparator values for the 5.7 ancl 7 .3 reference ¡:'cints on the rotor f¡om the axis cf rotation ' Factor 2 (EZI Ivtagmfication fastor for optical systcm of tire uliracentrlÍuge (0.0455 in niachine used)

Faoror 3 (F3) Arithmetic rnean of the 5 .7 antÌ 7 .3 reference points on the rotor frorn the axis of rotatfon.

Factor ¿, (F4) Displacement corresponding to I frlnge width (282v in machine used)

Factor 5 (FS) 2RT where R = gas constant T absolute temperature w2 (l-vp) = lv 5 .angular velocitY of rotor V = partial specific volume of protein P Í: derrslty of solvent

Thls program was written by Dr. D.I. Fennelf, Department of Physlcal and Inorgnnic Chemlstry' rJoiversity of Adelaide' kit)

ACKI{O}1¡LEDGEMENTS

It is a pleasure Èo acienowledge the assistance generously affordec{ by the J)epartment of Microoiology ciuring'tire course of thls study.

AlthouEh many members of the ciepartmenL contributed by their wllllngness to parli.cipate- in critical and siimulating discussions of various aspects of this work, special thanks are due to Professor

D. Rowley, Dr. K. ]. Turner, Dr. Ieva Kotlarski and D, C. R- Ienk:in for their encouragement and advfce when the end was not clear-

I would also lfke to acknowledge wiÈh thanks, the assistance and tuition given l¡y Dr. D. j. Fennellin carrying out the molecular weight determinatlons usÍng the Yphantls method.

The co-operatlon received from Mrs. Barbara Teasdale durlng the typing, and the sl

BIBtroGRAPrlv

ALCOCK, D. (1965). J. Path. Bact., 9Û, 3l-

BEEKEN, W.L., VOLWILER, W., GOLDSWORTTTY, P.D-, GARBY, L.8., REYNOLDS, W.8., STODSGILL, R. and STEMLER, R'S' (1962)' !. clin. Invest., L 1312. (1957). BENACERRAF, 8., BIo|ZZI, G., HALPERN, B.N. and STIFFEL, C. In ,'Physfo-pathology of the Reticulo-Endothellal System : A Symposium. " p. 52. Editors B. Bennacerraf and J'F' Delafresnaye, charles c. Thomas, springfield, Illinols.

BERRILL, N.I. (1955). In ''The orlgin of Vertebrates.,' Oxford Univ. Press (Clarendon), tondon-

BISSET, K.A. (1947). I. Hvg. (Camb.), a'5, 128'

BISSET, K.A. (1948). r. Path. Bact., lig, 87'

BISSET, K.A. (1949). I. EndocriDol., -0, 99'

BOFFA, G.4., FINE, t.lvl., DRILHON, A. and AMOUCH, P' (19671' Nature Gond -| , ry, 7 00.

BOGERT, C.M. (1949). Evolutlon,3, 195-

BOGERT, C.M. (1959). ScientiffcAmerican, æ!, 105'

BORSOOK, H. ôrtd KEIGHLEY, C.L. Ct935)' . hoc. RoY. Soc. Ser. 8., JfQ' 488'

BRYSON, V. and VOGEL, H.I. (1965). In ,,Evolving Genes and hoteins: A Symposlum." Editors V. hyson anrJ IJ.J. Vogel, Academic Press (N'Y')

BURNET, F.M. (1968). Nature (Lond")t 218,426' kiv)

QAMPBEI¿, D.H., GAR\Inr, ].s., ÇREMER, N.E. ôfid sussDofiF, D'H. (1964). In,,Practical Immunologty." XV.A. BenJamin Inc. (N.Y.)

CAMPBELL, R.M., CUTHBERTSON, Ð.P., I\4ATTHE\MS, C'M' and M.FA¡{LANE, A. S. (1956) - Intern. I. AppI. Radialion Isotopes, ! 66'

CANTACI'ZENE, I. (I9I9). Compt. Rend. Soc. Biol . &, I0I9.

CEBRA, J.J. anci Sn4ALL, P.A. ]r' (1967). BfochemistrY, 6, 503 '

CEPPELLINI, R.9!3!. (1964). Bull. Wor1ci i{ealth Organ ' 30 , 447 '

CHING, YI-CHUAII and- WEDG\'1/OOD, R'J' (1967)' J. Immunol., 99, I9l'

CLEM, L.W., DeBOUTAUD, F. and SIGEL, M'M' (1967)' J- Immunol.,99, L226'

CLEM, L.W- and SIGEL, M.M' (1963)' Fed. Proc . , 4, ll38 '

CLEM, L.\A¡. and SIGEL, M.M. (1966)' In "phylogeny of Immunity." p, 209. IJniv. of Florida Press (Gatnsville).

CLEM, L.W. and SMALL, P.A' (1967)' I. exp. Med-.ç Jë, 893' coHEN' s' and PORTER' R'R' (I964)' ,'l*dvances Ín ImrnunologY , 4, 287 ,

COHÎ.I, E.J. and EDSALL, J.T. (1943). In "Froteins, AÍnino Acids and Pepticies'" p' 375' Reinholci (N.Y. ). (xv)

COIBERT, E.H. (r9ss). In ,, Evolullon of the Vertebrates . '' J. WÍley & Sons, Inc . , New York.

COIVLES, R.B. and BOGERT, C.M. (1944). Butl. Am. Mus- Nat- Hist. 83 (5), 267 ' CRICK, F.H' and OR'GEL, L.E. (1964). I. mol. Biol., P, 16l. (I958) CRUMPTON, M.J., DAVIES, D.A.L. and HUTCHISON, A' M' ' I. gen. Microbiol.,-L9, I29'

CUSHING, I.E. (L942) - ,. Immunol', Æ, I23 '

DAIüIALS, T.M. and STAVITSICí, A.B. (I964)' p In " Methods in Medical Research. " Vol. 10, ' I52 ' Year Book Medícal Publishers, Inc., Chicago'

DAVIS, 8.D., DULBECCO, R., EISEN, H.N., GINSBERG, I{'S' and WOOD, W.B. (1969). In " Microblology." Harper and Row, New York'

DAWES, E.A. (I9OZ). In "Quantl,tative Problems in Biochemistrlr. " E.S. Livlngstone, Edinburgh and London-

ÐAY, ¡.9. (r966). In "Foundatlons Of Immunochemlstry'" The Willtams and Wllkins Co., Baltimore.

DETERMAilIN, H. (1968). In .'GeI Chromatography.,, Springer-Verlag, New York, Inc.

DIENER, E. and NossAL, G.I.V. (1966). Immunology, -l-.0, 535 . kvi)

DD(ON, G.H. (l$66). In "Essays tn Blochemistry." VoI' 2 , p. L47. Academlc Press (London) Íor The Blochemlcal SocÍety.

DREYER, V/.I. , GRAY, W.R. and HOOD, L. (1967). Cold Sprlng Harbour Symp. Quant. Blol' g, 353,

EDELI\.IAN, G.M. (195E). J.Amer. chem. Soc., Bl, 3f 55.

EDELMAN, G.M. and POULIK, M.D' (1961). I. exP. Med', ff3, 861.

ELEK, S.D., REES, T.A. and GOIIIIING, I{.F.C. (1962)' ComP. Biochem. and PhYsÍoI ', L 255'

EV/\\IS, E.E. (1963). Proc. Soc.. exp. BloI. Med., ]!!, 531.

EVAI{S, E.E. and CO\Â/LES, R.B. (f 959). Proc. Soc. et(p . Biol. Med . , I'ÙL , 482 , ' EVAÀIS, 8.E., KENT, S.P., ATTLEBERGER, fuI.H., STEBERT, Ç" BRYAI.IT, R.E. ANd BOCEH, B. (1965). Ann. N.Y. /\cad. Sci ., W, 629'

EVERHART, D.L. and SHEFNER, A.M. (1966). I. Immunol., W, 231.

FAITEY, J.L. (1965). f .A.M .A'. , 194, 71. FAHEY, J.L. (1967). In "Methods fn ImmunologY anci Immunochemistry"' Vol. l, p. 330. Editors villlliams, C-/\. and Chase, IvI'W' Acadenúc Press (N.Y. and London).

FAULK, w.P. and PONÐMAN, K'w. (1969). ImmunochemistrY, 6, 215' (xvii)

FINSTAD, J. and GOOD, R.A. (1964). I. exp. Med. , !2t, lI5I' FINSTAD, I. and C'OOD, R.a. (1966) In '' Phylogeqy of Immunlty..' p. 173 . Editors Smith, R.Î. , Miescher, P.A. and Good, R.A. Univ. of FlOrlda Press (Gainsvllle). (I964)' FINSTAD, J., PAPERM.A.STER, B.W. and GOOD, R'A' Lab. Invest., ]E, 49Û'

FTEISCHMAN, f .8., PAIN, R.H. and PORTER, R'R' (1962)' Arch. Biochern. Biophys ' , Suppl ' l , L-/ 1+ '

FURNESS, G. and ROI|úLEY, D. (1956). I . gen- Micrcbiol. , 15 , I40 '

GOLD, E.R. and FUNDENBERG, H.H. (1967)' J. Immunol.,99,859'

GOOD, R.4., and FINSTAD, ¡. (1964). Fed. Proc., þ, 285. (1966). C,OOD, R,/\., FINSTAD, J., POLLARÀ, B. and GABRIELSON, A.E' In ,,phylogeny of Immunity." p. 1119. Unlv. of Florlda hess (Gainsvllle).

C'OOD, R.A. and PAPERMASTER, B'VU' (1964)' Advances in ImmunologY , !, l '

GREY, H.IvI. (I963). J. Immunol., !!, 819-

GREY, H.M. (1966). In',Phylogeny of Immunity." Eclitors srnith, R.T., Meiscþer P.A. and Good, R'.4. Univ. of Florida hess (Gainsvflle)' (xviif )

GREY, H.M. (1967). f. fmmunol., 98, 8ll.

HAVEL, R.I., EDER, I{.4. and BRAGDOI¡, f.H. (1955). J. clln, Invest., 4, f 345.

HEIMBERGER, N., HEIDE, K., HAUPT, H. ancl SCHULTZE, I'tr.8. (1964). Clin. chim. Acta. LÛ, 293.

HILÐEMA¡ü, tr'l,¡.H. and COOPER, E.L. (1963). Fed. Proc.,22,1145.

HILL, XV.C. anci ROv"trLEY, ú. (1967). Aust. I. exp. Biol. med. Sci., 45, 693.

HOÐGINS, H.O., VfEISER, R.S. and RIÐGW,AY, G.f. (f 967). l. Immunol., $, 534.

HOOD, L.E. and EIN, Ð. (1968). Nature (Lond.), 220, 764.

HOOD, L.8., GRAY, -W'R. and DREYER, 1i1/.J. (1966)' Proc. Bãt.Acaci. Scl.,(Wash-), 55, 826. . HOOD, L.E., GR.AY, VV.R., SAIIDERS, B.G. and DREYER, !y.J.(1967). Cold Sprlng Harbour Symp- Quant. Biol. E?.,133.

HUNTER, \¡.M. and GREEI\ilMOOD, F.C. (1962)' Nature (Lond.), IlE 495.

KABAI, E.A. and N4AYER, M.M. (196I)- In "Enperimental Immunochemistry." Charles C. Thomas (Springfielci, Illlnois) .

KAWAHARA, K. ancl TAIiTFORD, C. (1965). f. biol. Chem., 24, 3228. (lox)

KRUEGER, R.G. and TWEDT, R'.VV. (1963). ]. Immunol.,9C,952.

KUNKSL, H.c. (1960). In,'The Plasma Protetns." vol. 1, p.279. Editor Putnam, F.W. Academic Press (N.Y.)

LAMM, M.E. and SMALL, P.A. Ir. (1966)- ochemtstry, þ, 267 -

LAMM, M.E. and StviALL, P.A. Jr. (1967). Blochtm. bioPhYs. Acta., l![], 519.

TAIVDSTEINER, K. (1945)' In "The Speclflcity of Serologtical Reactiorls'rr Rev' ed' Harvard Unlversity Press, Cambridge, Mass' (1945)' ReprÍnted by Dover Publlcations, New York (1962)' (f LERCH, 8.G., HUGGINS, S.E. and BARTEL, A'H' 967)' , Proc. Soc. e>

LENNOX, E.S. and COFIN, M. (19671. Ann. Rev. Biochem', æ, 365'

LOVtrRy, O.H., ROSEBROUGFI, N.J., FARIì, A.L. and RANDALL, R.J. (1951) . I- bfol- Chem., I93, 265'

LYIGKIS , I.I. (1968). Immunologr, Y, 7EE - LYIGKIS, I.!. ancl COX, F.E.G. G968). ImmunologY, L5, 429.

McE\MEN, C.R. (1967). Anal. Biochem., p, ll4'

Mcl(AY, D. and IENKIN, C.R. (1969). ImmunologY, I-!- I27 ' kx) NIARCHALONIS,I.!. (1959). Aust. f. exp. Blol. med. Scf . (in press)

MARCHALONIS, 1.1., EALE:í, E.H-M. and DIENER, E. (1969). Aust. I. exp. Biol. med. Sci. (tn press)

IVIARCHALONIS, f.f. and EDELMAN, G.M. (1965). I. exp. Med. , ]22, 60L.

IrIARCHALONIS, I.I. ancl EDELMAN, G'M. (Ig66a). f. exp. Med., 1241 901.

N/IARCHALONIS, T.T. ANd EDELIVIAN, G.M. (I966b). Science, ].[É, 1657. MARCHÀLONIS, r.I. and EDEIMAN, G.M,. (1968). I. exp. Med. , U-, Bgl.

I\4ARKHAM, R. (1960)' Blochem - 1., 77 , 516.

I\4AUNG, R.T., (1963). f . Path. Bact., Ë, 5I.

METSHNTKOFF, E. (1905). In "Immunfty in Infectious Diseases'" Gambridge Univ, Press London and New York.

METZGER, H., SHAPI}ìO, M.8., MOSIIMAIIN, I.E. and VIlitrTON, f'E' ( 19 68) . Nature Gond.l , ?I9, I 155 .

MITCHEÍ.T., F.J. (1950). Record. S. Aust. Mus' , 9, 275.

NIALL. H. and EDÌvIAN, P. (1967). Nature (Lond -'), 2J9' 263.

NOLAN, C. and IvIARGOLIASH, E. (1968). Ann. Rev. Biochem., 37, 727 '

NOSSAL, G.I.V., AUSTIN, C.M- and ADA, G'L. (1965)' Immunology, L 333. (xrd)

I oucHTERtoNY, o . (1967). In "Handbook of Experlmental Immunology." p. 655. Edltor D. M. Welr. Blackweli scienllflc Publicatlons (Orcford).

PAPERMASTER, B,W, (1966). In ,'Phylogeny of Immunlty." p. 118. IJnlv. of Flortda Press (Galnsville).

PAPERMASTER, B.W"' CONDIE, R.M,, rINSTAD, J' and GOOD, R'A' (1964). I. exp. Med., 119, 105.

PAPERMAffIER, B.W'. OONDIE, R.M' and GOOD, R.A' (1962)' Natt¡re þond.), 196, 355.

PERËV,. D.Y., HINSTAÐr 1., POIJ.ARA. B' and GOOD, R.A. (1968)' Lab. Invest., f!, 598.

PEIERSON¿ E.-4. ANd SOBER, H.à. (¡962).

[il. " Methods 1Ê EnAymology." Vcl- 5, P. tr. Genera.I E&,tors 6"P' Colowück, and N*O., I(aplan. Acadestsc Press tN.Y')

PHELPS" R.À. ar¡d FIITAfANT F-w. (lg0o)' fn "The PlaSma hoteinsr" VOl. I, p. 143. Editor F.lL/. Puhram' Acade¡nlc Fre-ss gü.y")

POLIARA, 8.," FINSTAD, t. and GoÖÐ, R.À,. (1966). In ,,Phylogeny of ImmunÍty.f p. 88, Edltors R.T. Smith, P.A. Miescher aÌld R.A- GOêd. univ" Of FIOnfda Press (Gatnsvi¡le) " POLLARA, B.â SURAN, 4., H¡NSTAD, I. and GOOD, R'A' (1969)' Proc. nat. Acad. Scl., (Wash.) 59. 1307'

PORTER, R.R. (1959). Blochem. !., !1, ll9' (xxli)

PIIINAM, F.W., TftANI, K., WIKLER, M' and SHINODA, T' (1967)' Cold Spring Harbour Symp' Quant. Biol' 32., 9.

RIDG\MAY, G. (1962). A,mer. Natur. 99, 219'

RIDGÍ\MAY, G., HODGINS, H.O, ôod KLONTZ, G'V1¡' (1966) ' In "Phylogeny of Immunfty." p. 195. Editors R'T' Smlth, P.A. Mieschêr, and R.A. GoOd. unÍv. of Florida Press (Gainsville).

ROMER, A.S. (I962). In ',Vertebrate PaleontoloÇlY." unfversity of chfcago Press (Chicago).

ROMER, A.S. (f 962a). In "The Vertebrate Body." 3rd Ed. Saunders, Philadelphia (Pa)'

ROVVT.EY, D. (1962). Advances in Immunology 2, 24L'

SCHACHMAN, H. K. (I-a59). In ,'Ultracentrlfugatlon in Biochemlstry." Academic ltess (New York and London) -

SOHoENHETMER, R- (1942). In ,'The Dynamic state of Body constituents." Harvard univ. Press, Cambridge, Mass.

scHI]LrzE, H.E. and HEREMANS, J.F. (1966)- In "Molecular Blology of Human Proteins." VoI. I. Nattre (U.K.) and Metaboll.sm of Extracetlular Proteins. Elsevier

SIGEL, M.M. and CLEM, L.W. (1965)' Ann. N.Y- Acad. Sci., re, 662' kxitl)

SIGEL, M.M. and CLEM, L.\M. (1966)' rn ,,Phylogeny of Immunlty." p. 190. Editors R.T. Smith, P.A. Mlescher and R.A. GOOd. univ. of FlOrida Press (Galnsville). SINGER, S.r. and DooLITTtE, R-F. (1'966)' Scfence, fÉ, 13 '

SINGER, S.J. and DOOTITTLE, R.F' (1967)' In "structural ChemÍstry and Molecular Biology'" p' I73' W. H. Freeman & Go. (San Francisco and London)'

SIROTININ, N.N. (r959). In ,'Mechanlsm of AntfbOdy Pormation." p' ll3. Proceedlngs of a Symposium, Czechos}ovak Academy of Sclence, Pragrue.

SIvIALL, P.A. Jr. anci LAMM, M.E. (1966)' Biochenús4/, g, 269 ' 726' SMITH, I/V.W. (f 94Ù). Proc' Soc' exp' Biol' Med " 45' SMITH, R.T., IVIIESCHER, P.A. and GOOD, R'Â'' (1966)' (Gainsvitle)' In "Phylogeny of Immunlty." univ. of Florida Press (1958)' SPACKÌVÏÄN, D.H., STEIN, W..H' and MOORE, S' Anal. Chem. Eg, 119Û'

STEPHENS CHAD\ATICK, IUNE (1967). Ped. Proc . , Æ, 167 5 '

suR.A,N, A. and PAPERMA,STER, 8.1/. (1967). Proc. nat. Acad. Sci., (wash') gg, 1619' (1967)' SURAN, 4., TARAIL, Vi.H. and PAPERMA^STER, B'\¡/' J- Immuncl. 99, 679' (:o

SVEDBERG, T. and PEDERSEN, K'O' (l!r'û)' In,,TheUlt¡acentrlfuge..,oxfordUniv'Press(Londonancr.N.Y')

TAIT, N.N. (1967). Ph.D.Thests,AustrallanNaLionalUnÍversitY,Canberra,Australia.

TIIOMAS, L. (I959). In,,CellularandHumoralAspectsofttreHypersensltiveState'" York' p. 529. Edfton Lawrence, H'S' Hoeber' New

THORUN, trV. and MEHL, E' (1968) Biochim' btoPhYs ' Acta" I60' 13?' (1963)' TURNER, K.J. and RO1 ¡LEY, D' Aust' I'exp' Biol' med' Sci" 41' 595'

uHR,].w.FII{KELSTEI}{,M.S.andFRA}'IKIII\T,E.c.(1962). Proc. Soc' exP' Biol' Med" !}l' 13'

BISTÊ;q' F' (1952)' V,/ESTPHAL , O., LUDERITZ, O' and Z. iiîaturf ' 7ç, L4E'

iArID/\],. F. and SICAIìD, A' (IS97)' C.R. Soc. Biol. (paris) , þ, 1047.

\^[LIIAMS, C.A. and CHASE, M'W' (IE68)' ' In,,Methocisinlmmunologyandlmmunochemistry.''Vol.fI. Acactemic Press (New York and London)' Analy Z' 279' WINZLER, R.]- (1955)' Methods Biochem' '

YOUNG, J.Z. (1950). In..TheLifeofVertebrates..,oxfordUniv.Press(Clarendon), London and New York' 3' 297 YPHANTIS, D-A- (1964)' Biochemistry' '