THE FRACTIONATION OF ISOIMiiUNR BOV IN hi SERA

BY ALOOHOL PRECIPITATION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Uraduate School of The Ohio State University

By

EDWARD J. LAZEAR, B.6., M.S.

The Ohio State University

1952

Approved by:

Adviser TABLE OF CONTENTS

Page

Acknowledgement ...... 11

Introduction...... 1

Review of Literature...... U

Materials and Methods...... 16

Production of Isoimmune Sera ...... 16

Serum Identification...... 16

Collection of Blood Samples ...... 17

The Hemolytic Test ...... 17

Complement Source ...... 16

Preparation of Serum Reagents...... iy

The Selection of the Serum Reagents... 21

Fractionation Procedure...... 21

Electrophoretic Analysis...... ?li

Results...... 25

Chemical Results...... 2$

Serological Results...... 27

Electrophoretic Results...... 3d

Discussion...... DO

Summa r y ...... 51

Tables...... 53

Bibliography...... ci*

Autobiography...... 70

i

91H272 AGKNOWL£DQ£M£lfT

The author is deeply indebted to Ur. L. C.

Ferguson for his valuable counsel and encourage­ ment. His helpful suggestions and stimulating

discussions during this study are appreciated.

The author also gratefully acknowledges the

cooperation of the staff of the Bovine Gellular

Antigen Laboratory. The technical assistance

rendered by members of this group has greatly

expedited the progress of this study.

ii THE FRACTIONATION OF ISOliUUNE BOVINE Se HA

BY ALCOHOL PRECIPITATION

INTRODUCTION

In recent years the workers in the field of immun-

ology have shifted the emphasis from the investigation of

untreated serum as an antibody source to a study of the var­

ious serum globulins as the antibody source. This shift in

emphasis has led to a broader understanding of the immune

response of different animals to the injection of antigens.

In general, the antibodies produced in response to an anti­

genic stimulus have the chemical nature of gamma globulins.

However, serum fractionation studies have shown that 5^-3

species, such as the horse, produce antibodies whose prop­ erties are not similar to gamma globuLin, but resemble aLha

and beta globulins.

The identification of the antibody-containing glob­ ulin fractions and the separation and purification of these fractions has become invaluable for prophylaxis and therapy.

Human gamma globulin is being used extensively at the present time for prophylaxis and therapy in many of the viral and bacterial diseases.

The technique for alcoholic precipitation of serum globulins has been used for the separation and purification of the globulin fractions of practically every mammalian

species. The separation of the globulin fractions of human

plasma and serum has received special emphasis since the

end of Aorld Aar 11. The processing of surplus human plasma

from blood banks has been responsiblef at least in part, for

this increased emphasis. however, little work has been done

on bovine serum. There are reports in the literature per­

taining to bovine serum fractionations, but these reports

deal with normal bovine serum and little space has been de­

voted to the antibody content of the various globulin fract­

ions. bince the antigens of the erythrocytes of cattle have

been studied so extensively, it is unfortunate that no more

attention has been given to the antibody content of tne dif­

ferent globulin fractions present in bovine umnune serum.

it is the purpose of this dissertation to shed

some light on the location, in the globuLin fractions, of

the isoimmune antibodies. To this end, the alcoholic pre­

cipitation technique has been used on certain of the ico-

imraune serum reagents now being used for the blood ty. ing

of cattle.

It is not the purpose of this report to review all of the literature which has to do with the use of tne alco­ holic precipitation technique, since the largest volume of these rej^orts deal with the separation of the globulin fractions of human sera. however, selected articles on human serum fractions will be referred to for the purpose of calling attention to similarities and differences between hiuiMii uf-i'u/n H itl.Ui'iillRn m id Llie 1 noljrimurm antlboillM i of c « tL lc , RFVIa# OF THL bl'i’iiKATUitb

Landsteiner (19PK>) reported that two cellular anti­

gens on the erythrocytes of humans could be identified by

means of normally occurring antibodies in the sera of other humans. He was able to divide humans into three brou, s on

the basis of the cellular antigen on their erythrocytes;

oturli and von becaotello (l9rJ2,f adned a fourth t roup. Von

Uui^em find Hirschfeld (191->) showed that groups A and

could be further divided when they identified antigen A^

and Ap, by absorbing serum from certain group B individuals with cells,

Landsteiner and Levine (1928) reported another

cellular antigen on the red blood cells of some humans,

ibis new antigen was called 1, on rare occasions, isoanti­ bodies against i have been found in normal human scrum, and may result from isoimmunization during repeated transfus­ ions. Antigens U and/or ti (Landsteiner and Levine, 1921,; occur on the human erythrocyte; however, naturally occur­ ring antibodies are rare.

Rh, an additional antigen on the human erythro­

cyte, was detected by means of iiomune sera prepared in rabbits immunised with the blood ceils of rhesus inonKeys

(Landsteiner and nelnor, l^UO .) *s in the case of factors

0, i , U and N, normally occurring rfh antibodies are rare but may result from repeated transfusions.

- U - Careful examination of serum from patients follow­ ing post-transfusion reactions has resulted in the identifi­

cation of still other antigens on the human erythrocyte

(Cellano, Kell, Lutheran, Lewis, etc.J

After the first reort of Landsteiner appeared in

19JO, considerable emphasis has been placed on the cellular

antigens of the many s pecies. Cince the lower animals are more readily available than humans for isoimmunizations, considerable study has been carried out on goats, dogs, cattle and fowls.

c-hrlich and tiorgenroth (191C; found that isoanti bodies were produced when one goat fin.s immunized witn the blood of another. Not only was this isoantibody active against the blood cells of the donor animal but it also was reactive against the blood cells of certain other goats. in no case was the serum hemolytic for the blood cells of the goat which produced the antiserum. Moreover, they found that the blood cells which would be lyse by tnis seriim were capable of absorbing the antibodies from the serum, while the cells of the recipient did not absorb the anti­ bodies.

Horses were divided into four groups on the basis of their reactivity with normally occurring isoantibodies by Hirszfeld and i-'rzesraychi (1923J. Lehnert (1939) used normal isoantibodies and isoimmune antibodies of hit.ner titer for the grouping of horses. he found that intergroup

- 5 - transfusions resulted In fatal hemolytic reactions and con­

cluded that these reactions were caused by the normally occur­

ring antibodies.

Ottenberg and Friedman (1911) found that rabbits

could be placed into four main groups on the basis of their

cellular antigens. Castle and Keeler (1933) recognised two

agglutinogens and Fischer (1935; identified three more anti­

gens. Uarcussen (19?6) and hnopfmacher (19U?> contributed

to the knowledge oi the cellular antigens of rabbits, the latter indicated that the strength of the reaction could be correlated to the genotype of the animal. The genetical­ ly homozygous animals reacted stronger, than did the hetero­ zygous animals.

By means of isoimmune sera, von Uin,;em and Hlrss- feld (1910) were able to divide dogs into four groups, bright (1936) reported that isoimmunization occurred as the result of transfusions with some bloods. The resultant antibodies persisted in the serum of the recipient for long periods cf time and were Increased by repeated injections.

Melnick et al (1936, 1937) also observed the antigenic dif­ ferences in dog erythrocytes, and concluded that repeated transfusions were necessary for the production of isoanti- bodtLes. Recently, Young et al, (19U9) reported a hemolytic disease in newborn dogs following isoimmunization of the bitch. Antibodies to four different antigens have been demonstrated in the sera of dogs by Christian et al. (1951)

Cellular antigen studies on mice (dorer, 1936;,

- 6 - sheep (Bialosuknia, 19 2k , and leas* 191*9), seine (Jiymanow-

skl and Aacklerowna, 1936, and Kuhns, 1950), cats (lngebrig-

sten, 1912), chickens (Todd, 1930), pigeons and doves (Xrwln

and Cumley, 19U3^> anc ducks (McGlbbon, 19UU) have been re­

ported.

Todd and flhljte (1910J, who were working on cattle

plague at the time, fc|und that Iso antibodies were produced

when cow to cow transfusions were performed. rthen a number

of these sera were pooled, it was found that lysis of all

bloods occurred in the presence of guinea pig complement.

Absorption of the pooled sera with the blood cells from one

animal, resulted in the removal of the antibodies for this

blood, but those antibodies for the other bloods were left

in the serum. Ibis absorbed serum reagent could then be

used to identify the blood from the one animal or a closely

related animal.

i Flshbein (1913) obtained strong agglutination with normal isoantlbocles bu, could not classify the animals

into definite blood groups. Little (1929) attempted to

classify cattle into three main groups by means of hamagglu-

I tlnation, but cross reactions indicated that additional

groups were present.

Ferguson (lSflil) identified nine cellular antigens

on the red blood cella of cattle by means of artificially

produced and selectively absorbed isoimmune sera. Addition­

al antigens were identified (Ferguson et al, 191*2) and now more than forty antigejns have been recognized on cattle cells,

- 7 - Ferguson (19Ul) postulated that the presence or

absence of each antigen was controlled by a single gene

pair. Jtonnont et al. (191*5) indicated that in some cases

a multiple allelic series might be responsible for the pres­ ence of many of the antigens, btormont et al. (1951) and

Stormont (1952) have proposed four blood groups in cattle.

The blood antigens in each of these groups are under the control of penes in an alleLie series. They postulate that all the seroLogically identified blood factors in the group are controlled by an allele of a single 6ene. accordingly, blood group B* made up of at least ei; hty alLeles and con­ trolling twenty one blood factors; grou> U, made up of twen­ ty two alleles and controlling seven blood factors; t,roup

F-V, made up of one set of alleles and controlling two blood factors, and grou . Z, containing one set of alleles and con­ trolling the presence or absence of one antigen, have been proposed by jtormont. Unpublished reports of Jtornont (1951) postulate the oressnce of seven more blood groups, in addi­ tion to the four mentioned above. The antigens in each of these groups are assumed to be under the control of the alleles of a single gene pair.

According to the reports of otormont, all of tne more than forty blood factors are under the control of eleven gene pairs. These blood groups or systems nave been established by observing the presence of the antigens in the progeny of selected sires. The interpretation of the authors (btormont et al, 1951) that the antigens in

- Q - the B and C systems are under the control of the alleles of a single gene is plausible, but the alternative explanation of closely linked genes is not excluded.

The apulication of physical chemical methods has contributed immeasureably to the advancement of immunology.

The chemical nature of antibodies and antigens, the quantity of antibody and antigen which combine and the response of animals to the injection of antigens are a few examples of the contributions made by physical chemistry to the field of immunology. (Kabat and ilayer, 19^3. )

Analyses of funified antibody preparations from hyperimmune horse sera have shown that the chemical nature of the antigen will in part determine the nature of tne re­ sponse of the animal. r’or instance, antibodies to the type specific pneumococcus olysaccharide have a molecular weight of nearly a million, (Heidelberger and Pedersen, 1937 ) while the antibodies to diphtherial toxin have a molecular weight corresponding to that of normal gamma globulin of the horse, electrophoretic analyses of horse sera, hyuerimmune to pneumococcal t>olysaccharides, had shown that the s. ecific antibodies are located in a component which has a .mobility intermediate to that of beta and gamma globulin (Tiselius and Kabat, 1933, 1939J. However, upon prolonged immuniza­ tion the antipneumococcal polysaccharide antibodies were confined to the gamma lobulin portion of the serum (van der icheer et al»19U0)

- 9 - Van der 3cheer et al. (19U1) reported that the antibodies to tetanus toxin were located in two serum components. They found that after short periods of immun­ ization, the antitoxin was in the gamma globulin, but on continued immunisation, antitoxin appears as a component migrating between the beta and ganma globulin.

Kabbit hemolysins for sheep erythrocytes have a molecular weight of nearly a million, whereas antipneumo- coccal polysaccharide and egg albumin antibodies of the rabbit have a molecular weight of autjroximately one hundred and sixty thousand (Uselius and Kabat, 1930.)

The isoagglutinins of human (Pedersen, 19^5) and portions of the human Was6eraan antibody (Davis et al., 19U5) have a molecular weight of nearly a million, whereas the remaining portion of the Wassernian antibody has a molecular weight Identical with that of gamma globulin.

Mfttebsky et al (19l*o) found that the Kh antibodies in humans were usually in the ganvna globulin fractions, and that the group A and D agglutinins were located in either globulin fraction. Deutsch et al (19^6) and Cohn et al.

(1950) reported that the Isoagglutinins and typhoid "U" agglutinins were associated with the gamma^ globulin while the other antibodies were associated with the gamrna^ globu­ lin fraction. Gann et al (1951) reported that a consider­ able nortion of the rth blocking antibody was associated with the gamma globulin of low mobility and that the cryptagglu- tinoid antibodies were distributed through the aldha, beta, and gamma globulins.

Tlselius and Kabat (1939) found that the pneumo­ coccal polysaccharide antibodies of the cow had a molecular weight of nearly one million and that these antibodies mi­ grated with a mobility intermediate to the beta and gairi.ua globulin. Smith (19b6) reported that the antibody activity of bovine plasma and colostrum was associated with two com­ ponents, one migrating between the beta ana gamma globulin and the other with the gamma globulin. hess and peutsch

C19U9) found that the major portion of bacterial and viral antibodies were associated with the gamma t.lobulin, but some antibodies were precipitated with the beta globulin fraction.

A variety of physical chemical methods hove been employed for the fractionation of serum globulins. iTact- ionation has been accomplished by the addition of neutral salts, salts of heavy metals, the saturation of diluted serum with acidification with acias, dialysis, elect­ rophoresis, and the addition of cold alcohol and acetone, ihe latter method has received the greatest amount, of atten­ tion because comparatively jure globulin fractions can be attained rapidly.

'Ihe use of cold alcohol for serum ,,lobuLi.n pre­ cipitation was first reported by iiellanby (1908). ihis original work w:s devoted to the isolation of antibodies

- 11 - against, diphtheria toxin from hyperimmune horse serum. Hardy and arwdiner (1910) compared the alcohol precipitation method with the 3alting out method for the separation of antibodies from diphtherial antitoxin produced in several animal species, with special emphases on horse serum. Hiey found that approx­ imately seventy percent of the neutralizing capacity of the serum could be recovered by tne addition of alcohol to a final concentration of twenty five percent.

Hie works of Wellanby and Hardy and jardiner were surported by Villa (1922) who separated the serum globulin fractions of the horse by the addition of coLd acetone. The pH of the serum w;is altered over a wide run. e. Ap roximate- ly seventy percent of the neutralizing capacity was recover­ ed by this technique.

Felton (1931) reported the use of the cola aiconol procedure for horse seru.u fractionation. In this a, tr, r’elton stated th?t the cold alcohol technique was much more satisfactory than the . reel itation of serum globulins by the acidification of diluted serum. Ihe procedure which

Felton used, in the above report, briefly, was to aad nine­ ty five percent alcohol at O b to serum at 0 b until a con­ centration of twenty percent alcohol by volume was obtained.

This mixture was held for eighteen hours at U b, after which time, the precipitate was collected and reconstituted to tne desired volume.

It was not until after the report of bohn et ul

- 12 - \19hO) that widespread application of the cold alcohol precipitation method took place. These authors were the first to emphasize the necessity of controlling the ph along with the variation of the alcohol concentration.

The serum fractions which were obtained from the beef olasma by these workers were subjected to electrophor­ etic analysis. These analyses showed that each one of the four fractions obtained was a comparatively pure protein.

A small amount of the beta globulin was precipitated along with the gamma globulin, but further treatment could sei.ar- ate these fractions.

Nichols and ueutsch (19hd) reported the use of this technique, with modifications, for the fractionation of scrum from many species; namely, humans, goats, dogs, rabbits, cats, chickens and guinea pips. In their research, the concentration of alcohol, and also the . n and ionic strength were varied. These modifications m.nae possible the purification of the globulin fractions.

Smith (19li6) reported the seoarat. jn of the glob­ ulin fractions from colostrum and plasma of cattle. oince the amount of gamma globulin which is present in the sera of calves after birth and prior to tne ingestion of colos­ trum is neglible, Smith referred to the ,,amma globulin of the mature animal as being immune globulin.

Hess and Ueutsch (19U3) reported the fractionation of pooled sera from Holstein cows by means of the alcohol

- 13 - precipitation technique. In this rcuort, the ph, ionic strength and temperature were carefully controlled. Ihese authors reported that comparatively pure globulin fractions were obtained by their technique.

rtecently, Hess and oeutsch reported the fractionation of hyperinumine bovine sera by the cold alcohol techniuue. selected cattle were hyperimmunized against brucella abortus and the virus of Newcastle L&sease. oera frorn the hyjerbnnune cows were fractionated according to the technique presented in their paper of 19Uh. after fractionation, each precipitate was reconstituted and test­ ed serologically. These tests indicated that precipitate A, the beta and gamma globulins, retained the full antibody titer of the untreated st,lutinating titer and one tenth of the bactericidal titer.

Oncley et al (19H9) have summarized the many var­ iations ana modifications wiiich have been used for the fractionation of human plasma. The autnors state that eacn species investigated must be considered as a separate prob­ lem. A given pH or ionic strength may be completely satis­ factory for one species and entirely unsatisfactory for

- 1 U - another species UATirtlALi *UD METHODS

rroduction of Isoimmune Sera

The isoimmune serum reagents were produced by in­

jecting the blood of one cow into another. ihe animals were

selected on the basis of previous laboratory tests so that

antibodies to not more than four previously recognized anti­ gens would be produced by the immunization. r’our weekly in­ jections of 100 to 500 ml of blood were given. Approximate­ ly one week after the final injection, a large blood sample, usually 1500 ml, was collected. ihe blood was incuoated for 2 k hours at room temperature and then placed in the refrigerator at h U overnight to attain a maximum serum yield. Ihe serum was decanted from the clot and the re­ maining olood cells were removed by centrifugation, ihe clear supernatant was aspirated and stored in 50 or 100 ml amounts in lusteroid tubes at -15 0 until used.

Serum Identification

Ihe isoimmune sera are identified by the numbers of the cows which were used in the immunization. Ihe num­ ber of the donor animal followed by the number of the re­ cipient, in parenthesis, was the standard terminology.

Any blood which had been used for absorption was encircled, i^or instance, 6 5 6 (514?)(^76) as a serum identification means that cow 5U7 was immunized with blood from cow 656, and

- 16 - that the resultant serum had been absorbed with red blood cells from cow 676.

Collection of Blood damplea

ihe bloods which were used for testing and ab­ sorptions were collected from the jugular vein of cattle into a 3*5 percent sodium citrate solution. Ihe ratio of citrate to blood was 1:10. These samples were collected as aseptically as possible and stored in the refrigerator at I* U. ihe bloods were used for periods up to two months for testing and absorptions.

Sixty animals, whose antigenic formulae had been established by repeated testing, were available in the exoerimental herd. Blood samples from at least thirty, usually forty, of these animals were on hand in the labor­ atory throughout the course of this stucty for testing and absorptions.

The Hemolytic Test

The hemolytic test described by Ferguson (19dl) was used for the titrations of the immune sera, the serum reagents and for testing all serum fractions. This test was perforrrLd as follows:

The suspension of red blood cells was prepared from the citrated blood which had been collected from the

Jugular vein of the different cows. The blood cell3 were

- 17 - washed three times by centrifugation in physiologicaL (u.y oercent) saline in graduated centrifuge tubes. ‘ihe su cr- natant was discarded after each washing and fresh saline was added. After the third and final washing, tne cell volume v.as adjusted to 0.3 ml. To these packed cells were added 9.7 ml of saline, making a final concentration of 3 oercent.

The hemolytic test consisted of 0.1 ml of the ser­ um reagent or the serum fraction, 0.0$ ml of the test cell suspension, and 0.0$ ml of normal rabbit serum as the com­ plement source.

To facilitate pipetting, I ml serolo, ical pipettes, calibrated to deliver twenty dro^s per ml, were used through­ out.

Hiree readings were taken on each test at thirty minutes, one and one half hours, and finally at three hours after the complement had been adoed. r^ach tube was examin­ ed, with the unaided eye, and the amount of lysis was ob­ served and recorded. The amount of lysis was r,raded into six categories. These categories ranged from 0*, a faint amount of lysis, tnrough tr, 1, 2, 3, and I*, or complete lysis.

Complement dource

The rabbit serum was obtained from normal rabbits by means of cardiac puncture. Approximately forty ml of

- 18 - blood were removed from each rabbit sltt the time of bleeding.

The serum was decanted from the clot .and the remaining cells were removed b y centrifugation. The clear supernatant serum was aspirated, pooled, and frozen In 20 ml amounts. These tubes were maintained at - 20 0 until used.

Preparation of derum Reagents

Ihe serum reagents were pre pared from isoimmune sera by selective absorptions. The iasotmnune sera were

Titrated in doubling dilutions, against the blood cells from a number of cows, usually thirty to forty. This titra­ tion was performed to determine the aaitibody level in the different sera. Ibe absorptions were carried out according to the proposed method of Lazear (19US^. briefly, the ser­ um was diluted to one fourth of the titer with 0.9 percent saline and to this was added one tent^i the serum volume of washed packed olood cells. This mixtwire was Incubated for thirty minutes at room temperature an k would be diluted 1/16 for absorption. Ten ml of the diluted serum were mixed with one ml o f washed packed cells.

Absorptions were ordinarily performed with each of the test bloods which gave a positive reaction in the preliminary test. tach of these absorbed serum samples was tested against the known bloods, rfhean analysis of the re­ sults showed that a n y one positive b l o o d removed the anti- bodies Tor all the other positive bloods, It was assumed

that antibodies wi„h a single specificity were present.

This serum was then called a serai: reagent.

However, when the results showed that bloods

one and two, for example, removed the antibodies for each

other, but left the antibodies for bloods tnree and four;

and bloods three and four absorbed for each other, but did

not absorb the antibodies which were reactive with bloods

one and two, It was assumed that antibodies for two inde­

pendent specificities were present in the serum.

The identification of serbloglcal subtypes was

established In the foLlowlng manner: when the test results

of the absorbed serum samp Les showed that bloods one and

two removed only the antibodies for each other, but blooas

three and four removed the antibodies for ail four bloods,

it was assumed that an antigen with subtypes had been recog­

nised. In this case, the former bloods were designated as having subtype 2 antigens, while the latter bloods were

designated as having subtype 1 and 2 antigens.

*Vhen the test results indicated that bloods one

and two removed only the antibodies for each other, blood

three removed the antibodies for itself and bloods one and

two, and blood four absorbed tne antibodies for all four bloods, it was assumed that an antigen with three subtypes had been recognized. In this instance, bloods one and two were designated as having antigens of subtype 3, blood

- 20 - three as having antigens of subtypes 2 and 3, and blood four as having subtypes 1, 2 and 3.

The Selection of the berura tie agents

The serum reagents which were used In this study were selected from the supply which was available in the

Bovine Cellular Antigen Laboratory at Ohio State University.

These reagents were used routinely in the laboratory for the blood typing of cattle.

derum reagents for antigens J, Z, C, T, U, 1,

0, L', and H were selected for fractionation and study.

These reagents were selected for a variety of reasons but mainly because they were representative of the serum re­ agents available. The anti-J serum was chosen especially because it i3 the only normally occurring isoantibody recog­ nized in bovine sera. Anti-W and anti-Z reagents were selected as representative sera for antigens which did not possess subtypes. Anti-C, anti-T, anti-U, and anti-i sera were selected because these antigens were reported as having two subtypes (btormont, 1950J. Anti-U and anti-L' sera were selected because each of these antigens were re-

(x>rted as having three subtypes (btormont, 195UJ. jinti-n serum was selected because it was characterized by incom­ plete reactions (Ferguson, 19^1)» even in the region of antibody excess with excessive com.dement.

Fractionation Procedure

- 21 - The fractionation procedure employed was that described by Hess and Deutsch (19^8). The method was as follows:

One hundred ml of the serum reagents were diluted with 300 ml of distilled water. The solution was chilled to 0 G and all materials used, and all procedures employed, were kept at this temperature.

The pH of the diluted serum was adjusted to 7.7

£ 0.1 with 0.05 N acetic acid or 0.2 M disodium phosphate.

All >>H determinations were made on a Beckman pH meter,

Model (i. fifty percent ethanol was adoed to a final, con­ centration of 20 percent. The resulting precipitate was collected by centrifugation at 2500 R.P.M. for thirty min­ utes at J G. Hereafter, this preparation wilt be referred to as precipitate A. Ihe supernatant was discarded.

rreciuitate A was dissolved in a small amount of p.9 percent saline and the ionic strength adjusted to

0.01. Ihe ionic strength was equal to one half the sum of the products of the molar concentration of each ion and the square of its valence. The pH was lowered to U.95 £ 0.J5 by the addition of 0 . 0 5 N acetic acid. Ihe precipitate which formed under these conditions was collected by centri­ fugation, as above, and the supernatant was collected also.

Hereafter, this second precipitate will be referred to as precipitate B.

The pH of the supernatant was raised to 7.U £

- 22 - J.l by the addition of J.2 *4 disodium phosphate and per­ cent ethanol was added to a final concentration of 2$ per­ cent. Ihe precipitate was removed by centrifugation and tne supernatant was discarded. This precipitate was desig­ nated precipitate C.

Precipitate C was dissolved in distilled water and 0.9 percent saline with an ionic strength of 0.1. Ihe pH was lowered to 5.0 by the addition of 0.05 N acetic acid, and then raised to 5.8 ^ 0.0$ by the addition of 0.2 * di­ sodium ohosohate. Fifty percent ethanol was added to a final concentration of 10 percent. The resultant precipitate was removed by centrifugation and the supernatant was collected.

The precipitate was called precipitate

uisodium phosphate (0.2 M) was added to the super­ natant until a pH of 7.U £ 0.1 was obtained. rifty percent ethanoi was added to a final concentration of 25 percent.

Th** precipitate was collected, as before, and the super­ natant was discarded. This precipitate was designated

In all steps of the fractionation, at least Id hours were allowed to elapse between the time of adjust­ ment and the time of centrifugation.

After collection, a small sample of each precip­ itate was removed, dissolved in 5 to 8 ml of saline at pri

8 . 0 ^ 0.2. This reconstitution of precipitates was cal­ culated so that tne concentration of this protein fraction would approximate that found in the original serum. This

- 23 - solution was tested in doubling dilutions against at least thirty (usually forty) different bloods. The remainder of the precipitate was stored at -15 0 in loosely stoppered tubes until used.

Electrophoretic analysis

Electrophoretic analyses were performed on sever­ al precipitates. Hiese analyses were made in the klett electrophoresis Apparatus making use of the Tiselius (1937J cell. Barbital buffer at pH 8 .I1 with an ionic strength of

0.1, was used for each analysis. Brotein concentration was approximately 1 uercent. approximately 1.2 volts were applied for 180 to minutes.

- 2 h - RESULTS

Ghentcal Results

Approximately 25 g (wet weight) of serum proteins were precipitated from lOO ml of serum in the first step of the fractionation. Upon subsequent fractionation, 1J g

(wet weight) of precipitate B and 12 g (wet weight) of precipitate G were obtained. further fractionation of precipitate G yielded 6 g (wet weight) of precipitate G^, and U g (wet weight) of precipitate C^.

In order to make a direct comparison of the hemo­ lytic activity of the serum fractions to the activity of the untreated serum, one fourth, one sixth, or one eighth of each precipitate was dissolved in a volume of saline at oH 8.0 ^ 0.1 equal to one fourth, one sixth, or one eighth of the original serum volume. As an example, lOO ml of serum 295(523) mere fractionated according to the method

Doubling dilutions were made from this sample and were tested against the cell suspensions.

Precipitate b from this serum was collected in

- 25 - oi* centrifuge tubes. Each of the tubes contained a p p r o x ­ imately 1.1* g (wet weight) of the precipitate. To one o f these tubes were added 16.67 ml of saLine at pH 8.0 ± 0 . 1 .

Precipitate C was collected in eight centrifuge tubes. Each of the tubes contained approximately 1.5 *5

(wet weight) of the precipitate. To one of these tubes

were added 12.5 ml of saline at pH 8.0 ^ 0.1.

There were approximately 5.6 g (wet weight) o f precipitate C^. This precipitate was collected in four centrifuge tubes. Twenty five ml of saline at pH 5.0

0.1 were added to one of the tubes containing the p r e c i p ­ itate.

Precipitate was collected in four centrifuge tubes. Approximately 0.9 g (wet weight) of the precipitate was in each tube. Twenty five ml of saline at pH 8.0 w e r e added to one of the tubes for the purpose of testing.

in order to avoid confusion, the reconstituted serum precipitates were termed fractions A, B, J, a n d

which correspond to the Drecipitates A, B, U, and

After reconstitution, fractions A, b, C, a n d

C g were tested in doubling dilutions with the cell sus­ pensions. The untreated serum was tested in similar d i ­ lutions at the same time. The strength of the reactions and the titer end points in each fraction were compared directly with the strength of the reactions and the t i t e r end points in the untreated serum. Generally, a one tubes

- 26 - reduction in the titer was observed when fraction A was compared with the untreated serum. Tiiere was no change in the speed of reaction in the region of antibody excess.

Further separation of the globulin fractions was accompanied by an overall reduction in the titer, however, no change in the speed of the reaction was observed.

Since tne titer w^s not a satisfactory standard for the interpretation of the test results, the hemolytic activity of the dilution was used in each case. .then fifty percent or more of the cells were lysed in the *s dilution, the reaction was considered as being positive.

Less than 50 percent lysis, regardless of now much lesb, was considered as a questionable reaction. No visible lysis was considered as a negative reaction.

Serological Results

Antigen J

Antigen J of bovine blood was originally ident­ ified (Ferguson et al., 19U?) by the use of a normally occurring antibody in the serum of certain cattle. llie antibody for J used in this study was obtained from a normal cow, LlOH. This serum was titrated in the hemoly­ tic test against forty different bloods. Fifteen of these bloods showed positive reactions, the remaining 25 bloods were negative.

One hundred ml of tnis normal serum were fract-

- 27 - ionated by treatment with alcohol at 0 0. Fractions b,

and were prepared by dissolving 0.38 £ 0.05 g (wet weightJ

of each precipitate in Li ml of 0.9 percent saline at ph 7.8

^ 0.1. c*ach of the fractions was tested with the same l»0

bloods. The results showed that nearly all the hemolytic

antibodies were associated with the G£ fraction. There was

no activity in fraction b. Only a very weak reactivity was

observed in the fraction. iTvree bLoods showed a very

weak reaction. On the other hand, all of the J positive

bloods reacted in the fraction and these reactions were

nearly as strong as the reactions in the untreated serum.

The remainder of the G^ precipitate from this

serum was dissolved in 10 ml saline at pH 8.0 0.1 and

retested approximately 30 days after the original test.

None of the bloods were lysed in this test, indicating that

the hemolysins in the C fraction were unstable at -15 G.

k comparison of the test results with the serum

before fractionation and each of the fractions is shown

in Table I.

Antigen C

The isoimmune serum, 656(5k7J» produced by the injection of tne blood of 65b into cow 5U7, was processed

as the one above. The hemolytic test f>erformed prior to

the fractionation showed that the homologous titer was

1/128. Xn this test, 27 out of the forty bloods showed positive reactions and the majority of these bloods were

- 28 - lyaed completely in a dilution of l / 6 U .

After separation, fractions A, B, and were tested in the hemolytic test. The results of these tests

Indicate that practically all of the hemolytic activity was recovered during the first precipitation. In general, a one tube reduction in titer was observed. Fraction B possessed some hemolytic antibodies, but the major portion of the antibodies were associated with the and fract­ ions. Tests on these fractions showed that the hemolytic activity was evenly divided between the two fractions.

The positive bloods which reacted more strongly in one fraction, reacted more strongly in the other fraction, also.

Those bloods which were weakly reactive, were weaKly react­ ive in both fractions. The blood cells from cows 512, 597,

612, 651, 668, 671, 6 7 6 , 711, 716, and 719 reacted with the G^ and fractions, while bloods, 272, 5oi*, 519, 537,

5U8, 601, 6 8 3 , 685, reacted with fraction b, ^ and C^.

Previous testing of these animals had shown that the animals in the former group fx>ssessed antigen C2 ** while those animals in the latter group possessed both antigens G^ and

Absorption tests were performed on the untreated serum to determine whether or not this serum contained anti­ bodies for antigens G^ and G^. Three of the bloods from

* There is no relationship between antigens and G„ and fractions and C2 .

- 29 - from group two, In every case, absorbed all the antibodies from this serum. On the other hand, when the serum was absorbed with cells from cow 6 7 6 , a group one blood, anti­ bodies remained for bloods of group two. These absorptions satisfy the requirements for the identification of a sub- type antigen.

The remainder of the C precipitate was dissolved in 10 ml of saline at pH 3.0 0.1 and retested approximately 30 days after the first test was performed.

None of the bloods were lysed in the latter test. This test, as did the retest on the fraction of the normal serum, showed that there were no hemolysins in these fract­ ions after storage at -15 0 for 30 days.

The results obtained from the studies of the isoimmune serum, 656(51»7)» are summarised in Table il.

Antigen U

The isoimmune serum, 512(553), which was pro­ duced by injecting the blood of cow 512 into cow 553, was fractionated by alcoholic precipitation. This serum was known, from previous absorption tests, to contain anti­ bodies for antigens H, Ui,U2 *** unidentified antigen.

After the fractionation had been completed, fractions B, and were tested against forty test bloods.

The results of these tests showed that all the hemolytic antibodies were associated with fractions B and C^. There was no hemolytic activity associated with the fraction.

- 3 0 - Blood cells from oows 673, 512, 568, 566, 560,

607, 612, 6l6, 662, 651, 661, and 671 were lysed in the

serum before fractionation and In fractions B and

These bloods were known from previous tests to have anti­

gen U. In addition, cells known to lack antigen U, from

cows 6 7 6 , 6 8 3 , 709, 732, and 765, were lysed in fractions B and C^.

In order to learn more about the U antibodies,

the untreated serum, and fractions B and were absorbed

individually with the blood cells of cow 6/6. After ab­

sorption, the serum and serum fractions were tested with the cell suspensions. These tests showed that antibodies remained for the bloods of the animals in group one, and that those blooas in group two were no longer acted upon.

The blood of 568 was lysed in fraction but not in the

B fraction. All the other bloods in group one were lysed in both serum fractions. Absorption of the untreated ser­ um with any of the bloods in group one, except 566, re­ moved the antibodies for all of the bloods in group one, but did not absorb the antibodies for the bloods in group two. These absorptions show that antibodies were present in this serum for antigens and U^, and an unrelated antigen.

The serum which had been previously absorbed with the blood of 676, was further absorbed with blood from 566. The test on this serum showed that the abaorp—

- 31 - tion had removed only those antibodies which were reactive with the blood of 51*8. Routine laboratory test9 for the identification of bovine cellular anti ens have indicated that all of the animals in group one, except 51*3, had anti­ gens and Ug. Cow 51*8 had only antigen

The results obtained with this serum are summar­ ised in Table III.

Antigen T

An anti-T serum reagent, obtained from isoimmune serum 585(51*0) by absorption with cells from cows 53l* and

653* was fractionated, fractions B, and G2 were tested with the erythrocytes from forty animals in the experiment­ al herd, fraction B contained the major portion of the antibodies. Fraction contained only a low concentration of hemolytic antibodies. Fraction G^ showed no reactivity whatsoever. The results of these tests are summarized in

Table If.

Lysis of the red blood cells from cows 1*73, 51*8,

561*, 577, 585, 596, 601, 615, 61*2, 657, 661, and 672 occurred in fraction B. Hie hemolytic reactions with this fraction were as strong as the reactions with the absorbed serum before fractionation. CSells from cows 1*73, 51*8, 561*, 577,

585, 596, 61*2, 657, and 661 were lysed in fraction

However, there was a marked difference in the strength of the reactions with this fraction as compared with fraction

B or the serum before fractionation. A very tow grade

- 32 - reaction was observed In each case.

The results of these tests are difficult to

Interpret, and will be discussed In greater detail later.

Antigen I

Anti serum 392(1*73)* which was known from previous absorption studies to contain X^, ^2 * other unident­ ified antibodies, was fractionated by the alcohol precipi­ tation method.

Fractions B, and were tested with the for­ ty test bloods. The lyslns were confined to fractions B and C^. There was no reactivity demonstrated with the fraction. The results of the tests with the untreated ser­ um and the serum fractions are summarized in Table V.

The animals could be divided into three groups on the basis of their reactions with the serum fraction.

Gkroup X consisted of those bloods which failed to react with this serum, i.e. 295* 1*7 3 * and others not shown in

Table V. Group II consisted of those animals known, from previous tests, to possess antigens X^ and I2* i.e. 1*1*3 ,

519* 6 3 6 , etc. Group III was made u^ of those bloods which reacted with the untreated serum but are known, from previous tests, to lack antigens X^ and X2» i.e.

1*23 and 560.

Since this serum was known to contain a diversi­ fied group of antibodies, absorption studies were performed in an attempt to learn more about the Y-j_ and X^ antibodies.

- 33 - fraction* B and wore absorbed individually with cells from cow 56O* ThiB animal was one of those in

Group 111. After absorption, the fractions were tested with the same forty bloods and the cells of those animals in Qroup III were no longer lysed in this serum. The cells of animals in group XI were uniformly lysed by fract­ ions 9 and C^.

Antigen 0

Antiserum 295(523)* which contained antibodies for antigens 0 ^, Oj* and 0 ^> was selected for fractionation since it was one of those sera which reacted with an anti­ gen presumably containing three subtypes (Stormont, 1950j.

fractions B, and C£ were tested with the for­ ty test bloods. The antibodies were confined to the d and

fractions, with no reactivity in the fraction. The test results on this serum are summarized in Table VI.

Serum reagents for the demonstration of subtypes

C>2 and 0 ^ antigens were not available in the laboratory at

Ohio State University. The bloods which were used in this study were tested in the laboratory at The University of

Wisconsin, where serum reagents for 0 £ and antigens were available. The results of the Wisconsin tests are

Included in Table VI along with the results of the tests on the immune serum, 295(523) and the fractions obtained from this serum.

The bloods, which had been designated as having

- 3U - antigen 0 ^ by previous typing, were reactive with fraction

B, while all the bloods which were designated as having

any of the 0 aubtype antigens were reactive with the 0^

fraction. There was no difference in the reactivity of the cells containing and 0 ^ antigens within this globu­ lin fraction.

Antigen E*

Antigen E' is another antigen which was ret>orted

(Stormont, 1950) as having 3 subtypes; therefore, the anti- serum 537(577) which was reactive against all E' positive bloods was fractionated by cold alcohol precipitation.

Fractions B, and C-> were tested for the pres­ ence of antibodies. These tests showed that all the hemo­ lytic activity wis confined to the B and fraction. No reactions were observed in the tests using the fraction as an antibody source.

Serum reagents for the demonstration of subtypes

E '2 and E'^ antigens were not available in the laboratory at Ohio State University. The bloods which were used in this study were tested at the University of Wisconsin where serum reagents for E'^ and E'^ antigens were available. lhe results of the Wisconsin tests are included in Table Vll along with the results of the tests on the imnune serum,

537(577), and the fractions obtained from this aerum*

The red blood cells from cows U73, 512, and 7 k S were lysed in this serum even though they were known to

- 35 - lack £' antigens. Since this serum contained antibodies

in addition to those for antigen , absorption studies were performed. The untreated serum and fractions B and

were absorbed with the blood celLs from cow 512 since

this animal's blood was known to lack antigen £' but was reactive with this serum. After absorption of fraction

B, the erythrocytes from cows hi* 3, 519, 560, 565, 598,

65l, 6 6 8 , 671, 677, 709, and 732 were lysed. The eryth­ rocytes from the above animals and 6 0 7 , 61?, 6 3 6 , and

683 were lysed in the untreated serum and fraction O^. lhese resuLts do not agree with the blood types recorded in the standard hemolytic test and will be discussed later.

Antigen Z

Serum reagent Z, which was obtained by absorbing serum 576(660) with bloods 6 3 6 , 6 7 5 , and 6 1 6 , was fraction­ ated by the cold alcohol technique. Tractions B, and

C2 were tested with the forty test bloods.

The results indicated that most of the hemolysins were located in the Cl fraction. There was no hemolytic activity associated with the fraction and only a very slight reactivity in fraction B.

The results of the tests on serum 676(660) VXIi.

Antigen W

The serum reagent ft, obtained from serum 692(392)

- 3 6 - by absorption with red blood colls from cow 6 1 0 , was fract­ ionated. Fractions B, and were tested with the for­ ty test bloods. Ihese tests showed that practically all the hemolytic antibodies were associated with fraction B.

Fraction caused a few weak reactions with certain bloods which contained antigen W and with blood 661 which was weakly reactive with the untreated reagent. The blood from 661 has been tested with A reagents from three differ­ ent sources and in each case a weak reaction was observed.

These reactions will be discussed later.

The test results for the serum 692(392)^61^) are sumarized in Table IX.

Antigen H

Serum reagent H, prepared from serum 552(553) by absorption with 52U, was selected for fractionation by the cold alcohol technique. Fractions B, and Cg were individually prepared and tested with forty bloods. The results of the tests with the untreated serum reagent and the serum fractions B, and are summarized in Table X.

The results indicated that the hemolytic activity of the anti-H serum was confined to the fraction. There was no activity associated with fractions B or C^.

The red blood cells from cows U72, 512, 5li0» 5oli,

570, 607, 6 1 2 , 6 li*, 620, 6 U 2 , 651, 671, 6 7 6 , and 732, were lysed in the untreated serum reagent and also in fraction

G-^. The animals listed above were known to possess antigen

- 37 - H. Ihe other bloods which wore tested did not possess

antigen H and the ceils did not lyse in any of these tests.

The nature of the hemolytic reactions was not

changed by the fractionation procedure. Incomplete re­

actions, which have characterized this reagent since its

original isolation were observed with every H positive

blood. In no case were more than 50 percent of the cells

lysed in any tube.

ivlectrophoretic Analysis

c.lectronnoretlc analyses were performed on the

isoimmune bovine serum, 656(5k7), fractions B, and

precipitated from this serum, fraction B from the isoimmune

serum, 295(523)* and fraction B from serum 692(392)

The serum components of the untreated serum

separated into four groups. The electrophoretic mobil­ ities of these four components were calculated to be:

Albumin - 6 .6 i* X lO"^ cm^ volts”^ sec"^. — ^ P — 1 i Alpha globulin - U.65 X 10“ 17 era volts sec~ .

Beta globulin - 3.2k X 1 0 ~^ cm^ volts”'*' sec”*’.

Gamma globulin - 2.29 X 10~^ cm^ volts-"*- sec"*.

Two components were observed in fraction B from each of the sera which were analyzed. Fraction b from serum, 656(5k7), had two components with the respective mobilities of 3.71 and 2.79 X 10“^

The two components in fraction B from serum 295(523) had

- 38 - mobilities of 3- 2 U and 1.06 X 10~^ cra^ volts-^- sec"^.

The components from serum, 6 9 2 ( 3 9 2 had mobilities of 3 . 2 8 and l.ij£ X 10“ -* cra^ volts'”^ sec-^ .

In each case, approximately 9 0 percent of the protein migrated with the faster mobility, while the re­ maining 10 percent moved with the slower mobility.

Precipitate from the isoimmune serum 656(6^7 ) contained two components which separated upon electrophor­ esis. Hie faster moving component had a mobility of 2.33

X io-5 cm^ v o l t s " s e c ^ and the slower conn>onent migrat— -j - 1 _ 1 ed at the rate of 1.20 X IO cvolts sec . Approx­ imately 80 percent of the protein migrated at the slower rate.

Precipitate from the isoimmune serum, £>5£>(b>h7), contained a homogeneous component which moved at the rate of 1.15> X 10“-* cm^ volts-^ sec“^.

Hie calculated mobilities are presented with those of Uann et al. (19U9) in Table XI.

- 3 9 - DISCUSSION

More than forty cellular antigens have been

recognised on the erythrocytes of cattle. These have

been identified largely by means of isoimmune sera. The

chemical composition of the cellular antigens is not known,

but oreliminary studies have shown that some of them may

be hexoseamlnes (Royal, 1 9 5 2 ) . Since the chemical nature

of the antigen has been shown to influence the nature of

the antibodies produced by many mammals, it was assumed

that some of the antigens on cattle erythrocytes would

stimulate the production of antibodies which would resem­

ble beta globulins while other antigens would stimulate

the production of antibodies which would resemble gamma

globulins.

l^eliminary serum fractionations showed that

the antibodies for antigen were located in the beta

globulin fraction, and that the antibodies for the anti­

gen Cj were located in the gamma globulin fraction. On

the basis of these preliminary findings, it was oostulated

that the antibodies for aLl the subtype 1 antigens would

be located in one globulin fraction and that the antibodies

for the other cellular antigens would be located in another

globulin fraction. This hypothesis was not fulfilled en­ tirely. Ihe antibodies for antigens C^, 0^, U-^ and were located in the beta globulin fraction, and did ful­ fill the hypothesis. Ihe antibodies for antigens 0^,

- U0 - O j , U2, £' 2* £ *3 > Z, J and H were located in the gansna glob­ ulin fractions. However, antibodies for antigens T and I, which are reported as having subtypes, did not fit into

the hypothesis; also, the reagent for antigen A, which has

no reported subtypes, possessed antibodies in both the beta

and gamma globulin fractions.

In the report of Hess and Oeutsch (lyhHjl the auth­ ors stated that precipitate A was made uo largely of beta and gamma globulin, precipitate b was largeLy beta globuLin, pre­

cipitate was gamraa^ globulin, and precipitate was gararaa2 globulin. The authors did not state their reasons for the designation of the subtype, but it is assumed that they were using the terminology which has been adopted by the majority of the workers in this field,

Most of the literature dealing with this subject designates the various globuLin fractions on the basis of their electrophoretic mobilities, the faster moving compon­ ents are designated as subtype one, while the slower moving components are designated subtype two. However, this prac­ tice Is not followed by all authors. dome authors reverse the usual procedure and label the slower moving component as subtype one and the faster component as subtype two. in most instances, sufficient information is giver, by the dif­ ferent authors so identification can be made, but there are

Instances in which the subtype designation is the only inform­ ation given. These inconsistencies in the literature make

- ia - the Interpretation of different reports doubly difficult.

Cann et al (19li9) reported the electrophoretic mobilities of the serum components in normal bovine serum.

They reported the separation of two alpha globulins, two beta globulins, two gamma globulins and a component with a mobil­ ity intermediate to beta and gamma globulin. In each in­ stance, the faster moving globulin fraction was labeled as subtype one, while the slower noving component was label­ ed as subtype two. Electrophoretic analysis of the d,, fract­ ion from the isoimmune serum, 6 5 6 (5U7 ), showed that the homo­ geneous component in this precipitate had the mobility of the ganma2 globulin of Gann et al (19U9)( therefore their terminology for the subtype identification was adopted and used throughout this dissertation.

The results of the hemolytic test using the serum fractions from the normal hemolysin indicated that the nor­ mal antibody for antigen J was located in the gai^T.a^ glob­ ulin. Ihese results are the opposite of tnose reported for human serum by Cohn et al (1951). 'Ihese authors reported that the Isoagglutinins in human sera are associated with the ganona^ globulin.

Deutsch et al (19ii6) characterized the ganuna^ glob­ ulin from human plasma as being similar to fibrinogen in thi.t this fraction is heat labile. In this same report, the authors stated that the gamma2 globulin was more heat stabile.

- 1x2 - The stability studies which were carried out in

connection with this work have shown that the garamaj globu­

lin of bovine serum was unstable on storage at -15 0. The

ganma^ globulin fractions which had been aged at -15 C

either as precipitate or as dissolved precipitate C^,

did not possess or rapidly lost all of their hemolytic act­ ivity. On the other hand, the beta and gamrna-^ globulin

fractions were more stabile and did not lose their hemoly­

tic activity after storage for three months at -15 0 .

Hemolysins were demonstrated in the gamma2 glob­ ulin fraction of only two sera. The two sera, U10H and

6 5 6 ($U7 )> were collected approximately one month before they were fractionated by cold alcohol precipitation. The other sera had been stored at -15 0 for from six months to five years. There was no hemolytic activity in any of the gammap globulins of these older sera.

In the paper by Ferguson et al (19U2) antigen T was reported as having a single specificity and that the antiser­ um could not be further fractionated by absorption, btor- mont (1950) reported that he had produced another anti-T reagent which had the same specificity as the original anti-

T reagent. However, he was able to produce serum reagents for antigens and T 2 by selectively absorbing the new anti-T reagent.

The anti-T reagent which was used for this study presumably resembles the original anti-T reagent reported

- U3 - by Ferguson et al (19l*2)„ Kepeated tests of bloods which

reacted with the original T antibodies showed that these

same bloods reacted with the antiserum which was used in

this study. Hie fact that the original T antiserum could

not be selectively absorbed; that the anti-T reagent of

this report could not be selectively absorbed; and that

the hemolysins were located in a single globulin fraction,

suggests that these sera did not possess antibodies for

antigen T^.

In this case the apparent exception to the hypo­

thesis may be explained on the fact that this serum con­

tained antibodies for only antigen therefore, no separ­

ation into different globulin fractions was observed when

the serum was fractionated.

Hie serum, 392(1473)» which was fractionated in an effort to characterize the and antibodies was unique.

This serum, which contained antibodies in addition to those

for the two antigens mentioned, was the only serum which had approximately the same reactivity in the beta globulin fraction and in the gamma^ globulin fraction after precipi­ tation.

This immune serum was produced in 19h7. A complete history of the immunization was not available, but the rec­ ords show that serum was collected one weeK following the fourth imnunization and another serum collection was made two weeks after the fourth immunisation. A portion of the

- W * - serum from the second collection was used In this study.

Two possible explanations for the unique nature

of this serum are obvious. One explanation may be that the

precipitation technique was faulty. This explanation Is not

very probable, since the procedure was standard for all

sera. Electrophoretic analysis was not performed on this

serum or the precipitates, but analysis of precipitates

from other sera showed that the globulins did separate

under the reported conditions. The second, and more tenable,

explanation may be that during the extended time between the

final Injection and the serum collection, the chemical na­

ture of the antibodies had been changed.

Ihe response of cattle to prolonged Immunisation

was not known, but it was assumed that the response in cat­

tle would be similar to that of horses. It has been shown

by Van der 5cheer et al.(19U0, 19bl) that prolonged immun­

isation of horses changed the chemical properties of the

resultant antibodies. The specific pneumococcal polysacchar­

ide antibodies had an electrophoretic mobility intermedi­

ate to beta and gamma globulin, but upon prolonged immun­

isation the antibodies were confined to the gamma globu­

lin portion of the serum. Similarly, the tetanus antitox­ in was in the gamma globulin, but upon prolonged immuniza­ tion the antitoxin appears as a component migrating between the beta and gamma globulin.

In the case of the isoimmune bovine serum, the

- US - increased time lapse between the Immunisation and the serum

collection may have had an effect in the c o w which would

parallel the observed effect of prolonged Immunization in

horses. The antiserura for antigens G, 0, U, and £' which

were collected one week after the final injection of anti­

gen had the antibodies for the subtype one antigens in the

beta globulin fraction and the antibodies for the other sub-

type antigens in the gamma^ globulin. Tne antiaerum for

antigen f was oollected two weeks after the final inject­

ion of antigen. In this serum, there was no separation of

the antibodies for antigens fp and Into the different

globulin fractions. These results suggest that the primary

response of the cow is to produce antibodies for the sub-

type two antigens in the gantoa^ globulins. However, anti­

bodies for the subtype two antigens may have the pro:-erties

of beta and gamma^ globulins, if the time between antigenic

stimulation and serum collection is extended.

Serum reagent for antigen A was the only one

studied which showed reactivity in the beta globulin fract­

ion when the antigen did not have a reported subtype. The

blood cells from cow 6 6 1 were lysed in the gmnma^ globulin

fraction but were not lysed in the beta globulin fraction.

Absorption studies have shown that the antibodies which

caused the lysis of this blood could be removed by absorp­

tion with this blood without affecting the antibodies which are reactive with the other bloods containing antigen

- U6 - W. Reciprocal absorptions with W positive bloods did not

separate these two antibodies. All absorptions with bloods which contain antigen # removed, in addition to the anti­ bodies for antigen W, the antibodies which were responsible

for the reactions with blood of cow 6 6 1 .

Blood from cow 6 6 1 was tested at The University of Wisconsin. The report from that laboratory coincides with our findings in that this blood reacts with reagent rt, although not as strongly as other bloods.

Ihe tests of the globulin fractions showed that a weak reactivity, which was present in the reagent, was present also in the gawna^ globulin but there was no react­ ivity for this particular blood, cow 6 6 1 , in the beta glob­ ulin fraction.

Reciprocal absorptions have not been successful in the separation of the two types of antibody which are present in this serum, indicating that an antigen with subtypes is present. Ihe serum fractionation studies snow that the antibodies of one specificity are located in the beta globulin, and the antibodies for the other specifici­ ty are present in the gamma^ globulins. Therefore, it is proposed that antigen # is actually composed o f two por­ tions, and # 2 *

Antigen hi • was reported (otonnont, 1950; as having three subtypes. All of the bloods which were re­ ported as having antigens and were lysed by

- h i - the antibodies In the beta globulin fraction. However, the

blood from one cow, namely 6 7 1 , which was reported as having

antigens £ ' 2 and was lysed also by these same antibodies.

All of the other bloods which were reported as having anti-

gena h,'3, or antigens £ ' 2 and ^'3 * were not lysed by the

antibodies in the beta globulin fraction. All of the bloods

which were reported as having any of the £' antigens were

lysed by the antibodies in the garnma^ globulin fraction.

The Bovine Cellular Antigen laboratory at Ohio

State University does not have serum reagents for the ident­

ification of antigens ^ and The bloods which were

used in this study were tested in the laboratory at The

University of Wisconsin where antisera for the differen­

tiation of these antigens were available. The results of

the Wisconsin tests were used in this study* The anti-m*^

reagents at the two laboratories have the same specificity.

Fraction B and the untreated serum, 537(577) f were absorbed individually with the erythrocytes from

cow 671* This blood was used for the absorbing blood be­

cause it was reported by the Wisconsin laboratory as lack­ ing antigen £'^, but was reactive in the beta globulin fraction. After absorption, the serum and the serum fraction were tested with the known bloods. There was no reaction with any blood in these tests. Absorption with this blood, had removed completely the antibodies for anti­ gen These results suggest that this animal has anti-

- U9 - gen The results of the absorption and precipitation

studies suggest that the report from the Wisconsin labor­

atory is in error. In the light of this information, the

original hypothesis is fulfilled by antigen L1.

The antibody for antigen H Is unique in the field

of bovine cellular antigens. This reagent is characterized

by the fact that lysis of a ous*tension of red blood cells

is never complete even in the area of antibody and comple­

ment excess. Cohen (19^1) postulated that this peculiar

reaction pattern was caused by the competition of two anti­

bodies, one non-hemolytic, for the same antigen.

Since the alcohol precipitation technique was

successful in separating the antibodies for the serolog­

ical subtypes of antigens into the different globulin fract­

ions, the technique was applied to the anti-H reagent in an

attempt to separate the non-lytic from the lytic antibodies.

After the globulin fractions had been separated

by the alcohol precipitation, hemolytic antibodies could

be demonstrated only in the gamma^ globulin. The nature

of the hemolytic reaction was not changed by the fraction­

ation procedure. The positive reactions were characterized

by their incompleteness.

lhe findings of this study do not support nor do

they disprove the explanation put forth by Cohen for the incomplete reactions. If there are hemolytic and non­ hemolytic antibodies in the anti-H sera, both are located

- U9 - In the gamrua^ globulin.

The electrophoretic analyses were performed to

determine the ; irity of the globulin fractions obtained

by the precipitation. Analysis of precipitate B showed

that 90 percent of the precipitated globulin migrated with

the mobility of beta globulin and 1 0 percent migrated with

the mobility of gamma globulin. eighty percent of the pro­

tein in precipitate migrated with the mobility of 1 . 2 0

X 10-5 crn^ volts-^ aec"^-, and 20 percent migrated at the

rate of 2.03 X 10~^ cm^ volts”^ sec-^. A homogeneous com­

ponent was observed in precipitate C^, which moved at the rate of 1.15 X 1 0 ” 5 cm^ volts-^ sec"^.

Hess and Deutsch (19U9) stated that the major portion of the antibodies for B. abortus and Newcastle

Disease virus were located in the gamma^ globulin fraction of hyperimmunized cows. In general, the isoantibodies were found to be present In a greater concentration in the gamma^ globulin fraction. In two serum reagents the antibody level was higher in the beta globulin than in the ganvna^ globulin.

In one serum reagent the antibody level was higher in tne gamraa^ globulin than in the gamraa^ globulin. In all the other sera, the antibody level was greater in the gaxoma^ globulin than in either the beta or gamraa^ globulin.

- $0 - SUMMARY

The beta, gamm*^ and gamaa^ globulins have been precipitated and separated from ten isoimmune bovine sera by means of cold alcohol precipitation. These globulins were reconstituted in saline and tested in the hemolytic test to determine the antibody content of the different globulin fractions. Serum reagents for antigens d, H, J,

0, T, U, H, Y, Z and S' were fractionated. 'These antigens can be placed in four groups on the basis of the globulin fraction which contained the major portion of the homol­ ogous antibodies.

Group I - those antigens which were reactive with the anti­

bodies which were located in the gamma-, globulin.

Antigen J is the only member of this group.

Group II - those antigens which were reactive with the anti­

bodies located in the beta globulin fraction.

Antigens G^, 801 would be in­

cluded in this group.

Group III - those antigens which were reactive with antibodies

located in the ganvna^ globulin. This group includes

antigens C2 , 0?, U?, £*2 » ^*3* rt2* Qroup IV - those antigens which were reactive with antibodies

located in both the beta and gamma^ globulins.

Antigens Y^ and Y^ make up this group.

- 51 - The antibodies in the gamraa2 globulin fraction were unstabile on storage at — 1 5 U, while the antibodies in the beta and gamma^ globulins were stabile at this temp­ er at u r e ,

electrophoretic analysis of the precipitates showed that there were two components in the beta globu­ lin fraction, two components in the garama^ globulin fract­ ion, and one component in the garama^ globulin fraction.

- 52 - T A B U I

Scrum Reagent for Antigen J Snwiry of the hemolytic tecta on the normal scrum, 4loH

Hemolytlo Test Results Serum before Serum Tractions cow* Tractlocation Diluted £ Diluted i B ci °2 504 4 _ .. 4 _ 519 4 — i. 4 — 524 4 - JL V — 573 •— ——— 581 —- - - — 590 4 — 4 — 601 _ _ • 620 _— * _ 636 4 — • 4 — 650 4 - ± 4 — 651 — - •• 663 4 — — 4 — 666 •* _ 6 7 1 4 • — 4 • 672 ———— — 676 4 — _ 4 _ 663 4 •_ 4 6 9 0 -— ——- 7 U 4 —- 4 — 7 22 4 -- 4 -

* Im ell, forty mmlmmls were tested. Twenty ere presented me representative. # Traction tested after 30 days storage at -15 C. 4 Positive reaction ± Weak reaction - negative reaction

Vote: Mere than 5 0 peroent lysis la the i dilution was recorded as positive; less than 5 0 percent lysis was recorded as a weak reaction; ne lysis was recorded as negative.

- 53 - TABLE II

Strua Reagent far Amtic*a C

Summary af tha hamalytic taata am tha isolmmuna serum, 656(547)

Ha melytic Test B e suits Serum Serum COM* i>9XBa/ama trt AOVCrD6u Serum Fractions Diluted & fraction­ with B C c C # ation 676 1 2 2 Diluted £ 272 f a ♦ ♦ a — 295 -- —-- - 443 ------504 ♦ a + a - 512 a - - a a - 519 a + ♦ ♦ a - 537 ♦ + + a a - 546 a ♦ ♦ a a - 597 + -- a a - 601 a + ♦ a a - 612 + - - a a - 651 ♦ —— a a - 666 ♦ —— a a - 671 ♦ -- a a - 676 a —- a a - 683 ♦ a ♦ a a - 665 V a + a a - 711 ♦ -- a a - 716 a - - a a - 7X9 ♦ - - a a —

* la all, farty amimals vara tastad. Twenty ara prasamtad ms representative•

f Cg fraotioa tastad after 30 days storage at -15 C.

a Positive react lam £■ Weak reaotlon - negative react lam Mata: Mara than 50 percent lysis la tha i dilution was recorded as positive; lass than 5 0 percent lysis was recorded as a weak reaction; no lysis was recorded as negative.

- Sh - TABLE III

Serum Reagent for Antigen U

Summary of the hemolytic testa on the isoinriune serum, 512(553)

Hemolytic Test Results Serum derum Serum Fractions IJiluted ^ CO,** Reagent 4 Before Fraction­ B *u£luEed^ £ "i U2 ation °1 C2 iJiluted i

U73 4 + 4 4 + + 4 512 + 4 4 4 4 + + 4 ShQ ♦ 4 4 + 4 56U + 4 ♦ + - ♦ 4 4 580 4 4 + 4 + 4 4 + 607 + 4 + + 4 + + 4 612 + 4 4 4 4 4 4 4 611i ♦ 4 4 4 + 4 4 4 6U2 ♦ 4 ♦ 4 + — 4 4 4 651 4 4 4 4 4 * + 4 661 + + 4 + + — 4 4 + 671 4 4 4 4 4 4 + 4 676 —_ + 4 + — _ 663 —- + 4 + — —— — 709 - - + 4 4 --- - —- 4 4 — -- - 7?2 4 7U5 —— 4 4 4 _ 585 —— - —— w —_ _ 598 ——— ——— — —

616 — ——— - — — — —

* In all, forty animals were tested. Twenty are presented as representative.

♦ Positive reaction + Weak reaction - Negative reaction

Note: Wore than 50 percent lysis in the A dilution was recorded as positive; less than 56 percent lysis was recorded as a weak reaction; no lysis was recorded as negative.

- 55 - T A B U IT

Serua Reagent far Antigen T 9 o m r j of tha haaolytic testa on the liolnune tenui, 585(540)(g9^52> Hoaolytlo Test Besuita So run COW* Before Serua Fractions Diluted & Fractionation B Diluted -J1 ci °2

**73 4 * t 5 12 ——— — 52** —_—— 5**8 ♦ 4 56** ♦ 4 ± 5 7 7 ♦ 4 ± — 585 4 ♦ * — 5 9 6 4 ♦ ± 601 4 4 _ 6 1 2 _ • _— 6 1 4 _ _ _ 615 ♦ 4 —_ 620 —__ _ 6 3 6 — —— — 6 4 2 ♦ 4 ± — 651 —— - 6 5 7 ♦ 4 ± — 661 ♦ 4 ± • 6 7 2 ♦ 4 — — 6 7 6 — - - • * Im all* forty anlaals were tested. Twenty are praeentod as representation.

+ Positive reactloa at Weak reaction - Begatire reaction Vote: Mere than 50 peroent lyela in the H dilution was reoardad aa positIra; lose than 5 0 percent lysis was recorded aa a weak reaction; no lysis was recorded as negative.

- 56 - T4BLI ▼

Serna Btafeiit for Antlfen T

SiiMnry of the heaolytlc tests on the lsolmanne ■•run, 392(^73)

Henolytic Test Besuits Serna Serna COW* Beagent Before Serua Fr not ions Diluted £ Fraction­ ation B Ti T2 Diluted £ ei °2 ( 5 ^ ? 295 __ _ _ 426 - - 4 4 4 - - - 443 — 4 4 4 4 — 4 4 473 ------519 — 4 4 4 4 - 4 4 560 -- 4 4 4 -- - 577 - - 4 4 4 — - - 585 — 4 4 4 4 - 4 4 596 — — 4 4 4 - -- 596- — 4 4 4 + - 4 4 607 — 4 4 4 4 - 4 4 614 —— 4 4 4 -- - 616 — 4 4 4 4 - 4 4 6 3 6 4 4 4 4 4 - 4 4 651 * 4 4 4 4 - 4 4 666 - 4 4 4 4 - 4 4 672 - 4 4 4 4 - 4 4 683 4 4 4 4 4 - 4 4 709 - 4 4 4 4 - 4 4 745 — 4 4 4 4 4 4 I ■

* la all, forty- anlnals were tested. Twenty oxo prosontod as representative.

4- Positive reaction ±, Weak reaction - Negative reaction

Note: More than 50 percent lysis was recorded as positive; less than 50 peroent lysis was recorded as a weak reaction; no lysis was recorded as negative.

- 57 - TABLE VI

Serum Reagent for Antigen 0

Summary of the hemolytic tost* on the iso Insane serum, 295(523)

Hemolytic Test Results Serum oov* Serum # Before Serum Tractions Diluted 4 Reagents fraction­ ation AB C c °1 °2 °3 Diluted £ 1 2

295 4 4 4 4 4 4 4 **3 - 4 4 4 4 -— *73 ♦ 4 4 4 4 4 4 — 512 - - 4 4 4 -— 5*7 ♦ 4 4 4 4 4 4 - 577 4 4 4 4 4 4 4 — 580 - wm 4 4 4 — — 585 ♦ 4 4 4 4 4 4 • 598 - 4 4 4 4 — 4 - 607 4 4 4 4 4 4 4 — 815 4 4 4 4 4 4 4 - 6 2 0 - 4 4 4 4 —_ 6*2 4 4 4 + 4 4 4 — 6*7 4 4 4 4 4 4 4 — 651 - 4 4 4 4 —— 661 4 4 4 4 4 4 4 — 671 * - 4 4 4 — 4 — 672 4 4 4 4 t 4 — 709 -- 4 4 4 - a — 7*5 4 4 4 4 4 4 4 -

* la all* forty animals war* tooted, twenty are presented m representative. # Tested at The University of Wisconsin f Positive reaction ^ Weak reaction - Veg&tlve reaction

Vote: More than 5 0 percent lysis In the ^ dilution was recorded as positive; less than 50 percent lysis was reoorded as a weak reaction; no lysis was recorded as negative* - $8 - TABLE VII

Serum Re aren't for Antigen Summary of the hemolytic testa on the isoimmune serum, 537(577)

Hemolyti c Test Results Serum Serum Serum Fractions Diluted £ c o n * Reagent # Before Fraction­ ation B b H Diluted ^ °1 s A Uh3 + 4- + 4 4 4 4 k ^73 —— — 4 4 4 — — 512 —— — 4 4 4 — — — 519 ♦ 4 4 4 4 4 — 4 4 560 4 4 4 4 4 4 — 4 4 585 ♦ ♦ ♦ 4 4 4 — 4 4 596 ——— — — 598 4 4 4 4 4 4 * 4 4 607 — — + 4 4 4 _ _ A 612 —— 4 4 4 4 _ — ± 6 2 0 —— — 6 3 6 — — 4 4 4 4 — — k 651 ♦ 4 4 4 4 4 — ± k 668 + -f 4 4 ♦ 4 — 4 4 671 — 4 4 4 4 4 — 4 4 677 4 + 4 4 4 4 — 4 4 683 —— 4- 4 4 4 — — ■A 709 4 4 4 4 4 4 — 4 4 ♦ 4 4 4 4 4 — 4 4 7 h $ — —— 4- 4 + — — —

* In all, forty animals were tested. Twenty are presented as representative.

# Tested at the University of Wisconsin.

-f- Positive reaction £ /teak reaction - Negative reaction

Note: More than 50 percent lysis In the £ dilution was recorded as positive; less than $0 percent lysis was recorded as a weak reaction; no lysis was recorded as negative.

- 59 - TJBIJE Till

Serua Reagent for Antigen Z

3n— ry of the honelytic teote of the Im Im m m serua. 676(660) (tSejwixlKt > [ Reaolytlo Toot Results ] Serua 'before Serua. Fractions Diluted % | co ir* Fractionat i on diluted £ B °1 295 4 £ ♦ 443 4 £ 4 - 519 4 £ 4 • 548 ♦ ± 4 — 560 • 570 4 1 4 — 596 ♦ £ 4 — 596 • _ 601 4 £ 4 — 614 ♦ £ 4 — 615 — • _— 62U 4 £ 4 — 647 -——_ 651 ———— 657 ———— 668 ♦ £ 4 672 4 £ 4 — 677 ——- • 6 8 3 4 £ 4 - 709 - — --

* la all, fortj asiaal1 war# to■ tod. Twenty aro proooatod aa representative. ■f PositIt# roaotloa -± Weak reactioa - Negative reaction Koto: More than 50 percent lyele la the £ dilution was recorded aa positive; less than 5 0 percent lysis was recorded as a weak reaction; no lysis was recorded as negative.

- 60 - TABLE IX

Serun Reagent f#r Antigen V

% i m u 7 #f the heaolytle teste oa the lsolnnunt> serun, 692(392) (618^

Henolytle Test Heoulto Vise. Ohio Serun Serun Fractions cov* Serua Serun Before Diluted & Reagent Reagent Fraction­ V V fron ation B 380(379) Diluted £ °i °2 < & & 295 ♦ 4 4 4 —— h*3 4 4 4 4 — 512 — - —— • 519 - ———_ 5 6 k --— - — — 570 -- —— — — 585 4 4 4 4 4 — 596 ♦ 4 4 4 4 607 4 4 4 4 4 _ 612 —__ * 616 4 4 4 4 6 2 k __ 636 4 4 4 4 ± 642 —_ __ 647 4 4 4 4 ± — 650 4 4 4 4 ± — — 661 * i - ± 4 668 4 4 4 4 4 * 709 4 4 4 4 - - 732 4 4 4 4 4 •

* In *11, forty animals were tooted. Twenty are presented no representative.

+ Feeltire reaction ^ Weak reaction - Begatire reaction

Bote: More than 50 percent iyolo la the £ dilution was reoerded no pooltire; lees than 50 percent lyeis was recorded aa weak reaction; no lysla was reoerded aa negative.

- 61 - T A B U X

Serun Reagent f*r Aatl^en H

•f t)M hens lytic testi ^ tha 1 so insane »«n», 552(553)«*t Heaolytle Test Be suits COW* Serua Before Serum Tractions Diluted % Traotloaatlen B Diluted & ci ° 2 4 2 0 __ 4 7 3 d - * - 5 1 2 d - * — 5 4 8 ± - d - 5 6 4 d - ± - 5 7 0 ± — d - 596 d - ± - 6 0 7 d - ± - 6 1 2 d - d - 6 1 4 d - d — 6 1 6 — - - 6 2 0 d - d — 6 4 2 d - d - 6 4 7 — -- - 6 5 1 d - * - 6 6 1 • -—— 6 71 d - d — 6 7 6 d - d - 7 3 2 d - d - 7 0 9 — — ——

* Im all, forty •alwlo voro tested. Twenty are presented aa represent at lee.

t Incenplete reaetlone - Begatlee reactions

Bets: Mere than 50 p e r c e n t lysis In the J dilution vas recorded as positive; lees than 50 percent lysis was recorded aa an incomplete reaction; no lysis vas recorded as negative.

- 62 - TABLE U

The Heotrophorotic Mobllltiet *f the Serum Component! from I t e l m e Berime Sera,

Serum or Mobility Z 10"^ cm2 relta"1 tec”* Seram fraction Albumin Alpha globulin Beta globulin Gemma globulin 1 2 1 2 1 2 Borml Senm Cean et el 6.53 5.76 4.62 3.86 3.08 1.97 1.28 (1*9)

I to Immune Seram 656(547) 6.64 4.85 3.34 2.29

Fraction B 656(547) 3.71 2.79 295(523) 3.34 1.08 692(392)^18) 3.28 1.45 fraction 656(5^7) 2.83 1.20

fraction 656(5^7) 1.15 BIBLIOGRAPHY

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- 69 - AlTTOBlOQrtAPHY

I, Ldward J. Lazear, was born in Louisville, Ohio,

June 5, 1922. I received my secondary school education in

the public schools of Louisville, Ohio, hiy undergraduate

training was obtained at Mount Union College in AlLiance,

Ohio, and I received the Bachelor of Science degree in 19Hi.

After completing training in Northwestern Midshipman School,

1 served in the U. S. Navy for three years. Upon being

honorably discharged, I entered The Ohio State University

to work on advanced courses in my chosen field, and received

the Master of Science degree in 19U9. From 19^7 to 19b 9 1 worked as desearch Assistant in the Department of Bacteri­ ology* An appointment to Instructor in the Department of

Dairy Science came in 19b9 and 1 have held that position while completing the requirements for the degree Doctor of

Philosophy.

- 70 -