Immunologic Pregnancy Tests Evaluation of Pregnosticon Dri-Dot and Pregnosticon a Cci~~Pheres

Immunologic Pregnancy Tests Evaluation of Pregnosticon Dri-Dot and Pregnosticon A cci~~pheres

EMMET J. LAMB, MD, FACOG

Urine specimens from 50 pregnant aiid 50 venicnce in use, Pregnosticon Accuspheres nonpregnant women were rested with five and Pregnosticon Dri-Dot. immunologic pregnaiicy tests. Pregnosticon Ac- cuspheres, a new hemagglutination inhibition MATERIALS AND METHODS test, was found to be more convenient when compared with UCG, an established tube test. Selection of Subjects s Compared with two established rapid slide tests, Pregnosticon and Prequest, Pregnosti- Urine specimens used for this study were con Dri-Dot, a new latex agglritinarion inhibi- submitted to the gynecology laboratory be- tion test, was the most convenient to use, had cause a pregnancy test was clinically indian endpoint that could be detected easily and cated; none were collected specifically for acceptable degree accuracy equal to the an of this study. An aliquot of each was stored Pregnosticon Slide Test from which it was derived. The latter would be our choice as a at -20°C until clinical records had been re- pregnancy test for routine office use. viewed 8-1 2 weeks later. Approximately half of the urine specimens tested were positive for human chorionic gonadotrophin MICfUP;oLoGIC TESTS for human chorionic (HCG). Those selected for study had been submitted I gonadotropin (HCG) are popular and practical pregnancy They are more by 50 pregnant and 50 nonpregnant women. With the following exceptions, they were scnsitive, specific and convenient than bio- consecutive specimens received by the labo- logic tests in general use, before 1960. Since ratory: a) subsequent specimens the little formal training is needed to use the from same patient were not tested, and b) no immunologic test kits now available, many pregnancy tests are performed in physicians' urine specimen was tested if supplementary offices rather than in hospital or clinical clinical infomation or followup data in the medical record were not suffcient to estab- pathology laboratories. This is a report of lish with, reasonable certainty, whether or our evaluation of two new reagent kits designed to provide the greatest possible connot the patient had been pregnant at the time the urine was obtained. The technician performing the tests was not aware of the From the Department of Gynecology and Ob- clinical information. stetrics, Stanford University School of hledicine, Stanford, Calif 94305. Principle Support by a Grant-In Aid from Organoa, of the Tests Inc, West Orange, NJ. All tests for HCG used in this study are Address reprint requests to: Emmet J. Lamb, hl D based on the same principle: inhibition of Submitted for publication Sept 15, 1971. agglutination of indicator particlcs, either Vol. 39 No. 5 May 1672 665

-.. LAMB

sheep red blood cells or polyst3rene latex rial dilutions of urine to prevent the loss o€ particles. Two reagents are used: a) an HCG by adsorption on glassware, Twofold aqueous suspension of red cells or latex par- serial dilutions of the undiluted urine, a ticles the surface of which has been coated 1 :10, 1 : 100 or 1 :500 dilution, were tested. (sensitized) with purified HCG, and b) an In tests with diluted urine, a phosphate anti-HCG serum derived from sensitized buffer, prepared from a concentrated solu- rabbit’s serum. When the two reaeents are tion supplied by the manufacturer, was used mixed, the antiserum reacts with the HCG instead of water to reconstitute the antiserum coated on the particle, causing agglutination. and red cell suspension in the Accusphere When performing the test, if urine contain- tubes. The buffer restores a proper ionic en- ing sufficient HCG is added to the anti- vironment which, in the qualitative tcst, is serum, urinary HCG neutnlizes the anti- provided by the undiluted urine. The last serum, thereby inhibiting agglutination of dilution with clearly discernible agglutina- the indicator particles. At.glutination is not tion was recorded and the titer calculated. blocked by urine from nonpregnant women, since HCG is not present and, therefore, cannot neutralize the antiserum. RESULTS Pregnanf Patients Qunlilative Tests * All five tests for urinary HCG were posi-’ All urines were tested according to the tive in 37 of the SO pregnant patients. Of manufacturer’s recommendation, using each the 13 urine samples for which one or more of the 5 reagent kits listed in Table 1. The of the tests were negative, 4 were from pa- sensitivity of the reagents was verified using tients with threatened or missed abortion serial dilutions of two freeze-dried urine whose excretion of HCG is expected to be samples containing 10,000 and 12,000 In- abnorma!ly low (Table 2). Five other urine ternational Units (XU). All urines were specimens had been obtained within SO days tested for sugar and protein using Combistix of the last menstrual period-ie, at a time reagent strips. when HCG excretion IS normally low. The test results with the remaining four urine Serniquanritdce Titers specimens must be considered false nega- If the qualitative test was positive, a semi- tives. The two new test kits, Pregnosticoi’i quantitative titer was determined with the Dri-Dot and Accusphcres, gave correct posi- Accuspheres reagents. A urine diluent pro- tive rewlts with these and with all other vided by the manufacturer was used for se- urine samples from women with a normal

TAB- 1. SEXSmVlTY OF THE REAGENTSUSED 1N THISSTUDY Semiticity (IUlliter)

Reugenr Manrrfocturer Stated Obstmed htu (slide) Prcquest . Parke-Davis m >m <6OOo Pregnosticon Organon 1OOO-xx)13 >I500 <2500. Dri-Dot Organon low-2Ooo >Its0 <15m n Red blood cell (tub4 UCG Warnpole lo00 >375 <625 Pregnosticon Organon 750 >750 <12M Accusphertf -. / ObSktfiCS Jt?d 666 Gynecology

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IMMUNOLOGIC PREGNANCY TESTS

TABLE2 NEGAT~EI\c\t~OLS1C TEsrs FOR HCG: URWEFROU ~EGNAX~WOVEN Pregnonry tesr

RBC liarex Accuspheres Pregnosiicon UCG (lU/rnl) Prc9ifest slide Dri-Dot Weeks Followup Spontaneous Abortion - - - - - 6 Aborted 2 weeks later; not bleeding a', time of test - - - - - 10 Missed abortion-curetiage - - -8- - 10 Missed abortion-Curettage 3- +(I *a - + + 11 Aborted 3 weeks later; bleeding at time of test Early Pregnancy - - - - - 5 Clinically normal 7 weeks later + +(lo) - + + 6 Clinically normal 8 weeks later I- +(IO) - + + 6 Clinically normal 7 weeks later + +(2.5) - + + 6 Positive Prequebt 2 seeks later + - - f + 6 Positive Prequest 2 weeks later Fake Negative + +(I. 5) - + + 7 Ciinically normal 7-12 weeks later *-- + +(I .5) + - + 7 Clinically normal 7-12 weeks later - +(I 5) -t + + 8 Clinically normal 7-12 weeks later + +(I 5) - + + 18 Clinically normal 7-12 weeks later 5 5 11 5 4 Tmu 10 10 22 10 8 NEGATIVE(A) 1 0 2 1 0 Normal pregnancy after 6 weeks 2 0 4 2 0 NEGATIVE(YC)

pregnancy bcyond 6 weeks. The other rea- thus, less concentrated than a morning urine gents had a 24% false-negative rate. As sample. expected, the least sensitive (Prcquest) had the highest number of negative results. Amorig the pregnant patients were 7 with Nonptegnnnt Puiients abnormal pregnancies. Each of the 4 who All fivc tests were negative for HCG in aborted within 8 weeks of the test had a 39 of the 50 urines obtained from nonpreg- negative result with at least one of the five nant patients. One or more of the rcagcnt tats (Table 2). Each of the 3 women with kits gave a positive test with urine spcci- ectopic pregnancies excreted enough HCG mens from 11 women (Table 3). Most of at the time the urine specimen was obtained these patisnis were taking tranquilizers, cor- to have positive tests with all five reagent ticosteroids or progestational agents. In 9, kits. only one of the test kits gave a positive test Semiquantitative titers were determined which would indicate that these were false- from scrial dilutions of urine using Pregnos- positive tests. Considering the sensitivity of ticon Accuspheres (Figure 1). The back- the slide tests and reported values for pitui- ground shading in the figure depicts the tary gonadotropin, it is not likely that the normal range determined hy Wide using a positive reactions are due to cross reaction hemagglutination test on morning urine spec- with luteinizing hormone. Although it seemed imens. Many of the values in our samplc are unlikely when the rccord was reviewed be- "below this range, probably because most of fore the results of thc pregnnancy tests were the urine specimens were random samples, knowpr an early abortion cannot be abso- Vat. 39 No. 5 May 1972

I LAMB

i f 1 i 1 fig 1. Scmiquontito!ive tilers during the firs! trimester of prcg- nony os datermined from ron- dom urine specimens using Preg noilicon Accurpheres. The shaded arcas represent thm fiducial lim- its of error (P = 0.05) of the immunclogie HCG activity in mornlng uriner from women with normal pregnoncy as determined by Wide. Note log cmle. Open tirclo = abnorniol prcgnancy; N = negative tart, less than IO00 lU/liter.

WEEKS OF PREGNANCY

lutely excluded in the 2 patients who had when the slide containing the suspension of positive tests with more than one reagent kit. reagents and urine is rocked gently and ob- r served unde? a good light against a black DISCUSSION background (Figure 2). We noted no marked - difference in ease of readability among the. Practicality three slide tests used in this study, although A new immunologic pregnancy test must a pale green dye added to the Prequest and be judsed in terms of its use in a physician's Dri-Dot latex reagents aids slightly in de- ofice where only a few tests are done each tecting aggIutination. day, usually one at a time, by sonieone with- Without question, of the five reagent kits out special laboratory skills. The ideal test tested, the Preyosticon Dri-Dot is the most should have a definite end point and be quick convenient to use. The reagents are dried on and easy to perform. The reagent kit should a black cardboard slide and are brought into be self-contained and require a minimum of suspension with a drop of tap or distilled additional glassware, equipment or prepara- water. Only 30 seconds are required to

-- IMMUNOLOGIC PREGNANCY TESTS -.. TABU3. Pam\€ IMWJSOLOGIC TESTS.URIXE FROM NOSPRFGSAXTWosirN

Pregtruncy test RBC Lotex

ACCKUpllrfCs Prcgnoslkon UCG (1Ulrnr) Prewest slide Dri-Dot Drugs Disease +(0.7) - + - Narcotic Proteinuria, pelvic infection - Barbiturate Progestin +(0.7) . - - - None Oligomenorrhea, bled after + progesterone \ Corticosteroid Autoimmune disease @ ii1.S) - - - Diuretic (vasculitis) - Tranquilizer +(1.s) - - - Corticosteroid Menorrhagia, dermatitis +(O. 7) - - - None Menorrhagia, bled after progesterone - - - + Barbiturate Oligomenorrhea. age 48 Chloral hydrate - - - - + Progestin Lactating (post padurn), bled after progesterone - - + Propstin Oligomenorrhea,Proteinuria, bled after progesterone - -. - + - None Post pill amenorrhea - - + - - Progestin Amenorrhea, age 50, Tranquilizer hot flashes - - + - - None Granulosa cell tumor of ovary, age 6 1 5 2 2 3 TOTAL 2 10 4 4 6 POSlTfVE (%) the tube tests requires 1 to 2 hours. Despite serum and one of red cells. These are con- the obvious disadvantage of the longer time tained in a screw-capped round-bottom tube requirement, the overall reliability of the which is used as the reaction test tube, as- tube test makes it preferable whenever a suring that the precisely correct quantity of clinical decision is to be aided by the test antiserum is present in each test. Thc rea- results. The clarity of the end point and the gents are reconstituted

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LAhlB . A - - uc At Prt Prt Dr- .. ti\ I’ I, to sir te, wi 8 .. , dc. ... . of A B C tes Fig 3. Pregnorticon Accuspherer tub. k8t. A. Freezm IfiL dried pellets of ontiserum and of red cclls. The reagents are reconstituted by adding 0.1 ml of urine .- ond 0.4 ml of tap water. 8. Positive lest: in the ab- PO rence of ogglutinotion the cells settle to the bottom Prt as a ring. C. Negative test: agglutination of the cells ni, results in their settling 10 the boftom of the tuk as - a mat within 2 hours. -.a m all .I tes .. Relinbility 1:11 The idcal reagent kit should contain an re : Wil C antiserum capable of detecting the various .. concentrations of HCG present at each stage lo,. , .- n;.i of pregnancy. Its end point should be specific enough so that it is not ‘influenced by as c other urinary proteins, drug metabolites, etc. 03- Since more than 5000 XU of HCGfliter crc are required to give a positive test with Pre- su: quest, this test is often negative early in the Sei’ first trimester or late in the sxond or third ceC trimester of normal pregnancy when the uri- Gcj‘ . a:. nary excretion of HCG may fall below this be level. This lack of sensitivity was evidenced rre in this study by the large number of false- .. of cli- Fig 2. Dri-Dot latex test. A. Dried ontiserum and latex negative tests (Tables 2 and 4). The other particlcr. The reagents are reconstituted with o drop reagent kits, each of which is designed to 0 so:. of top water (latex) and a drcp of urine. On. end 10; the plastic is used io detect levels in the range of 500-2000 XU/ of hollow stirring rod dispense 1 the wine and the other end to mix the reagents. a liter, had far fewer false negatives. The Ac- Positive led: in the obsence of ogglutionation the la- cuspheres and Dri-Dot tests gave correct lo(. tex particles remain in a milky suspension. c. Negative positive tests for all women with normal US. test; granular clumping of ogplutinoted latex por- sen, tides is evident in 2 minuter. pregnancies beyond 6 weeks. A falsenega- -..-1 Vol Obstetrkr and .._. - MZY 670 Gynecology ._ .. _. -.

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I IhfhlUNOLOGIC PREGNANCY TESTS

TABLE 4. kRC€Nr FALSERESULTS able, but some commercial kits, not those Negafice tan Positice test used in this study, are known to vary in Test (pregnunt) (not prcgnunr) stability from lot to lot.2#' The clarity of the UCG 10% 2% end point obtained with thc commercially Accusphercs 10 10 available Accuspheres reagents was superior Request 22 4 to that obtained with the lot reported here, pregnosticon slide 10 4 . Dri-Dot 8 6 which was testcd before the Accuspheres were marketed.

tive pregnancy tcst is usually less distressing SUMMARY AND CONCLUSIONS to the clinician than is a false-positive one, 1. Random urine samples from 50 preg- since, in most instances, he can recheck thc nant women and 50 nonpregnant women test several weeks later, induce progesterone were tested with five reagent kits designed - -withdrawal bleediiig as a pregnancy test, or 2s immunologic tests for pregnancy. The depend on his clinical findings. The number Pregnosticon Dri-Dot and Preposticon Ac- i of false-negative immunologic pregnancy cuspheres, two new kits, were evaluated and tests can be minimized by using a first, morn- compzred with two other slide tests and one t ing specimen. other tube test. A number of factors account for false- 2. Dri-Dot with dried reagents on a card- i positive immunologic pregnancy tests. a) If board slide was the most convenient to use, I present in large amounts, certain nonhor- had an easily detected end point, and an i monal urinary proteins or drug metabolites accuracy equal to that of the Pregnosticon i may interfere with agglutination. Therefore, slide test from which it has been derived. The all urines tested for HCG should also be Dri-Dot would be our choice as a pregnancy tested for protein. b) Errors in interpreting test for routine office use. the end point account for some false-positivc 3. The Prequest slide test is relatively in- reactions. c) Anti-HCG serum will react sensitive and, therefore, gives a high rate of with pituitary gonadotropins. The immuno- false negative results. logic pregnancy test may be positive if uri- 4. The Pregnosticon Accuspheres test kit nary excretion of gonadotropins is increased, has freeze-dried reagents contained in a - as during menopause or with the use of vari- screw-capped tube which is used as the re- ous psychotropic drugs.'~J The immunologic action test tube. This format is quite ccn- cross reaction of LH and HCG permits mea- venient especially if only one or two tests surement of pituitary gonadotropins; using a are to be done. Its sensitivity is such that sensitive antiserum, urine concentration pro- there were no false-negative tesls with urine cedures, or more sensitive detection nieth- from womcn with normal pregnancies after ods (radioimmunoassay), gonadotropins can 6 weeks gestation. be detected at the level excreted by normal men and nonpregnant women. If the results of tbe pregnancy test are in conflict with . REFERENCES clinical signs, the physician usually can re- 1. Bell JL: &rnparalive study of immunologic solve the conflict by using different immuno- tests for pregnancy diagnosis. J Clin Pathol 22~79-83, 1969 iogic tests or other pregnancy test methods. 2. Cabrera HA: A comprehensive evaluation of The consistency of reagents from lot to pregnancy tests. Am J Obstet Gynccol 103: lot is an extremely important factor not eval- 32-38. 1969 3. Hobson BM: An evaluation of immunological uated in this study. Slight variations in the tats for pregnancy. Practitioner 202: 388-392, sensitivity of antiserum are certainly accept- 1969 Vol. 39. No. 5 May 1972 671 , -IC- ..--

LAMB

4. Kcrber IJ, Inclan AP, Fowler EA et al: Irn- pregnancy tests. Am J Obstet Gynecol 105: munologic tests for pregnancy. a comparison. 1222-1225, 1969 Obstet Gynecol 36:3743. 1970 6. Wide L: An irnmunologicd method for the 5. Ravel R, Riekers HG, Goldstein BJ: Effects assay of human chorionic gonadotrophin. of certain psychotropic drugs on immunologic Acta Endocrinol 41: Suppl 70, 1962 . c

Tlie Fourteenth Annual Emil Novak Memorial Gynecological Pathology and En do criiio logy Course (5 Days-October 23-27,1972) CONDUCTED BY Pathology: Drs. J. D. Woodruff, E, R. NOVS~,R. F. Mattingly, J. K. Frost, and H. J. Davis Endocrinology: Drs. G.S. and H. W.Jones, Jr This course represents a complete and up-to-date review of endocrinology, cytogenetics, and cjtopathology in addition to a thorough resume of gynecologiczl pathology. While Ccsigned primarily as a refresher course for American Board applicants, it is appropriate for any gynecologist of pathologist desiring a rapid resume of these dynamic facets of gynecology. A collection of appropriate 35 mm kodachrome slides will be furnished for study prior to the commencsment of the Course. A $250.00 enrollment fee will - - include the slides. No registrants accepted after September 1 or unless accompanied by check. For details write to: EDMUND R. NOVAK, MD, Hopkins Apartments, 3100 St Paul St, Baltimore, Md 21218.

672 Ails tlcn Bchriiigu LrLcn AG, llarburg a. d. 4.ihrr lh. lsol ierung 11n cj Ch a rakte risieru ng des sch wang er - sctiafts-spezif ischen Fl-Glykoproteins crh ilt r Von I lans Ihhn Abspii! In clcr nicnsch!ichcn Plnzcnta uurdc ein Protein aufccfundcn, &IS riorimlcr\vcise in1 Ulut riiclir vorI,r,mint, sich nlm Ici CrnvitlitSt .ini miiitcrlichcn Sciurii imiiiuno- logisch nnchwciscii liilh unt! mit kcincm d-r Imcits I)ck;imtcn scl~.,~nn~crsch;ll’rs- spctifischcn I:nz!.nic odct 1 Iormonc idcntisch ist. ,4uf Grund scincr clcktropliurcti- schen Ilcucglichkcit und scincs I;o~lcnti).dr:!rgchalts wurdc cs nls schwnngcrschafis- spezifischcs sl-G1\koprotcin bcxichnct [ 71. Das sclrwrngcrsclr;iits-spczilischc p,-Glykoprotcin wid uuhrschcinlich in dcr Plazcnta gclildct ;cs crschcinr 1~1dnach Beginn dcr Schw;mg:er.zchaft im ~niittcrlich~,n nlut und Idh sich auch im L‘rin gravider Fraucn ininiunologidi nach\rciscn. Sriti Nachwcis im Scruin hnr. C‘rin kann tur Erkcnnung dcr Sclrw.mjicrxli.ift vcr- wcndct wcrdcn. Xlit iorrschrcitcnder Graviditit nirnnit tlic Konx~iitriti~~iidicscs Glykoprotcins in1 Scruni stctig zu. \Yahrschcinlich bestcht Hhnlich uric hcim huma- ncn Plazcntr-I,aktogcn (Hl’12) und bci geu%scn sctru~nnScrsclrnits-spcziflsclicli ISnzy- men (Histaminnst., Olytocinasc) auch bcim sch\\anScrschnfts-spczirisclrcn P,-GIyLo- protcin cinc Kurrclation zwischcn dcr Zunahinc dcr Kowcntration in1 Scruni unit dcm Waclrstum dcr l’lnzcntn Iuw. dcr lijtop1;izcntarcn Eiiilicit. I-3lic qunntit.ltivc Rcstirnniung dicscs Protcins kiinntc daher evcntucll flir die cbcrwachung der Schwangcrzcliaft yon Ucdciitung werdcn. Im Nachfulgcnclcn ucrdcn dis Isolierung ails der Plnnzcntn sowic die Ligen- scliaiten clicscs sclr\\~nScrsch;rits-spezifischcnI’rotcins ndrcr Ixschricbcn.

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1- 1. Isolicrung Die im methodischen Tcil bcschriehencn Isolicrungsschrittc sind in Tal>.1 nochmnls I~US~ICUIC 1- schcmaiisch daryxtcllt. IXc ’Tabelk enth5lt auncrclcm Angnl~cniillcr und It Rcinhcit dcs schu.rnScrschnits-spctjfischcn P,-Glyiioprotcins in dc11 einzclncn I:ra L t i oncn . 7- Abb. 1 zcigt dic crstc Adtrcnnung an Scphndcx G-150, wohci dcr vortlcrc Pcnk T Xlakroglcibulinc, der wcitc icn wcscntlicl~cnlmmun~lobulinc und @,A-Globulin .5 cnthil~;anschlirllcnd kornmt SP,, gcfolgt van ~,llS-GIyk~prntcinunci nntlcrcn nicrlri~:crmtilckiiI.~r~nPlismn- bzw. 1’1.7zcnu-Protcincn.

. * Lingctr3gcncs \‘i’xcnzcichcn dcr lkhring\vcikc AG.

1015b01 Falll;: ithO\ bci ci Ah:, durci mit d priiai; snlzc:, PUS ti aIgc? einc Ncu: su:r;,:

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Abb. 1 : Gcltilirariun an Scphadcr G-150. Siulc 20 x 100 cni. PulTcr: 0,l hI Tris-I IC],pH 8.0, mit 1 mol/l NaCI. - Bci der pripnrativcn Zoncnelektrophorcsc lassen sich die noch vorhmdcncn Immunglobulir~csowic dns z2 HS-Glykoprotein vom SP, abtrcnncn. Dic nach- folgcndc 1oncn;iustauschchromatopphie an DEAE-Cellulose trclint SP, vom p,h-Globulin. Xach cincr nochmaligcn Aufrrennung an Scph.tdcs G-150 crhilt man cin Produkt mir cincr Rcinhcit von iibcr 95.7;. I%\ imniunologiscli vollkonimcn rcincs, nbcr chcmisch modifizicrtcs Pr?iparat 1'1111 crhilt man nncli Alxinltung dcr Ncurnminsiurc mit Nc1lr;Iminidnsc. I,csitzt SI', >Lt..O' cinrn holicn I;ohlcnh!.drltSch3It und \vircl durch Ncurnminidnscl~chnn(llungwcscnt- c1.t .I Iich stirkcr basisch als die noch in geringcr hlcngc vorhnndcncn llcglcitprotcinc. nii:i, Dis modifizicrtc I'rotcin lilh sich so durch 1lcchromatogr;Iphic an DliAl LScphntlcx A ! rcin isolicrcn. scdii; I3ic I~iillii~i~sci~~nschnftcndcs schwnnscrschnfrs-spc~i~schcn fi,-Glykoprotrins ticrt indcrn sich lxi tlcr .\uhrl~citungmi[ zunchrncntlcr Kcinhcii dcs I'rutluktcs. Sic sind nrniii nnch tlcr Elution von dcr DEAE-Celldosc andcrs nls im rohcn I'hzcntcncxtrnkt bci ( . bzw. in dcr Plazcnta-Fraktion 1'. SP, licgt im Ausgilngsmntcrinl ofTc1cnsichtlich s:~lz- irtig en Garninngiol~u~incgcbunden voc und zcigt dnhcr zunichst dns glcichc = '.

0 I . F.illungs\-crhnltcn wic dicsc, d. h. cs blcibt lei dcr Fraktionicruns niit Ihniino- itlios);icritliii-l.alirlt* zur I Inuptsxhe-ini ubcrstand, wird nbcr mit .‘\th,inoI bcrciis 1x5 cincr 1-ncl~orizc.ritrationdcs hlkohols ton 25:; quantiirativ aiizgcf;illt. Nach LICK AbtrcnniiriK tlcs Scliwangcrschnftsproreins von dcn basischcn Ganiniaglol,iiliiicn durch l~i~.\l~~-~~c~l~~l~~s~~licxcn tlic \‘crhiiltnissc F;crAddc urngckrhrt ; SP, wird ~I;IIIII rnit tlcr i\cridiiil)-isc aus!:ci.illt, abcr nicht nichr durch r\lkohol (nucll nicht Iwi 407,) prizipiticrt. \Y:cnixcr stirk iinclcrt slch das Fillungsvcrhnltcn gcgcnul,cr Ncutral- sali.cn. hlit hinnir)iiiiinisiilfat wird d3s schwnn~crschafts-spc.zifisclicfi,-GI).koprotcin aus dcm I’lnzcntc.n-l;str;ikr Lzw. aus Fraktion V bci 50‘); Siittigung vollstiiiidig abgcjchicdcn. Ihs gcrcinigtc isolierte I’rotcin ‘senijtigt zur quantit;iti\.cn Ausfillung cinc ctwas hiihcrc Silzkonzcntration (607; Sattigung). Ihdurcll I\bspaltung der Neuraminsiure niodiiizicrtr Schwangerschaftsprotein dagescn fillt mit Arnnionium- sulfat crst zwischcn 50 Lis 70‘>; S5ttifiung aus.

2. Physikalisch-chcrnischc Charaktcrisicrung +

Ahb: 2. Elcktrtrphorcsc irn PAA-Gcl bci pH 8.6. SP, + Nil =: SI’, n:rch Ein\virkuiig ron Ncurnrninidasc.

I Abb. 2 zcigt dic Wandcrung des aus dcr Planzenta isolierten Schwangcrschafts- proteins im PAA-Gel ini \‘crglcich mit dcn Plasmnprotcincn. Kiufig ist aukdcr Ijaiiptkonil’onciitc, die sich langsamcr als Transfcrrin bewcgt, noch cinc schw5chcrc Zonc, dic ctwas schncllcr als Transferrin wandcrt, zu schcn. Dicsc schwichcrc Komponcntc prizipiticrt rnit cincm spczifisch Gcgcn SP, gericlitctcn Antiserum und I. ist wahrsclicinlicl~cinc Untcrcinhcit odcr cin Spaltsiuck dcs Schwangerschdts- protcins. ‘1 Nach Alispaltiing dcr Ncuran:insiure mit Ncurarninitlasc wird das Glykoprotcin I in sciner lkwcglichkcit stark vcrindcrt; cs blcibt dann I;lst am Start zuruck (Abb. 2). In dcr Ultrazc~itrifiigczcigeli die Pr5paratc dcs Schwangcrschaftsprotcins cbc~ifalls cinc Aut‘trcnriun:,. in mindcsrcns 2 J

!* schifts-spczilischcn k,-Glykop:otcins scdimcnticrt rnit 4,6 S. I:ur das rnit Ncuramiiii- dasc hclrandcltc GIy Loprotcin wurdc cin Scdirncntationskocffizicnt von 5,6 S cr- . mittclt. . Ahb. 3 zcigt dic Auftrcnnung cincs solchcn Priparatcs. Dic klcincre, Iangsamcr scditncnticrcndc K;rmponcIitc ist vcrniutlich \vic+r dic Untcrcinhcit ; sic scllinicn- ticrt init 3,O S tinct vcrh;ilt sich danii: ihnlich wic dns in 5 A1 Harnstoff gcliistc neur- aminidasrl~cliantlc.ltcGlykoprotcin, das cinhcitlich niit 2,8 S scdinicnticrt. huch Ixi clcr clcl;troph~~rctischcnAuftrcnnung in 1iarnstoflh;iltigcm Polyacr)-lanli+cI

. * Von dcn Fnrbwcrkcn I-Iocchst AG untct dcm \Yarcnzciclrcri RivanoI* in1 Flnndcl.

t " -.--

oeigt SP, nur cine Zone. Aus dem Vcrhnltcn in dcr Ultrazcntrifilgc, Ixi tlcr Gcl- hltr;rtion tirid in1 l';\A-Cicl Idk sich schlickn, ddl rlas scl~win~crsclint'ts-spc~i~schc ~,-Glgtoproiuinciii .\lolcliuInrgcu-iclit von ctwn 120006 bcsitzt und ails zuci oflcn- 3 sichtlich itlcnrisclicn Untcrcinlicitcn xusnnimcngcscizt ist. ... Auf Gruncl tlcr smkcn \'criindcruti,c, tlic das ~ch\\~nnRcrschaftsproteinin dcr clcttropli,,rctischicn \'i'antlcrung mch Alqnltuiig tlcr Ncuraminsitirc crlcidet, war 4 XII crv.~.irrcii.clnl: clcr ~ohlcnh~~dmrgclialtiii dicsciii I'rotcin schr Iioch ist. Ilic l\nnlysc 1wst;itijitc tlicsc Vcrinutring. \Vic tlic Lrgcbnissc in 'fiih. 2 zcigcn, I)ctt;ist die Sumnic dcr K;ohlcnlrydr.itc 38,lYA.

I Idcroscn 10.8:; 5 I Icsosamin (N-acctyl-) 3.3:o Ncurxninsiurr (N-ncrryl-) 6.8'7; 1:ucosc 0.6"b

. a. 2H, lo/; Tab. 2. 6

Dic spczifischc Exrinktion des p,-Schwangerschnftsl~rotcinsist trotz des groOcn ~olilcnh~drat.tnt~il~crstaunlich hoch; der Wcrt fur E;:, bcirug 11,2, Acniesscn in 1/151[ I"hosphatpu:~tr,pH 7,0, bcini hlaximum dcr Ex:inlitioli voii 278 mg.

3. lmmunologischcr Nachwcis 1: Das nus Plnzcnrcn,isolicrtc schwnngerscliafts-spc~i~sclicGlykoprotcin \vandcrt bei An: der Elcktrophoresc auf Acctarfolic und in Ag.tr(osc) wic dic B,-Globulinc, nach tli: Al>spaltung der Scuraniinsiurc alicr uic dic y-Clolniliiic tlcs nicnschlichcn Scrums. tCll Abb. 4 zcist dic tlurch Immunrcaktion sichibar, gcnxictitc Auftrcnnung dcr hcidcn I. Protcinc in Agar und Agarosc. Das Schw~rn~crscli~ftsprorcinirn Sch\vangeren- I SI', scruni watidcrt gcn.iuso wic dns isolicrtc Protein. sc I Ilic Imniiinrcattion nncli huftrcnnung der I'rotcinc im Ph A-Gcl ist in hbb. 5 VCi dnrgcsicllt. Iin ScIi\\.angcrenscru~nwird SI', uahrschcinlich clurch dic hiisischcn

' y-Clol)uline ct\vns zuruckgclialten. Rcim isdicrtcn SI', ist dcurlich vor dcr Haupt- N*i liomponente dns scl:ncllcr windcrndc Spnltstiick (Untcrcinhcit) zu crkciiiicn. d :I

/' . I 297 I

2 r ..

:. L. 3 ,. a

-. .. . ’ -1 ._ r 4 Se+ ND .-

I 5

6 .SP,+ ND -

. i

23 8 t Iaru Ilobn I I

1 I 3. ti I .. 1 I i I I I

I 1

! 1 I

Abb. 5: Aufrrcnnung in1 PAA-Gcl rnit Immrlnrcnkrion. SS = Schulnjic.rcnsrrum. N1) = Rcuraciiirlidasc. A = Anti-SP,.

..... -...... - . ,. :-I i !. .. ;I .*. 1 I * .. ! 1 j .. I

.@ i. i-3. I i .! ' I I .. I I

. t

* I ...... -...... Alh. 6: Gcldiflusionstrst. A = Anti-SI',, . I = Scli\\.alifi'rcns'rum, 2 = Sch\vangcrcllurin (konz.).

Dns hfolckiil in1 llrin hat ofTcnhar rinc 5hnlichc IidunS, al>cr cin klcincrcs hlolcku- Ii~rp\vicht:iIs tlns Sch~,.nn~crschlhsprorrinim Scrum. I Scrcn von trichrigcn 'Ticrcn (1 Itlnd, Rattc) zcigtcn, so wcit sic untcrsucht \vurdcn, in) <;cltliflusionstcst kcincrlci Rc:lltioll rnit dcm htiscrurn gcgcn rlns rncnsclllichc sch\\~ingcrschlfl~-sl,czilischc~!,-C;lykoprotcin. huch cine Ilcilic on rncnschlichcn Scrcn yon Paticntcn rnit dcn vcrschicdenstcn ! I Tumurcn wurdcn in1 GcltlillLsionstest rnit Anri-SP, gcpruit; sic warcn aIle ncptiv. I Es standcn uns alxr lcidcr noch kcine Ltrzinomscrcn von Paticntcn mit tropho- I ..

blastischen Tumorcn fur dicsc Untersuchung zur Vcrfugung. Das Auftrctcn von SP, in1 Serum solchcr l'nticntcn \vice nahclicgcnd und zuglcich ein I [inweis dnfiir, dafl dicscs Schwnngcrschaftsprotcin tatsichlich im Gcwcbc der l'lazcnta gcbildct w i rd. 0 bc r die '1 un nt i tit i vc i ni iiiu no iogi schc Best i m mu ng des sch \v a nSc r sc hn !is -spcz i - fischcn p,-Glykoprotcins im Serum schum-tgercr Fraucn wurdc bcrcits Lcriclitct [I]. Dic Konzcntration irn Scriim zcigte dnbci cine stctigc Zunahnic mit fortschrcircndcr Schwangcrsclxiit his mr Itntl>indung hin. AIS Uczugsscrum wurrlc dimils tin Rctro[,I.izcntarscriim mit iil~crdi~rchscl~r~ittlicl~hohcni SP,-Gchalt aiisgcwililt. hlit Hilfc cincs hoch~crcinigic~iSch\\.angcrschaftsprotcins koniitc jctzt die absolutc hIcnge in diescm Bczugsscrum ermittelt uwdcn. Fur das Uctugsserum crgnb dic Absolu~bcstimniung cincn Gehalt an schunngcrschafts-spczifischcn] pl-Glyko- protein von 33 mg pro 100 ml. Abb. 7 zeigt nochninls den Ansticg dcs p,-Schwanjicr- schafrsprotcins in Ycrschicdcncn Scrcn wiihrend dcr Graviditit, jctzt alxr mit Ab- SOlUt\bTftCll als ()rdinatc dnrgcstcllt.

Schwongtrrcholfswochrn Wochrn noch drr fnrfrndung Abb. 7: Gchilt an SP, irn Scruni schwanqrrer Fraucn.

Wic niis dcr hlhildung crsichtlich, crrcicht der SP,-Gchnlt im Scrrlnl bcrcits zwischcn tlcr 8. und 16. Sch\vangcrschaftswochc I mfb urid licgt im lctztcn Tri- mcstcr zwisclicri 5 irntl 20 mg pro 100 ml. Sich dcr 1:ntbindung vcrschwindct dns sch\\,in~crsch.iI'ts-sp~zilisclic::,-Glykoprotcin inncrhalh von 5 \Vochcn wicdcr voll- stindig ius dcm muricrlichcn I3lut.

Uiologischc Tcstc \Vir vcrmutctcn, dall das schu.angerschnfts-spczifischc @,-GIykoprotcillcvelitucl] mit der von flix und F/OWI[-f] aus mcnschlichco Plizcntcn isolicrtcn und als utcro- trophcs Plazcntnhorrnon bczcichneten Subsinnz identisch sein konntc. In cincm

. 300 JJurtr /h,bn

I-lormonscrccning an Rattcn wurdc gcpruft, ob SP, cine Wirkung nuf den Uterus, Bci d. nuf Oraricn, Hodcn, S.lmcnblnsc, Prost,ita, Nclcnnicrcn, Schil&lrusc odcr Th) mus nuf )lor. \vir die besitzt. Iki \'cru.criduiig dcs hochgcrcinistcn Protciris zcigtc sich jctloch bci cincr J. Srh t5gliclicn lhsicran: voii 0.7 ins SI', pro T:ig und pro I'icr nac!i I:! 'l*n[utter Dcr h abgcstolkn wird. \'crniiitlich spiclcn solche Lohlenli~clrairciclicnI'rotcinc (Xliiko- (IICSOSC protcinc) aucli Ixini Auflxiu dcr soscnanntcn ,,I~il~rinoiclscliicI~t"in tIcr I'Imcntn, Dic .I wclclic dic hlirttcr imniunologisch vom 1:ctcn abschiriiicii soil, cinc lhllc [6,71. sc h n I; st i nini t 1 rng110 Bcsprcchu ng 25 rng; 11

I Die I) Diis sch\\,angcrsclinr:s-srczi~sclicp,-Glgkoproicin ist lnit kcinciii tlcr bislirr 1)ckann- noch ut1 tcn scliwnns:crschat'rJ-spc/.irtsc\lclici~I:.nzymc, Inhibitorcn odcr 1 lurnionc ikntisch I I). Scinc I~iologisclicI:iinktion ist iiocli iinbckannt. In cincm an iritiiit ilcn I~2rtv1iclurchgcfiihrtcn I Iormontcst zcigtc das hoclixcrciciigtc Protcio ucdcr cinc \\'irLiing aiil'dcn Uterus nocli auf Ovnricn, 1 lodcn, Snmcnhlasc, Summa: Prostnta, Ncl)cnriicrcn, Schiltlclriisc odcr Thymus. Zwischcn SP, und tlcm yon Jlmm kind I:/orrs 1.11 in dcr nicnsclilichcn l'l:~tcnta nachgewicscncm iiicrot roplicn I lormon Thc is0 sclicint tlilicr ot1cii:;ichrlich kciii %wunmrnhnng zu Iicstclicn. placcntn, Das scli\\,.in::crsch,iits-spc~iIischc p,-GIykoprotcin I)csitzt cincn ihlilich hohcn womcn, Gclialt an Kohlcnli!tlrnccn wic cl.is humanc Chorion~onntlotro~~in.llic Kolizcn- In the tratioii voii SI', iin Stmni schwnngcrcr Fra~ciixist nlxr cincn antlcrcn llonni tlcr Scli\\.;rnscrscli.~~c,wvihrctid dic I

. prscliai[swoclic hcrcits 1 niCS/I 00 ml i:nd licgi in den Ictztcn 1)cidc.n Ncinnicn tlcr (9,90,3 I Scli\\,nri~crscli.litz\&rhcn 10 und 25 rii~/100nil. In 3~ciro~~I.iz~iitarscren\vurcicii arno u n !5 \Vcrtc his 211 33 nix,'[OO nil gcnicssrn. IXc Konzcntrnrion yon SI', iibcrrrifl't damit The r dic allcr atltlcrcn Imticr Iwl:inntcn schu.im~crschnfis-spczi~sclicnProtcine in1 I3l~1t. protc in AIS i3iIchiii~~tirtIiir Sl', ist in Aiinl~Jgicxu anckrcn Scliu~nli~crsclinIi'Ispr~iici~icninit incrcnsc ilmlirhriii Kiirvciivcrlauf w;rlirsclicinlich dns Synzytiuin tlcr nicnsclilichcn l'lnxcnta \\,CCJi 0:' anzunclinicn. lk\vcisc clafiir stchcn noch aus. rite

.- / I **

2u sa n1 nic n fn s s u n g

13cschricbcn wird dic Isolicriing und physi~alisch-chcniischcChnraktcrisicrung cincs liohlcnltydratrcichcii Protrins nus dcr menschlichcn Plnzcnta, das such im Scrum schwangcrcr Fraucn vorkonimt und als schunnjicrschnfts-spc~i~schcsp,-Glyko- protcin bczeichnct uurdc. Dicscs Schwangcrschnfts~roteinsedimcntiert in der Ultrazcntrifilgc mit 4,G S bzw. nich :\bsp.rlttlng dcr Ncuratninsiurc mit 5,G S. Es bcsitzt cin A[olcl;ulargcwicht von etLi.3 130000. In 5x1 Ilarnstoffzcrfillt cs in zwei offcnsichtlich idcntischc Untcr- einhcitcii, die mit 2,s S scdinicnticren. 'lh Kohlcnhydratgchali dcs Clykoprotcins ist schr hoch und bctragt 29,1% (Hcsoscn lO,SyL, Mcsosaniin 9,976, Ncurnminsiurc G,81;, Fucose 0,G;h). Die ilhsolut\vcrtc dcr Konzcntration des l'rotcins wurdcn in mchrrrcli Scren schnmgcicr Frnucn quantitativ nut der cinfachcn radialcn Ininiundilfusion bc- stimnit. Sic licgcn in dcr 8. his 12. Scli\~~angcrschnftsu~odicim lhrchschnitt bci 1 rng/100 nil und errcichcn nin Ende der Schunngcrschaft \\'crtc z\vischcn 10 bis 25 mg/100 ml. Dic hiologischc Funktion dcs schwangcrscliafts-spczifrschcn pl-CIylioprotcins ist I noch unbckannt.

- .. Summary

The isolation and characterization of P carbohydraic-rich protcin froln human placentas is dcscribcd. This protcin which was found to occur in scrs from prcgnnnt womcn, too, h:is bccn tcrmcd pregnancy-specific fi,-giycoprotcin. In thc ultrazcntrifugc for thc pregnancy protcin a scdimcntntion constant of 4,6 S was dctcrniincd. Its nio~culsr~~~~~~.~_tol~~O~.i\ftcr trcitmcnt with ncuraminithc thc molcculcs scdinicnt with 5,G S. When tlissol\.ed in 511 iirra thcy .dissoci;itc into subunits \vhicIi havc a scclinicntation conJtant of 233 s. . Thc pccgnancy prnicin \\'ns found to contain hcsoscs (10,8yo), hcsosiimincs (9,90,1),sialic acid (cj,X:.h) and fucosc (O,GO/,); thc totid cnrlxd1ydr;itc contrnt thus amounts to 2R,17b. 302

. ..

15b I b Carv D. I!ody.en and Criff T. ROSS with the technical assistancz o€ C. K. Tu ner, 3. 1,. Barber, and A. If. O'COnnOT. Reproduction Yesearch Branch, Kational Institute of Child llealth and llunan 3evelopnent, '!aticnal Institutes of llealth, Bethestla, 'Iaryland 20014.

Q @T!?~CT. An antiserum to the Bsubunit of ovine luteinizino hornone has heen used to develop a sirple, rapid, and specific hema?nlutination inhihition test for urinary macaque chorionic RonadotroDin (3CS). Reliable diap,nosis of 20 qregnancies were made usinp. neat urine ssecinens collected on all days betveen the 18th and 23rd dav after fertilization. Onlv 0.2 ml of urine were needed for the test vhich required 2 hours to connlete, llhen urinar.7 concentrates were tested, diamostic results were ohtained on the 16th day of premancy. ?he rate of "false positive'' tests was less than 12.

lle have produced an antiserum prinary imunization, a total dose of . (A/S) which reacts tilth antigenic 10 pp, of the inmunoc.en susnended in deterninants comon to chorionic 2.5 nl of an emulsion consistinr: OE r,onadotropins of hunans, gorillas, 0.15 ?! saline, arlacel A (Hilltop oranEutans, chirinanzees, baboons and Research, :!ianiville, Ohio) and nacaquas, but not to follicle marcol 52 (Hunhle Oil and Aefininr! stimulating hornone (FSI!) and Co.) in the proportions of 1 to 1 to luteinitine hornone (LII) of the 3 was qiven at multiple intradernal latter tve species. This h/S has sites. been used to develop a simple, For 35 consecutive vee1:s after inexpensive, rapid and specific the booster iniection,' the h/S henaqglutination Inhibition test for harvested remained soecif ic and urinary nCC. Only 9.2 ml of neat produced anclutination at a naxinal urine (or an equivalent amount of dilution of 1:3009 under the assay urine extract) was required for conditions described below, testing and results vere 2. Anticen and diluents. Apart interpretable ti:o hours after the from the AIS and the reFerence renccnnts had been nixed. preparation, all other reaxents were obtained from nrtho Qiaenostics 'UTERIALS A!rn 'ETlOnS !ksearcli Foundation. These consisted of sheep red blood cells to which . A. Reazents: 1. AIS. The AIS hunan chorionic Eonadotrooin had teen was 1,arvested fron 1 of 5 rabbits attached hv a modification of the (11-26) each Riven 150 j~gof an ovine nethod of Onkelinx et al. (2), a . luteinizinp, hornone B-subuni t 0.025 'I phosphate buffer solution (pR preparation (3HE.I 8-161 generously 7.3). used to produce susaensions of provided by Dr. '3. N. Ward, Y. D. these cells, and a bulfered AlS Anderson liospital and Tumor diluent containinc. ?rotein. Institute, l!ouston, Texas) at 3. Urine specincns, All urine multiple intrademal sites (1). The was collected daily or intermittently imunop,en had been nixed with from cyclinR c.=tp.ed female rhesus conplete Freund's Adjuvant (Difco) to monkeys (Yacaca mulatta), fron which an additional 5 ny, of heat prey.nant nonkeys heforc, durinE and killed, desiccated nycohacterium after biolocicallv active chorionic - tuberculosis were added. At a Ronadotropin could he detected in separate subcutaneous site, 0.5 ml of / urine or serum, and fro3 castrated, pertussis vaccine was riven on the juvenile or hypophysectonized same day. On the 140th dav after the monkeys. Urine specimens were 927 -..

I O-15b I1 T collected three wavs: 1) rinc. annrOXlnatel*? 5 nn in dfaneter cantinuously for 24 hr periods wkere \*en a similar rinn forned in hot?, the voided urine innedtatelv drained the absence and t!ic presence of .\is into pl.a".tfc bottles packed Fn drv the test wau recorded as ''poSitiVc?-. 2. 11 test%, ice, where it Froze 2) analo?ous 24 Inconclusive" cor hr specincns collected at Toon reasons prescntlv unknown, riny temperature (26C) and 3) sinzle fornation failed to occur in voidinqs collected on sheets OF approximatclv 8:: of the urine nlrminun foil and imediatelv specinens tested without A/S and recovered bv aspiration with these teAts were re?arded as disposable pipets. "inconclusive", Qihtion oE these 'kat urine and urine extracts' snecimens 1:3 (V/'J) with AlS diluent prepared either by a nodification (3) resulted in rinc? formation vhen all of the kaolin-acetone procedure of such sandcs were retested tJlthout Alhert (4) or by the acetone A/ so precipitation nethod of Prank and 3. "*!egative" tents, A test Salmon (5) were tested. All was interpreted as "neffatlve" vhen specinens were centrifuqed at 1000 X cells formed a rinr! nattern In the 9 for 10 minutes and onlv the absence, but a diffuse, natted supernatant Was used for testinr?. pattern in the presence of AlS, Kaolin acetone extracts of n. Thin? fertilization and pooled nregnant rhesus monkey urine diagnosing premancv, For tinine with a bioloqical specific activity events in cvcliny: female rhesirs of 5 IU/nr, (rat uterine welqht assay, nonkeys, the onset of nenstruation, Second International Standard hCG) detected hv dailv vazinal smears, were used to orepare an adti aCC counted as day 1 of the menstrual reference preparation. A ninimal cycle. On the morninn; of the 11th effective dose of this ?reparation day of the cycle, feales to be nateci was included as a positive control in were cafied with males for 43 hr and all assays. removed earlv In the 13th dav. B. Conduct of assays. Detection of coamlated semen in the nisposable Rlass culture tubes vaplna indicated that inseminations (Scienti€ic Products), 12 m by 75 had occured. Since ovulation occurs nm, were placed in a rack over a most freauentlp on the 13th day of mirror so that bottoms of tubes could the menstrual cycle of rhesus be seen without disturbing the monkeys, we have arhitrarilv contents. The volume of mixed desiltnated day 13 as the day of reactants vas 0.5 nl and contained fertilization. 0.2 nl of either a 1:2400 dilution of To provide independent evidence the A/S or AlS diluent, 0.1 nl of of prer,nancv, serum collected on the sample and 0.2 ril of a suspension of 18th day, and concentrates of 24 hour hCP, coated sheep red blood cells. urine specimens collected on the 2Oce After shaking to thoroughly mix day of pregnancv (vide supra) were reap.ents, tubes were left undisturbed bioassayed for mCC (6). at anbient temperature (22 to 25C) for 2 hr. RESULTS C. Interpretation of results, Test results were read as either Ilemacrlutinction failed to occdr "posit ivc" , 'I inconclusive" , o t at hlS dilutions in excess of 1:3000, "negative" according to the following To allow sme marp;in for variation criteria. from lot to lot of hCG-coated 1. "Positive" teats. In erythrocytes and buffer, a 1:2400 control tuhes, lackiny, h/S I dilution of the AIS was chosen for henaffElutination did not occur and esaays. the red blood cells settled out in a Specimen6 known to contain high

/ Table 1. Hemagglutination tests on urine sauples collected from female rhesus aonkeys.

Nonpregnant Pregnant

Days after Onset of Days after Fertilization Others** Menses ---20 -----<12 12-17 18-23 24-30 >30 * Number 100 80 91 94 42 34 22 17 31 Positive 0 0 1* 1* 13 31 8 0 0 Negative 100 80 90 93 29 0 14 17 31 Initially Inconclusivet 8 6 7 6 4 3 .2 2 '. 2

t All "inconclusive"resu1ts were resolved by fetes ting after diluting - 1:3 with A/S diluent so that final results were either "positive" or "negative" for all specimens tested.

* Interpreted as "false positives".

8 ,/ - ** Specinens collected randomly from castrated, hypophysectonized and juvenile animals. A

concentratlons of rS11 and L!1 were *'inconclusive" tests were encountered testccl for cross-reactivitv of t,/S in this series. with pirultary ronadotronlns. To arrive at some estinate of . Specinens fron both unnated cycliny: the day of nreqnancy on which'neat nonkegs and fron preynant fenales 6 urine niqht first cive '*positive" weeks after bioloricallp active teats, specinens collected seriallv yonadntropin vas no lonqer measurable from 4 rhesus monkevs that conceived in urine were tested to deternine were tested, The earliest "positive" susceptibility oc the test to urinary tent vas found In a spechen nubst anccs 3iv in I: '*fa 1s e posit ive" collected on dav 14 after results. Test results, classified as f ertilizat ion . A11 specinens "positive" , "inconclusivcO, or collected fron davs 18-23 after "neqative", osinn the criteria fertilization were 7osit ive for all described, are sumarized in Table 1. monkevs and fron days 13-28 in one of It can be seen that fever than 1;: these. This reflects the knocm . "false positive'' reactions were variabilit:? in urinarv nCC excretion obtained in assavs of 511 specimens. fron aninal to animal (6-8) rather False Dositive tests were never than unreliabilit:? of the test, observed in opecinens collected fron Becau.se the peal: period of mCC the sane aninal on two consecutive excretion is so brief, this - davs. diaEnostic teat becones prooressively hllquots of a total of 36 less definitive when opolied to neat specinens vere te.sted with and urine specinens collected either without acetone precipitation of prior to day 18 or after day 23 *urine. There were two apparent Po 11owing f e r t i 1i z ation, The t e s t advan tapes to t hi 9 orel ininar v uay not be reliable when exanining precipitation: 1) pregnancy vias . neat urine snecinens collected before diacnosed about 48 hr earller than in or after the reconnended period tests usinE neat urine; and 2) no because of the Incidence of false /

-.- 930 RA PI D CO11 h I US[CATIONS JCE h V 1974 \'ol 34 .so 5 ne-,.itivcn crhcn nC': lc\*els arc lor. cnllcctlon and the delrrta in urine 71c period of rcliahilitv can ha sncc irlens. cxtvndcd two d,-ws or more ::lien urine Ice believe collearue.: yvill find conc e 11 t r 3 t e s il r e t e s t e d . the ans.iv .iseful in research r;hcrc 1\11 accurate clIaccmis of dtacnoainr. or nnnitnrinr the course prc.rnnuc!r '-3s -dc b-: tests 0' urinc oc earlv nre-nancp fn mcanucs and specinens fro- tr-o b;l!won!i, trro hntwxi.; io irpnrtant. .lccordinpl**, clitnpanzccs , one t-orflla and one *?e have prepared 3 ~oolor 4f)q -1 of ormytAn. the tindjluted antiserun, ?\e renainder of the rcar.ent s have seen purc!iased and kits contatninc! a quantity sufficient for Sn dtaclnostic Traditional ncthnds for tests have- been prenared under the dia9,noainc preynanc.. in nacarlues and aeqis of t5e Contraceptive balmxvi depend upon either rectal Development nranch of the Center for nalpation' or IJ~O~SS~\*Sof aerun or nopii 1at ion Research , '!Tat I anal concentrated urinar-r extracts. Thc Institute of Child I!ealth and Yunan tine iAicli nust elapse nost-coitalll- i~C\rclopl.rent. ?:its can be obtained by beCorc reliahle rcsult.: can be writine to The !!ornone 3ist;ibution o!rtaincd linits usefulness of both Officer, nffice of the Director, tests For sone nurposec,. 3iay.nosis :!1.":DD, Ruildin? 31A, ?.oon 9.\47, of prevnancy ir.tt!i hioloyical assa-rs Xatlonal Insti tutes of Iicalth, for -.onadotro?tns in urine or scrurl !kthesda, 'id. 20014). frm nated rhesus oon1:el.s hecones reliahlc about t!ie 2'3th day and :'.)-.7r?xIms djawosis by rectal palnation about 1, Vaitukaitis, .I, l.,, .1, R. the 3Qth dw acter fertilization. . RobEins, E, Xeschlar an1 7, 3ioasoays are cnstl-r, require a T. ?.ow, .I Clin Xndocr !letah ninimum of 72 hours to caplete, and 33: 1941, 1?71. suitable anindr; are not alwavs 2, hkelinx, E,, I!, 'leuldernans, ?I, available , Joniau and 2, Lontie, I-r-unolo?v In contrast to these traditional 16: 35, 1969, tests far preqnancy, thc nethod 3. ?lixon, U, re,$. 0, i?odgen, 11, i!, described here has heen shown to !liemann, C, T, ?os.; and 11, -7, provide a dcpendahle diarnosis by t!ie Tullner, EndocrinoloTv 30: 11q5, 18th Jay after fcrtil?zation vhen 1973. . neat urine vag tested or on dav 16 4. Albert, A,, %cent ?ron,r '!ornone when 5 .'fold concentrates CEKe tested. Zes 12: 227, 1956. !ksults viere obtained less than 3 hr 5, rrank, 2; and ?.. Salqon, Proc after the spccinen had been Sac -Ex? Diol Xed 32: 1666, 1935, collccted, Each of the urine 6. Tullner, !'. '), and T!., l!ertz, collection procedures used here was Endocrinoloqy 78:204, 1%6. satisfactory, l!nr.rever, the 7. Arslan, i!., R, K, 'lever and collectf.on of aliqunts of freshlv R. C,' Yolf, Proc SOC Exp Aiol voided urine vas nost satisfactorv !!ed 125: 349, 1967, because it nininized hoch the effort (I. Tullner, 11, I?, , Lndocrinolozv and time invested In sanple 82: 874, 1969...... - .- . , Evaluation of four slide test kits for the detection of human chorionic gonadotropin in urine

Michael Dietrich, M.D., F.R.c.P.[c] arid J. A. French, F.I.M.L.T., L.C.S.L.T., IVindsor, Onf.

Summary: Three "indirect-type" I'bpreuve directe dtait la plus prbcise. can be further divided into two types slide tests utilizing the principle of Avec ce dernier test, il importe - indirect and direct. hemagglutination inhibition and one d'exercer un contr6le pour diceler The indirect type of slide test uses new "direct-Pjpe" slide test employing des substances susreptibles the same principle as the tube test 21- direct agglutination were evaluated d'interference et. le cas Bchbant. ready described except that lates par- for the:r sensitivity in detecting recourir B un autre test. Tous ces ticlc, coated with HCG pie substitutcd hwnan chorionic sonadotrcpln (HCG) types d'bpreuves posddaient for the coated erythrocytes. in urine. The results of paitive tests waimmt la sensibiliti rehtive The interpretation of resulu for both in a group of woman in vwy early mentionnbe par le fabricant. indirect-type tests is expressed in the pregnancy were correlated with following statements: the "days ahlast menses". In this 0 No agglutination of coatcd erythro- swics the direct slide test was The presence of human chorimi: gona- cytes = HCG present. tho most accuiate. A control must dotropin (HCG) in serum and urine 0 Agglutination of coated tvthrocytes be use6 with each direct test to of a woman is gcnerdly interpreted as = no HCG present. indicate interfering substances and an indication of pregnancy. As is well ?he direct type of slide test uses when such are present a different test known, it is also found in the urine latex particles coated with anti-HCG. mst be used. All tests were found in certain other conditions such as Urine ssmples containing HCG will to be of the relative send:iv;ty stated chorixarcinoma and hydatidiform agglutinate the latex particles, and those by the manufacturer. mole. There are two types of immuno- samples with no HCG present will logic test availabie to dctenoine the leave the particles unagglutinated. Rbsumk €/ahation de qiratre Cpreuves presence of HCG in wine - tube Tbe interpretation of rcsults for sur lame pour la dPtection de la tests and slide tests. direct-type tests is exprejsed a3 follows: gonadotr>phine chorioniqve humdine The tube test uses the hemagzlutina- 0 Agglutination of coated latex par- dons rurine tion-inhibition assay method first in- ticles = HCG present. troduced by Wide and Genmll in 0 KO agglutinition of coated latex Nous avons Bvalub la sensibilith 1960.' The principle of this method is particles = no HCG present. de quatre Bpreuves sur lame quznt as follows. \Vhen HCG antiserum is As the nianufacturers' instructions RC- B IS dktection de la gonadotmphine mixed with cnthrocytes coafrd with companying the tcst kits indicate, tub$ . chorionque humaine (GCH) dims HCG, agglutins!ion of these ce!is takes tests for HCG are more sensitive.and I'wine. Trois de ces Cpreuves sur la place. If, however, 3 urine sample more accurate than slide tests. Tube lame (du "type indirect") utilisaien! containing HCG (e.g. onz from a pwg- tests in gcneral require a two-hour in- le principe de I'inhibition de nant woman) is first added to the anti- cubation period hefore a result is avail- I'h8magglutination et ulie nouvelle serum neutrali7ation of the antiserum able tvhile slide tests require only one Cprcuvo sur lame (du "type direct") occurs, and when the coated erythro- lo two minutes. Conscquently, labor.,- ernployait I'agglvtination directe. cytes are added there is inhibition of tories thnt perform tests for HCG are Nous avons conilationnb les rBsultats agglutination. If the urine snmplc con- faccd wirh a choice between speed and des eprcuves positives ct IPS "jours tains no HCG, neutralization of the increased accuracy and sensitivity. aprbs Ics dernikres mens:rues" chez antiserum does not take place and the Some use both types, a slide test for un groups de femmes tout au dBbut coated erythrocytes. when added, ag- "stat" samples and a tube test for roil- de leur grossesse. Dans cette sdrie, glutinate. Characteristic settling pat- tine smiplcs. If a slide test of com- terns occur Wii5 &e at the bottom of parable accuracy could De found the the tube and retlect whether agglutina- tube test could be eliminated. From the Departnient of Pzth~lopvmd Combincd Laboratories. Crs~rOZJ Windror tion has or has not occurrcd. This eyaluation was performed to de- M'c,lern Hospild Centre. NinGlor. Ont. Slide tests, or latex paticle tests, termine the.accuracy of fow slide tests Reptin1 requests to: Dr. hlik-hie! Dictrirh. were first dcscribed hy Robbins et (11 for HCG - Brevindexa (Ortho Phar- Stratford Ckiicral Hospital, 5:raIhrd. Ont. MAIY6 in 1962.' These tests for HCG in urine maceu tical [Canada] Ltd.). PregnosisP ChlA JOURNAL/AUGUST 3, 1974/VOL 111 1045b2 I 235 ll4~1Cl~~'L., I'c' Gb- 11,; :c.-. SIIJL' (Ihn\c.i L.i>t~i~iiuil~~,Ldii..~. 1 ;

< TC>U~:> L>~~~I!>CJ Id IO ..: Ltd.) and D.ip (mncro)9 (Dcn\rr). \vith all the test kits and thc results sensitivities of the test kits used. l3-1~ Urevindcx, l'rcposis and UCG-Side of furtlicr investigations pcrfornicd on sensitivities reported in the manufac- ace indircct-typr: t~',t> .IIIJ DJi> (macro) thcsr: samples arc listcd in l'ablc 11. turcrs' instructions inserts arc' given in is a dircct-typc tcd 1\11 thc test kits l'he 16 siinrples werc divided into six Table 111. for HCG uscd in this survey were groups according to thcir reactions The sensitivities of all the test kits nrdc available by tlic' nianufacturcrs. ~iththe test kits. used arc relatively close with the exception of Brevindex, so some othcr Grorrp 1 - spt-cirtient 1. 17, 84, 164, factor must be involved. The niethcd- Methods 307 and 372 ology for indirect-type slide tests for The urinc saniplcn were all rcceivtd These specimens were shown to con- HCG requires the addition of a $!and- at the lnborarories lor prcgnniicy test- tain HCG only by Dnp (macro). Only ard amount. depending on the sensiti- ing. Thc sources were prcsur~icalpa- one specinien (no. 372) contained pro- vity of the tcst tit, of anti-HCG. This tients, outpatients and cniergcncy ad- tein and blood, so the failure of the is neutralized by HCG present in the nrissions, e.g. for thrcatcncd abortion. other test kits to detect the HCG If testing was not perfornird the s3me present in these spccimens must be due Table Ill-Sensitivities of the test kits day the spccinien \\'as received, the to some othcr cause. The spccific as reported in the manufacturers' sample was stored at -2O'C with known gravity of all thesc specimens \vas also instructions inserts positive and negative control speci- normal, excluding 'insufficient concrn- Brevindex 3.5 IU/ml nicns to ensure that storage did not tration as a cause. The presence of affect thc results. In 129 cases the DarvonB. in Specimen 1 was not im- - Pregnosis 1.5-2.5 IUjml samples were clinically confirmed as portant; Lang' has shown that Darvon Dap (macro) 2.0 IU/mi containing HCG and in 271 cases as does not interfere with the accuracy containing no IiCG. of slide tests for HCG. The variations Each sample of urine was testcd with each of the kits, following the Table I-Percentage accuracy of the fow pregnancy test kits manufacturers' instructions. The results were related to the clinical evaluation Total Brevindex Pregnosis Dap (mauo) UCG-Slide of the patient including, if possiblc, Clinically no. confirmed of No. No. No. No. thc date of the last menses. Any 5ani D I es samDles detected R detected % detected % detected % saniplcs that showed disagreement in the results of any of the kits were HCG-positive 129 115 89.1 121 93.8 127 992. 115 91.5 further tested by determining specific HCG-negative 271 270 99.6 271 lG0 271 100 271 1W gravity using a hydronietcr and the 'Calculrled cn the basis of 128 samples; one test sample was excluded becausejts control sample g2ve a presence of protein or blood using positive reactton, iiidicating the presence of interfering substances, and invalidating theresuits. Bili-labstixg (Ames CO.). Because we believe that accuracy Table Il-Results of further investigations performed on 16 urine samples giving should be the most important con- variable results with the test kits or with the clinical evaluation sideration in routinely using a test kit - for HCG, the following conqiderations Drys were disregarded: (a) ease of perform- after Specimen Specific last ancc of the test, (b) readability of no. Results of tests gravily Protein Btood menses Medioticn the reaction, and (c) price of the kit. Regarding case of performance, the - 1 Positive by Dap (macro) only 1.015 negative negative 29 Duvon pap (macro) test kit requires filtration 17 Positive bv Dao (macro) onlv 1.017 netzalive neaative 31 none v.ad an internal nicthcd control must 84 Positive by Dap (macro) only 1.010 negative negative 31 none be set up with each urine sample tested ~ to rule out the presence of interfering 164 Positive by Dap (macro) only 1.014 negative negztive 31 none substances. In our e\pcriznce these 307 Positive by Dap (micro) only 1.014 negative negahve 32 none steps take about 1% minutes extra Positive by Oap (rna:ro) only moderzte none time. . 372 1.011 + 31 111 Positive by Dap (macro) and Proposis only 1.012 negative negative 33 none Rest ills 243 Positive by Dap (micro) and Prignosis orlly 1.011 negative negative 34 none The percentage accuracy of the 35 Negative by Brevindex only 1.012 negative negatit,e 42 none HCG test kits is shown in Table I. Negative by Brevindex only negative negalive NA NA The figures agree quite closely with 67 1.013 those for Gravindex' and Pregnosis 72 Negttive by Erevindex only 1.012 negative negative 42 none obtained by Driscoll et UP but arc 201 Negative by Brevindex only 1.015 negative negative 41 none considerably lower than the figures for UCG-Slide obtained by Goetschel.' 64 Negative by UCG-Slideonly 1.014 + large 39 none amount Dap (macro) is a new product, so no othcr percentage accuracy figures are 292 Negative by UCG-Slide only 1.014 + large 39 none available. amount For 16 urine samples there were dis- 357 Posilive by Brevindex only 1.010 ++ small NP none agreements either between one or more / imount 91 ?milive specimen not picked up by any test 1.011 negative negative NA none mcthod *Gravindcx and Brcvindcx are lhc same prCduct. B=vin&x king the Dame uud for marketing NA not ptrporcr in Cmnada. NP = not pregnant I 236 CMA JOURNAL/AUGUST 3. 1974VOL. 111 tOlSb22 i aborlion I hrrs hours beforc curettage was performed. The surgical specinicn showed dccitlual tissue only. .-regnancy iherc may not be enollgh iiCG present in the urine saniple to ficiently h;gh to be detected by Brcw Coiiclusions xutra'ire all of the anti-HCG added, index until 41 days after the last In this survey the Dap (macro) was ~3 thc amount reniainillg agglutinates nienscs. The intervals since the last the most accurate of the HCG test IIC coated latex particles, indicating menses of three of these four patien!s are all close to 41 days (not availablc kits, followed in order of accuracy, by '0 tlic observer that no HCG is present Pregnosis, UCG-Slide and Brcvindex. .II that saniple. for no. 67). In direct-typc slik tests no neutral- Thc direct-type Dap (macro) test re- Crortp 4 - specinicns 64 and 692 quires filtration of the urine and use Edrioli of anti-HCG is required, so thc .~bove problem docs not arisc. This These specinlens showed absence of of a control to detect interfcring sub- :odd explain why these six specimens HCG by LJCG-Slide only. Both speci- stances, and when such substances are cactcd only with Dap (macro), the mens contained a large amount of present a different type of test must Tnly dircct-t)pe test of the four kits. blood and it has been thc authors' be used. In this series, in only 1 of All were cases of early pregnancy ac- experience that in the UCG-Slide test 400 tests was an interfering substance cording to "days after last menses". agglutination of the latex particles oc- dctec!ed by the Dap (macro). The c curs in the presence of blood, giving four tests werc found to possess the Croup 2 - specinlais I I1 nnii 243 a false-negative result. llie interference relative sensitivity claimed by the m3- These specimens showed the pres- niay not be caused by the blood alone nufacturer. vice of HCG by Dap (macro) and but by some product formed by interaction of blood with other constituents 'regnosis. No protein or blood was References Jrtected and the specific gravity of of the urine.' -0th was normal. The problrni is prob- 1. WinE L, GEMZUL CA: An iminunolopical Group 5 - specimen 357 cgnoncy test. Acla Enduninol (Kbh) 35: lbly thc same as that already dis- F61. 1960 This specimen, which showed the 2. RORBISS JL HILL GA. CAUE RN. ef P: :used for Group 1. Because Preynosis L.rex r$glul/n&:'on reactions bctwten human ias greater sensitivity and therefore presence of HCG by Brevindex only, chorisnit pnnrtotropin and rabbit anhbody. Proc SOC Exp Bid Mrd 109: 321 lYbl equires the addition of less anti-HCG was from a clinically confirmed non- 3. DRISCOLLSG. STPIUSS WF. ALm'M. et .I: pregnant woman. The specimen con- Evaluation of a ticw 5liJe test for preg- han the other indirect-type tests used, nancy. Am I Obrrcr Cgnrcol 110: IO'i3. 3971 tained a sniall amount of blood and 4. knver hborvtories: Personal communi-- :ailurc of agglutination as described ,;An br Group I wil! bc observed at B 2+ protein. The instructions insert of 5. KEG L: A rapid immunologicnl test for Brevindex states that interference by regnancy. Ani I Mrd Tdtnol 30: 345. 1964 'owet urinary level of HCG with Preg- 6. 6uisEY TF. SPIERSbf: Protein intcrfcrcna protein is unlikely. However, there in a lalex rkdc prcrnurcy test. Cm Mcd -,osis. Thc "days since last menses" of ASSOCJ 102: 97.5, 19iO hese two patients, again, show them appears to be no other cause for the o be cases of relatively early preg- false-negative results than protein inter- lancy. Thc datc on which a sample ference. s obtained niay be significant; for Group 6 - specimen 97 rxample, the level of HCG in thc urine This, specimen, which was negative luring pregnancy increases rapidly be- P ween the 30th and 40th days since for HCG by all four test kits, was he last menses (Fig. 1). shown by quantitationt to contain 1000 IU HCGIlitrc of urine. This is simply Group 3 - specinlens 35, 67, 72 and too low a level of HCG to be detected 20 I by any of the test kits used. The speci- These specir.iens showed absence of men was taken from a patient con- XGby Brev.ndes only. No protein or sidered to be a case of incomplete dood was present and the specific .iavities were normal. The reason for lUCG3 lube test, Dcnvcr Loboratone$ (Canada) he false-negative results obtained is Ltd.

HCG 1 u/1 00000 0 so000 f

50000 25000 10000 5000 2500 1000 so0 p-=-=- 250 100 /

DAYS ArTER LAST MENSTRUAL PERIOD 3G. I-The level cf HCG in the urine of pregnant women at various times fler 1861 menstrual period (after Widc'). 1015b23 CMA JOURNAL/AUGUSI' 3. 1974/VOL. Ill 237 J. ch.Parh. (1969), 22, 79-83

Comparative study of iiniiiuiiological tests

for pregnancy diagnosis z.

JOYCE L. BELL ... _, .. . ..

.. .. - -...... I -:. . )psis The reliability of five conlniercially produced ~mrnunologicnl pregnancy diagnosis '1. .: . .. I& has been investigated. Thc tests used wrc Pregnostkon (a tube test) and four slide tests, _- .. . .. Illd, Pregslide, Gravindex, and Plnnotest. In the series described, Planotest gave 0.5 :/, false . .. lives, Graviiidex had 2.1 %, Pregnostkon 2.6%; and Presslide 4-7 x, Hyland A (sensitivity _. .j iu/].) gave 3.6 and Hyland R (sensitivity 2 10 3,000ia ].)had S-7 76 bise positives. Pregnosticon 0.5% false negatives, Planotest 2-0x, Gravindex 3-5%, Hyland B 6-5%, Pregslide 9 %, and .. ;nd A 19-59; false negatives. .. ,lnoiest. and Preposticon wre found to be less influenced by protein and blood in the urine i the otller pregnancy tests investigated. ..

...... J&I many papers have been written on indi- diagnosis tests: Pregnosticon' (a haemoagglutination a1 immuiiological pregnancy tests, such as the inhibition tubc tcst); and three-complenient fixation slide tests, land HCG-test,' Preglide.J and Gnv- index slide test,' and such tu& tests as HI index.' ln addition, 233 of these wines were tested with -: :iiosticon,2 UCG? Prepucrin,' and the Ortho Planotest,' another complement-Bxat ion slide tar. there is a lack of papers comparing these tests Tuo huxdrcd further wines were tested with Planotat .-_. .. each other. In niany papers also, clinical con- alone. .-. .. .:tion of the diagnosis was not obtained and the The rensitiritia of the tests were: -t - L -. :*itswere not divided according to thc state of Grab-index, 3,000 ta 3,500 iull. (stated on the product). 1 ?...... 7;egnancy. Recently, some ncw slide tests. which Pregnosticon, 1,OOO iu;l. (stated on thc product). P1ano:cst. 2,500 iu,l. (manufacturer's personal com- 5 advantages ovcr thc tubc tests in simplicity _. .--. :imc takcn to obtain a result, have been produced munication). .. Prcgrlidc. 3,000 iu/L (manufacturer's personal com- these have not beeii adequately in\-esligated. .. municzi ion). .. thesc reasons it {vas tlioirght that it would be of Hyland, 2.030 to 6,000 iuil. (manufacturer's personal ._ .cst to compare all tlic availsble slide tests with communica!ion). :ubc test, Pregnosticon, which had becn in use It wss noted during the preparation of this paper that '>islaboratory for somc years. the pa:tem of resuits with the Hyland HCG test had -. changed during the courle of the work, thc earlier results .: LmKODS showing feutr false positives and more false negatixs than the law resulls. Bacter laboratories were contacted ::sncy tests were carried out on 6S6 early niorning and infcrmed us that they !lad changed thc sensitivity of .. . .. irncns of urine. Final Gizgnoscs on 311 patients w'crc their 12;: from 6.000 iu.'l. through 4.500 iu/l. io 2 to 3,000 . _. ixd cithcr from the czse notes or by correspondence iujl., which they 2re cnrrent:y supplying. U'c wcie unable ihe gencral practitioner. Four hundred and eighty- to obt2i:i the exact dates Hlien these changes were made xines were tested with four dirferent pregnancy and arc thus unable lo include all the results with the Hyland slide test in this pap. Few tats were carried out o Dirpnosria. Rarhm. New Jersey, (English addrrn. VSA with the Hyland test ai a sensitivity of iu,'l. and ..-. 3tnon. R~IcLs.). 6,000 . Orgdnan. 01s. Hulland (English addrw. Orcanon bbralorin. thesc hire been exc1udc.d. Thc results obtained using the 1 How. hlotdcn Surrey). Hyland test at a sensitivity of 4,500 iu/l. arc referred to . .. ?ole Lboraturss. Stanford. Conncclicm. USA (EnplkS as type A and with a sensitivity of 2 to 3,000 iu/l. as :A, Drnwr bbor-tor*. 11 Cnrlirle Road. London. N.W.9). cw.phsWcllcontc Xi Co.. Euston. London. KW.1. +&gland Laknlorks. Los Anfrlrr. Californh. USA (English iddrm. ,cd for publication 16 May 1968. Barter L2borrtoritd!fd, Thciford, Norfolk). 79 I 80 Joyce L. Bell

type B. The importance of pregnancy diasnosis kits result with Hyland A when tested 21 days 1:i- carrying an apprcrxinlste ehtimare of their scnsitivity Planotest and Gravindex had a similar senbiti.. annot ty overerrph;rjized and users should be notified Planotest giving 2.0%false negatives and Grab.- jfa change is made. \Vithout this. mistakes and confusion 3.5%. Pregslide had 9% false negatiws. HyLr can arise when interpreting results. was the most insensitive test, giving 19.5% f All urines were filtered before testing and the urines were tested for protein by the s3lic)l sulphonie acid test negatives, Hyland B gave 6.5 % false nrg3tivej. and Albusrix,' blood by Haernasrix' and for snlicylates with ferric chloride. urine was tested earlier than No PREGNASCIES AXD PATHOLOG:; 35 days after the stated dire of the last men~1rus.lperiod. COMPLICATED All urines not tested immediately were refrigerated and CONDITIOXS All pregnancy tests were posiii\ : all glassware used in the tests has thoroughly washed in the patient with a hydatidifcrm niole and nezr: tap water followed by distilled water. Soap and detergents in the follow up patients who were tested be:\\: were not used. three and 18 months after the original diagnojis .. treatment of the mole. The latter patients had WOE The tests were carried out according to the TESTS evidcnce for recurrence of the mole. manufacturers' instructions. It wsfound easier to read the result in daylight but uken artificial light was used Four patients were diagnosed c!inically as EC:: it was found best to use a Tungsten bench lamp, the slide pregnancies. With one of these patients no choc!; king held near, but not directly under, the light. The tissue \vas found histologically and the dia,wcs. results were read as positive (no agglutination). negative therefore in doubt. This patient had a ne:..: (agglutination present). and inconclusive (doubtful prqnancy test by all five methods. One patient i agglutination). a positive test with Pregnosticon and a ne;>: TUBE TEST The test was carried out according to the result with the other tests. This was presumab:) . manufacturers' instrktions. The result was read as to the grcater scnsitivity of Prepnosticon.. i A positive (definitive, well formed brown ring), negative patient was positive with all tests and the other \ (no ring formation), and inconclusive (a very open, thin all tcsts except Hyland A. Sixteen patients hr. or irregularly formed ring). The end point of the tube threatened abortion. The patient with a ne;:. tcst was easy to rad and tests could be carried out result by all tests was tested at 40 days after the . tither in daylight or artificial light. - of the last menstrual period; this patient is still F: RESULTS nant. Of the 11 patients with a positive result .. cach of the tests carried out. seven aborted withi UNCOMPLICATED PREGSASCIES Table I shows the 10 33 days the tcst, while are still piesr results in 203 uncomplicated pregnancies. Preg- of four nosticon was the most scnsitive test used, giving The reniaining four patients were positive .. only one false negative (0.5%). The patient giving Prepnosticon and had varying results with the 0- tests. A single pregnancy test, thereforc. appca:. the false negative result was tested at 37 days after the date of the last menstrual period, and gavc a be of little help in thc management of a thred!:. positive result with Pregnosticon and Gravindex, an abortion. inconclusive result with Pregslide and a negative The four patients with incomplete abortior.. had positive results. while patients with con?;- a~~~~ co.. IW.. hart. Ir,dian>. US,\ (Englkh rddrcu, Di\-ision ,,( abortions generally save nsative resuhs. Pres::; Mila Laboratories Ltd. Stoke PugeS, Slouch. Buck.). con and Prrglidc, however, gave inconclusive re. .

TABLE I RESULTS OF PREGNASCY TESTS is PKECSAKT PATIEXTS (EXCLUDING URINFS COSTAINING BW~DASD~OK PROTEI>, Days aJwr La: nicnstrwl Prrtnorriron Njland A . Hy&d D Period -. + , - & FolJr IC- + - * Folx In- + -_= Fabc In- NrgaIirr conclusive Nrtorirr rodurirr tirgorin conr! (%) (%I (%) (%I (7;) c, 3549 41 I 3 2.2 6.5 10 6 I 35 6- I521 11 6 5M9 5201 0 I .9 13 2 1 12 6 20 3 1 87 4? -9 4500 0 0 13 4 0 14 0 I1 I 0 8.3 0 7&79 IS00 0 0 510 17 0 400 0 ( 8W9 1300 0 0 401 0 -XI 500 0 t 9&99 700 0 0 200 0 0 SO0 0 0 IM)-199 I100 0 0 300 0 0 100 0 P >m 300 0 0 100 0 0 200 0 G LonptanJingaincnorrhau ' 2 0 0 0 0 100 0 0 100 0 C No amenorrhoea too 0 0 100 0 0 000 0 C Posipartumamenorrhwa ,A 0 0 0 0 100 0 0 '1 .o 0 0 @

.. and frochloropcr~zine and h'itrazepani. Eight .ihOK pt;cni each. two and three days respectively patients had infertility, six menstrual irregularities, zhc ,k abortion. and tigo chronic actke heprtitis. Thcre wcre, houeber, 75 patients on barbiturates, patients yrr-CRf(j~~\~pATlt.STS fSCLC'DISG URISIS WITH seven a contraceptive pill, four on chlordia7epoxide *,rlTJs A?r~BLOOII This group of patients H~Son W .. @~cdinto n1c.nopall-l and prernrnoiwml hecausc: and three on penicillin, \\ho did not givc false dl,,,,~r\aiion (\VI& ROOS,and Genizcll, 1961; posititc results, so thesc compounds do not appear Lacnfcld, lscrsky, and Shslesnvak, $962; Ham- to bc the cause of the fdlse positites seen. No falsc FhrFC and Arquilh, 1963; l'aynlor, Goss, and positives aerc found in paticnts on salicylates. wqlendorp, 1963) that antiscra to huniun chorionic Cmr,.jotrophin will crossreact Will1 pituitary gonad- $OX-PRFGSANT PATlChTS WITH URINE CONTAIMNG dro;rhin. patients H,ilh postpartum amenorrhoea BACTERIA, PROTEIN, ASD,'OR BLOOD In this group rm conti&red separately ~C~USCof the obser- of patients, Planotest \\as tlic only pregnancy test (~cnini,1965) that false positives are found which did not give a false positive result. False ar t* patients. There were eight paticnts with positives ith the other pregnancy tests apimred F'(t~~rlumantenorrhw; all results were negative to be related more to protein in the urinc than to ._- one inconclusive with Prcgnosticon. bacteria but some urines with an E. coli infection wgnosticon gavc two inco~icluriveresults in the gave a positi\*e or inconclusive pregnancy test even *:- mpusal group which became negative on in the absence of protein with Pregslidc or Grav- ...: &luting the urine. Thcue werc presuniably duc to thc index. .. viersensitivity of Pregnosticon causing pituitary .. PREGSASCY TESTS ON BACTERIAL CULTURCS Prep pmdotropliins to be detectcd. In the premeno- arations of cultured staphylococci, streptococci, and .. pual group, 0.5% false positi\es and incon- .. 05% of an E. coli-Proteus mixturc \sere prepared with a d&t were found with Planotest; 2.1 false % bacterial count several times higher than that which pn\iti\rs, 4-1 inconclusi\le with Gravindex; 2.6% % would bc present in a highly infected urine. These fdw positives, inconclusive H,itli Prcgnosticon; 6.2% cultures were then tested undiluted and at a dilution 4 7%false positives, 8% inconclusive with Pregslide; 1 in 10 and 1 in 20. The pregnancy test to ?+:.:false positivtsnone inconclusive with Hyland A, of only give a positive was Hyland B which reacted down to and 8.7% false positives. 1.4% inconclusive with a 1 in 10 dilution with the staphylococci and gave liyhnd B. an ... - The 32 patients who gave false positive or inconclusive result with undilutcd streptococci. .... imnclusive results with one or more of the pxpnancy lesls were thoroughly investigated to find EfFECT OS PREGNANCY TESTS OF ADDlTlOK OF PROTEIN TO URISE Human albumin,' y globulin,' and if any cause for these falsc positives could be found. lhrc~patients were taking steroids, two patients glycoproteins were added to urines at varying 'AB. Kdbi. Stockholm. Sweden (English address. Billon Hourc UUC on contraceptive pills, two on barbiturates, Uxbridge Road. W.5). .,on penicillin, and one each on chlordiazepoxide 'Koch-Lipit bbwatorirr Ltd. Colnbrobk. Bucks. k1-...... T A B LE l-contirzucd RESULTS OF PREGNANCY TESTS IS PRECSAk7 PATIENTS (EXCLUDING LRl%zS COKTAlNlSC BLOOD Ah'DIOK PROTEIN) kp*fer &st Mraitruol Prrcslide reil Cra rindrr Planarrsr .. -. + - f Fahe In- -- *, Fakc ln- 4- - I Falx In- Nrzarilu ranrluiivc h'rgoriw ronrfuiiir Nega rirr conclusive (72 c:, (;o (:;I (%I (%I 1WQ I. .M64 13 9 41 s 0 11 0 4534 6 8 sw 44s4 9 7.5 52 1 0 1-9 0 5510 2 0 w*9 3661 I3 6.7 44 I 0 22 0 3700 0 0 mT9 1800 0 0 1800 0 0 1000 0 0 Iwp , 1102 0 IS 1300 0 0 I800 0 0 -99 100 0 0 700 0 SI99 0 600 0 0 11 0 0- 0 0 I300 0 0 900 0 0 .so 210 - 0 300 0 0 200 0 0 *8Wundinprnamorrhou 2 0 0 0 0 200 0 0 100 0 0 .. '0 amnonhou 100-0 0 100 0 0 000 0 0 'mlrnwn amrnorrhw 200 0 .o 200 0 0 200 0 0 .- .., ..

.. - .. -. .. . .> . . . ..

, ..

-. . -. .. . 82 Joyce L. &I1

concentrations to hestigate the relative non- 1 g y g1obulin;lOO rnl. Since a fairly high propor:;, specificities of the pregnancy tcjts. Using human of pregnant fema!cs may h3ve non-symptorr. . albuniin at a concentration of 2 &'IO0 ml urine, bacteriuria and,'or proteinuria, a preg~ancy;. positive results nwe obtained with Hyland B, which does not givc false podites under t'.. Prcgslide, and Gravindex but not \vi: h Presnosticon circumstances is \ranted (Goclts and h1iyhc.r or Planotest. At a conccntration of 1 g/lW ml 196-1; Kew, Seftel, and B!oomberg, 1367), otherw. negative results wtxe found with 311 tests. Using it is necessary to maintain a biological test. T:- human y globulin, Planotcst and Prcgnosticon gave loiter limit of sensitivity of immunological pre_eni:;- inconclusive results at concentrations of 2 and 1 g tests necds to be set at such a level that crojj reac:.:: y globulin/100 ml. Yregslide and Gravindex gabe with pituitary gonadotrophins do not intec.::. positice results down to 250 m:!100 nil and Wide and Gemzell (1962) found that the lcvel Gravindex gave a further two incoiuAu4ve results luteinizing hormone in fertile and rnenopac.. at 175 and 62.5 mgllOO ml. Hyland B gave positive urine would be detected as 100 to 400 iu of FlC<' results at concentrations down to 125 mg/100 ml. and it has been suggested that thc lower limi: ._ Human glycoprotein (Colin fraction VI) gave sensitivity should therefore be set at 1.000 iu positive results only \vith Gravindex and Hyland €3. which is thc limit for Pre-mosticon. It would appt: Gravindex and Hyland B gave positive results down however, from the two inconclusive results seen to 500 mg/100ml. Gravindex also gave an incon- the menopausal urine and the inconclusive rC-jL' clusive result at 250 mg/l00 ml. seen in some patients with infertility that even at 1 , level pituitary gonadotrophins may sometir;. TESTSON FILTERED AND UXFILTERED URINE Although interfere. all tests in this series were on filtered urine, a series A tcst which does not give a large number of f:I.. of tests was carried out with the slide methods to find negatives is also required. especially in such c::. if filtering was absolutely necessary. Six urines with as eztopic pregnancies and thrcaiened aborti- varying degrees of' cioudines \\ere testcd. The only when the level of I ICG may be much !o\ver than ;? 0 difference found was with one nioderately cloudy in normal pregnancy. In this series Pregnosticoa L urine, which gave a positive result with Prcgslide on the most suitablc method, giving only 05% E.' the filtered urine and an inconclusive result on the negatives in normal pregnancies. Pianotest k.. unfiltered urine. It would appear from this small 2.0%. Gravindex 3.5%. Prcgslide 9%, hyla^.! series that filtration may not be necessary before 19.5%, and Hyland l3 6.5%. False negative re>-' pregnancy testing with Planotest, Hyland, Pregslide, using immunological pregnancy tests have b..

or Gravindex. reported in ectopic pregnancies (Southarn. Sul:~:-, and Cohen, 1963; Gusdon, 1964; Tslami, Fisher, :.; DISCUSSION Kupfer, 1964) and in threatened ahortions (Hu:cL. son, Schwartz, and Rates. 1964; Sato and Green?! : To be completely satisfactory a pregnancy test 1965; Fink and Frie, 1966). Pregnojticon pave :. should give neither false positive nor false negative siiiallesl number of false nengtives in these condiiic r results. in the present work. Tn this survey Planotest \vas the most satisfactory The type of pregnancy test used will depend on t' method for eliminating false positives, giving only type of pregnsncy being tested. As a gr.r:-. 0.5 false positives in protein and blood-free urine presnancy test, Planotest would zppear to he :'. from non-pregnant patients compxcd with 2.1 7; most efficient. In ectopic pregnancies and threz:i.- - for Gravindex, 1.6 :< with Pregnosticon, 4.7 % with abortions it may be preferable to use a more s- Prcgslide. 3.67; with I-lyland A, and 8.7% with sitive lest, such as Pre-mosticon or a siniilir t: Hyland B. False positive inimtinological tests could test, but the number of falx positives viill thc2 ! be due to a number of diRercnt reasons: technical greater. factors (Noto and hliale. 1964). impurity of reagents (Lunenfeld e! nl, 1962; Loewitt, 1966), cross reaction MYthanks are due to the local general practitioners .,' ' of the antihuman chorionic gonadotrophin (anti- were so helpful in supplying final diqnoscs and c' HCG) preparation with antigens other than HCG relevant inrorninticn on their pitienis. to thc OSjt:; Department for ~CCCZSto their note, and to Pro:';.\-.- (Wide e/ nl, 1961 ; Wide and Gemzell, 1962; Goss D. Baron for valuable help and criticism. and Lewis, 1961), and otlier as yet undetermined N. causes. Tcsts using albumin, y globulin, glyco- RLTERESCES protein. and various bacteria ha\ e shown that Prcgnosticon and Planotcst contiiin the purest Fink.H.. and Frk, A.61966). ObJttr.adGjwr.. 18.66 Godtr. P.. and h1i:ho;u. 1. C. A. (1964). Anirr. 1. Obrre. Q - ': reagcots, only giving inconclusive results with 89. 290.

...... -- .. .. -.. .. . ~- Luncnfc!J, 8.. 1w\l;y, C.. and Shclernyak, hf. C. (1961). 1. din. Fndot-r.. 12. S5.5. Nolo. T. A.. and hlde, I. B. (1964). Atricr. 1. r.'in. Parh.. 41. 273. Sam.T.. and GrccnWim. R. 11. (1965). Anrrr. J. Ob$/rf. Gprc.,91.31. Southxn. A. L.. Suliicr. I). 51.. and Cohcn. Ii. (1962). Anrrr. 1. Ohfrf.G~wc.. BS. 495. P Tdymor. PI. L.. Corr. D. A.. and BuywnJorp. A. (1963). Frrril. ad 9crd.. 11, 603. Wide. L.. 3nd Ccnrzcll, C. (1942). Acra ridicr. (Kbh.). 39. 539. -. Roor. P.. and Gemxll. C. (1961). /bid. 37,445.

The November 196s Tssue THE h.OVEhlBER 1965 ISSUE CONTXISS THE FOLLOWING PAPERS Mitral stenosis togcthcr. with il giant cell myocarditis limited to the lcft atrium 1. GILLIF. and 11. rox Porotic hyperostosis and the Gellipaer skull JOHN CLU and I. LmN EVAKS Scmi-autoinated method for the differential determination Of phna catecholamines HELEN MCCULLOUGH A stable starch pi cparation for amylase detcrminations ~~al&~lstudy and a recommended technique for G. P. FRASEK and J. c. B. FENTON minirig the euglobulin lysis tinie CHAKKADARTI, R. Assessnient of thyroid status in pregnant wonren and in 1L wmir.c, J. F. EYASS, and G. R. FEARXLEY patients taking or31 con!raceptives by a free thyroxine factor V activity in plasma cr).oproteins A. VON index T. M. D. GlMLElTE and A. PIFFASCLLI mLns. p. w, STRAW, and P. G. FRICK Residual fornraldchydc after low-tcmperature steam and formaldehyde sterilization G. L. GIBSON, H. P. IOHNSTON, -utomatic method for routine evaluatinn of and V. E. TUKKISCTON c)rinolytic com~onents D. COLLEN, G. TYTGAT, and U WXSXAClE Teclrrricnl nrcrhods Effect of unconjugatcd bilirubin on the eshation of Vatuc or pcl filtration on Sephadex G-200 in the analysis serum iron by a modified Sanford technique PAUL M. d Nh3d poup antibodies F. STRATTON, D. s. svm, and SMIlH, JOHN E. MIDDLLTON, and KOGER WTILIAMS +. I. nAWUKS0S Improved automated methods of protein de(eminalion D. F. GIBBS C. M. 1. HRlGHT &~mtms slripIloidrs in South Australia R. G. and Qooni and GERAUI~Xw. BROWN Micro modification cf the dilute blood clot lysis time for detemiining fibrinolytic activity c. SPI~LE, ESm of dilution on the growth of bacteria from blood R. N. M. LASSDO\\% and C. hl. HAWKTY cdt-ua A. r. c. H. ROOME and R. A. TOZER Eqqhrocyte mechanical fragility test R. G. COOPER, Antibiotic raistancc of coagulase-negativc staph) lococci R. A. KAHN, c. N. CORSELL, and M. E. MUHRLR snd micrococci JEAN COME and R. E. 0. WILLIAMS Diffeientiation of foetal and maternal ctghrocytes in bf.mlogiml tesh and onychomycosis R. R. DAVICS fomiol-fued tissues c. G. PAISE Akntage of a routine Reitcr protein complcrncnt- Mechanical ro1a-y device for plating out bacteria on htion lest in the serodlagnocis of syphilis in pie,"nancy solid niedium R. FRASER \VILLIANS and JESXFER Y. c L(0KRIs BAMBURY hurt and significance of metaplastic nodules in the Letter to the Editor mlmiicou J. F.ARTHUK - Book revicws hthology of hereditary nephritis V. V. iosiii The Association of Clinical Pathologists: 81st general meeting Gbgmethod for the examination of the small Index for 1968 afalinal villous pattern in necropsy material IAIN w. COCK and HRCWA GRAY Contents for 1968 - copies arc still availablc 2nd may be obtained from the PunLisHIxG MANAGE& .. BRlTlSH MEDICAL ASSOCIATION. TAVSTOCK SQUARE. W.C.1, price 18s. 60.

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. . ./ .. - ... - e- l,;, r:lll,~,,imL,ltir* tht. +r'tiii. .,----.,.,., tk C.o.~g~~l;~~ionof in-oluhlr and h.i\ic. vrotrins froni r:,t .iemin:ll vCbic./t, st*crction with \i.>i..illiiat Irrflui.nt~~*'If coilaccnascb-lil,q~ ppp;idahc frcini r:it ta'sils *\a*: 'i 1'.

,.,.! ,,,. E.; I,,~L,,. .; t1.,1t3. fi:~!). ..!.,' ( ti" i:,t~ri~i~.;:.IC-. *, ,>.-,(., I...I l!!;.i. ;-I i,)!-,

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i-[ :\\,.f'. ')e ,,,-I. ..,.r\ .1!11 l)e,,Llcl ir!,,n i.1' tt..:i- r+,-r.id&,,llicilll prcal*.~ntri(.11.tti. ;!:i.[ prpvcnted c :*:i;~!ll:!~! :t.r:!i.,ricill. 2.j.j.i.im 1sol:ition and characterization of Iactcifrrrin from SOW milk and honr seminal pla5nia Itiilii~rt~.'r, li : ~~~~~r-l~~ll..I. (' it i:ii k:I~,rid l'h::~-i(~l. Hi(' t,em.. Acric. Re\. cclullc.. i'airhridzi. E:LLI. 1. lii.procI. FI .ti!. 1973. 421,:). .j72-+2 ~Rng! Ltctid~rt-ini.t~lalcd film .xwniilk i -\I.(; mg nil) \,US chcg.nomti~enecius. .tn c,hservatinn suppirted i)v elet,[riq)horv>i>ar.d iiv reac!inil aFainst moncl;p. ant i.ictderrin antiwriirii. Isibclec. !iicusii:k showed multiple tornis I\! the proreiii i-tiplw. pint 3.:;- l!) 111 converted hy neuraininida-e to 1 torm firi~lec.poitit 9.6.ii. Hiiar seminal plasma rcint;iincd irnrnunoli~pic~allyidentirai !actofcrr!ii 10.1 til 0.5 mg ml) which was hound striin;!y to Iwar sperm. 25536n ldcntification and estimation of plasma proteins acids in hriinan fallopian tubal tlti-id s~gp ed hot!] transudation ' in human dentine. Thomas. 51.: Leaver, A. G. (Sch. Den!. and 2ctive secretion. as supported hy a milar diqtribution ot SurE:.. I.niv. Liverpool. Liverpool. Engl.). Arch. Owl Rial. major protein iractiiini. the alxence of r.unogkt ruin. imrniinoglohulins I IgG. IgA. and IgSI was demonstrated t)y 25.563n Placental -glutamyl transpcptidasc. Landon. \I. i druble diil'usion on Ouchterlony plates. Quantitation of the .J. thled. Res. Corinc. Growth I'nit. Princes Mary Jlatern. ' amts. of these serum proteins in human dentine was achieved by Hasp.. Xewcasile upon Tyne. Engl.). IT! J. R,(,c hrm 1973. the single radial immunodiffusion technique. One gram of tjt;~, 341-4 t Eng). ;-Glutnmyl traiwpcptitiasr Jc,ti\.tt> wn- dentine NCXl rontained 20.1 mg of albumin. 2 1 mg of transferrin. measured in a scries of normal full term ;il.izentas. The pt-i and 40 mg ol' I&: 18.4 and IgM each occurred at a level of 024 optimum of this enzyme was S.5 and the acti\ity ranged trglc. mp:g. 10: to 333 munrTs,'p wet wt. 2.5557~ Quantitative histochemistry of y-aminobutyric 25564~ Mineral content of various tissues in cattle. 1. acid in the human substantia nigra and globus pallidus. Dry matter, calcium, magnesium. sodium. and potassium Kanazawa. Ichiro: Toyoklira. Yasuo (Fac. \led.. L'niv. Tokyo. contents of mucosa and muscular layers of various segments Tokyo, -Japan). ('onfin. .VPurol. 1971. :3fiI4-6\, 27% 91 iEngl. of the gastrointestinal tract. Ilittrich. Bernd: Kt4b. Eric5 In the normal adult human substantia nigra. there was a marked (Sekt. Tierprod. Veterinaernied.. liar: \Iwx t.niv. LeipriF, unevenness of the vaminobutyric acid (1) distribution, with I Leipzig. E. Ger.). .Arch. E.rp 1.; tcrincirmt,a. 1975. 2kt:,. being most highly concd. in the pars reticulata. In the human 171-9 iC;erl. Among various par% of rhr ea.-tr(lintestinal rr3i- plohus pallidus. there were high levels of I both in the external of cattle. there uere considerable difterenir; it1 mineral ccmtc:;! and internal segments at almost the same concn. The I level was Forestomach mucosa contained high con:ns (11' Ca and \I<. higher in the dorsal than in the ventral parts of the pallidum. attributable to accrmulation and seiin. 111 ::iinc.rsl salts in th:. Caridate destructicw in the monkey derreased the I rcrncn. in tiscite. There ~3spenerally rnore \la 153:; ",I iri the :: -ccmrn;- bilth external and internal segments oi the pallidum and the of' the small intestine. and high r~wn: ,I' \,i 21x1 1.; were medial 67% of the nigra. suggesting that the caudate nucleus pre-ent. The re.;ults are intended as a h.:-tor cc~tnp.iri~onw::; >ends I-operated fibers to these brain regions. paihiil. crates. 2.5.55Xq Comparison of C-. Q-. and EM-banding patterns 2.ii6.iq Alkaline phosphatase ar exhibited by the chromosome complement of the Indian and urine of rattle and calves. muntjac. Muntiacus muntjali with reference to nuclear Er!ch {Sekt. Tierproid. Vetertna DNA content and chromatin ultrastructure. Grecn. iiichnrd Leipzi;, LeipziC;. E. Ger.1 .4r,'li. F.I,!I '. ~~:,-~it:o,~rv~~,~19;:. ,J.: Hahr. fiunter F. \.Armed Forces Inst. Pathol.. LVashinpton. 29121. 181- 1 tGeri Alk. phtl+pha?a-- 111 nc.ti\i:it. .: I>. ('.I. Chromosoma 19i5, 5O(ll. 53--67 (Eng). M'hen ihc. Kinp-~.\rrnstriingunirsp in the serrini <#! ,:!:\r-. cart!e. sriti his;*- chromosomes of the Indian muntjac were compared tdlorvinp were 909. l.".]. and 32.5. resp.. and thew \d!iie.- \vtre stati-tirali.. treatment with 2 presently used banding method>. trypsin-(;iemsn significant. Yali:ec for bile I a~,tivity\\ere: rahe.; 411.. ~317'. (GI and quir.acrine-HC'1 t(11. with structural bands as seen in the elrrtrtm rnicrosciipe. definite correlatiiins were ohsd. with reqpect CicTificant. In itrinr, I ar:ivitie< wrt: ,.?'. e' (I y-. <.atilt to the nos. and positions of individual hands. Wts. obtained tor and hulls 1.69. The relations httwern t!w I at.:iviry in rke .t 711 : the individual chromosomes were: Xu. 1 9.d9 pg. No. 2 4.11) pz. and urine are discussed. No. 3 4.43 pg. No. 3-X 5.05 pg and 1- 0.55 pg. Avg. diameters 2.i.i66r .4ctivities of potassium and sodium ions in rabbit and WLS. for individiial fibers were 193 A and 8.74 x 1(1 16 E,b. heart muscle. Lee. Chin Ok: Fozzard. Harrx A (Prit-lker S 6 resp., for stimulated metaphase chromosome.; and 185 A and S.73 \led.. I.niv. Chicage. Chirnpo. Ill.). J (;t,fi, Phvsioi. 1975. x 10 16 g'u. rrsp., for unstimulated chrcirniwimes. Fihera of fi.iihr. 69.5-70s (Knpl. Activities 01 ntraiellular K* and Sa- in interphase nuclei exhibited an av. diameter ot '31 .iiind a vt. 0' rabbit ventricular papillary muscle< wcre detd. with cation--selecti\-e Ih g, y. The total amt. of nuclear I)%.\ present in C~FYmicroelectndec and k'oncns. werr .-td uith flame phntometr nuclei was :L88 pg. The rmcn. and activit!- of li- ivf the mu+c!c:: wtre ~14.9and ,?:.e 35539r Immunochemical determination of soluble antigens inM. resp.. at 25'. The orrespondin,: rwLxand activity of Ss- in protein fractions from plnccntal tissue. Brock, .I.: were X.7 and 5.7 mM, renp. The apparen: intracellular activiry Stranbe. \V.: Hofmann, R.; Klausch, B.: F ' mel. H. (Inst. coeffs. for K* 1-h) and Na+ were 11.612 anti (1.1;:;. re-; I'hysiol. Chem.. Vniv. Rostock. Rostock. &er.,. Zcnfralbi. Similar results were obtained at 35O -,K Fa.; sulistantially low- (;\,naekol. 1975. 97i5), 281-7 (Ger). Protein iractions were +hen the activity coeff. 10.7453 of extracellular fluid \Tyrode = prepd. from placental tissue by homogenization. (NH,)SO, sdn.). which might be expected on the basis of a differen? fractionation. gel fi!tration, and ion-exchange chromatog. Some intrarellular ionic strength. 3s. Was much lower than that uf' of antigens detected in these fractions by absnrhed antiserums extracellular fluid. thus much iif the Na+ was compartmentalized against placental tissue and pregnancy serum were present in or sequestered. For external K+ concns. >5 m.M, the rwting pregnancy and fetal serum, also. The other antigens, which were membrane potentials agreed well with the potential differen- found neither in pregnancy serum nor in fetal serum. could be calcd. from the K+ activity gradients acrw the cell membrane as specific for placental tis: SJe. Their possible significance is a K+ electrode. Thus. K+ equil. potentials in heart muscie

discussed. should be calcd. by activities rather than concns. ~ .-1- _.,.I .P l':ir*i.,! rc.\crciil !I\ 3dt.i o*in*. 'r'p\nsoha!e Of th, c' i'i (mi '%-r' rlcor -ir.dirct i *cr,>:t.(. !n thv f -I,PI...,,I , '2 =us, :* . I 1' i;l.t.r. I:,: .. I' r. I! .I:? .,. . , . :..-, 3. ij;: '

*, I!Y , .-,II< -1 ; \ jl. ;, ..,f.' , L,;. . -.I- .. rnt r-,]! ,>f . .. /. .,.~ . . -,. . ? ,,, ,. ,.. *,- ",i, - _. .., . . . . .! .I. , .~~ , I. ... I. .. ; <, ' , ...

'. . + " 11' I .. <~ 1 * .. :. ri., r: .

. and cyclic, AhIT'

hvii.t,!\;k ~i:;\i:!: th.iti Cilnnin;ham. V. .J.: El-on. Pecricia 11 : vitro p<-.+!i ii?en-.t~r3nti Cpit $led. Ht 5. COUIIC.. Ciirsha!toil. Ez: 1iydrt1:eni tab mt-nii,rane c 1-151.;).&:g-er' t!?ngi. .Inanlinear relarion \vas t,wnd between cy-specific ji-glycoprotein (SI'-i ). n(:ne.;torifje(! :AII*; n,-id$ concn. in h!a*td plasma ind the a. G.. Plt~:hr:d, P.. firtite:ie(.k 'r. (;. tli. rer.'+r.:ai;e ;?re plnrtl:a tr:;p!,,phar. in r.;t> i!* d:!:+re::* . i'niv. i;r.tuenk!in. \Vien. Yienria. Austria). \\*it,n. kilt;. nutri:iouai stJtes. bu~this was not 5een :n rats injured by limb ,"r \t'ot.hccqciir. 1973. 87(8,. 279-81 (Cerl. Single radial ischemia. (-!-lcoprenalinc bitartrate 4 3'0 ,UP k;. s.c.), u-hich is immunodilf'usior. provided a simple and highlb reproducible clin. knt.wn to increase plasma iionesteritied fatty acid concn.. tY . ass.,. for preennncy -specific 31-glycuprotrin SP-1 in maternal decreased tota! plasma tcptnphan coiicn. Dic~~croisoprenalinrHCI 'n!fi ' serum. The half-time for elimination of SP-1 from the serum (17 mgt kg. i.v.) proauced a sustained increase in nonesterified was .7: hr pobtparturn. There was a direction correlation hy i fattk- acids accompanied an increase in free plasma tryptophan : I-' I" bet\ceen SP-l concns. in maternal serum and placental wt. and concn.. which was followed by a fall in total tryptophan concn. birth wt. of the infant. The IUSS ol' plasma tryptophan was attributed to a1te:cd : 'i25703h Role of potassium ion loss in the anoxic distribution in the extracellular space rather !han to increased impairment of respiration of rat cerebral-cortex slices. metab. Patel, Ncshiru an .J.; Fixter. Leslie 51. iDep. Biochem.. I-niv. 25710h DNA-to-protein crosslinking in synchronized C;lak$im. Glas.goa-, Scot.). Blochem. Sur. Trans. 1975, 31 I b. HeLa cells exposed to ultraviolet light. Hen. hntun: 1.11 2 rEnpj. Preincubation of rat cerebral cortex slices with Korhelik. 5tladen: Ban. .Jasna (Lah. E!p. Cancerol.. Cent. Inst high .peed brain homogenate supernatant at 37 for 60 min Tumor Allied Dis., Zagreb, Yugoslaviai. Int. J. Rad;at. Hu:. prevented the mmic impairment of subsequent respiration obsd. Rciat. Stud. Phis.. Chpm. Med. 1975. 27111. 63-74 rEnpi after ;iii:ierohic preincuhation. ii+ was involved as preincubation The max. 5-ield of DNA-to-protein crosslinking 11: uv -irradiated iii isimniot:<- KCI Tria-HCI IpH 7.4, 30 mM KCi) was nearly as HeLa cells was obsd. during the S phase with much smaller eft'wrive 3- the homogenate. K+ was not replaced by Sa+. Li+. vields in the G: and G: phases. Exposure LI! mid ~5cells tu 256 or I'rk .\erI~IFCUH. L.dSR,. 7'.1fd~~&\ ti rat liver mitochondria for undergoing peroxidn. increased - 2.3 197.5. 171.3). 31-5 t Russ). .4 cclnipi+(,n uf !hc chrcimostmes i.111d A- !he age of the rat increased from 3 to 24 months, whereas banding patiern after G- and C staining with the rime tri DS.4 ihr 4tqree t)t unhain. of the mritochondrial lipids fell from 3 to 12 replication a!id rhti degree of c.hrtmostirne c4lndensati:in. w.1. niiti1th.G (11 ape and rose again from 12 t0 24 months. This may carried out using Chinese hamsre. 3WtJphaSc chrclint;soin~;. he due to an age-related increasr in the proportion of polyunsatd. Chromosome condensation was studied during :> hrom(,dc~,~.\.iirinin~ inirv acida and a decrease in the proportion of monounstad. faltv and ~;-hrornodec~*S.cytidinetreatment. XI! the chromcwmi! wid> in the mitochondrial lipids. segments stained with the C-technique were also 3tainab:e wirh 2570.ik Effect of confinement of the circadian rhythm of G-technique. whrreos only some G-pos. ippmentc sverp abb to ovinc cortisol. Holley, D. C.; Reckman, D. A.; Evans. .I. LV. he C-bands. C-bands were heterochromatic seementz characieri;wi i Ilep. Anim. Sci., L'niv. California, Davis, Calif.). .I. hy extremely late replication with great delay in condensaririi! Endocrind. 1975, 6311. 147-8 IEng,. The circadian rhythm of under the action of the DSX nuclr3)tide anaiocs, while G-bands ~ilnsniacortisol was similar in sheep before and during a IO--da>- were segments with e:.rlier labeling and irregular dernndensation. period (if cage rsstraint (single animals in small cages). Two There is apparently a close correlation between chromasoma! C;- peaks in cortisol concns. were obsd., at 4:OO a.m. and 4:M p.m.. and C-staining ahility of different regions and the degree of the latter possihly resulted from feeding at that time. he1erochromatinization. J. R. Mase 2.5706m Role of plasma volume in t# increase of 25912% Study of renaturation of the DNP [deoxyribonucl= 1 aldosterone secretion rate during sodium deprivation. eoproteinj complex of cellular nuclei by means of microfl- Dalakos. T. G.; Screeten. D. H. P. (Dep. Med.. State L'niv. uorimetrg. Arryushjn. S. K.; Lideman. R. R.: Zlobina. G. P. New York. Syracuse, N. Y,). Clin. Sci. Mol. Med. 1975, @I@\, (Lab. Gen. Pathol. Physiol., Inst. Psychiatry. Moscow, USFR:. 161-5 (Eng). Redn. of h'a intake from 200 to 10 mmoles,day Byul!. Eksp. Biol. .Wed. 1975. 79(3). 44-6 (Russ). The increased the aldosterone secretion rate without alterin placma intensity of tluorescence of the DNP of Iymphoc-yte cell nuclei vol. With a Sa intake of 10 mmoles/'day, the airfosterone upon transfer of the cells to a cold citrate salt soh. after secretion rate was unchanged by salt-poor infusion of albumin previously heating tu various temps. was measured as a function I 175 g in 150 ml). although plasma vol. was increased by this of time by a modification of the method of R. Rigler and D. infusion. This indicated t'iat Na restriction did not stimulate Killander (19691. No renaturation olmrred in prepns. heated t4~ aldmterone secretion by a redn. in plasma vol. 6.5". but renaturation did occur in prepns. heated to 75". 85". or loo", as judged by tht characteristic fluorimetric profiles. A. Kuby Q cock

----.

1 Brodc u. a, Narhweis luslicher Antigene in Proteinfraktlonen 381

I ZbLGJnPk.87 (1@76) 3J1-3l)l ]

Aus der Forrcfiungsabteiluns Immunologic (Leiter: Prof. Dr. SC. med. H. P r Ie m e 1) em lnstitut fur Physiologiwhe Chemic ' (Direktor: Prof. Dr. x. med. D. M J c k e) und der Fauenklinik (Direktor: OMR Prof. Dr. sc. med. H.K y a n k) des Sereihr Medizin der Universitiit Rrtoet

Inimui~eltcmischerSocli\wis liislicher AntIcJene in Prorcinfraktioncn nus Flazcntaue\wbe Voa J. Broclr. W. Strambe. E. Aolmann. B. Klavrdr und €I.Frkmd Mit 5 Abbildungen * Zuram menlawung: Pmtcinfnktionrn anmPlucntagrwck rurdcn dumb Homogrnisii.Ammo. - H'wnmn- ~iiomru~f~t-~iitung.Cclfiltrst ion und 1onrmunhusrhchromatcltolFpbie gwonnen. Die in dieen Fd- tiotm mil abmrbiden Antirvrrn gEpn khranprcncrmm bzr. Plamtarrtnkt narbrcirhren And- gene lind ;om Tril aueb im miitkliehrn der fct.len &Nm vorhrndrn. Die itwigen Proteine.die tsb in1 muttrrlichen nab fct~kn&rum ucrkommrn. k&ntm plauot;p.ibwb rin. Ibre m6glicbe Bc rleutuag riddi&8li+rt. Summary: Protein fnetionr rere prepared from plactntrl thus by bomogcniutioa. ammonium ailfate frnctionation. pel liltration and ion exchangr chromatography. Tbc antigrr dsLrcted in tbw fraaionm by rborbcd antirra aprinu piamntat time aid pqnancy ICNm partly mrtr io the pw lune? and f-1 mmtoo. The other antigmm. hich arc found neither in pregnancy auum a* in &tal cNm. could bc ipfific for p1.Smt.l titme. Their pcuiblc rignifica~ei

Die !5dwangerrdraft kann kCh B i 11 i n g h a m I41 alr cine parabiotlrche Verbia- dung von 2 genetis& differenten Individurn aufgefal3t werdcn Mutterliha und fctrler Organismus befinden rich in einem ZuslJnd der immunologls&cn Hom6cstw; die pk- zenta eeint hierbei vine zrntrile Rolle LU spielen [ZO. !U, 26,31). Die immunologische Bedeutung der Platenta ist im wesentlicfen durh 4 Aspektc ge- kennzcichnet [MJ: 1. Plazenta ah immunolngi~eBarriere fiir Antigene, antigentragende und immun- kompetente Zcllen rowie ImmunglobuUnc 2 ImmunogeniOt der Plazmta. 3. Immunologiwhe Komptenz der Plazenta. -. 4. lmmunologie der Trophoblasttumarm. uci Die vorliegenden Unkrsuchungen rollen ein Beitrag zum 2 Aspkt, d. h. sur Rape der Imrnunogenitiit der Plazenta. darstellen, In diesem Zusammenhang is1 besonden dk Immunogeniut des Trophoblasten von Interesse. der fetalen Urrprungs irt und dadt wie der Fetus per definitionem ah allogenes Transplantat im mutterlihen Organimw mgesehen werden konnte. Auf der Ekne der Trophoblaslenschlht diirikn si& fetale 4ntigene. die tu 50% vaterliohes Erbyut enlhalten. und die mutterlichen imrnunolod- <&en Erkennungsmechanismen unmittelbar gegeniikrstehen. Na& unserem heutigm Nissen enthfill die Plazenta Transplantationsanligene,mutterliche und fetale Serum- Jroteine sowie organspczifis&e und ni&torganspeziflsdw zytoplasrnatirche Protcinc, roki letztere moglicherweisc ebenfalls immunogen wirken kannen IS. 13. 22, q. a o h n 171 konnte in Proteinfraktionen aus PhzcntaBewek inrgesdmt 6 l&li&e Anti- gede immunchemirch darstellen. die si& weder im m liaen naeh im fetalcn Sem =&weisen lieBen. Unter den in der Pluenta rei& FI& vorhandenen Serumprote@n> ..

;-. ,

. _. >. ... e. .-...... -- .* .. \OI5b3t . ' . .-

I - .- '. - . .... >. f' d

.I 1 E n Y Y, u .b

-.. . w

.. .

I i -. 1 Charackrization of four hum-an

I pregnancy-associated plasma proteins

TSUE-MING LIN, P1r.D. SESBIOUK P. IIALDERT, M.D. DAVID KIEFER, D.S. I \VILLIAXf N. SPELLACY, M.D. STANLEY GALL, M.D. Afionri, Florida :I

I i ‘I I

i I! Ij constitiicnts haw hccn found diich arc not clctcctablc in mritial urilrcntcd nonprcginnt irlnalc siibjcrtr or in mnlcs. IViih the usc of inirnl~nocl~c~iiiiralrncihods, onc or two such I”egn.7ncy-n~~ocintcd pl;istnn protcinr had bccri previously rcportcd with nniiwra

223

I !. I I

I i

i 1

I !

i L

? i i Four presnoncy-osroci~tcdplormo proleins 225 1;

with distillcti. wntcr ;rnd scanlid at 2110 niii. Fractions wcrc pootccl at tcri-tiibc inter- val\, c.o~icc~itr;~tcdby 1)rcripit:itim widi sat- ura lcd alnmoii i11 I 11 I i;~IC, ;I id rvc! issolvcd to ihc original volunic with distilled wiitcr lor iiiimrinoclirrusioii assay. 1,ipid staining \\.it11 Sudan IhLand iron s (ai ni ng \vi ih I3atIio;~licnnntli roli iic wc IC done as dcscribcd by Uricl.'" 'To stain for iron with ferrocyani

c 9P

1015b38 . . - . . .-. ... . -..-... J

Four prcgnancy-o5sociorcd plasma protcinr 227

..r

1.i

I 4; 0 0

. .. ._. .I .-.- . _- _. , . . , . . ._ , ...... ~. .. -...... _... . I‘

Four pregnancy-ossocioted plamo pfole;m 229

1 I 12 !

Yc ? ”a !*

12a 4 5 6 7 19101112 Fraction no.

‘II !

Y -.

4-'i 3 02

'4 1.7 1.6

,- ,- 1.5

f.4 * 1.3 i -i P 12 \ 3

1.l

t I I I 111111 I 1 I11111 / 10' lo5 10' M. W.

n I I

I fU75b42 _- I.

Four pregnancy-orroc;c:cd plosmo proteins 231

I 0 Q N 0 0

1 12 -I---- ii I I 10 ,I

'6 0 Q N 04 0

IO 20 30 SO 50 60 70 Tube no.

I - O1Sbh4 ~ _. .I...-... ,- - . . .. , . . _.._ ...... --..- - ... - .. -..- _...... Four prcgnoncy.osrociotcd plorrna pto:eir,r 233

1 I i. i I

1 234 lin cI 01.

P :‘ e !; ”‘1 7

51 I

I 41,

I L I t I 1 I 10 20 33 40 50 60 10 80 Tube nb.

1015334b ...-... ..--_...... -.. -..- ...... - ...... _._...... _, .-._ - --.

I1 236 Lin ct 01

0 ORh'L-tr-2623 * Arch. Cynak; 208, 187-195 (1969)

' OZ~L-TR2 6 2 3 Immunochemical Investigations on the Problem cif the "Pregnancy Zone"'

11. Antiplacenta Irmune Serum

iI by

R. Hofnann, H. Friemel and J. Brock Universitzts-Frauenklinik Rostock and institute fur '. Physiologische Chemie der Universitat Rostock .. rr Submitted May 27, 1969 1

. i I

'I .. I /

IC . l'ramlated frorn the German by' '6 ' R. &egg E!msfieZd Inform tio n Diuis ion

Oak Ridp itutionul Laboratory-d . July 2972 *. ' t015b51 /. 1 . l frmuno-Chcmical Investigations on the Problem of the "Pregnancy Zone" i 11. Anti-Placenta XmmuncSerum

Summary. The placenta is suitable for obtaining antlg1 ns induced by

pregnancy. With use of an absorbed antiplacenta immune Berum from a

placental extract at least 7 lines of precipitation can be obtained by

means of immunoelectrophoresis. I

The placenta immune serum precipitates alto ether three pregnancy- I' si 1 .4 specific antigens when sera of pregnant women, amniot c fluid, sera i 1 from the umbilical cord and from puerpera3 are used. i

The principzl usefulness of absorbed antiplacenta immune serum for I the quantitative radial immunodiffusion of pregnancy-induced antigens is .. *' being studied.

Introduction

Pregnancy can be considered as a speciai case of homologous trans-

. plantation (14, 23). For elucidation of the immunological significance * of the placenta many authors have dealt with the analysis and detection

I of antigens of the organ (3, 4, 6, 8, 9, 10, 19, 22). According to more t recent investigations, the marginal layer of the placental trophoblast

I assumes-the significance of an immunologically neutral buffer zone between

, the mother arid the fetk (24). Investigctfoas 0: the placents, in tha normai

fur.ctidni;rg stars icvoive tne acnger of injury ro the chi?d, Also, prlzgnancy serum is not available it. sufficient qtlaatity for ?roductio-=l of imune sztum, ---- -cI ------. ___ . ---.--- .- - ,-. - . wh5l.e piacenzas afCez birth caa ecsily be ptocessel for obtaining antigens. - -- -_ -- -- . -- 'i Since in the preceding 8 tudie8- we found a relationship of the "pregnancy .; I zonett to antigens of the placenta, tha purposz of the present work is to i fl I I

i Q15b52 I ./ /'

test the suitability of absorbed antiplacenta immune serum for detecting and 1 . ;. . correlating pr 'ex;?.J-induced serum antigens. '. r;. '. 4 * b,.oj' +,.'; .* . P. . I- . '- Methods .'

.' /; I I a) Extraction of protein. Placentas from women with a pregnancy of normal L term and spontaneoug birth pt the end of the carryinglperiod are examined and .' I then inmcdiately,freed. of the ovular nembrane ana the umbilical cord. For *. *. - extraction of the'cyeoplasmic proteins of the organ, the organ is first I t rinsed fp~~O-mip.,.in,cold_~u~ni_ng-_tapwater_tThen as much as possible the --. I I fat and connective tissue which is not associated with the parenchyma of the .\ .. f organ is removed. The placentas are then cut up into %feces th?-.size of beans \ 0 ------h

and the pieces of-the-organuinsed -- in-..---... 0.14 I-_ M NaCl-sqlution-_-- at- 49C-b the rinse \ 1 v-; I solution appears clear and colorless, whereby up to 5 b renewals of the rinse

solution are required. After each washing step the moist pieces of the organs fl------____a are allowed to drain on a Perion sieve before they are again stirred. in fresh I I washing solution. Contact with base metals must be avoided under 611 condi-

- tions during the.Gholeprocess in order to prevent inactivation of enzymes

and le/b:le proteins by metal ions. Once the washing solutiori is clsGr, the / pieces of the organ---- &remixed,~ith_twicetheir-vol~me A€ 9K 7.4 MI15 Fhospnate) -----.-----____- 0 buffer'and Sonogenized for i ain at 0 C in the iJltrh-Ttir:cx (Jznice and t-----.-----. t-----.-----. Kunkel KC, Staufen). I Formula of buffer: Surensen M/15 phosphate, pH 7.4 (2).

Solution 1: NaH2P04 Hi69.i9F g per 1000 ml dlst. water I Solution 2: NaH2P04 . 2 H20 11.876 g per 1000 QI dist. water

Mixing 18.2 parts of solution 1 with 81.8 parts of solution 2 results in /'

I I I -3-

7.:A" 7E.3 Instead of the potassium dihydrogen phobphate of the original Sbrensen buffer,

sodium salt was used, as potassium ions interfered in gnimal immunizations. * The.ibinly fluid slurry of the organs is place2 in a graduated cylinder,

the total volume determined and the mixture tirm ixnediately centrifuged for <-----a 20 min at 20,000 g. and 0' C in the preparative untracentrifuge (M. Christ,

Osterode, Type bmega, red ro or 6 x 100 ml). The sup-ernatan&(solution A) -t /- e%? are combined In a graduated cylinder and after detdrmination of the total-'.. -

volume of solution A, the determination of hemogiobin is made (0.29 Hb/100 ml). --7 From solution A the proteins,,ar.e-precipitated with ammonium sulfate, p.a. e------.. . For this, solid (NA ) (SO) is added to solution A to full saturation, and 42 4 gc;.---"--,

the solution then allowed to staniF4-47. hours '.'-7-'-.r- at 4' C> The precipitate is

filtered off and the filtrate discarded; the residue is dissolved in y/lS --e --- 0 phosphate buffer, pH 7.4. In order to eliminate the (Nti ) SO , Ciaiysig is . -..-7--:-'.4 2 c-4---- then carried out against the same phosphate buffer for '8 nours at 4-C.--I (buffer 4-* . -- -.---l.-- renewed after 25 hours). A solution B result (2.i 3 proteifi per cc which are contained only (NH SO - precipitable protein3 from the organs. In -4-2.-4--- e--- #order to increase the protein concentration of soiution it concentrtited B, is * -. I to 4 g protein/100 ml n a dialyzing tube ageinst ti 2OX (w/v> soiution of poly- '- i p- ethylene giycoi (Fluica, acc;ls, Switzerland; toLcncurs =t-,C3 This solution -Wn-7-4-

D - C serves as inoculating nat2rla: for preptiration of #;ne bwaknc ancipiacenta c-- 2 1 . -sera (4.0 g protein/100 ali. ?or tine electropnoracic 3a6 imcnocheaical angiyses the proiein content is raised to

vacuum uitrafiltrstion (for detaiis eee article I). 'The highly concentrated

protein solution Is then centribged In the preparative ultracentrifuge 6; (13. Christ, Osterode, Type' Omega,. green rotor 8 x 10 ml) for 60 min at 100,000 8 and 00 C, 6nd the clear supernatant (solution D) uyd for electrophoretic and immunochemical analyses. I IO15G54 I

t -4-

b) Determination of protein. The determination of protein in solutions

A to D from the organs was made by the biuret method (12). The calibration

I curve was plotted with beef serum albumin as a protein standard (VEB Serum- I t and Inpstoffwerk Dessau). ._ .. ' c)' Determiaation of hemoglobin. 'The determination of the hemoglobin 'I . concentration in placenta solutions A to D was used as a relative measure for I determining the admixture of blood In the extracts. The determinations were

carried out according to the proceiures of Ruckpaul et 1. (18) by measure- *. 7 .merit of the extinction of cyanohemoglobin.

11. Preparation of Antiplacenta IrrJnune Serum For the Immunization 10 rabbits (Chinchilla 9 ', 6-12 months old) I were used. 1 ml of placenta extract C emulsified in the proportion of 1 : 1

in incomplete Freund. .: adjuvant was injected subcJ taneously weekly over a period of 10 months. After checking the antibody titer (ever+ two weeks by the

Ouchterlony teet and' imunophoresis) the an1Eals were bled and the sera

stored at -20' C for later absoiption. . III, Absorption of Antiplacenta Immune Serun

Aftez the equivalent proportioits htid been determined In a preliminary

experinsnt, the polyvalent zctiplasenta imons eerc ware absorbed and

lyophli zed aicording to the procedurz given'ir. article I. .I. ! $:, IV. Agar Gel Diffusion, Immunoelectrophoresis, Mancini . Technique . The procedures have been described in detail in the parr on methods in

the first paper of this series. . / 3 . -5- P I

;Results and Discussion I 't After immunization of the rabbits, a precipitating immune serun is r # I obtained which reacts both with pkegnancy serum and placenta extract. After res-, 'n absorption of the antiqlacenta immune serum with human serum, the antibodies -* 1 c - - which are directed against proteins removed. In immunoelectro- [email protected]. "7- serum are 7-- -- f+rr---' phoresfs zit least 7 precipitation lines were obtained against placenta I extract 20.D with this absorbed antiplacenta immune serum which are dis- w tributed over the whole range of electrophoretic mobility of 0 to 7 x 10'' .\ 2 -1 -1 cm V sec .. - In the reaction between absorbed antiplacenta immune serum and

pooled pregnancy serum imunoelectrophoresis shows at least one precipitation -5 2 -1 .. line with tne medium electrophoretic mobility of 3.2 x 10 cm V sec-I (fig, 1).

It ie known that during pregnancy components of the placerits may pcss over into the Grganism of the mother (10). It is prob able that the protein found 4" 5n pregnancy serum with absorbed antiplacenta inmune serum is of placental I orinin.origin. This findinnfinding is contradictorvcontradictory to earlier results of other authors (22) ' HS 0 -La \.- A---_ -..--. . 4 t .- -4------._--__. . Anti 1I.y s '. .. war- . 9s GY

.. W Fig. 1 Immunoelectrophoreticanalysis of human (HS) and pregntincy serum (SS) using anti-human serum (anti-HS), /antiplacenta (Anti-PL) Hnd absorbed . serum 0 antiplacenta serum (a. Anti-PL) at pH 8.0 (Verona1 acetate buffer). -6-

..I and may be explained by an inadequate gethod for placenta extraction or 0' *. L d variable quality of the immune sera. Bayer (3) found in immunoelectro' ;tc.- \$ ,,1: J phoretic analysis of pregnancy serum against "neutralized antiplacenta c .*4 ..

3 serum a precipitation line in 'the alpha-2 range which is similar to 1- . "pregnancy-induced".proteQn and may be a metabolic product of the mother, i * .. I . It is possible that Bayer's antigen is identical with the placental protein

', ' which we..found. \ I. . ! * Id the diffusion test of Ouchterlony the suitability of absorbed

' antiplacenta immune serum for precipitation of antigens in umbilical cord

serum and puerperal serum as well as in amniotic fluid and placenta extract

' !. -7- 8 not precipitated by absorbed antiplacenta iumune serum; the absorption .

ir therefore to be regarded as complete. In the reaction of pregnancy

serum at.least 2 precipitation lines are obtained which are inmunologically '?. identical with 2 of the 5 precipitation lines of the placenta extract.

Between,umbilical cord serum and aborbed antiplacenta immune serum at

least 1 precipitation line can be demonstrated which shows a ciossing * phenomenon with the neighboring antigens of the pregnancy serum. From

this finding it may not be concluded that the umbilical cord antigen is not

.'contained in pregnancy serum; the detection of precipitation lines by iromuno-

diffusion is.decisive1y influenced by the concentration of the reaction

partners (2). ..In the case of tne reaction between amniotic fluid and , .. . -,.absorbed >titiplacents immuile serum at least 2 precipitation lines may ' b 4 , .* 4be.detected whicn likewise show immunological identity with antigens of *; 0 v . .the placenta extract. On the contrary, a phenomenon of crossing occurs

with 1 precipitation sickle, which Is visible between puerperal serum i.and absorbed antiplacenta imune serum. *. From the literature it is 'Known that with sufficient concentrating of the amniotic fluid nearly the whole spectrum of the blood serum proteins .*. may be obtained immunoelectrophoretically. The fact that the high-molecular

~er~rnproteins IgP. ana IgEwere not found in the maictlc fluid is ewlained / by 8 sieve effect (i7). Bevfs (4; reports an is~u~oei2ccrop~o=eticszudies

of -;liotic fluiZ tind placenta extrzct with aitlhunazl s2zuin. iie fomd tWG

I A

*. '.4 I

Fig. 2. Diffuslor. test for detection of antigens in umbilical cord serum (2), c; pregnancy serum (3) placenta extrect (4), amniotic fluid (5) and puerperal serum (6). Control: Human serum (1). I absorbed antiplacenta serum. I1 Non-absorbed antiplacenta serum. 'I I , !I - 8- D # precipitation lines which, however, were identical with antigens of

* 'human serum and placenta extract. Lambotte and Salmor/ (13) in electro- '. phoresis with human aminiotic fluid and antiaminlotic fluid immune serum %. of rabbits which had been absorbed by human serum found 2 precipitation lines.. in the a1 and a2 range, the latter showing lipoprotein character .-as evidenced by staining with Sudan black. IP some cases the authors could detect the al-globulin post partum in the serur of the mother (13a). . - Fig. 3 shows immunopherograms of pregnancy serum, aminiotic fluid, placenta extract, umbilical cord serum 6nd puerperal serum which were

developed with absorbed antiplacenta immune serum, In the placenta /-= ------extract at lyt 7 precipitationUines may be seen. Since absorbed anti- / * 0 I/ placenta inmune serum was used as antiserum, it is highly probable that 0 c - . the antigens of pregnancy serum, aminiotic fluid, umbilical cord and 4 --..--- I puerperal sera demonstrated with this afitiserum are contained in piaccnta v -c------.- __ extract. Just .as with absorbed antipregnancy imnune serum, here also the I ccc-- precipitation lines were formed mainly in the u /B, range, so that possibly 2 the same antigens are involved that were also demonstrated with absorbed

antipreinancy immune serum (see article 1 in this journal). Work is being

done on the descriptiori of :he pre;aration ana 03 the chsracterization

of ti e 2lzcenta tintigens. I # These findings provo, t!x existence of several pregnancy-sgscific

antigens. X pregnarxy-spzcific srayms is oxyto:haSs (il) which 2s c 4 separable into 3 zones (20) by means of starch gelelectrophoresls. The I #. / HCG fdrmed in the placenta shows a partial immunological identity with .--5c./ 0. LH (5, 11, 15). 'A further factor which 1s typical of pregnancy, is presently the subject of many studies (7, 16, 21). -9-

HS 0 I t . -- - a.Anli-l'L ss 0

J-*

XS 0 0

Fig. 3. Immunoelectropk,oretic detection, of the antigens in pregnancy serum (SS) , aminiotic fluid (FW) , placenta extract1 (PL) , umbilical cord Berum (NS) and puerperal gerum (W) by means of absorbed antiplacenta serum (c Anti-PL). '1 ! During pregnancy the placenth 6ssunies central importance. For this I

reason ahsorbed antiplacent? imniuns serm should be used to axamine the

problem as to whether the prsgnzniy-inducad serum antigem of the nother / are dekectabh with .his imune ssrum. Moreover, 8 quantitative deter-

mination of these serum antigens Chrr indicate new possibilities of diagnosis for the clinic in the recognition of pzthological r ourses of pregriancy. kig, 4 shows the suitability in principle of sbsorded antiplacenta immune / serum for the vihncini method of quantitative radial immunodiffcsion. The c* preparation of monospecific precipitating imaune sera from placentalextracts offl I 1 -10-

the possibility of detcrmining pregnancy-specif IC an tigens quantitatively . by immunological 'methodk. 1

I HS Fit' NS 6s ..

Fqg. 4. Mancini radial immunodifussion of human ser1 c1 (HS), pregnancy serum .(SS), arciniotic fluid (FW), placenta extract ( L), umbilical cord (NS) and pueFpsral serum (W> against antiplticznta serum (Anti-PL) snd against absorbed antiplacenta serum (a. Anti-PL),

c' L ITZ.RATiJRE I I I -1 1- I

Imrnunchcmischo Untcnuchiingcn rumProblcm dcr ,.c cgnancy zone". II, I$s I 7. CROSS,A. D.. and J. LEWIS:Immunologic diffcrcntatioii of IiitciniLinq hornionc rnd huninn chorionic gonadotropin in conipoitiicls O[ hiah piirity. Endocrinology 3,83 (1DG-I). 8. HASKOVA.V.: Tran5plirntatiori non-antigenicity of thc fCCh1 plnwnto. Saturc (Land.) 133. 278 (19G2L 0. JItRscnr'Etu, J.. and U. SODERB~~G:Immuno-clcctrophoretic dcmonqtrstion of prccipitnting cornpoiirnts in sera from prcgnant wonicn. Sature (Lond.) 197, 332 (19GO). J I 10, HULK*,J. F., K. C. Ilqcr. and S. >I. HEISER: Antihadics to trophoblast during (hc post-parturn pcriod. Saturc (Lond.) 191. 510 (1901). 11. Josnro\xrr, J. B.. and J. A. NC~AKEX:Prcstnce in thc human p1accnt.z and tcrm seriim of n highly 1ncto;cnir substance irnmunologically rclnted to pituitary growth hormonc. Endocriiro!o;y il. '101) (19G3). 11r. - Human placental Inctozcn (HPL). a tro1)hoblastic hormonc syncrgiring . with chorionic gonadotropin and potentiating tlie enabolic eflccts ol pituitary gmdh hormonc. ;\mer. J. Okitct. Gyiicc. SS. 86i (19G.l). 12. KLIL, B., 11. 2. SORJIOVA: I~borntoriunistcclinikfur Biochernikcr. Ltipzig : Akad. \'crle6.sgcscllschaIt Ciccst S; Portig 1DG5. 13. LAlnnomE, It.. et J. SAIAOS:ctudc iinrniiiroclcctrophorC.tiqr/o du liquid0 rmniotiquc humnin C. R. SOC.Diol. (Pnris) 1.X 530 (1962). 1311. - Les antiphes spkifiquc9 du liquide otnniotiqiie. Yroticlcs of tlic biological fluids. Xmsfcrdam-l-ondon-Sc\r York: Elscvicr I'ubl. Co. 1965. 14. JIULLER.RL'CIII~OLTZ,\I*.:LLis .Sch\\.an~~rschoftsprodul;t ah honiologcr Trans- plsntat. 2. Ckbiirtsh. Cyndl;. IC4. 257 (19G5). 1.). PAUL,\Y. E.. and G. T. ROSS: Irnnninoiogic cross-reaction between hunlnn c;iuciuiiic guiiducroyiir viiri iiuiiinn pliUiKJry gonndorropir,. Lnriocrinoiogy ij, 3.52 (1964). . IS. KEISS. A. 31. H. 0. SfSCER. J. B. HAWK,and R. J. JAKORS:1mrnuno:ogic crossrcactio bctwecn liitrinizing lioririonr nnd onticcra to human chorionic gonndotrop;.B . Int. Arch. Allergy 30. 5Gl (IDGG). li. I~OCLET.D.L. -4.. 11. G. V. NCRALT:Antigcnanalysiwhc Untcntlchiingcn an Fniclit\vas!er. und Jlcconiitiiil,rotrinc~.Schwciz. nied. \\*scIir. 31. 74 (1961). 1% I?UCKPACL, K.. K. jYEttsf!{. 11. \V. C~TIIE:Bcstirni~iung des Hirnoglobins im RIitL. Arzn.-Standsrd IO. i IS (19t;i). .' 19. SI>IXOSS.2. L.. 1'. CRUSE.and D. C. XC-KA~:Thc inimunologic probicm of pregnancy. Anrcr. J. Obs!rt. Gyncc. I.;. 213 (196i). 20. SJOHOLN.I., niid L. Y~s:Prepr~tio~i oi highly puriiicci oxytocinnsr (:\.stint rnrinopcptidasc) froni tcIroid.ircntal srriini. .Acta phnrn~.succica 1. 355 (1966). ?I. \\'nsm. P.. u. R. \Vrmiiot-r: L'ntrrsiichuncen ziir \\'nrli~tuinrfordcnin~dtircli huninncs PInccntn-hctozcri tt11'I4). Klin. \Vschr. 41;. 1167 (~gctj). 3.'*~KKES, 11.: XnbiL-Sch:.. >id. Pahitiit RostocL iDG3. 3.ZI.\IZIEH. F., \I. Z. PARKS:nil. Sclit\\wn;ersrliaft LIS iainirinologischcs Probicni. 1. Fortschr. JIeci. P.i, 973 (IW?). I / ~II- i)ic Scbwnngenchaf: als imrnirnologischcr Froblcn. Fortschr. Sled. 63,

I Ol'Fibb2 United States Patent (191 (1 11 4,065,445 Bohn et al. [45] Dec. 27, 1977

PREGNANCY-SPECIFIC Chemical Abstracts, vol. 55, No. 23724f-i. Centonze. BI-CLYCOPROTEXN AND PROCESS F'OR 1957. ISOLATING IT Chemical Abstracts, vol. 65, No. 9157a-b, Mansfield. 1963. Inventors: Ha08 Bobo, Marburg, Marbach; Chemical Abstracts, vol. 74, No. 6232Or. Boenisch. Ferdiruad Stutziogcr, Waldstetten, 1970. both of Gemy Chemical Abstracts, vol. 78. No. 9549511. Bohn, 1972. Assignee: Behriogwerkc Aktieogesellscluft, Chemical Abstracts, vol. 77, No. 1181841 and 118185f, Marburg. Lnhn,Gennany Bohn & Zwisler, 1970. Chemical Abstracts, vol. 76, No. 32096w, Bohn, 1971. Appl. NO.: 569,416 Chemical Abstracts, vol. 77, No. 59673q, Bohn. 1972. Filed: Apr. 18,1975 Chemical Abstracts, vol. 76, No. 70559g, Bohn. 1971. primary Examiner-Paul R. Michl Rehtal US. Appliatioo Data Attorney, Agent. or Finn-Curtis, Morris & Word Continuation-in-putof Scr. No. 292,735, Spt. 27. P71 ABmcr 1972. abandoned. A pregnancy-specific &-glycoprotein, characterized by Foreign Appliation Priority Data an electrophoretic migration speed in agar gel in the Sept. 29, 1971 Genruny ...... 2148587 range of the j31-globulins. a sedimentation constant 4.6 S A 0.5 Nov. 20, 1971 Germany ...... 2157610 of S, 8 molecular weight of 100 OOO f 15 OOO, Int. CI.2 ...... A23J 1/06 an extinction coeficient ElmlS of 11.6 A 0.5 (278 US. CI...... 2W112 B 424/12 mk; 1/15-molar phosphate buffer, pH 7.0). Field of Search ...... 260/112 R, 112 B a carbohydrate content of 28.05 k 1.55%, compris- 424/12 ing 10.7 A 1.0% of hexoses. 10.0 k 0.5% of hexos- amine, 0.55 & 0.05% of fucose and 7.0 & 0.5% of Referencer Cited neuraminic acid, U.S. PATENT DOCUMENTS and process for isolating it from the placmta or blood or 3,310,471 3/1%7 Parcella ...... 260/112 B urine of pregnant women. This protein may k used as 3,687,833 SA972 Pucellr ...... 260/112 R 1 reagent and for the preparation of antisera as reagents for the detection of pregnancy and for pre-natal super- OTHER PUBLICATIONS vision. Chemical Abstracts, vol. 51, No. 16804f-g, Ccntonze, 1957. 12 cI.imr, No Draw

1015bb3 4,065,445 1 2 analytical method and not to a non-uniformity or con- PRECNAN-SPECIFlC B&LYCOPROTEIN AND tamination of the product submitted to analysis. PROCESS FOR ISOLATING IT The pregnancy-specific B,-glycoprotein of the invention serves for the preparation of antisera. With thesc, a This application is a continuation-in-part application 5 pregnancy can be proved by immunological methods of application 9r. No. 292,735 filed Sept. 27, 1972 and (hemagglutination - inhibition test, complement binding now abandoned. reaction, radio-immuno-assay, latex agglutination). The This invention relates to a pregnancy-specific BI- proof can be carried out with the serum or urine of the glycoprotein and to a process for isolating it. pregnant woman. A quantitative determination of SP,, It has not been known hitherto that the placenta and 10 for example by radial immunodiffusion, may become the urine of pregnant women contain a pregnancy- important for pre-natal supervision. specific fll-glycoprotein. Although a protein in the For Preparing the Pregnancy-specific flI-glYco- &range has already ken proved immunologically in Protein, comminuted placentas are extracted with water the serum of pregnant women, a process for isolating it or a weak salt solution. A weak salt solution in the sense has not yet been described. 15 of the invention means an aqueous solution of one or The object of the invention is a process for isolating more physiologically compatible salts having an overall the pregnancy-specific B ,-glycoprotein (abbreviated concentration not essentially below or above that of a SPI)by fractionation from the placenta and the blood or socalled physiological saline solution. Accordingly, urine of pregnant women. Another object of the inven- suitable concentrations range between 0.1 and 5% by tion is the pregnancy-specific BI-glycoprotein which is 20 weight of solution. Suitable salts are those which are characterized by usually used in the aqueous extraction of tissue mated, an electrophorectic migration speed in agar gel in the preferably sodium chloride, sodium phosphate, sodium range Of the BI-glObulinS citrate, tris-(oxymethyl)-aminomethane/hydrochlorc a sedimentation constant of 4.6 S -+ 0.5 S, acid, glycine/hydrochloric acid. a molecular weight of 100 OOO f 15 OOO, 25 The aqueous extract thus obtained or the blood serum an extinction coefficient El c,,,l% of 11.6 zk 0.5 (278 Or urine of pregnant women is then precipitated at an mp; 1/15-molar phosphate buffer, pH 7.0), acid, neutral or weakly alkaline pH by addition of acri- a carbohydrate content of 28.05 f 1.55 %, compris- dine or quinoline or of a derivative, thereof. ing 10.7 f 1.0% of hexoses, 10.0 f0.5 of hexosam- The most preferred pH for this precipitation is pH he, 0.55 f 0.05% of fucose and 7.0 +, 0.5% of M 7-8 with acridine derivatives and 5-6 with quinoline neuraminic acid. derivatives. However, higher or lower pH values can An analytical amino acid assay of the pregnancy spe- be used with almost the same success so long as they are cifx Bl-glycoprotein was carried out according to the in the range between pH 5 and pH 9. method of Moore et al. As acridine or quinoline compounds used in the pre- (m.Chem. 30 (1958) 1185) using the liquid chro- 35 sent invention there are suitable, in addition to the basic amtograph Mdtichrom B (Fa. kkmann) and the ion compounds per se, the derivatives obtained from them exchange resins M 71 and M 82. Cystine (half) was by substitution. The substituents may be alkyl, alkoxy determined as cysteic acid after oxidation of the pro- and/or amino groups in various positions. Since the teins with performic acid (S. Moore; J.Bio1.Chm. 238 acridine and quinoline bases are sparingly soluble in an (1963) 235) and subsequent chromatography as men- 40 aqueOUS medium it is preferred to use slightly water-sol- tioned before. Tryptophane was determined with the salts. E.g. 2*thoxy-6,9diamino-acridine, prefer- direct photometric method according to H. Edelhoch ably in the form of its lactate, and bis-(2-methyl4 (Biochemistry 6 (1967) 1948. aminoquinolyl-6)carbamide suitably in the form of its The analytical amino acid assay of the pregnancy hydrochloride, have proved especially suitable. specific &-glycoprotein shows the following composi- 45 The acridine or quinoline compound is added to the tion: placental extract or body fluid in form of an aqueous solution. The concentration of the acridine or quinoline compounde in the final stage shall be between 0.2 and Aminorid on duespel 1.0% by weight calculated the volume of the solu- 100Minorid, 50 tion. ebkrund.rd The acridine or quinoline compound should act on Ammouid WUI devuuon (* 2S) the extract or the body fluid for a period of time which LydW 4.37 f 0.28 gives the components sufficient opportunity react Histidine 1.77 f 0.07 to Ar#ininc 5.07 f 0.22 and to form a precipitate. In general, a contact time of AspMic.cid 9.85 f 0.17 55 about 10 to 30 minutes is sufficient. Longer times may Thnwine 8.24 f 0.39 Serine 9.07 f 0.55 be used without hesitation. A shorter contact time is not Glutamic acid 8.97 * 0.22 advisable. although under certain circumstances it may Proline 7.96 * 0.31 also give good Glycine 7.07 f 0.83 results. AlUlk 3.74 f 0.33 The preferred temperature is room temperature i.e. Cystine (half) 1.57 f 0.14 60 - but higher or lower temperatures in the VdiW 5.84 f 0.32 20' 25' C, Methionine 0.88 + 0.19 range betwem 0' and 40' C will be suitable, too. The Mkucinc 5.79 4 0.40 contact of the liquid with the acridine or quinoline Leucine 9.60 f 0.28 Tyraim 5.75 * 0.20 compound can be improved by intensive mixing, for PhenyWanine 2.12 f 0.21 example by stimng. TWplopW 2.39 2 0.70 65 The precipitate that forms, i.e. the first precipitate in ' this process is then separated from the solution. This The ranges given' for the amino acid contents in the can be done in known manner, for example by decants- above table are mainly due to the imprecision of the tion, filtration or, preferably, by centrifugation. The 4.065,445 3 4 precipitate is discarded. In the remaining liquid contain- In addition to the fractionation steps that take advan- ing the SP,,the precipitating agent which is not bound ti age of its precipitation behavior and its electric charge, in the precipitate can be removed, if desired. according 11 he pregnancy-specific P,-glycoprotein may be ob- to known methods, for example by adsorption on suit- t;ained by a process which is based on the interaction able adsorbing agents such as silica gel, bentonite, active S between the pregnancy-specific P,-glycoprotein and charcoal or by precipitation with alkali metal halides. t he antibodies obtainable therefrom. In this process, the After separation of the adsorbing agent or of the precip- Fregnancy-specific P I-glycoprotein is removed from its itate formed by precipitation of the excess acridine or Solutions by specific immune adsorption to a carrier quinoline compound, which separation is carried out E:ontaining an insoluble antipregnancy-specific PI- according to the above-described known methods, or 10 eLlycoprotein, and eluted thereafter. even directly for separating the first precipitate, the An antibody that can be bound according to known SP,remaining in the supernatant is precipitated together mcesses to a solid carrier can be prepared by immuni- with gammaglobulin by salt addition. For this purpose ation of animls with the SPI, for example the product an inorganic physiologically acceptable salt e.g. ammo- ,btained according to Example 3, recovering blood nium sulfate, sodium sulfate, potassium phosphate is IS iom the so-immunized animals and isolating in known added as a solid to the liquid up to a salt concentration nanner the immunoglobulin fraction from the serum of of 50% calculated on the volume of solution. After his blood. standing for a while a precipitate (the second precipitate The thus obtained anti-SP, serum is bound to a suit- in this process) is formed. It contains the SPI together rble carrier according to known methods as described with the main quantity of gamma-globulin. This second 20 :.g. in Cuatrewas, P., Ann. Rev. Biochim 40, 259 precipitate is collected by a suitable method, e.g. decan- :1971): Feinstein, G.. Naturwissenschaftcn 58. 389 tation, filtration or centrifugation and is dissolved in an 11971) or Axen, R. et al., Nature 214, I302 (1967). Suit- suitable aqueous solvent for further separation of the ible carriers are high polymer carbohydrates such as SP,.This solvent may be pure water. More suitably, :ellulosc or agar, synthetic resins, for example poly- however, the solvent is one of the known saline or 2s icrylamide or ethylene-maleic acid anhydride- buffer solutions used in the isolating procedures for :opolymers, or even glass particles. proteins. Especially suitable solvents in this stage of the A preferred carrier material is purified agarosc in the process are tris-(oxymethy1)-minomethanelhydro- form of small spheres of a diameter of about 20-200 pm chloric acid buffer solutions or sodium phosphate buffer to which antibodies against pregnancy-specific BI- solutions. 30 @ycoprotein may be bound covalently according to the The solvent is used in an amount sufficient to dissolve method of Axen, inter alia. the whole second precipitate. A slight excess of the For the isolation of the pregnancy-specifx pl-glyco- solvent is not deleterious. However, a large excess protein a solution which contains still other proteins or should be avoided. The solution thus obtained, if de- carbohydrate impurities in addition to pregnancy- sired, is dialyzed against a saline or buffer solution for 3s specific /3 ,-glyoprotein, for example a placental extract further purification. or Serum or urine of pregnant women, is brought into Subsequently the SP,is adsorbed from this dialyzed contact with the specific anti-pregnancy-specific solution on an ionexchanger, preferably an anion ex- glycoprotein adsorbent, the pH of the solution being changer such as diethylaminoethyl @EAE)cellulose. adjusted to > 4. &cause the upper pH value is limited the adsorption may be carried out in known manner in 40 by denaturation phenomena of the protein, it is advanta- a batch process or continuously in a column. After geous to choose the pH within the range from 4 to 9. adsorption, the SPIis eluted from the exchanger with a The salt concentration of this solution is not critical. weakly .acid buffer solution. e.g. a tris-oxyme- Conditions that are known to dissolve antigen-anti- thylaminomethyl-hydrochloric acid buffer, preferably body-bonds must be avoided. Advantageously. the of pH 6.5. Generally, all buffer solutions having pH 4s aqueous solution contains neutral salts and/or buffer values between 2.0 and 9.0 are suitable for elution. The substances commonly used in biochemistry, especially buffer solution may contain as additional ingredients sodium chloride, phosphates suitable as buffer sub- physiologically compatible neutral salts such as sodium stances, trishydroxymethyl-aminomethane or buffer chloride in concentrations between and 0.1-10% and- substances that are enumerated by Good et al., Bio- /or as stabilizers e.g. alkali metal azides in amounts so chemistry 5, 472 (1977), preferably in a concentration ranging from 0.01 to 0.2%. The eluate may be precipi- range of O.O1-0.2M/I. The ratio of protein solution to tated again by salt addition, e.g. by ammonium sulfate, adsorbent is advantageously within the range from 1 : 1 whereby the SPIis obtained in enriched form. It can be to 10 : 1, preferably 2 : 1. After the adsorbent has been further purified by a molecular sieving method like in contact with the pregnancy-specific glycoprotein ultrafiltration or gel filtration for example with the aid 5s solution for 0.1 to 5 hn., the pregnancy-specific BI- of Sephadex @ G-150. For further purification, prepar- glycoprotein is specifically bound by the antibodies. ative-zone el&trophoresis with PVC as a carrier in a The adsorbent is then separated from the solution by suitable buffer solution, preferable in a solution contain- centrifugation or by filtration and washed several times ing volatile buffer salts for example 0.075% ammonium- with a neutral salt solution to eliminate excess salts and bicarbonate may be used. After the electrophoretic 60 unspecifically adsorbed impurities. separation, the SPlcontaining &zone of the carrier is The elution of the pregnancy-specifx B,-glyco- cut out, eluted with ammonium-bicarbonate dyophi- protein can be carried out using measures which are lized. Other suitable fine purification methods comprise known for their capability of dissolving antigen-antibo- chromatographing on DEAE-Sephadex and subsequent dies. elution with a sodium chloride gradiemt, and fraction- 6s Thus the elution can be carried out using a solution ation with alcohol, preferably with ethanol. The preg- having a pH value of <4, preferably within the range of nancy-specifc P,-glycoprotein is isolated in similar from 2 to 4. This procedure is effected by contacting the manner from the blood or urine of pregnant women. adsorbent bearing the SPIwith a weakly concentrated I O15tsb5 4,065,445 5 6 aqueous salt or buffer solution. preferably one contain- ml, the pH-value was adjusted to 8.0 by means of 0.1 N ing 0.2 to 8 moles per liter of neutral salts or buffer sodium hydroxide solution and stirred with 250 g of wet substances. Most simply a dispersion of the adsorbent in DEAEcellulose for 1 hour. the solution IS stirred for 0.1 to 2 hours, whereupon the The DEAE cellulose was then separated by filtration pregnancy-specific PI-glycoprotein is desorbed from 5 from the solution. washed twice with 1 liter portions of the immune adsorbent. After separation Of the adsor- 0.01-molar trishydrochloric acid buffer having a pH- bent the solution containing the pregnancy-specific value of 8.0 and then eluted thrice with 500 ml portions p,-&coprotein is adjusted to a pH near the neutral of a 0.02-molar trishydrochloric acid buffer having a point. Le.. between 6 and 8. Thereafter further PUnfica- pH-value of 6.5 and containing 0.85% of sodium chlor- tion. for example with hydroxylapatite may follow9 if IO ide and 0.05% of sodium azide. The combined eluates desired. were combined with 30% of ammonium sulfate. re- Likewise the elution can be carried Out by the Use of ferred to the weight of the liquid, and the whole was solutions of protein-dissociating agents, for example, stirred. The precipitate which contained the SP, was urea solutions or solutions of so-called chaotropic salts, dissolved in 300 ml of distilled water and subjected to for example NaNOs* NaBr* NaCIOd* CF3CmNq 1s gel filtration by means of Sephadex G-150 and using a NaSCN and cc13COONa having a molarity of02 to 8 O.I-molar trishydrochloric acid buffer having a pH- m/l. TO remove the ChaotrOPic salts from the solution value of 8.0 and containing, per liter, 1.O mol of sodium after desorption of the pregnancy-specific 8i-glyco- chlofide (100 : 20 cm column). protein and separation of the adsorbent, the solution is ne eluatff Were then tested with specific sPI- dialysed against a neutral salt or buffer solution having 20 antiserum, the splcontaining fractions were collected a concentration between 0.1 -1 MA.Further purifica- and the proteins were precipitated again therefrom as tion may follow also in this case, for example with hy- described with the aid of 30% solid ammonium droxyl apatite as described below. sulfate. Generally, the PregnancY-specific gbcoprotein ob- A further was effected by preparative tained according to the process step has a 25 zone &ytrophor&s with polyvinyl chloride (pvc)as degree of purity within the range Of from to 95%. 9o buffer, a 0.075-molar ammonium-bicarbon- An especially adequate method Of highly purifying ate solution (PH was the electropho- the product is an adsorption step using a hydroxylapa- 8.0) used. AR~~ tite suspension as adsorbent choosing the retic separation, the SP,containing &zone was cut out, tion conditions such that the pregnancy-specific 30 eluted with the ammonium-bicarbonate buffer and the p,glycoprotein is not adsorbed, but only the impurities were lyophilized' still present are bound by the hydroxylapatite. The dry substance was dissolved in a 0.01-molar tris/- It is essential that the adsorption takes place in an hydrochloric acid buffer having a pH-value of 7.0 and aqueous medium containing phosphate ions, preferably containing 0.2% of sodium chloride. chromatograhed in a buffer solution containing phosphate ions. for exam- 3s On DEAE-Sephadex 2o cm) and a solution of potsssium phosphate or diu,,, phos- with a linear gradient of 0.2 to 2.0% of sodium chloride phate in a p~ range of6 to 8 and with 0.001 to 0.01 mols in a O.O1-JTiOlar trishydrochloric acid buffer having a phosphate ions. For the adsorption of the impurities, pH-value Of 7.0. Then, precipitation with 40% ethanol batch processes as well as column chromatography are was carried Out at -5* c. After the Precipitate Was suitable. According to these processes, the.pregnancy- separated by centrifugation, the supernatant was di- specific fl,-glycoprotein is obtained in a purity of above 4.0 alysed against distilled water until it was free from salt 99%. and lyophilized. Purity 90 -95%. This preparation The following Examples illustrate the invention: could be used for the manufacture of antisera. EXAMPLE 1 EXAMPLE 2 45 Isolation from placentas Isolation from blood 150 kg of deep-frozen placentas were comminuted 5 Liters of the serum of Pregnant women were di- and extracted with 150 liters of a 0.5% aqueous solution luted with distilled water in a ratio of 1 : 1 and the main of sodium chloride- The extract was adjusted to pH 8 by quantity of the Plasm Proteins Was Precipitated there- means of 2N-sodium hydroxide and combined with 50 50 from with the aid of 2.5 liters of 3% of Rivanol at a liters of a 3% aqueous solution of diaminoethoxyacri- pH-value of 8.0. For removing the Rivanol, the super- dine lactate (Rivanol @). After 1 hour the supernatant, natant obtained by centrifugation was combined with which contained the SPItogether with gamma-globulin, 5% of sodium chloride. The rnulting precipitate was was siphoned off from a first precipitate which had removed by centrifugation and the supernatnnt was formed, combined with 5 % of solid sodium chloride 55 separated from the sediment and combined with 30% of (I Ikg) in order to separate the Rivanol which still bad solid ammonium sulfate. The precipitate contained the remained in solution, filtered and combined with 26.5%, gamma-globulin and the pregnancy-specific B,-glyco- referred to the weight of the liquid, of solid ammonium protein. The precipitate was dissolved in a 0.01-molar sulfate and well stirred. After 1 hour the precipitate trishydrochloric acid buffer having a pH-value of 7.0, which formed was filtered Off. 60 dialyzed against a 0.01-molar trishydrochloric acid 500 g of the precipitate that had deposited on the buffer having a pH-value of 7.0 and then purified with filter were dissolved in 500 ml of distidod water and DEAEcellulose. The treatment on DEAEcellulose dialyzed against a 0.01 molar tris-(oxymethyl)- was camed out in a O.OI-mohr trisflydrochloric acid aminomethane (hereinafter referred to as "tris")/hydro- buffer having a pH-value of 8.0. chloric acid buffer solution having a pH-value of 7.0 65 Elution from the DEAEcellulose, gel filtration with and containing 0.05% of sodium azide. The dialyzed Sephadex, preparative zone electrophoresis, purifica- solution was centrifuged and the supernatant was filled tion on DUE-Sephadex and precipitation with alcohol up with the same buffer solution to a volume of 2.000 were carried out as described in Example 1. 4,065,445 7 8 an extinction coefficient EI,,I* of 11.6 +- 0.S (278 EXAMPLE 3 mp; 1/15-molar phosphate buffer, pH 7.0). Isolation of pregnancy-specific PI-glycoprotein from its a carbohydrate content of 28.05 2 1.55%. compris- solutions by immune adsorption ing 10.7 f 1.0% of hexoses, 10.0 -C 0.5% of hexos- 5 amine, An immune adsorbent suitable for specifically bind- 0.55 o.~5% of focose and 7.0 o.~%of neura- ing pregnanc y-specific /3 ,-glycoprotein was prepared minic acid. by binding 1.75 g of protein Of an immunoglobulin frat- 2. A for isolating a pregnancy-s~fic p,- tion obtained from anti-pregnancy-specific BrglYco- glycoprotein, which comprises protein rabbit =rum to 175 B Of ~P~~ (registered 10 a. combining the blood *rum, urine 01 an extfaCt in a trade mark) 4 B, (cf. Handbook of Biochemistry, x- weak salt solution of the comminuted placentas of lected data for molecular Biology. Edited by Herbert pregnant women with an acridine or quinoline A. Sober, 2nd Edition, 1970, page K 73) that was acti- compound at a weakly alkaline pH whereby a first vat4 with 21, 8 g of BrCN (cf. Axen R., Porath, J., precipitate is formed; hbach, S.: Nature 214, 1302 (1%7)). By m- of this 15 b. discarding said first precipitate and combining the immunoadsorbent, pregnancy-specific flI-glycoprotein remaining supernatant liquid with an inorganic was isloted from the extract of placentae according to physiologically compatible salt, whereby a second the following process: precipitate is formed; 1 liter of a placental extract containing pregnancy- c. collecting said second precipitate, dissolving it in specific-/jI-glycoproteinthe protein dontent of which is 2o an aqueous medium and dialysing the resulting 5%. is dialysed against 10 liters of 0.1 molar trishydrox- solution against a saline or buffer solution; ymethyl-aminomethanehyd~hloric buffer (PH 8) d. filtering the dialyzed solution on anion-exchanger, which contains 1 mow1 of NaCI and lg/l of sodium whereby the pregnancy-specific flI-glycoptotein is mide. Thereafter the adsorbent is obtained above is absorbed on said ion-exchanger and added to this solution, the solution h stirred at 4' C for 25 e. eluting the 41-glycoprotein from said ionex- 2 hours and the adsorbent is suction-filtered together changer. with the bound pregnancy-specific B,-glycoprotein. 3. A process as in claim 2 wherein slid eluted PI- The adsorbent is washed with 2 x 250 ml of 0.1 molar glycoprotein is further purified by pncipit.tion with trishydroxymethylaminomethanc-hydrochloric acid- ammonium sulfate, by gel filtration, or by preparative buffer solution and then with 2 X 250 ml of a physiolog w) elec~Phoresis* idsodium chloride solution (0.9%); then the absorbent '* A process as in 2* an &dine is six for 20 minutes each with 400 ,,,l compound diaminoethoxyrcridine lactate is added as a portions of a 0.5 molar glycine-HCI buffer of pH 2.5. so'ution in an one- ne are adjusted to pH 7 1 N fourth of the VOhmC Of the blood SenUn, & Or ex- NaOH and concentrated to 10 ml on an ultrafilter. The 35 tract '' which it 'added* wn-trate is dialw against a 0.005 molar dium '. A ~OceSsIs in clrim 2. u an hrwcsalt phosphate buffer of pH 6.8 and further purified by chro- solid sulfate is to suvmt matogmphy on hydroxylapatite as described in Muid fmm 26*5 to u, percent by weight of Em- the supernatant liquid. plc 4. 6. A process as in claim 2, wherein said buffer soh- The same process can be when the prcwc~10 tion is a 0.01 to 0.1 molar tris-oxymethylrminome- specific fll-dycoprotein is be Obtained from the thane/hydr~hloric =id solution. Serum of the urine of pregnant women. 7. A process as in claim 2, wherein said fl-glyco- EXAMPLE 4 protein is eluted from said ion exchanger with a buffer solution. For hiBh1~Purifying PreWanCY-spcCifiC fl1-BlYcO- 45 8. A pr- for isolating a pregnancy-spcclfic pI- Protein. 50 mg Of the product obhkd glycoprotein which comprises conwting the blood Exmpla Or 2* hrving a purity Of 9f%9596' dis. mm,urine extract, in a weak salt solution. of the solved in of 0.005 molar sodium phosphate buffer or an IO ml comminuted pl-w ofprrgnant with u1 anti- @H 6) and applied to a column ofa didonof2 x 8 Myof & pregnrncy specific fl1-g]y~pro~bound Cm filled with hydroxylapatite Waft. SCwh 50 to a solid at a p~ between 4 md 9 suk- berg) which quilibnted &fore with 0.m q-dy eluting the glycoprotein fmthe carrier at a molar PhoJPbte buffer. upon the pH bctwecn 2 and 4 or by treatmmt with a protein with 0.005 molar phosphate buffer, the PremY- d~~gcompound. specific h-glycoprotein migrates much the WIUmn, 9. Recess of claim 8, wherein the antibody is ob whereas the impuritic~remain adsorbed 00 the hydmx- 55 by &ministering the prepancy-spcclfic bl- ylaptite. The Proteincontaining elute Was glycoprotein to rabbits and isolating the antibody there- against IO tima its volume of water to eliminate the *. salts and then lyophilircd. Yield of P~panCY-~fiC 10. Process of claim 8, wherein the Carrier is I poly- B,-glycoproteia about 40 mg; degfe OfpdtY: > 99%. meric carbohydrrte. That is according to immunological processes, none of 66 11. procesS of claim 8, wherein the protein hiat- the admixtures ascertained before purification could be hg -pound is rtascertained. 4 12. Process for the purification of an isolated preg- WeCh. nrn~yapecific8I-glycoprotCin. wherein said glycopro- 1. A purified, isolated p~gnmY-S@fic 81-dYCo- tein in an 4ue0us solution containing phosphate is con- protein characterized by an electrophoretic migration 65 tacted with hydroxylapatite and subsequently the hy- speed in agar gel in the range of the &-globulin& droxylapatite is separated from the 4ueous medium a sedimentation anstant of 4.6 f 0.5 S, containing the glycoprotein. a molecular weight of 100,OOO f 15,000, I+*+* I .... , Ik: ..-...... - . . . ,

469 mVll0,DT.v YZTOYLI.CE:I?AL COtiThIHUTIC:: 70 MATEIlNAL ;ift:hOS'J PCOL %AX. >:.;,-T.-r,'~,: ;. . Pci -.~o~~lnn..,r.~!It~.?~ml :.neb= IN I --Knr~t.~nnrn.*kpt .of ;nJ.tGmy.Univcruxty of Ce:iforniu. Snn Yrr.ncioco, Cn. 9.;143. Ylnomn ontrocfn wr.d >rodnutaronu *ere detorrnineiz by, r,diolmunow,or.y on d#hy 15.17 .19 a0 21 of procno- ncy An tho follocliti i:rnupai 0: Mnc-Evbnn ri.tn:nnrmnl prccnant-- (::>, fotcctori-cd--- (F) ,ov#,rircrooired (6).i.nd Yorrdrennlcctomice.1 i?uA/ on dry 11 of prrcnuncy; in 811 inotnnc,lo pl*.cuitt,* who left in oitu, Altnouch prncraterone drcrnnond drnr.txcnlly in N rmtu on dny 21. o&,tr'o~ondAd not cti~n,co throu~hauf jcnthtAon. :;O chnrLo uno ohooivod in Y r,.rn. IroCcutorono dccroro- ed drnmnticdly. but ootrocun noderotoly. rn C knd f3 r#&tc,,nlthouch P3 rnto <$nowed a ell(:ht ancroano in procontorona ovar 6 rnto. AlrLoudn both-nrocnutorono Rnd entroden YO;-0 further docronoed in YOA rutb.thay remnincd in rpprrcinhle lcve;e, throudhout ecntation. Thnao reoulto indlcrtn thnt aurine pr6r;nfmcy in tho rat tho rnnAn COUTCO of >ro,!b6torone iu the Orc6ry;hor- ever, the motornel adrer.h;o 4 the plreentt. ulno co- ntributo both rotrocon and proceeteronr into tnc. E*- tornnl steroid pool_Thr CNIOO

490 IIyIv*u.oc:. ALPlU-2 PRLCH0tLOIUL:N LEVELS * A TEST OP tU..ZZJiK;AL MlCTION IY 1ATLY PREGNANCY. Bcmard H. Bema. Ked. Scl. Fror. , lndlsna Ilnlv., Rlnt-nIn.ton. 1:; L7LOl Alpha-2 DrcRnOSAOhlln (u2P) 1s a unlqw protrln (m1.w. 376.000) greatly eltvatcd In ssmm Io pregnancy and In estrogen and ore1 conrraceprfvt uaeri (Icrn.. B.H.. 1RCS kd.Scl.(73-10) 10-26-L. 1973). L'a mrasuie Irvrla by radla: lrsunodlffuaino. In control#, 452 of collrge wown and 51 of men have measurable level# up ta 5 unlta. At tcm ln prrjnaney. 241 wwn'! Ievala dlatrlbutcd wldely on an aeymctrlc curva: -6. 20 unit# at the 30th percontile; ncdlao (5) 31; mean 32; rrnpe (E) ?-iWl Some have only n.m-precnant lavsla. The Itteraturc b..g&cata the mean to b. 90 m%/lnO rl. 70 evaluate hvcla durlnl( spontananua ahortlon: Yorrel ?rem. SDont. Abnrt. Veeka o II n 5 I t (Xrnn-Uhltney) 4-6 66 Ozl8 10 2 of5

/' k* mwoLOcv h -1 4: :. '*" 4tiD IIIGliLY SEKSlllVE W'TIItW TO OCA..TITATE 1SDIVlQU- a. '.'" "l~i;XY,qirmrCENS UTILlZISt TllE TESSOSTLROSE-D;NDIEG , !' '\vllC) Or R\OHIT FUSW ,\SI) TllC RESPECTIVE hI-LICICYDS. '. 3 .. . . I-* (sIu:;: *I. PcynolJ,;). Unlv. of Pcnnrylvanla. 1 .I .. ,I, \et. !((..I..Vcnoctc Squorc. PA. 19348. I. ,- lI~..aiTc:lC tnnr rccrntly hrcn dcnonscrated to have "~~r!co-cl~rnlcalpropcrtlo that of husan TebC. 19. ,,., 3. :#..,, xcera to rnhblt pl~srrsand the avsllabllity of - le.,, ., tr3tn%tcronc (T), dlhyJrotc5toarrrnne (Dlil) and .*.e.., ' -I101 (kl) prompted thls study. Followlnp rthercal '..* b, ." of rmplcs T. Dill and Ad are individually fract:on- ' .t 'lhadex Lll-20 COluM ehtcwtorcaphy. TeBC-Isotope . t., **ploycd 18 prcptcd hv rplklne 0.51 rrbblf plasma 'I.. - of rlrr 2r.Irrd lipwvi (7, Dlff or Ad) In phosphate '.L) and i':crLnll Is urcd to separate the bound ,,: .I? I.... Sc:i-i~lvIfy for car IC0 p~ and " . T .,. .' ,- T .a )I' .'.I. A., ,,N lndcr of preclaton thc .,,, 'vhr~.,L:llty lor all rhrcc Ilpanda the cocfflclcnt .aa #',.n was Id;. Slncc IPO~IIrpecific hctlvlrics na well ., l-lon constants of thc..~. 1IGanJs to TcUC ale differ- ,, '-us mctltodn vatnu on~vIO-T and hwan TcEC to _. ''I and Ad Icvcls art InOCCUrOle. The prcicnt etudy : la tire above problem9 and thus Is s more rcllable ... I a~m~ltaneousquantltntlon of :, DItT and Ad fro- '\ sample.. -1

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! \ ' 3,17 1,783 United States Patent Office patented Mar. 2, 1965

1 2 ddccled with fonds and frors only if adeauntc volumcs 3,171,783 ol urinc arc grossly concentrated. DIAGSOSI'IC PHOCE1)URE The sensitivity of various animals to HCG is dcpendent Roy Tholna4 I%k, Glendale, Calif., auigncr to won a number of facrors, including species, maturity. I Iyland Laboratories No Drawing. ~il~dAug. 15, 1962, ser.so. 216,962 5 weight, season, environment, reuse, health and routc of 1 Claim. (CI. 167-64.5) administration. Perhaps the most serious handicaps in- cident to the use of biologic pregnancy tests is the propen- This invention is concerned with an iinliiuno-chcn>ical sity of such tests to give falbe positive reactions. Such pregnancy test. false positive reactions in pregnancy test aninialj are pregnancytests, in general, are based on the determina- 10 Positive responses to Substances other than HCG. For tion of hormones which are produced by [he developing example, fake positive reactions are noted with mice and placenta= gonadotropic hormones similar to those rabbits when high levels Of follicle stimulating hormone produced by the anterior pituitary gland and steroid (FSH) are prc5ent. Such FSH may be Present in large hormones similar to those of the ovary and adrenal gland. in nlcnoPausal Patients or patients with primary The majority of currently employed pregnancy tests are 15 ovarian failure. It is generally believed that a false posi- based upon assay for the placental hormone, human Ilve reaction in a Pregnancy test may have serious conse- chorionic gonadotropin (HCG ). quences because of the possibility of considerable psy- Human chorionic gonadotropin (HCG) is a hormone chological trauma 10 the Patient. For this reason and which is found in body fluids (blood serum and urine) for the other reasons, hereinbefore mentioned. attempts only during pregnancy and when rare hormone- roduc- 20 have been made 10 develop non-biologic pregnancy tests. ing tumors arc present. An Internationali&of One class of non-biologic pregnancy tests involves the HC='adopted in 1938 and is defined as the specific use of Or immunochemical techniques. gonadotropic activity of 0.1 mg. of a dried standard kept Since HCG is a Protein hormone, it acts antigenically in at the National Institutes of Health, London, England. a heterologous species. That is, when HCG is injected Human chorionic gonadotropin (HCG) is generally 29 by suitable techniques into a suitable animal, as for ex- considered to be detectable in the urine about the twenp a rabbit* antibody to HCG is produced. This fEhday of the gestation period. However. one%ould antibody to HCGI under Proper conditions, will combine be mindful that the "detectability" of HCG in urine with HCG to form a visible precipitate. In theory, this is influenced by the concentration of the HCG and the describes a classic antigen-antibody direct precipitin re- sensitivity of the particular test system employed. The 30 action* However* when related to the practicality of twenty-fourth day of the gestation period is usually about , the determination of Pregnancy in a human patient, this the tenth day after ovulation, but is called the twenty- Perfection becomes imperfect. The direct fourth day because gestation is conveniently calculated precipitin reaction is not sufficiently sensitive to detect, from the 6nt day of the last normal menstrual perid. in unconcentrated urine, the relatively low levels of HCG By the fortieth day, HCG in urine may reach a level 35 that are Present in such urine during early stages of prcg- of about 5,000 I.U. per twenty-four hours; from day nancy* in order to increase the I.U. of HCG sixty to day seventy there is a very high peal;, which may Per volume of urine, the urine is concentrated to reach 500,000 1.U. per twenty-four hours. While this 3 smaller volume, substances which interfere with the peak more commonly occurs at sixty to seventy days, it 4o Precipitation reaction likwise become more concentrated. may be present at any time between fifty and ninety days; Difficulties attendant to the use of a direct precipitin it rarely exceeds ten days in duration. For the remainder Pregnancy test have led to the evolution of so-called in- of pregnancy the level normally ranges be:ween 4,000 direct tests- Such indirect tests are based essentially on and 11,000 I.U. pcr twenty-four hours. an inhibition phenomenon. In such tests, ;intiserum 10 Thc variety of tests currently used for the diagno,is of 45 HCG is added to a Pirtient's urine. A urine specimen pregnancy affords ample evidence that an "ideal" pro- from a Prefnant woman will contain HCG. The HCG cedure has not yet been achieved. Historically, the most in such a specimen neutralizes the HCG antibodies in commonly employed test procedures have been based the antiserunl that has been added Lo the urine. Thus upon the response of various experimental animals to a carrier (latex Or sensitized cells) which has been coated hormonal substances contained in the body fluids (urine 50 with HCG is not agglutinated following addition of the .

and blood serum) during pregnancy. The first of these coated carrier to the urine specimen. Hiid the urine ' so-called biologic tests to be developed was designated specimen been from a non-Pregnant woman, the added the Ascheim-Zondek or "A-2" Test. In this test urine HCG antibodies from the added HCG antiserum would from a suspected pregnancy is injected into immature fe- have been Present at the timc of the addition of the HCG male mice. There then followed the Friedman Test in 55 coated carrier, and agglutination would have occurred. which mature, non-pregnant and non-estrus female rab- Although indirect tests such 3s these are useful for the bits replaced mice as the test animal. In both mice and diamosis of Prcrmncy. they have certain limitations. In i rabbits, the HCG causes ovulation and corpus lutcuni general, the Urine to bc testcd must be thc first morning formation, a responx which is observed upon the sacrifice urine. It is in such first morning urine that the HCG and examination of the animals to determine the presence 60 appears in the highest conccntration. Urine passed after of corpora lutca in mice and hemorrhagic follicles i the first morning urine generally contains toa low a con- rabbits, both of which indicate a positive test. 3 centration of HCG to be useful for indiiect diagnostic In recent years, various species of frogs and toads of tests. For the Same reasons as pertained for the direct both sexes have been used for pregnancy testing. AI- precipitin test, it is not possible to artificially concentrate though these animals are relatively insensitive as com- 65 low specific gravity urines for use in indirect tests, pared to mice and rabbits, they are at this time possibly It is an object of this invsntion to provide 3 direct (as the most commonly used animals for biologic tests since opposed to indirect or inhibition) test for pregnancy. they pose fewer problems in storing and handling. It is a further object to provide for the diagnosis of The A-2 mouse and Friedman tests are capable of de- pregnancy a direct test that is based upon irnmunochemi- tecting levels, of HCG of about 750 I.U. of urinary HCG 'O cal principles and that includes the use of gel diffusion per twenty-four hours. HCG levels of this order can be techniques. 3,171,783 .. 3 4 It is anolhcr objcct to provide an immucozhemical di- Thc antiscriini to HCG thus ohlained is thcn admixcJ rest pregnancy test th.it cniploys urine ;IS a test qxeirncn. with the ag,~. HCG of thc orJcr of xyLLw.U. pcr but does not limit the spccimcn to thc first morning urine. niilligram produccs antisera wilh a sufficiently low non- It is yet another objecr to provide novel equipment and HCG antibody content that adsorption is not rcquired methodology for immunochemical direct pregnancy tebt- 5 for the succezsful application of thc antisera to the gel ing. dw~~~:hniqueof his inwlion. It is yet another object to provide a pregnancy test Also admixed with the agar is glycine in an amount which minimizes the likelihood of false-positive rcsults. sufficient to prevent the furnlalion of precipitatcs which Other objects and advantageous features will become can result from the interaction of the agar and certain apparent from the following detailed description and 10 substances that are normally prcsnt in the body fluids illustrative examples. employcd for diagnosis OF prcgnancy. Such interaction me instant inventive concept is based upon the im- results in the formation of precipitates which can be mis- munological reaction between HCG and antiserum to interpreted as evidence of pregnancy, a so-called false- HCG, the reaction taking place in semi-solid media utiliz- positive. Seventy-five grams of glycine per 1,000 ml. Of ing the technique of immunodiffusion. In the diagnosis 15 agar will function to inhibit this interaction. of pregnancy employing this inventive concept, urine is Additionally, when it is inadvisable to autoclave and employed as the test substrate. The HCG, if present in thereby heat-sterilize the agar with the admixed glycine the urine, is concentrated by adsorption on an appropriate and HCG antiserum, microbiological contamination can adsorbent and by elution from the adsorbent preferably be avoided by the addition to the agar of a preservative using a total volume of eluting agent substantially smaller 20 agent such as sodium azide. than was the volume of the urine specimen. A small In the practice of the invention, melted agar, to which amount of the HCG-containing eluate is then introduced has been added antiserum to HCG, glycine, and a pre- into or onto a defined space in or on a semi-solid medium servative, is added into an appropriate container and is which contains antibody to HCG. The HCG in the allowed to solidify. To be appropriate, a container pref- eluate diffuses through the semi-solid medium and there- 25 erably will be capable of receiving the molten agar and by contacts the antibody to HCG. This contact between restraining it in a form such that the agar will have a HCG and antibody to HCG results in the formation of a uniform depth throughout. Also, the vertical walls of visible precipitate, indicating pregnancy. this container preferably will be of a height such that Several types of =mi-solid media can be used for im- when the container is completely filled with agar the muncdiffusion. Representative of gelling agents for such 30 depth of the 3P.r does not exceed about '/s inch. In a media are agar, gelatin, pectin and chemicals which preferred embodiment. small wells of, for example, about polymerize to gel when catalysts are added to their aque- ?hinch diameter are punched in the agar. For quantita- bus solutions. Undoubtedly, many other gelling agents tion the wells preferably are circular, but they may could be used, since precipitin tests can be carried out in assume any conveient form for qualitative purposes. In 1 or on any substance through which antigen or antibody 38 other embodiments adsorptive disks are placed in or on ' can diffuse in aqueous solution. The semi-sdid medium the agar. It is Only necessary that the structure em- ishould not substantially interfere physically or chemical- pb'ed be capable of accepting and retaining a rest speci- ly with uniform antigen (HCG) diffusion. Optimally men in such a manner that any HCG contained in the the gelling agent is. therefore, uncharged or negatively specimen will diffuse into the agar upon standing. charged and the resulting gel is of a cohesive structure, 40 If the agar-containing containers are lo be covered with having characteristics such that it will permit the passage a lid and stored for any period of time, as would be the of macromolecules such as antigens. Agar is the pre- Case with a commercial embodiment of the invention, the ferred gelling agent in the practice of the present inven- deposit Of water vapor condensate on the surface of the tion, because it satisfies all of the above criteria. In the agar or in the wells must be prevented. It has been description which follows agar is employed as illustrative found that such deposit of condcnsate will not occur if of satisfactory gelling agents. 45 the surface of the agar is covered with a material such in the instant invention, agar which has been as Saran Or laminated cellophane. Additionally, to per- fluid by heating, and antiserum to HCG are admixed. mit visual observation Of the PreCipitate which indicates TWantiXrum is prepared in a conventional way Pregnancy, the container is constructed Of transparent Or by inoculating an appropriate animal with Hiwhich 60 transhxnt material. Polystyrene. L! The urine specimen employed for diagnosis in the can either be relaitvely y-or relatively impure. instant invention need not be the conventionally employed Rabbi& horses, goats and s eCp have &en found to be appropriate animals. Perhaps the highest specific activ- first morning urineyhe Use Of Urine other ban first ity reponed for an HCG preparation is 12,300 1.u. per morning urine is made possible by the application of 3 mi(1igmm. Such a preparation is essentially a laboratory 65 rapid scmi-micro concentration tecbnique which forms curiosity. The nonantigenically specific HcG prepara- a Part of this invention. In this technique, to a moderate tions normally employed for the production of HCG volunlc of urine. e.&. Onc to 10 ml., in a reccptable such antiserum contain on the order of 2,000 to 9,000 1.u. 3s a test tube, is added an WkOrbcnt Under acidic condi- per milligram. The HCG antiserum produced through tions. An acidified suspension of kaolin has been found the use of nonantigenically specific HCG, of the order of 60 lo be a useful adsorbent for this purpox. The HCG about 2.500 1.u. contains substantial amounts of anti- in the urine under these conditions is adsorbed on the bodies to henon-HCG antigens contained in the H~G kdn.. The urine-kxdin mixture is then centrifuged and preparation. lnese anfibodies to non-HCG antigens the suspernatant fluid discarded. To the kaolin in the miemoved from the HCG antiserum since they test tube is then added an amount of alkaline eluting +. :::\ may give a false positive pregnancy indication by form- fluid which is Of lesser o!.u.me than was the volume . of the u;ably~con~~~s~*,-a~se f 2%'. ' ' ing a precipitate when brought into Contact with their ' corrrsponding non-HCG antigens which may be prewnt indicator to assure that the resulting mixture is alkaline. nonuavid urine. Normally, this removal would be Under these conditions Of alkalinity tbe HCG is eluted by conventional means, using a WnCentrate from the kaolin. Although tbe alkaline kaolin-HCG .i ~tmenopausaland nongravid premen* 70 mixture can at this juncture be applied directly 10 the t urine to adsorb the non-HCG antibodies. agar. as by Placement in the wells, it is preferable to first invention, when an HCG preparation of centrifuge the mixture and transfer the supernatant to relatively low IU content (on the order of 2,500 I.U. per the agar, as by pipette. milligram) is employed for the production of antiserum, The instant invention will be more clearly illustrated it is preferable to adsorb with nongravid human serum. 75 by reference to the following illustrative examples. 1015bl8 3,171,783 5 6 EXAX1I’I.E I EXAStI’LE IIT Picparatioil of tlic utitiscrutti Tesf procedurc Twenty mg. of a commercially availahle (Organon) (1) Place 2 nil. of clear urine in 10 x 75 mm. test tube. HCG having 2,275 1.U. per mg. are dissolved in 2 ml. (2) Add two drops of kaolin adsorbent and mix. of water. The resuliing solution is emulsified hy the addi- (3) Allow tube to stand one minute, remix and centrifuge tion of an equal volume of Freund’s Adjuvant. This for one minute to puck the kaolin adsorbent. eniulsion is then injected into a horse subcutaneously at (4) discard the supernatant and drain the tube against multiple injection sites. After three weeks the multiple adsorbent tissue. subcutaneous injections are repeated using a solution (5) Add one drop of eluting fluid, flick the tube :a prepared by dissolving 10 mg. (22.750 1.U.) of HCG break up the kaolin button and centrifuge for one in 2 ml. of water. After two more weeks the HCG minute. antibody concentration is sufficiently high to permit the (6) Use a capillary tube to collect the supernatant use of the antiserum in the practice of this invention. eluate and transfer enough to fill a well of a precipitin Five liten of blood were obtained from the horse. This plate. volume of Mood provided 2,700 ml. of serum. To this l5 (7) Add the positive control to a second well and a 2,700 ml. of horse serum was added 2,700 nil. of pooled negative contra1 (the negative control being the same nongravid human serum. The mixture was permitted to as the positive control. but without HCG) to a third stand overnight at 4. C. at which time a precipitate bad well. formed. This precipitate was removed by centrifugation. 2o (8) Incubate at 37’ C. for four hours and observc for To three parts of the resulting antiseruni were added an a precipitin reaction which will be seen BS a sharply additional two parts of pooled nongravid human serum defined circular mne of precipitation around a well. and the mixture allowed to stand in the cold (4’ C.) The zone surrounding the test specimen well may be for twenty-four hours. The centrifugation was repeated larger or smaller than that surrounding the positive and the resulting centrifugate passed through a Seitz-type 25 control well depending on the level of the chorionic filter pad. The resulting filtrate was satisfactory for use gonadotropin in the specimen. in the subsequent gel diffusion technique. For the most reliable test results, the urine specimen EXAMPLE I1 will not be more than one day old and will have been kept under refrigeration until tested or will have been hfateriuls enrplojed in lest procedure knolin adsorbetrt 30 foxn if prolonged storage is necessary. The HCG con- Sodiuni acetate ______gni_- 12.3 cenlration is usually higher in the first morning specimens Glacial acetic acid ______nil__ 75.0 and for this reason such specimens are preferred for use Water ______--___-ml__ 100.0 in the practice of this invention, although they are in no Kaolin-Celitel ______gnl__ 35.0 sense essential to the successful practice of the invention. 35 The eluting fluid contains an indicator to demonstrate 1 liatilil~of thc Oxliird-Eugllrh t)-p and Cellte 545 in a 1 : 1 n./w. ruisture arc used. that the elution has been conducted at the proper alkalinity. The eluate, when bronithymol blue is the indi- Eluling fluid cator. must be blue or greensih blue (not yellow) for One normal 3mmonium hydroxide containing 10 nig. satisfactory results. of bromthymol blue per 100 ml. 40 Overfilling a well may result in a surface deposit which might be mistaken for a positive reaction or which may Positive coirtrol mask a truc precipitin mne in the agar below. This control ion:ains 200 I.U/ml. of HCG (a stock While in the foregoing specification, a detailed descrip- preparation, Orgmon. of 2,200 I.U./nig. purity was used) tion of the invention has been set forth for the purpost in glycine-saline buffer (0.1 Molar glycine in 1% sodium 4j of illustration, it will be apparent to those skilled in the chloride) at pH 8.2 with sodium azide 0.1% added 3s art that many modifications in the details of these em- a preservative and with 10 mg. of bromthymol blue per bodiments may be niadc without departing from tbt 100 ml. present as an indicator. spirit and principles of the invention. What I claim is: A trtihumati choriotiic goiiadotropiir precipitin ngar 50 In an immunochemical test for diagnosis of pregnancy Difco noble agar ______gm__ 20 based on the observation of the visible precipitate which Glycine ______gm__ 75 develops when a specimen derived from urine and containing human chorionic gonadotropin is combined wilh Sodiuni chloride ______pi__ IO Sodiunr azidc ______gnL- 1 an anti-serum containing antibodies to human chorionic Water ...... nil__ 1000 59 gonadotropin. the improvement which consists essential- pH adjusted to 7.1 with NaOH. ly of the combination of the following steps: (0) preparing crude anti-serum to human chorionic With 3.5 parts by volume of the molten agar prepara- gonadotropin by injecting an appropriate animal with tion at a temperature of 57’ C., there was thoroughly human chorionic gonadotropin and after a period of admixcd 0.5 part by volume of the antiserum of Ex- 60 time bleeding said animal to obtain said crude anti- nmale-. 1. . and the admixture was used to fill a rectangular serum, transparent plastic plate. This plate was internally 1-inch (b) treating said crude anti-serum with human non- in width and 3 inches in length wtih vertical sides gravid serum so as to remove from said anti-serum inch in height and with a Vi inch flange defining the those antibodies which are non-specific for human outer upper perimeter of the vertical sides. The agar765 chorionic gonadotropin and recovering purified anti- the plate was permitted to harden and a series of ve serum. circular wells YI,;inch in diamcter and 95 inch apart (c) combining said purified anti-serum with a liqilid were made down the center of the long axis of the plate. gel-forming material and a minor amount of glycine Each of these wells had a 10 microliter capacity. The and depositing a thin layer of the combined ma- surface of the agar was then covered with a sheet of 70 terials in a suitable container, Saran and the plates stored in the refrigerator until (d) after a gel has formed io said container, providing needed. at least one well therein adapted to receive a test Hereinafter. in these examples, when reference is made specimen, to precipitin plates, such plates are the plates of this (e) admixing a urine sample taken from the test example. 76 subject with acidified kaolin whereby the human i015b13 3,171,783 7 8 c5orionic pn3dotropin is nbsorbcd on said kaolin. Chorionic GonaJolropins," Expercn:ia 15, pp. &+&I, Drc. (f) >cparalir,

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. insrference of aureomycin with penicillin oc- hypothesis that the presence of a tact&* ats 2ot only in :.irra but ahin mice in- static concentration of chlormpbcn~, oculated with either S. pyycircr nr IC. pnru- aureomycin. or trrrsmycin may inten'ue wjb 7mmiw(7). Mice treated with both penicillin the multiplication of bacteria. Since pc& and surmnyin showed a hiher dratb rate cillin principally affects dividing mi- than did tho= treated with penicillin alonr. organisms such an interference with multipli- It is of interest that not only chlornm- cation micht result in interference with peni- phenirol but a11 3 of the '.newer"- anti- ciliin action. Future eqxriments nil1 hjw biotics that are wide!. uud c1inic:dIy. intcr- to esplore this h>pthesis. fere with ihc hxtrricidal action of penicil!in. \\'bile striking mnnifefiativns ai antihitic IYhile these nzrnrs hare ctrtain Irxtitrcs in :in t ~pnisrnhave been produced exprimen. cvmnwn thrrc is no rvidencr th:it they re- tally in niice it cannot beMrmed that Lbc sernhlr one mother chemicslly. To date no phenomenon may be of importance in the r\irlcnce hit; apprJred siqestinx that thr antihintic therapy of human pitirnti. It interirrcncr ni thrw drugs with pmicillin sh41uld be noted. however. that the drur Icvels aclirm is due tv a direct' phyiul or chrmicfrl xhowiin_u antibiotic antagunism in tdro snd intcrxtion. (Mer posiible mcch3nismi for in experimental animdr fa11 in the nnye com- tbii ..;intibiotic mtagonim" have hrrn pre- mrtnly olit.iined in human hotly duids and \+rdy su;:c-:ed(4.6). It might hc that the tisatrs with ordinar?. theripeutic dose. ? interirrin?: r1ru.p compte with one ancttfitr S?tmmm~.Aurmmycin and terramyin in- fttr -receptors" on the hrctrrid cell. So e+ trrfrred in :.ih n-ith tht bactericidal action tlrnce ha5 lmn protlurcd directly favoring tbis of penicillin apainst S. pp,pez and K. pnru- pmsibility. One druq miFht alter the dc- trrnniur. The early bactericidal rate w vzlopmental characteristics of the mirro- dainer with mixtures of aurcomj~nor tm- or:sni.im in such a way as to diminish their myin with penicillin than with pcniallii by su.

7. Jawrtz. €. Gunninn. ]. H., Speck:, R. S, and - -I-- Ctikman. V. W.. un9ublkhrd obwrvatiolu. Rccdvrd October 1s. 1950. PS.E.B311, 19% rt3. .

-A Serologic Study of Chorionic Gonadotrophin. (18263)

LEOXIRDH. Sctxvurm. KITRIDGE.-\SDERSOII, S~vvrrSasuw ASD CYRCSc'. Enic~;sos.(Introduced by Douglas H. Spruni) . From :kr Drpucwt o! Parhdnty. Drkr C&mity .Ilr&ul Schd, bz)o+lnrd 01 B).cim&gy, Army Yrdiol Drpwtwmt RueurrL adGrduair Shod. .-- ..-

The antigenicity of chorionic gonarlotro- $munized witb gonadotrophic hormone IC- phin' hu been suczestl by Leathem( 1 )t act=with-thse Eonnona in a sariety of Leathern and Raknff(2) and others. C'huc(3) immunoloqical test procedures CG in urine has demon-tntd that tbc wrum of rabbits, ------2. btbcm. J. R. and Rakol. A. E. Am. 1. 'Wcmftcr desimkd as CC Obrrrr. end C-mec, 1948. vSb. Sa. 1. ktbra J. H, 1. Cdndnrrhd. 1%. -4, 3. Ch.s,'J, Pa& 1. lid. #d Yd, 1943. TI?. .w. 517. sera any fac~urswhich reacted with urine from normal nonpregnant women. t!!e fol- lowinx absurption prwrdure conducted. Each *rum ihoning detectable antibock \vs ietd uith a group oi urines from pie- nant and nonpreunmt wonicn. Known noa- pregnancy urine5 which reacted with the CG antisera were u.4 in absrptjon. Thee uriws ncrr sterilized by riltraticm and added

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sults of !hi* cmpariwn are hhonn in Tahlc 111. It wa.i drsirnl to deternine whether the antihdirr which z3vr a pxitivc reaction with *..\l'L" Rere thr samr as tho* which reactd with thr urines [rum pianant women. Srru51 was &orbat aitb ".4I'L" usinc the mcthld dwribzd atme. This al).wrbecl serum nu loncer reacted with -.\PL" or I*iB the 19 presnsncv urine tetcd. Similuly, j~mm abwrbed with prcznancy urine failed to rc- 3ct with the IO prmancy uriw uml or with *..U'L.*' Dirrussinn. The specificity of chorionic gonadotrophin antiserum is of paramount im- pvtancr if that serum is to be used to dctm the presence oi the hormone. Tbc first wia of SS urines were tested smlogdly mtb unabsorbrd antiserum. Positiw precipitin tests were ohtsind with 42 of thc 48 urincs positive by the rat ttst: and fdsc @ti\- te.ctr were found with I? of 37 urine nea- tiw by the biolovic methotl. In tbe next wries. XOurine specimens were tatd with abrwl,ed *rum and the results were corn- 1wtd with tho- of the froK test. One hundred and three of thr 1Du prtpnuq urine zavc psitiw precipitin resulu. while 6 of the 91 urine n-tiw by the fmy t-t reacted ~rnloyically.tbus constituting 6 fslse psi- tivs TS- results indicate that the mr- relation betwen the biokgic and urologic rnethwis or dctrcting CG in urine is greater ahrn ahwrbd ;intiwrum is u.d. C'onsr- quently. brfnrr the sntiwrum is med for tFt pury~ses. it is nccrjury to determine the potenc? 3nd qwcially the specificity of the serum. It is nut to be implird that the antigen u-4 in prgarinz t+is xmm was purr. It is cnnceivahlr that the antism~mprepared with r more highly purified hormone prepa- ntion rnivbt give serum of higher gaency and .pmrr specificity. The prrcipitin test wpas cornpard with the Salmon rat tbt and the frog test. It bas hcrn found that the Salmon test pr- fomd at Duke Hospital has an accur3cy of 987. .\t t!e present time, it is not posible to .+Ute r.uctly the accuracy ol the irq test For our putpow in this study we haw used

.. .4oirs~ias11 LSYPHOaux ~lrr4~01.rsu 359

t~,ebiolo4c te>t 52 a buis of cuniparis)o: ducd detecbble antibodies. 2. Two hun- i!in1\ith the biuiai+caI wp. pouible to use rhis serologic method for the detection of prcgmancy; the develojment of Summery. 1. Seventeen rabbits wen im- .. 31) mtirrum of somewhat grater spcrihcity niunired with .'.\PI-,'' which a combined is would be advantageous. cstrxt oi hum.tn placental tissue and peg------trinsy urine. kenrabbits (6 iem3lcs) pro- Rcc~ivrdjcple~~bcr 25. 19-W. P.S.E.R.JI, 1950, v75. ., . : ..I: .

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int.tors ani: h:: r;tir.eyhrine upon the storage, (9-11). i brr3kiitnvn ani! atilizlrtiim of csrlwhydnte .I knowldxe of cartrthydrate chances h.i\-c btvn dr~c+A:it length( 1-5 1. .\lttrouuh drhrn the lymphoid ormu. induce41 by d- i 8 >:!itlirs ha\? uiadc of the inhence of itratiuna in ailrenal honnone levels. might tbe adrmn! g!nnd upon the quantities of hrlp to eliicidate further the role oi this end+ c:, r b-~hy d r 3:c 5 : ih I t mce, pri nci pdy &CC+ crinc gland in drienu rnechaniims This ?' Zen. in the iiver. muscle and CeRain other rcpm &scribes exprinients tledinu with the orsans(o-S). mi attention has knpaid to chrnnic effects oi adrenaktorn~and cortical the parr pla:;cc! !?' this gland in the c3rb rqrac t or epinephrine ~dmini+tratinnupon hl-drale nirtdfiJism of lmphirl tissue. This the dycopen content of Ipphoid organs. th ia surprising in -.iew nf recent work which li\er z!>cuum and the respiratory activity oi h.4~ snoun the inrprtmce UT the I>mphoid tbz?+ tissues. organs in 3 vxrkt:: oi defmw rmctions within .V~~tt<and m.thods. 1. ..Inimdt. Fe- the body and w.hi:h hudresrd :he influence -.- --_- - --- male r3ts of a hsrdy:cloxly inbred strain, 1. B;ittun. S. IV... AIM~ Silvct~c,Xi.. Symp. QUI~.~. aduhinp: 140-170 p 3t the time oi operation. Bid Cold .ipv Harbor. 1937. v3. 337. were d.The 36 adrenalectomized nu 2. !%IAITX~. R.. .In.3. PhFSiul.. 1940. \-lfl. 36. employed were divided iotn three pn,ups, 3, Lrmu. C. S. Ldorridurlnsy. 1942. vJ0, SiO. ti.. br first of which consist4 of 10 mts. to 4. RwU. J. A,. .in. J. PhydCJ, 1943. vWJ. 98. --- ~____ 5. Incrk. D. J.. Proc. Am. fib. AWL, 1% vg 3. 9 hugbefly. T. F, and Whne. .4, EEndo~n'n~l~g~: . :.-. b. .bdrrula. E. and BcrrtU. V. V- p.uC. Suc IW. v35, 1. : Esr. Rior. ASD VzD. 1910, TU. 362. 10. Dourbtrtv. T F. ud W%itr. A, 1. Lab. ad > .... . 7. Con C. F, EaiaUgy. lplQ vZb. 285. CkYrd. 1W7. ~32.5k. kupi. Ham. W, GmmiU. C. 11. Gurdols A. md dam. S. Y. . -. S. ti F.. H. t. S, ha.C. F, .I and Thorn. G. K.. Am. 1. Plyd, 1%1, VI)?,175. dcad. sci, 1949, vs2. 1. 7. I. ...\

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-7- ,)I l1irl 111 a tiirl ionlrtil. 'l'lic I :\(Eaiid Patent and Trademark Office lucl conrrols scrk ICI Irgiil.~t~*tucl Board of Appeals ,5 a lIiiictIon cdthc. ilid\s of;iir flc)wiiig ,gh rhr enainc. Both coiiirols ;ire rv- Ex ~idi.ti-Ki.cii/w ,~\eto vanutioiis in eiigiiie spccd to &a signal that is a function of spcrd Opiiuon dated .1iiI! Y 1. 1978 ,n dddition. both dre responsivr IO en- qx-rational paraincxters b.id 011 PATENTS Craiurr and pressurr of the air to pro- 1. Patentability Utility ($51.75) bel How as ai iiltiniatt. function 01. - air flow. Iiifrin enient is clrar. as Significant use for invention claimcd hr MFC and ISt 7 fuel controls per- must exist in order to satisfy uiiliry '- mbstantially the same fiiiiclion utiliz- rcquiremcrir of 85 1I.S.C. 101; claiiited ubstantially the sanae mechanism in compounds that are described as exhibit- mtially the same manner. ing herbicidal activity are disclosed to be re. in this case, the claims of the "useful." ,ptciit are not to he narrowly con- 2. Patentability Utility (P51.75) din light of the prior an or the Mock - kation and tile wrapper. the trial Exainiiwr inav rrquirr frirrhcr assurancc PX- :'I original opinion with respect in in- of usetirlness whcrc rcasotiahle doubt ment will not be disturbed. Inde- ists as to whc-thcr in\ rniioii will liiiictioii as mi of that conclusion, the issue has stated. notwithst;lnding asserted utility. reviewed in the li ht of the record in 3. Patentability - Utility (351.75) s,including thetriefs of the parties ;were before the court. I hold that in- Heading and practice in Patent Office ment has been established. - Rejections ($54.7) Ires ect to the broader claims I, 3, Examiner who disavows any finding that 1. inhngement does not de asserted utility which, on its face is not resort to the doctrine of equivapend ents to generally accepted scientific these claims do not contain the '""'"7princip es, is unbelievable. im ropcrly im- a'' language. These claims, being puted burden of proof to appP Icani; exam- cr than the claims sought to be nar- iner's unsupported skepticism as to 1. are infringed by the accused de- claimed invention's utility docs not provide legally acceptable basis for rejecting claims. qplemcntd Conclusion of L.w 4. Specification - Sufficiency ofdisclo- sure (062.7) n the findings of fact and opinion bne 19, 1975 and the supplemental S cification that discloses inveniion in a. supplemental and additional sucrmanner that one skilled in art would Q of fact filed herewith. all of which be able to practice it without undue Jde a part of the judgment herein. amount of experimentation need not con- U~Iconcludcs as a mater of law that tain working example. ms 1 I, 24. 25. and 28 of Kunz Par- 0. 2.720.75 1 are invalid, wherefore 5. Pleading and practice in Patent Offa- 5ition is dismissed as to that patent. - Rejections (OM.?) :~clairnsl,S..I,5.7,8.ll,1?.l3,14, Specification - Sufficiency of disclo- ;of Mock Patent No. 2.58 1.275 are sure (562.7) ad infringed, wherefore defendant t for infringrnieiit of that patent. Patent and Trademark Ofice Board of dinglv. plaintiIris entitled to recover Appcds docs not sustain rejection under dgment is entered to that effect. The 35 U.S.C.I 12 on ground that specification a of recovery is reserved for further fails to set forth bcsi mode contemplated rdirigs under Rule 13 1 (c). by inventor for anying out invention, ab- hgs omitted.] sent cause to surpccl inventor of concealing any infomuon a# to what he fech is preferred embodiment. Particular patenta - Hdik Krenzer. 1-Thiadiazoly L-5- Acylimidazolidinones. claims 1-10 allowed. 228 E\ \Nil tv

.-\pp~alfrom ~~Iotip122. ,Ap 1ic.1tion lor pa~ciiiol .lohri krciuer. Scriar Vi), 57 I ,Mi. tilrd hpr ?5. 1975. Frcim dc-c-istonrej~ctitig daims I- IO. iip jli- cant Appeals (Appeal so. ~IO-L'O). kc- versed. Rohcrt J. S

\ The Exanliner'$ o5iIion herr is tlia ;ip- plicant has iailrb)ro pro\ idc I he record ! with atiy \,a\ir at all hr011s linding. oiie wa! or ttir oiher, dbout thc brlic*vabilitv of IllC 11t1111\ ." (31 Regarding the abobe. the examiner has improperly iinputed the burdcii of proof to aplx4ant. Howeber. the court has All~ol'ihc clams also stand rrpiccl already passed upon esseniiall\~this bame "uiidrr 35 1'SC I I?because thr s1)vciIita- matter. albeit' with respect to "enabie- rioii herein fails to set fimh the besi modr ment,'' and its holding appears to be contemplated by the invetitor of carrying uniquely applicable herein. Thus. atten- out his invention" (Al1swc.r. page 4). tion is directed to the decision in In re Mar- [I) From the examiner's explanation of' i zocchi et al, 58 CCPA 1069, 1073, 439 his osition, it appears that he is errone- F.2d 220, 169 US 367,369. cited by ap- ousP y equating best mode with workin ex Ilant, wherein JuPg ge Baldwin. speaking amples. However. a specification nectfiio; ' Er ilic court, stated: contain a working example if thc inbention "tis a matter of Patent Otlice practice. is otherwise discloscd in such a m;inner i then, a specification disclosure which that one skilled in the art will be able to . contains a teaching of the manner and practice it withotit a11unduc. aniouiii ofcx- process of makin and using the inven- primentation. In re Borkowski et al. 57 , tion in terms whict correspond in scope CCPA 9.16.950.422 F.2d 904. 164 LSPQ to those used in describing and defining 642.645. We havenodoubt that theinven- the subject matter sought to be patented tion at issue is disclosed suficientlv to must be taken as in compliance with the comply with the criteria set forth in Bor- enabling re uirement of the first para- kowski. ;' graph of 3112 unless there is reason to [S] In response to a s citation of doubt the objective truth of the state- In re Glass, 492 F.2d f)'$$!!r:i USPQ3 I, . ments contained therein which must be the examiner points to the statement 3 relied on lor enabling support.'' therein that failure to set forth any mode is Although the Marzocchi et a1 decision equivalent to non-enablement. We invite subsequently acknowled ts that. in some attention, however, to the very relevant o,a statement. on itsyacc. may be con- discussion immediately followin the sen uuy to generally accepted scientific prin- tence to which the examiner refers. Said apks. such exception is not involved here- discussion uotes with a proval from In re a. Instead. we have before us the type of Cay. 50 &!PA 725, 74. 309 F.2d 7G9. +ct matter and associated circum- 772. 135 USF'QSI 1. 315, which states. in mnces described in In re Gauve. 54 part. that the best mode provision la]: CCPA 1524. 1530-1531, 379 F.2d 973. "* . . requires an inventor to disclose I54 USPQ92.96. as follows: the best mode coninplaid by hrm, as of "Appellant's discovery here does not the time he executes the application. of ar to us to be of such a 'speculative,' carrying out his invention. Manifestly. rmseor esoteric nature hat it must the sole purpose of this latier require- bc considered unbelievable. 'incredi- ment is to restrain inwnton from apply- bk.' or 'factually misleading.' Nor docs ing for patents while at the same tiine opemtiveness appear 'unlikely' or an as- concealing from the public preferred xrtion thereofappear to run counter 'to embodiments of their inventions which they have in fact conceived." what would be believed would hap n the ordinary person' in the art. Er "As we view portion [B].we think that docs appellant's field of endeavor ap an invenlor is in compliance therewith it r to be one where 'little of a success- he docs not conceal what he feels is a !referred embodiment of his invention Pnature has been developed' or one 0'' rhich 'from common knowled e has bng been the subject matter oB much Since, in this case, we have no cause to binbuggery and fraud.' Nor has the ex- suspect the inventor of concealing any per- miner presented evidence inconsistent tinent information he may possess, we shall not sustain this re'eaion. rnrh the assertions and evidence of rativeness presented by appellant" me examiner's decision rejecting %notes omitid). claims I-10 is reversed. Gtnsonant with the foregoing, the ex- RMd dr's unsupported skepticism as to the v DEVELOPMENTAL DlOLOCY (includc s EmbryOloCY) 7945549461

a . .!cr, and [unrtlon In lhc drv*lo ins rrlh of Ihr froc IadpIC. dbbns conhins bony lunula. In niu this nirniscus )us nicrgrd -i:& 1{UCT IGS 30(1 2): :;-IC?. Ulumu,--Thc 1st wllh the trlinylar articular disk. forinin,: a coiiflurnl proxinial arlirular ., .rrllT&phrc rcsponsc IO 11cli1 in dcvcloying t,u!;wlcs surlacc and conslrlcliny. Olf a di\crliruluili of thr synovial rac!icxarpal (e cavity, thc prcsiyloid rccc:.s. uliirh niay Idl:r Uir ulnar slyloid proc,.ss. ,=) a prcly cornca-ncir;rtivc slow pntcntid .I,ICII --,;rndcd in time of lsl aypcaranr+' ai13 thr 1st aiywarancc ol The prcscnt sludy rrz~scsscsUie dcvclop:iwiil of Ilw h;*nr.in Ii 1.11 join1 VI thc IIGhl of lhcsc fiiidin!:s. Inilia;ly >clt.ir.tlt. r:ii!i,*,:.%,', ..I. J rrccpfor wter sccnicnls rl vt,.ilrd by rlcclruii nircrosropy. .. ,ruge (6-7 dlys lllcr lCrli!i?-ili

t.79461. SALZEIt. PETER,and KARL TtIEILER. AN^. Lnsl. Unlr., Zurich. Srilz.) Ubcr einc bcsondcrc K!a~x.. am Ductus vcnosus Aranlil hi drr Irlaus. [On a sp-cia1 ralvc h Ihc duclus venosus Aranlll of lhc mouse.] 7. AN'A'I' EKTH'ICKLUNCSGESII 1m: 31-94. Illus. 19~0.Ik:nl:I. sum.i--A vatrc IS urcscnt on ttie rtdt side 01 thr duclus vcnisus. a1 vlc sllc d 11s )unr.tion vim lhc inl&or VCNc~va. It scpralcs Ihc duclus vcnosus Irtm UIC niiddlc and Irfl 1015'1110 52053. TOYAMA, RYOKO and TO\lO:;ORl IXUhllCIII. (Ec. Vet.- ZmtWh. Nippon, Sippon, Jap.) [Rcchcrrlics lordamcnulcs sur IC Rat d' "Hvdro-lcslis': I. Dcfaillancc dr Viril,lr chcz IC Rat d' "Ilgdro-

as virile in aplvarancc as normals, but thcir tcsticlcs showcd sicns .. of cflrcmc atrophy and had a browiiish color llkc tl~atobscrvrd in hy- prrscntalion Uicrc was rw) Icnlcncg for llic frcquriry, duratiuii md in- poyhysW~oniisct1animals. Thc niativg ol 2 malcs with h!dro-.tcslis tensity ol ulcrinc contraclions lo Incrcasc with Ihc procrcssion 01 prrg- 30 fenialcs did not rcsult in any iwc{:rrancirS; 25 Of llicsc Icnialcs mncy. Thc frcqucncy was considerably lcss Ilian In parlclal prcwnu- Yere lhcn niatcd with normal malcs aid bccainc prrgnanl normallv. lion. and in .wmc C~SCSof pelvic prcscntation contractions wcrc not ! The 5 other fcmalel had a gcililal Iracl lc?r whch disscctlon rcvcalcd rcgistered durlnc prccnincy. With labor the frcqucncy rcachcd the abnormalilics. Allhough malc rats with hydro-testis manilesl nor- mlucs rccistcrcd for paricfA prcscnl2lion. Discoordlrutcd contnclile -1 copulatory urges, lhey do no1 induce pregnancy in Ue fcmales. activity occwrcd in -la,and myomctrtc insdficlcncy in thc form of -4.L. R. vcak labor aclivitv was morc pronounccd in 37.17, comparcd lo plrie- tal yrcsentatlon. 'Jilwn thc fetus was lurncd from thc pelvic IO thc pric- 52054. mFFG\, G., J. SCIIVLZ, I. STUUUE and A. RICHTER. ul cntl the naturc of contractile activity did not chngc. Thc hck of (h~arg~ctc-D1mk-S1.8, 701 Lcipzic:, East Gcr.) I)CZiChunpcn zwischrn growth in rnyomrlric activity and Lck of dilfcrcnlllion bctwccn dil- plachgclmrlsvcrhIltung und Schwcrgcbwl bcini 1lir.d. Rclilions bclwcrn fcrcnt forms ol pclvic prcscnlation and prcrcntivr rotation may bc as- rclcntion of Ictal mc-nilirurs ml rlifficui! birlhs in cabIOKATSIT sociatrnl with inconiplctc hnctioniiy of lhc ncrve rcccptor apparatus VETERJNI\F.JWED 26~l'ETT33:8Tl-57. [Enpl. and rtuss. summ.1 of Ihc uterus, uslLllly due to Structural-morp~lopicalchanges in the --The frequency (if rrlaincd Ictal nirintiraiics IS rcimrtcd with reference tissues of ule uterus.--N. L. G. io evaluationof i82 tlillicult births. A Iiricf iiitroduction conccrning mc etiology and p1thof:rncsis of rctriilion is iuilowcd b:; an attempt lo PHYSIOLOGY Ah'D UIOCIIEhllSlRY eslahlish corrclalions bclwccn Ihc pcrccnugcs 01 obscrvcd cases of rclcdton of Ictal mcmbranco in coruicclion wlh diflrrcnt obstetric t 520CI. CUPTA, P. K., PARVATI K. hWLk;\SI and KAAWL DIUSIS. diagnoses as well as with rclcrcncc lo various thcravics, absence of (All-India Insl. hlcd. Pci.. NCW Dclhi. Dclhi. India.) Crlluhr rrsLnwr bruccllasis, vitality, Irerhncss, and wciChl of Calvcs.--P. L. W.

52055. YAhIACALlI, TAKESHI, TOSlllAhl IVISIIIhIUM, MASAKATSU ~ -.__ __ .. . . ABE and AKITSU TSUNAWAU. (Dcp. Obslct. cynccol.. Kurunie Unir. Ihc objL;(ivc of cluriihtinl: thc mode of aclion of an Lntraulcrlnr con- traccplivc dcvicc [IUDi. The lwsc ccllular inatcrial nccu~iiu~a~cdin the ulcrinc cavils and on Ihc surlacc of the IL;D was studied in 50romer. Thc dcvicc hail bccn in usc for pcrlds ol linic varying from 24 hr lo ahrmal dctivrry in clinical praclicc arc discussed.--P. L. W.. 84 mo. Thc prcsunicd biolgicallr incrl IUD material sccms to pro- voke an acule inflammatory rcsponsc which 1s probbly the IorerunMr 52056. RITTE;.'BACII. P. (Srk. Tirrprd. Vet., KArI+larx-Univ., of rnacroph.age accumubtlon. Tlic macrophages appcar about the third Lcipzic, East Gcr.) IMphalllr bcieim Ilau~hNnchcn. IDiphallin in Ihc day alter lhc IUD inscram and prqrcssivcly Increase in numbcr in &mlcstic nl9Jit.i 2 VERSCCIITILRK 13(3): 167-1mus. l9c proportion IOthe duralion of usage. Cytornorphaloglcal changes were 1Encl.,- ..~. sun1m.b-A ram obscnation of diphal:ia loulis s. pcrlcch in obscrvcd In thc endocervical smcarr. Structural chawer in Ule ILQ Ihc domcslic iabbil is dcscribrd. This anomaly was accompanied by ltscll also occur. a.uMotalduplicationofthc urcthra.md was not Iollowcd by functional rc- hction of reproductive ability or morphological alterations.--P. L. W. t 52062. DOHN, 11. (Uchrimcrkc AG. hfarlrurp, Wcsl Cer.) ClchrTl! und Charaklcrisicruq von Scliw~crschaflsprolclncnIn dcr nirvrh- 52057. TERZI, ICIXO and SEnGlO KJRDISO. (Osp. Maria Viltorh, lichcn Phccnta bowic ihrc q&intitativc inirnunohclschc Bcstimmc-z im Dlv. Ostct. and Cinccol., Torim, Italy.) Limili c pnssibilih wlh diag- nosi di cravidrrnza protratta con la studio rlrlla ci10:oci~t dcllc nicmbrane

987--6G. 1970!rccd. 1971 ].--Followins 3 drsrription 01 ihc c>

5ZOSB. DAYANOV. F. V. K voiirnsu Irchrniua iirvolwtivno-atroli-

,871) vypbIS!iCll VOP PATOL OtiKOi,, 1370, So. 4.53.1121.-- -rr.ty-nnc piticnts wcrc trcalcd fnr invo~utio~u~-atroy~:lcdisrascs -''L: wl,.a, lasliw Lrtwccn 3 mo. and 23 vr. Thc Ircatniriil 01 ncuro- *..*kr"\~~~il\ulml iiivolution inclulcd cor.scrntivc mcaxurcs ad ' '*.'r21-~vocalnc6cnrrvation of tlic rulm. Thc kttcr metlid was *-4 in 11 ~ollcntsuxiflrclcd by consrrvalivc Ircalmcnt. Thc usc of . -.,~~~-novi~crine block protlucrd a lull cllccl in 6 palicnls, manilcsled ' q.ipl%.ar.iwc 01 pruritic parcsthcsia, normalization nl thr wcll- *,f p.sIlrnls, nornial ronristrnry and sofllrss 01 Ihc vulva~sucs, * 4 **

Aktcnxcichcn :

Datum:

- .) ...

. Alrtmxcichcn: . Hoe 7118 020 - IU .141

hitun: 39. Novemb8r 1971 'L.

..._

. -3- 2157610

f Y -c

8 e' OWL-tr-2622

, Arch, Qnak 208: 266-274 (1970) .

ORNL-TR 216 2 2 .. INNUNOCHEMICAL INVESTIGATIONS ON THE PROBLETI OF THE "PREGNANCY ZONE" .

'ReHofmann, J. Brock, and H. Friemel

Univereitats-Frauenklinik Rostock (and Institut fur

Physiologische Chemie der Unlversitat Rostock)

.I .I

'* 8

/ Transiated from the Geman by .I R. Cregg MoxtficId Office q? Language Services Infonction Division Oak Ridge National Laboratory July 1972 I .. 0

i 1015118 I . ~OCHEPIICALXNVESTIGATIONS ON THE PROBLEEI OF THE "PREGNANCI ZONE'^ *' IV. Demonstration of Pregnancy-typical Antigens in Protein 1 Fractions of Human Placentas 3 R; Nofrnann, J. Brock and H. Friemel .. I .I - ' I./-'/ Universitats-Frauenklinik Ros tock (and Ins titut 'fur Physiologische I Chemie der Universitat Ros tock) fr'i .. i*.t I

Abstract : I. I , An extract from a human placenta can be fractionated by means of I salt-precipitation, gel-chromatography with Sephadex C-200 and ion ex- . .. . change with DUE-Sephadex A-5.0. Thereby, in 4 placental fractions i' ' altogether 5 proteins typi,cal of prignancy have been found. For obtaining

. antigens and production. of mono-specific immune sera against pregnancy- typical proteins the human placenta is suitable. I ! .' _- Introduction

In the preceding studies total 5 pregnancy-typical antigens .. - a of

were obtained in pregnancy serum, puerperal serum, serum from the a 1\ L ' &/.- pt-fyrurwc- umbilical cord and in the amniotic'fluid by means of Ouchterlony's SfrJ- a method of immunodiffusion and Scheidegger's technique of immunoelectro- ,i'nk ?;A ' G.r 4 phoresis, using absorbed antipregmnty and absorbed antiplacenta immune .4-= ,A-q**f'9 serum and a mixture of both immune sera (2c). jq&4.: *nf# . Wilkin (4) found no identity of the "pregnancy zoce" (pregnancy- / typical antigen no. 2) with antigens of certain placental extracts.

Bayer (1) sgrees only with reservation with the statement that protein I produced by pregnancy Is present In the plecenta, since, according to his opinion, In sddition to interferences by serum components the exact

c 1015119 t -2-

..-. I .- , position of a line is difficult to objectify when reference lincs are . )' I lacking., The purpose of the present work is the detection of pregnancy- i p or fi0JC typical antigens in protein fractions of human placentas Py comparative 4.0

imnunochcmical experiments. At the same time the problem is investigated ei 6: I p9. 1 r..C: as! io the extent to which the separation of placental proteins by gel fd* ,p hu*@ chzomatography and ion exchange can meet the requirement for the prepara- f >. ' *' I. - ' tion of pure antigen and hence for the preparation- of monospecific antisera _against pregnancy-typLca1 antigens from placentas, ----_ ---- c -.- - .. 8 Materials and Hethods 8' . Data on the preparation of immune sera, their absorption and mixing,

dn the technique of immunodiffusion and imrnunoelectrophorcsis , on con-

centrating of solutions of antigens, determination of the electrdphoretic

. mobilities of the pregnancy-typical antigens and preparation of tne pla-

cental extract may be found in the parts concerning methods of papers I,

. XI, and I11 (2, 2b, 2c). 1. Precipitation with ammonium sulfate. To placental extract C at e-. . ..-.._ -. -----.a- .. -.. . - ,. ~ a pH of 7.0 and at 4°C ammonium sulfate (analytical grade) is added to p-f I 8 degree.of saturation of 0.4 (3). After 12 hours the precipitate is 9

I i centrifuged off in a cooled centrifuge at 20,000 g (K24, Janetzki, '. e, ' Engelsdorf), dissolved in pH 7.4 Sarenser. buffer and again salted out / with ammonium sulfate (degree of saturation 0.4, pH 7.0). The precipitate

is centrifuged off at 20,000 g and 4OC, after ~i further 12 hours, then

dialyzed against pH 7.4 SYrensez buffer hnd coacentrated in Sarensen

buffer with polyethylene glycol 20,000 (Fluka, Buchs).I

/ I *

.. 1075120' ? I e

I 2, Gel chromatography. Sephadex C-200 served as carrier material. rl-c.., ---" - .) 3. Chromatography was performed by an ascending method using SISrensen

buffer, pH 7.4 at 4OC. The gel column had a height of 870 mm and a I diameter of 25 mm. The flowrate amounted to 10.1 ml per hour and was

# 4 - maintained constant with a peristaltic pump (4912 A-2, LKB). 5-ml fractions were collected. The photoncter for the dikicharge -- Uvicord

I1 with recorder (LKB) -- Served to record the extinction at 280 NP

(discharge cuvette 3 mu).

3. Chromatography on DEAE-Sephadex A-50. Fraction 91, after chroma- +.. -. -- tography on Sephadex G-200, was concentrated in a dialyzing tube against .

polyethylene glycol 20,000 (Fluka, Buchs) and then dialyzed against 0.05

trie (hydroxymethyl) a nomethane/HCl pH 8.0 for 48 hours, the buffer H- .. 7 I being replaced after 24 hours.

The chromatography'on DEE-Sephadex A-50 was carried out'with a linear gradient 0.05 M tris-(hydroxymethyl) aminomethane/HCl pH 8.0 - 0.5 M tris-(hydroxymethyl) aminomethane/HCl pH 8.0 ati4.C. An automatic measuring apparatus served to produce the grad1t nt (3). The bed of gel had a diameter of 24 &, a height of 370 mm at the beginning of the

'chromatography. The flowrate was 13 ml. per, hour. Fractions of 6.5 ml i were collected. / . .! I.* +i P I Results and Disc.jssior,

The 5 pregnancy-typical axhigens (2t) were obtsined from placental

extracts with ammonium sulfate (degree of saturation 0.4, pH 7.01.. If .- these proteins are separated by gel chrornatogr&hy oil Sephadex C-200 , / then 4 peaks are obtained (fig. lj. Zmunochemical analysis of the 4 O? 1 'I . .. IO1512 I , fractions showed in the diffusion test the presence of pregnnncy-typical

protcins in fractions 89, 90, and 91. Fraction 92 contained only blood

serum ,proteins. 8 Fraction 91 was further pruified on DEAE-Sephadex A-SO (fig. 2).

Hereby pregnancy-typical proteins were contained in fractions 106 and 108.

"t0 90 Fig. 1. Elution diagramI after chromatography on I Sephadex G-200 of placental-extract proteins separable by salting out with ammonium sulfate at a degree of saturation of 0.4. Fractions: 28-40 (FR.89), 41-53 (FR.90), 54-74 (FR.911, 78-91 (FR.92).

Pig. 2. Chromatography of placenta fraction 91 on DEAE-Sephadex A-50. Elution profile. Fractions: 50-58 (FR. 106),, 65-70 (FR.108) -

The 4 placental fractions (89, 90; 106, lq8) wnich contained 1y if;,id\ pregnancy-typical proteins were tested for immunological identity w,,I against serum of pregnant wcmea and puerperal serum, umbilical-cor6 serum t.": A I (EC',:, .and amiiotic fluid by xeas of

I .' I

1015122 . Fraction 89 contains at least 3 proteins; two precipitation lines I show immunological identity with antigens of pregnancy serum. In fraction 8 90 two precipitation lines are obtained; the innel- line of these proves *

to be identical with proteins of the pregnancy serum. The antigen of i fraction 106 is not immunologically identical with preghancy serum

serum antigens. Of two proteins of the fraction 108 on? also contained 'I is In Pregnancy serum. '," the~~~acenta!~fi-o-n~,--th_eFefore,- 3 pregnancy; I

typical proteins are detectable which also occur in pregnancy serum.

~ ---*.-_I_ -_._I.--- * The result of an immunodiffusion of the 4 placental fractions

compared to puerperal serum is shown in fig. 5. Fraction 89 yields two

.. precipitation lines which are immunologically identical with antigens

of the puerperal serum. Of the 2 proteins of fraction 0 one is not contai ned In the puerperal serum. The precipitation of fraction 106

crosses the,lines of the puerperal serum, from which it may be deduced

3. that there is no immunological identity. Fraction 108 shows no identity I I with the antigens of the puerperh serum. In the_.- 4---- placental fractions there are thus contained 2 pregnancy-typical proteins which can also ...-.... C. I .-- - ".. . . ----.. . ,-.,.-- .. be detected in puerperal serum. *+----- . . The identity of the 4 placental fractions with antigens of the . amniotic fluid is evident from fig. 6. In fraction 89,2 precipitation

lines are Identical with those of the amniotic fluid; one protein of ./ fraction, 90 is likewise ldestlczl. The precipitates of fractions 106

and 108 show no iaentity. In---.--- th# placental fractions- --_---_ 89_----- and --, 90, therefore, are contained 2 mtigecs of the smniotic fluid. i ____._--..--. --- - -_ *.

! .. -. -7 3

1015123 -0-

I .. 3 1 i I ‘1

.., .

Fig. 3. Diagrammatic summary of fractionation of ‘placental proteins by salt precipitation, gel chromatography and ion exchange.

.. The \roof of immunologic$l Identity of the umbilical-cord serum 0 I antigen with the pregnancy-typical protein of fraction 106 of the .. .. placenta is presented in fig, 7. Fractions 89 and 90 are not identical I wlth antigens of the umbilical-cord serum. Fractiorr 108, in addition tc

a crossing protein, contairs a trace of the umbilical-cord serum antigen.

In the four placental fractions therefore. B protein is contbined which .--I. .--e--- ~ , -.------*-.1-”-.- ,A,---,.... __ is imunologica.f$y identical with the pregnancy-typicai antigen of the -. . ._.... , ., ’ &bilical-cord serum. The bulk of this piotein is present in fraction < , ----,.t. -.-.’. 105, free from other pregnmcy-typical proteins.

The occurrence of tho, pregnency-typical antigens In the 4 placentai fractions is aumnarized in the teble. All 5 of the hitherto described I I pregnancy-typical antigens (Zc) can be Isolated from the placenta. H.

IO15124 c’ FR. 1OG

i I

Figs. 4a and 4b. Innnunodiffasion. a Antigens: human seium (HS), placeqta protein fraction 89 (FR 89), pregnancy serilffi (SS), placenta protein fraction 90 (FR SO), placenta protein fraction 106 (FR.106). Antiserum: mixture of absorbed antipregxncy ana absorbed antiplccenta immune serum (a. ASP). b Antigens: humsn serum (tis), uinbilical-cord serum (KS) , pregnsncy $serurr (SS), placenta protein fraction 108 (FR 108) puerperal serum (W). Antiserum: mixture of absorbed antipregnancy imu serum and absorbed antiplacsntg immune serum (a. ASP).

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b PR. 90 I HS

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Figs. Sa and Yo. Immunodiffusion. a Antigem: human serum (HS), placenta protein fraction 94 (FR 89), perperal 'serum (W) , placenta protein fraction 90 (Ell 501, placenta protein fraction iG6 (FR 106). Antisercm: mixture of absorbed antipregnancy and absorbed antiplacenta imnune serum (a. ASP). b Antigens : human serum (3s) pregnancy serum (SS) , puerperal aerum (W), plancents protein fraction 108 (FR 1081, amnii otic fluid (FW). ' Antiserum: mixture of absorbed antipregnancy and absorbed antiplacenta lmmur,e serum (a. ASP). ./

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a . . PR.80 ! FIS .. ... I . .. -..

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.. . . - ... - -. u-,. .. b Fll. l0.i / I I . Fig. 6a and 6b. Immunodiffusion. a Antigens: human serua (lis), placenta protein frsction 89 (FR 891, amniotic fluid (FW), placenta protein fraction 90 (FR 901, placenta ?rotein fractior, 106 (FR 106). Anti- serum: mixture of absorbed antiprcgnancy and absorbed antiplacgnta serum (a, ASP), S Antigens: human serum (HS), pregnancy scrum (SS), miotic fluid (FW), placenta protein fraction 106 (FR 106). Umbilical- cord serum (NS). Antiserum: 'mixture of absorbed antGpregnancy and

absorbed antiplaccnta immune sw(a- ASP). ' .

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Fig. 7a and 7b. Inmunodiffusion. a Antigens: humar. serum (HS), placenta protein fraction 89 (FR 891, umbilical-cord serum (NS),' placenta protein fraction SO (FR 90). ?lacents protein fraction 106 (FR 106). Antiseriun: mixture of aSsorbed 2ntipregnan:y and absorbed antiplacenta immune serum (a.'ASP). b Antigens: human serum (%), pregnancy Serum (SS), umbilical-cord serum (NS), placenta protein fraction 108 (FR loa), puerperal serum (W). Antiserum: mixture of absorbed antipregnancy and absorbed antiplacda immune serum (a. ASP).

iJ I

1015128 -11- / 'I ,' Table. Summary concerning occurrence of pregnancy-typical antigens in . placenta protein fractions with use of a mixture of absorbed 1 antlpregnancy and absorbed antiplacenta immune serum (a, ASP).

4 , Placenta Immune Pregnancy-typical antigen no.

fraction serum I no. 1'4 3 4 1, 83 a. ASP .+ +. '+ I

90 B. ASP +. i- I 100 a. ASP +. + loa a. ASP + +

By absorption of the immune serum used in nonpregnant serum, all

antibodies against normal blood serum proteins were eliminated; so'in

the present illustrations only pregnancy-typical proteins are precipitated.

The blood serum proteins contained in the fractions are not shown. For

' preparing a monospecific immune serum for the quantitative determination

of the pregnancy-typical protein no. 4, placenta fraction 106 is already . 0'' ;4 suitable ;,the $sofation of the other pregnancy-typical antigens from e* 4 placental fractions 89, 90, and 108 requires further steps of preparation. b The separatios bf placental proteins Sy gel chromatography, ion a/ exchange .and other preparative steps is auitable for obtaining pregnancy-

typical proteins €or .the preparation or' mnospetific imune sera. c ., !'. .i .. . t / I. . ..!

4 c t 07512'9 /"' -12-.

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I- .. .. . -., Evaluation of a New Pregnancy Test Preg n os ticon Dri -D ot?

CHARLES A. HORWITZ, MD, MELVIN SINYKIN, MD, DAVID HILL, MD, ELIZABETH JEROME, MD, and M. DESMOND BURKE, &ID

A new latex agglutination-slide test for preg- were obtained froiti nonpregnant female pa- . nancy in which the reagents arc incorporated tients (ages 12 to 45) in whom pregnancy onto the surface of the test slide was evaluated. had been excludcd on clinical grounds, by The te5t accuracy ranged from 94.GSS in preg- chart review, or by a review of available . nint patients (31 IO 100 days after the last normal menstrual period) to 98.65% in non- endometrial tissue. No specimen was ex- pregnant patients. The advantages and limi- cluded from this study because of pro- tations of this latex aS9lurination-ii~liibition teinuria, hematuria or hyposthenuria. dide test for pregnancy, which is stable at room Group temperature, are discussed. 2. The second study group consisted of 13 1 1 spzcifically selected urine specimens from males and non-pregnant fe- 131s PAPER describes our evaluation of males in the following categories: a) speci- new pregnancy test (Pregnosticon Dri- T B mens specifically selected because their pro- based on well-known principles of Do@) tein content could bc detected with Labstixa agglutination-inhibition. Unlike most avail- (377), grossly bloody urine (22), or mod- able agglutination-inhibition slide tests for erate to large amounts of hemoglobin de- pregnancy, reagents in thc Pregnosticon Dri- Dot test are incorporated in dried form onto tectable only by Labstix (16); b) there were specimens specifica!ly . selected the surface of the slide-in this case a dis- 61 from patients in tliz early menopause, 33 posable ardbonrd slide. in late menopause, 54 from patients on oral MATERIALS AND METHODS contraceptives, and 506 from paticnts on Group 1. The fust study group con- mcthadone; c) specimens (2 17) specifically sisted of 1616 urine specimens collected selected from patients with various im- at random from patients from either Mount munologic disorders in which elevated " Sinai Hospital, attending physician's offices, serum proteins might be expected, including or the Minneapolis Teenage Medical Service. conditions such as rheumatoid arthritis, in- OE these patients, 724 had unconiplicated fectious mononucleosis, primary atypical pregnancies; 72 had known complications pneumonia, infectious hepatitis, etc. These (ectopic pregnancies or spontaneous abor- specimens wcre then further subdivided into tions, threatened, incomplete or complete). 42 in which protein was present and 175 in Eight hundred ninety-two urine specimens which protein was absent. In addition, 22 From Mount Sinai Hospital and the Minneapol& specimens were included from paticiits with Teenage Medical Service, hlinneapolis, h''Iinnesota. multiple rpeloma. Address reprint requests to: Charles A. Honvitz, MD. Associate Pathologist. Mount Sinai Hospital, Test Procehre 2215 Park Avenue, hlinneapolis, hlinn 55404. Submitted for publication March 10, 1972. The test procedure was carried out is... vel. 4Q No. 4 Octob.; 1972 563

.. I13115132 e -- -.-

HOR WIT2 ET AL

directed by the manufacturer. A positive perature until thcy were again used in test for HCG was recorded if no latex par- February 1972. At this time these lots were ticle agglutination was seen at the end of a used on 30 specimens of known HCG con- 2-miniite reading period. A negative test tent. Fifteen of the 30 specimens testcd result was recorded if agglutination of latex were from patients known to be prcgnant particles was clearly defined within the 2- while the other 15 were from nonpregnant minute readins period. An inconclusive test patients. Neither hemoglobin nor protein result was recorded when true agglutination was detectable in thelatter specimens with was uncertain. Hemoglobin. protein and uri- Labstix. nary pH were determined on cach specimen RESULTS by a simple indicator strip (Labstixa). Test accuracy is recorded in Tables 1 Eight hundred and forty-two specimens to 3 and is based on the number of correct included in the first study group and 142 positive or negative results in the particular ’ specimens from the second study group had category under study. been stored for periods of 1 week to 15 Correct test results were also recorded months at -10” C were rethawed for test. utilizing the three aforementioned stored lots These frozen wines had been used in a on the 30 previously described specimens. previous pregnancy study of five other im- There were no appreciable differences be- munochemical tests. Prior to inclusion in tween endpoints (intensity of latex particle this study as an incorrect or inconclusive agglutination) when test lot 371134 (re- ; test result, the validity of a particular received Junc 1971) wa’s compared with a thawed specimen was reconfirmed by re- recently marketed test lot 871134AB (re- peating the other tests as indicated. The ceived February 1972). - rcmainder of the study was performed with fresh wines+. Wlicnever there. were discrep- DISCUSSION ancies between the known clinical diagnosis The availability of this latex agglutina- .. ‘or other immunochemical test results, the tion-inhibition test for detecting human tests were repeated as soon as possible to chorionic gonadotrophin in urinc offers -. esclude technical error. When such error certain advantages over other more corn- . .. was excluded and an incorrect or incon- monly used test forms: a) it is stable at ..i clusive test result seemed likely, follow-up room temper:lturc and does not require re- specimens were requested and obtained in frigeration for storage; b) the disposable most first-study group cases. Follow-up slide, sealed individually in moisture-proof spscimens were only used to confirm or ex- foil envelopes, eliminates the problem clude pregnancy in particular problem cases. causcd by residual detergent on glass slides At least eight different lots of test main other la lcx aggl utin ation4 nhibi t ion slide terial were used in this study, including lots tests; c) the prenieasured test reagents in- D2436, 271134 and 371134. The latter corporated onto the card surface obviate were received in our laboratory in Decem- the inherent inaccuracies of dropper pipettes. -. ber, 1970; March 1971; and April 1971, This change should be a step in the right respectively. As they were received, por- direction towards eliminating some of the . tions of cach lot were stored at room tem- technical errors that can occur in perform- .) At least four other ae~lutination-inhihi~ion ing slide tests for prcgnancy. Such errors prcgnancy tests were performed on cach of the can also bc attributed to an improper mixing specimens from this study. The Pregnosticong technic. .. tube and Pregnosticon@ liquid slide tests were performed on most of thwroblem specimens We confirmed thc stability of a small from this study. number of Dri-Dots tests kept in our lab- 564 Obstetrics and L Gynecology ..

.. .. 1015133 . 1: I

NEW PREGNANCY TEST-EVALUATION

- ~ No. ol Arcumcy* Specimen speciinerrs False-posifice Itrconrlrtsice (5%) Total uncomplicated preSnancics 723 33 2 95.17 31-40 dqsfrom LNXIP 25 16 I 32.00 41-50 days from LNMP 101 8 0 92.31 51-100 days from LNMP 451 5 I 98.68 14530 weeks from LNMP 90 0 0 100.00 31-40 weeks from LNMP 51 4 0 92.16 Total complicated pregnanciat 72 14 1 79.17 Threatened or incomplete abortions 58 11 1 79.31 Ectopic pregnancies 14 3 0 78.57 LNMP = last normal menstrual period Based on the number of correct positive test results. 1 An additional 12 specimens not included in this table were obtained from patients Hith missed abortions or the dead fetus syndrome; newtibe test results uere obtained in all cases with the Dri-Dot tat as well with all five other inimunochcmical tests. oratory at room temperature for periods done per day. One of the false positive test ranging from 8 to 14 months. Further results in the first study group was recorded studies, howzver, are indicated to detirmine on a protein-free .specimen from a patient sensitivity alteraiions over longer periods. on 130 mg of methadone per day. A warn- In the first study group, test accuracy in ing rcgarding methadone intcrference with uncomplicated pregnancies (31 to 100 days the Ptegnosticon Dri-Dot, however, is in- after the last normal menstrual period) was corporated into the test brochure. Metha- 91.68% and in nonpregnancies, 9S.65%. done has been reported lo interfere with The results from the second study group other latex agSlutination-inllibition slide tests suggest the need to exercise caution in in- for pregnancy.' terpreting Dri-Dot test results on specimens Results in the second study group also received from patients who are taking suggest that Labstix-detectable protein may methadone. False-positive or inconclusive engender some false-positive tests. Further, test results mere recorded on 27.9% (141 of 12 false-positive results among 892 of 506) of the specimens from patients on specimens in the first study group, S con- methadone. A review of dose records tained Labs t ix-det ect able pr Jt ein. This prob- showed that 87.9% (124 of 141) of false- lem has also been encountered with other positive or inconclusive test results were latex agglutinalion-inhibition slide obtained with specimens received from pa- The reported accuracy of a particular tients receiving 100 mg or more of melha- pregnancy test varies considerably with the

TABLE2. FUE-POSITIVEAND INCONCLUSlVE DRl-Dor TESTRFSLLTS FROM 892 SPECIUESSSUBM~ED moy NONPREWWX~FEVALE PATIENTS (ACES 12-48) No. oj Accuracy* Specimen apecinieis False-posiiices Lcoticlrisice (%I Protein and hemoglobin-free 735 4 0 99.46 Proteinuric 111 8 .o 92.79 Moderate or large amounts of only hemoglobin detectable by lnbstix 46 0 0 100.00 TmAL 892 ' 12 0 98.65 . Based on the number of correct negative test rcsub

Vol. 40. No. 4 October 1972 A65 -.-

.. HORWITZ ET AL

TADLE3. FALSE-PO~ITIVEAND IKCONCLUS~VE DR1-W T~srRESULTS OBTAI~ED ON SO5 SELECTEDSECOND STUDY GROUPSPECI\IEYS No. oj Accurocy Specimens specimens False-positive Iticotrclusice (%I' Total proteinuric specimens 377 18 6 93.63 Trace 126 4 0 96.83 1+ 132 4 2 95.45 2+ 88 5 4 89.77 3+ 31 5 0 83.81 Total grossly bloody specimens 22 5 0 77.27 Moderate or large amounts of only hemoglobin detectable by Iabstix@ 16 0 0 100.00 Multiple myeloma 22 1 0 95.45 Early menopaiue (ages 4655) 64 0 0 100.00 Late menopause (ages 56-90) 33 0 0 100.00 Patients on oral contraceptives 54 0 0 100.00 Immunologic disorders' (total samples) 217 0 96.77 protein-Tree urine 175 3 0 98.29 Urine with protein detectable by IabstixB 42 4 0 90.48 Patients on methadono 506 138 . 3 72.13 Accuracy based on the number of correct nezative test results t Spetimens obtained from p3tients with diseases such as rheumatoid arthritis, infectious mononucleosis, primary atypical pneumonia. polyclonal gammopathies, etc

source of case materid.' In the initial phases sensitive 2-hour Pregnosticon hemagghtina- of this study, many low-titcr early preg- tion-inhibition tube !est in eorfy pregnancy. nancy specimens were received from hos- During the test, occasionally a yellow pital ernployecs, in contrast to the latter color persisted on the test slide after the phases of this study when specimens wcre latex Dri-Dot was reconstituted. Despite received primarily from thc Minneapolis this rather striking appearance imparted to Teenage Medical Service where patients are the test-card, the presence or absence of cncouraged to bring specimens only after agglutination could be interpreted without they misscd two menstrual periods. A re- dificulty in ?.;I but a few cases. In the view of the test results from this study re- latter instancrs, the tests were repeated using flected the source of case material, as the another test card. majority of false negatives came from sources at Mount Sinai Hospital who have REFERENCES been encouraged to bring in early specimens 1. Horwitz C. hfaslansky R, Waldingcr R. et al: for testing. The effect of methadone on pregnancy tests- An analysis of SO6 specimens with four cur- Although this study was not a compnra- rently available immunoassays. Procredings of tive pregnancy test evaluation per se, the Fourth National Conference on hlethadonc problem specimens were tested by other Treatment, San Francisco, 1972. Edited by the National Association for the Prevention of immunochcmical tests. The Pregnosticon Addicticn to Narcotics (NAPAN), 1972. pp Dri-Dot seemed slightly less specific but 111-116 more sciisitive than the liquid form of the 2. Bell JL: Comparative study of immunological tests for prcgnnncy diagnosis. 1 Clin Pathol 22: Pregnosticon slidc test. Neither the Preg- 79-83. 1969 nosticon slide tests nor any of the other 3. Knight RA, Kilpatrick L, Porter hlM: Evalua- currently available latex agglutination-in- tion of two new pregnancy tests. Am J hled . Technologists 397-399. 1 hibition immunochemicaL4lide tests for 37: I97 4. Hobson BM: Pregnancy diagnosis. J Reprod , pregnancy were as accurate as the morc Fertil 12:3.3-48, 1966 .

Obstetrics and 5 66 Gynecology 8,-

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