IMMUNOLOGICAL STUDIES WITH A COMMERCIAL PREPARATION OF HUMAN CHORIONIC GONADOTROPIN
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Journal of Clinical Investigation Vol. 42, No. 4, 1963 IMMUNOLOGICAL STUDIES WITH A COMMERCIAL PREPARATION OF HUMAN CHORIONIC GONADOTROPIN * By SHINICHI HAMASHIGE t AND EDWARD R. ARQUILLA (From the Department of Pathology, University of California School of Medicine, Los Angeles, Calif.) (Submitted for publication October 5, 1962; accepted December 20, 1962) Immunological methods are being used to de- ml of complete Freund's adjuvant.2 Identical concen- termine human chorionic gonadotropin (HCG) in trations of HCG 1 in isotonic saline were used for booster preparations. urine and serum during pregnancy (1-4) and also Antisera were produced in male and female New to localize it in tissues and tumors (5). The anti- Zealand white rabbits weighing 2.5 to 3.0 kg and in sera used in these studies (1-5) were produced by guinea pigs weighing 300 to 400 g. immunizing rabbits with HCG preparations of Immunizing procedure. Immunizing doses were 5 and widely varying specific activities. The HCG-HCG 1 mg of antigen injected, respectively, into the toe pads antisera characterization studies that were per- of rabbits and paw pads of guinea pigs. Three weeks formed primarily with agar gel immunodiffusion, later, the animals were given two booster injections of antigen at 3- to 4-day intervals. The booster injection or immunoelectrophoresis, or both, have demon- of 2 ml for rabbits was given intravenously and intra- strated the presence of two or more antigenic com- muscularly in equally divided doses. The guinea pigs ponents (4, 6-8). were given subcutaneous booster injection, 2.5 mg of Two ways of producing HCG-specific antisera antigen, in the groin. Nine to 10 days after the course have been reported. McKean (2) produced them of booster inj ections, a test sample of blood was ob- tained from the ear vein of the rabbits and by cardiac by using a purified HCG preparation as antigen. puncture from the guinea pigs. If the test sample An alternative is the adsorption of crude HCG contained an adequate titre, blood was withdrawn from antisera with urine or serum extracts to remove the lateral ear vein of the rabbit within 1 to 2 days. precipitins and extraneous antibodies (3, 6, 7). Guinea pigs with elevated HCG antibody titres were This study presents methods for the production exsanguinated from the carotid artery. The rabbits were allowed a minimal recovery period of 1 month after of antibodies to HCG in rabbits and guinea pigs. bleeding. The usual course of two booster injections and The detection of HCG antibodies and HCG by harvesting of serum was employed for the subsequent hemagglutination and hemagglutination inhibition production of rabbit HCG antibodies. By this immuniza- methods have been described. It has been possible tion procedure, HCG antibody titres of 1/5,000 or greater to demonstrate several protein contaminants in a were consistently produced in 3 rabbits and 24 guinea pigs. All HCG antibody titres were determined by the commercial HCG preparation by immunoelectro- hemagglutination method. phoresis and electrophoresis. The adsorption of Processing of antisera. The collected blood was al- nonrelated antigenic components in the HCG anti- lowed to clot at room temperature for 2 to 4 hours. sera with normal male urine extract has been Antisera were separated by centrifugation and stored, studied by immunoelectrophoresis, hemagglutina- frozen, in samples at - 50° C. All sera used in hemag- tion, and bioassay methods. glutination studies were inactivated at 56° C for 30 minutes. The rabbit antisera were also first adsorbed with an equal volume of packed, washed, sheep erythro- MATERIALS AND METHODS cytes. Antibody-rich globulin fractions were prepared Antigen preparations. The antigen used for immuni- by ethanol fractionation (9). The precipitated fractions were dialyzed against distilled water and lyophilized. zation was prepared by suspending 5 mg of HCG1 in 1 Neutralivation of HCG activity by antisera. The pres- * Work supported in part by U. S. Public Health ence of antibody in sera of HCG-immunized rabbits and Service grant 2G-802. guinea pigs was demonstrated by neutralizing the bio- t Research Trainee in pathology, University of Cali- logic effect of HCG in rats and frogs. fornia. Rat prostate and accessory sex organ method (10). 1 Vitamerican lot PR360 and 90125 containing 2,570 and Intact, 21-day-old Long-Evans male rats weighing 40 to 2,790 IU, respectively. Vitamerican Corp., Little Falls, N. J. 2 Hyland Laboratories, Los Angeles, Calif. 546
IM'MUN OLOGICAL STUDIES WITH HUMAN CHORIONIC GONADOTROPIN 547 65 g were given a subcutaneous 0.5-ml injection of test Extracts of acetone powder from urine. Human urine material on 3 consecutive days. Each injection con- powders from normal males, pregnant females, and cas- tained 0.1 ml of HCG (10 IU) diluted to 0.5 ml with trate females were prepared by acetone precipitation (15). saline, normal serum, or antisera. The rats were sacri- All powders were prepared from two or more sources and ficed on the fourth day, and the prostate and accessory pooled. An aqueous preparation was made by mixing sex organs removed by dissection and fixed in 10% 1 g of acetone powder with 1 ml of buffered saline, ad- formalin for 24 to 48 hours. The organs were weighed justing the pH to 7.0 to 7.4, and further diluting with and the results expressed in grams per 100 g body 4 ml of buffered saline. The water-insoluble residue was weight. discarded after centrifugation. Saturated solutions of Preliminary studies with the Long-Evans strain of these urine extracts were similarly prepared by using a rats with 1 to 5, 10, and 150 IU of HCG per injection large excess of the acetone powder. Solutions of these showed a sharp extinction of biologic response. This extracts were prepared immediately before use. change occurred between 1 and 2 IU of HCG per in- Adsorption of HCG antisera was carried out by add- jection. The extinction point, 2 IU of HCG, was there- ing 30 mg per ml of finely ground, acetone-precipitated, fore used to bioassay HCG. normal, male urine powder, mixed and allowed to stand Frog test (11). Adult male frogs (Rana pipiens) at room temperature for at least 3 hours. The antisera were inj ected with 1 ml of the test material into the were obtained as clear supernatant fluids by centrifuga- dorsal lymph sac. Cloacal smears were examined mi- tion. Second and third adsorptions of antisera were simi- croscopically just before and 1 and 4 hours after injec- larly performed. tion of the test mixture. A positive test consisted of the Agar gel methods. Double-diffusion studies in agar presence of large numbers of spermatozoa in the smears. were accomplished on microscopic slides with 0.5% Test solutions contained 150 IU of HCG (0.03 ml) and Ionagar no. 2 5 in buffered isotonic saline at pH 7.0 or in 0.97 ml of either saline, normal serum, or HCG anti- barbital buffer at pH 8.6, ionic strength 0.08. sera. Immunoelectrophoretic studies by the Scheidegger Preparation of HCG-conjuigated sheep erythrocytes. modification (16) were performed on the LKB 6 electro- Washed, packed, sheep erythrocytes (0.1 ml) were sus- phoresis apparatus 6800A with Ionagar no. 2 5 in barbital pended in a solution containing 1.5 mg of HCG in 3.0 buffer at pH 8.6, ionic strength 0.08. Electrophoresis ml buffered saline,3 and 0.6 ml of freshly diluted 1: 15 was performed at 4.5 ma per slide for 1 to 3 hours. Af- bis (diazo)benzidine solution (12) was added. The ter electrophoresis, 1-mm troughs were cut in the agar suspension was shaken gently for 10 minutes at room and filled with the appropriate antisera. All incubations temperature and then washed three times with barbital- for development of precipitin lines by double diffusion or buffered saline with albumin (13). The washed, packed, immunoelectrophoresis were done at room temperature HCG-conjugated cells were resuspended in 5 ml of barbi- for 16 to 40 hours in a moist chamber. Slides for perma- tal-buffered saline with albumin (2% suspension) and nent records or photography were washed in several 0.05 ml of this suspension was used for titrating anti- changes of saline, air-dried, and stained. The dehy- bodies to HCG. drated slides were immersed in 1% acetic acid for 30 Heniagglutination and hemagglutination inhibition meth- minutes, stained with phloxine-eosine for 1 hour, and ods. The hemagglutination method employed in titrating washed in running tap water. The stained precipitin HCG antibodies has been described by Arquilla and lines appeared as distinct, magenta-colored arcs on a Stavitsky (12). Hemagglutination inhibition was used light gray, agar gel background. for the estimation of HCG (12). With this technique, Staining solution. The solution used to stain precipi- fractions of a microgram of the antigen significantly in- tin lines obtained in the agar gel studies contained 0.5% hibit hemagglutination of HCG-conjugated cells by anti- phloxine B7 and 0.5% eosine Y 7 in 95% ethanol. serum. Cellulose acetate electrophoresis. Electrophoresis us- RESULTS ing cellulose acetate strips (14) was performed on the Shandon Universal electrophoresis apparatus 2548 4 in Rat test. The presence in sera from immunized barbital buffer at pH 8.6, ionic strength 0.08 for 1.5 hours rabbits and guinea pigs of antibodies that neutra- at 0.4 ma per cm of strip width. The 12 X 2.5-cm strips lized the physiological activity of HCG was dem- were impregnated with barbital buffer, blotted, and al- onstrated with the rat test (Table I). The effect lowed to equilibrate for 1 hour before electrophoresis. A of 10 IU of HCG on the weight of rat prostate and Heathkit model PS-3 was the current power supply. The samples were prepared as saturated solutions and seminal vesicle was completely neutralized (p = applied as streaks with a 1-gl micropipette. After com- 0.0001) with 0.4 ml rabbit or guinea pig HCG pletion of the electrophoresis, the strips were dried and antisera. Similar results were obtained with the stained with light green S.F., or Ponceau S and nigrosin. 5 Consolidated Laboratories, Chicago, Ill. 3 Buffered saline solution contains 0.15 NM 'NaCl and LKB Instruments, Inc., Washington, D. C. 6 0.11 M phosphate buffer, pH 7.4. 7Allied Chemical Corp., National Aniline Division, 4 Consolidated Laboratories, Chicago, Ill. New York, N. Y.
548 SHINICHI HAMASHIGE AND EDWARD R. ARQUILLA TABLE I The average standard deviation for the rat Effect of rabbit and guinea pig antisera on the biologic activity tests (Tables I and II) was 0.02 g per 100 g body of human chorionic gonadotropin (HCG) in the rat* weight with a range of standard deviations from 0.01 to 0.03 g per 100 g body weight per test Mean wt, prostate group of rats. No. of and ac- cesory sex Frog test. The presence of HCG antibodies in Test sample rats organs p the serum of immunized rabbits and guinea pigs g/100 g was also shown in the frog. Antisera, 0.97 ml, body wt Saline 9 0.15 completely neutralized the biologic effect of 150 HCG 15 0.25
IMMUNOLOGICAL STUDIES WITH HUMAN CHORIONIC GONADOTROPIN 549 Veronal buffer, pH 8f6, II 0 083 0'4 ma/cm of strip width. 15 Hours. Vitameric an HCG Lot No. 90125 FIG. 1. CELLULOSE ACETATE ELECTROPHORESIS OF HUMAN CHORIONIC GONADOTROPIN (HCG). Veronal = barbital. Cellulose acetate electrophoretic stadies. Pre- ration 9 produced an electrophoretic pattern not liminary electrophoretic studies of HCG prepara- unlike that seen with the acetone-precipitated tions were performed on Beckman filter paper urine extracts. These electrophoretic studies sug- strips no. 320046 or 300028 in the Beckman Dur- gest the presence of at least three different elec- rum cell with various buffers. The results were trophoretic protein components in HCG prepa- unsatisfactory. A single, continuous, broad, pro- rations. tein zone overlaid the origin and migrated toward both the cathode and anode. Immunoelectrophoretic studies Subsequently, electrophoresis of HCG was tried Studies of HCG with guinea pig HCG anti- with cellulose acetate membrane and the result is serum. Immunoelectrophoretic studies of HCG shown on Figure 1. Three definite bands mi- with guinea pig HCG antiserum showed only two grating toward the cathode were consistently ob- precipitin lines. A corollary study using HCG served. Protein-staining zones were not seen and rabbit HCG antiserum showed five precipitin on the anode side of the origin. Nigrosin staining lines. The results show that substances present of electrophoretic strips can detect as little at 2 in the HCG preparation produced precipitating ,ug protein (14). These results indicate that the antibodies in the rabbit, but did not produce de- HCG preparations do not contain human serum tectable amounts of precipitating antibodies in proteins detectable by electrophoresis. This study the guinea pig. These results might be related also shows that Vitamerican HCG preparations to the number and duration of immunizations, but contain three electrophoretically dissimilar pro- this problem was not investigated. teins. Studies of HCG with normal rabbit and rabbit Cellulose acetate electrophoresis of a Squibb anti-insulin sera. Immunoelectrophoresis of HCG preparation 8 of HCG showed an electrophoretic with normal rabbit and rabbit anti-insulin sera pattern indistinguishable from Vitamerican HCG showed complete absence of precipitin lines. The preparation. Electrophoresis of an Ayerst prepa- results suggests all precipitin lines seen with rab- 8 Follutein lot P055561, 2,734 , per mg generously 9 Ayerst APL A6996FI, generously. supplied by Dr. supplied by Dr. Aleck Borman, The Squibb Institute for Daniel Michell, Jr., Harbor General Hospital, Torrance, Medical Research, New Brunswick, N. J. Calif.
550 SHINICHI HAMASHIGE AND EDWARD R. ARQUILLA In~w FIG. 2. IMMUNOELECTROPHORETIC STUDIES. RAH = rabbit antihuman se- rum; NHS =normal human serum; NHS AA= acetone-precipitated extract of normal human serum; and NHS B = ethanol-precipitated extract of nor- mal human serum. bit HCG antiserum were the result of antibodies retic studies of HCG and acetone-precipitated due to immunization with HCG. urine extract (Figure 3) with rabbit antihuman Studies of human serum teith rabbit anti-human serum 2 were performed. All studies were com- serum. Normal human serum diluted with an pletely negative for precipitin lines. The absence equal volume of serum protein-free urine was of serum proteins in HCG and acetone-precipi- precipitated with acetone (15) or alcohol (17) tated urine extracts were strongly indicated by and studied by immunoelectrophoresis with rab- these studies. If present, serum proteins were in bit antihuman serum.2 Serum protein-free urine, undetectably minute quantities, or else altered be- tested by immunoelectrophoretic studies, was ob- yond recognition. tained from normal young adults. The acetone- Study of normal male serum with rabbit HCG precipitated extract of serum consistently showed antiserum. In an attempt to demonstrate serum more precipitin lines than the ethanol extract proteins in HCG preparations, immunoelectropho- (Figure 2). The results indicate that acetone is retic studies of normal male serum with rabbit less destructive to serum proteins than ethanol. HCG antiserum were performed. No precipitin The rabbit antihuman serum would detect signifi- lines were ever observed. The negative results cant amounts of unaltered serum proteins in indicate no detectable serum protein antibodies in urine extracts. rabbit HCG antiserum and serum proteins in the Studies of HCG awd urine extracts zewith rabbit antigen preparation. antihuman serum. Numerous immunoelectropho- Studies of HCG with rabbit HCG antiserum. FIG. 3. IMMUNOELECTROPHORETIC STUDIES. RAH = rabbit antihuman se- rum; CFU = castrate female urine extract; NMS = normal male serum; and NMU = normal male urine extract.
PROCE PROCEDURE . IMMUNO- . . ~ ~ ~. IMMUNOLOGICAL STUDIES WITH HUMAN CHORIONIC GONADOTROPIN ELECTROPHORESIS HEMAGGLUTINATION TITRE * DUREUNABSORBED ANTISERUM Gu A ---t -l 5,000 ...=......... . II . ABSORBED ANTISERUM 5,000 551 NEUTRALIZATION IN 0.20 0.19 THE RATI 0*19 0 * The highest dilution of antiserum capable of agglutinating erythrocytes to which HCG was conjugated. H The degree of neutralization of 10 WU. HCG caused by antiserum in 6 rots, expressed as mean weight prostate gland in grams per 100 grams body weight, was not significantly different. FIG. 4. EFFECT OF ADSORPTION OF RABBIT HCG ANTISERUM BY NORMAL MALE URIN-E EXTRACT. To demonstrate the number of antigenic compo- HCG antiserum, the double-diffusion method in nents present in the HCG preparations,' 7 nu- agar gel demonstrated two precipitin lines (Figure 6, merous immunoelectrophoretic studies with rab- 5). bit HCG antisera were performed. These studies Studies of urine extracts with rabbit HCG anti- showed five precipitin lines with sera from two serum. In a final effort to determine whether com- rabbits and three, all of diminished density, from the third rabbit. Of the five precipitin lines, two were moderately dense, one was faint, and two were very faint. A photographic montage (Fig- ure 4) composed of the immunoelectrophoretic slide with superimposed sketch is included to il- R52J lustrate the precipitin lines which were not repro- : ~ ~ duced in the photographs. The two moderately dense lines were easily discernible within 24 hours :i : . of incubation; the three fainter lines were usually i *-R92Jf 2. ....... visible within 48 hours of incubation. Immuno- electrophoresis of Squibb and Ayerst HCG prepa- rations with Vitamerican HCG-immunized rabbit serum showed five, two, and possibly three pre- cipitin lines, respectively. All these precipitin lines appear to be identical with those seen with Vitamerican HCG. This study shows Vitamerican HCG to con- tain a minimum of five antigenically dissimilar FIG. 5. STUDY BY DOUBLF DIFFL SION METHOD IN AGAR proteins. It also shows the presence of at least RB2J rabbit HOG antiserum GEL and RB2J Al two or more common antigenic components in all rabbit HOG antiserum adsorbed with male urine once three HCG preparations. With identical rabbit powder.
552 SHINICHI HAMASHIGE AND EDWARD R. ARQUILLA mercial HCG preparations contain extraneous erythrocytes and 2) multiple adsorptions with proteins, urine extracts were studied by immuno- normal male urine do not remove any significant electrophoresis with rabbit HCG antisera. A amount of antibodies from rabbit HCG antiserum. single precipitin line was noted in immunoelectro- The second possibility appears much more prob- phoretic studies conducted with pregnant female, able. The results of the hemagglutination study normal male, or castrate female urine extracts have been duplicated several times. against rabbit HCG antiserum. This precipitin To determine the effect of adsorption on the line appears to be dissimilar from any of those HCG neutralizing activity present in antiserum, seen when HCG preparations were used as anti- thrice-adsorbed and unadsorbed samples of iden- gen against the same antiserum. This precipitin tical rabbit antiserum were tested in the rat. The line usually required 24 or more hours of incuba- equivalent point between HCG and HCG anti- tion to form visibly. serum was used to show no loss of HCG neutrali- It appears that HCG preparations and urine zing activity after removal of precipitins. The extracts do not contain common antigen or anti- equivalent point was achieved, and was reflected gens detectable by immunoelectrophoresis or he- in the organ weights of 0.19 and 0.20 g per 100 g magglutination. The single precipitin line seen body weight in the control and test groups; these between urine extracts and rabbit HCG antiserum weights are midway between HCG-positive and was easily eliminated by one or more adsorptions HCG-negative groups (Table I). The organ of rabbit HCG antiserum with normal male urine weights between the test and control groups show extract. no significant difference (p = 0.45), indicating The effect of adsorbing rabbit HCG antisera that the removal of precipitating antibody with with normal male urine extract. Numerous im- normal male urine extracts had no effect on the munoelectrophoretic studies of HCG with unad- HCG-neutralizing activity. sorbed and once-adsorbed samples of identical rab- These adsorption studies of HCG antisera with bit HCG antiserum showed no discernible change normal male urine extract indicate no significant in the number, relationship, rate of formation, or increased specificity for HCG. The disappearance density of precipitin lines (Figure 4). The Ouch- of precipitin lines seen with adsorbed antiserum terlony study showed comparable results (Fig- was also seen with an identical sample of unad- ure 5). Multiple adsorptions of rabbit HCG sorbed antiserum that was refrigerated at 40 C antiserum with normal male urine extract before for a similar period. Antisera frozen and stored immunoelectrophoresis decreased the number of at - 50° C did not show this loss of precipitating visible precipitin lines to three. These multiple antibodies. We have shown that the precipitation adsorptions were carried out over a day or two, seen with normal male urine extract added to rab- and the unadsorbed sample refrigerated for a bit HCG antiserum is not related to HCG. similar period also showed the disappearance of two precipitin lines. In no instance have we been DISCUSSION able to show disappearance of one or more pre- cipitin lines that is directly attributable to one or In this study, HCG antibodies have been pro- more adsorptions with normal male urine extract. duced in two experimental species, and demon- To study further the effect of adsorption of rab- strated biologically by neutralization of the physi- bit HCG antiserum, hemagglutination studies were ologic activity of HCG, by hemagglutination, and performed. Unlike bioassay, hemagglutination by precipitation studies in agar gel. The sensitiv- measures a sum of immunological reactions, since ity of the hemagglutination method used in this the HCG and homologous antisera were previously study is of the order of 0.5 ,tg or 1.2 IU of Vita- shown to contain several antigenic components. merican HCG. For measuring HCG, it is there- The hemagglutination study using unadsorbed and fore at least as sensitive as bioassay methods. thrice-adsorbed samples of identical rabbit HCG The formation of a precipitate between rabbit antiserum showed no change in titre (Table IV). HCG antiserum and urine extracts shown by im- The result can be resolved by concluding that 1) munoelectrophoresis required that all precipita- HCG is preferentially conjugated to the sheep tion methods for identification or assay of urinary
I.NIM-UN OLOGICAL STUDIES WITH HUMAN CHORIONIC GONADOTROPIN 553 HCG be performed with adsorbed HCG anti- demonstrated by immunoelectrophoresis of urine serum. Several investigators (3, 6, 18) have extracts with rabbit HCG antiserum. We were previously stated this necessity. We have demon- unable to demonstrate this "urinary protein" in strated the independence between HCG-neutraliz- human serum or in our HCG preparation by im- ing activity and precipitins removed with urine munoelectrophoretic studies. We therefore pos- extract, confirming the work of Van Den Ende tulate that this "urinary protein" is highly anti- (19). This independence enables adsorption of genic and is present in HCG preparation in in- HCG antiserum without significant impairment of sufficient quantity to form visible precipitin lines. HCG antibody activity. It appears to be the sole antigen or cross-reacting The purity of Vitamerican HCG preparation has substance shared by the HCG preparation and been studied by cellulose acetate membrane electro- acetone-precipitated urine extract. The antibody phoresis (Figure 1) and agar gel immunoelectro- to this protein can be easily removed from anti- phoresis (Figure 4). These studies showed at sera by adsorption with normal male urine extract. least three and five protein components by electro- It was not possible to demonstrate any com- phoresis and immunoelectrophoresis, respectively. mon antigens between Vitamerican HCG and The antiserum produced by immunization with human serum or human urine extracts. This HCG is, therefore, a mixture of antibodies di- deficiency suggests that the specificity of HCG rected toward several dissimilar proteins. antiserum cannot be increased by adsorption with It was possible to show consistently three elec- human serum or urine extracts. Studies were con- trophoretic and five immunologic components in ducted with antiserum adsorbed 1, 2. and 3 times. Vitamerican HCG, but we were unable to show Samples of identical but unadsorbed, HCG anti- more than two components by the Ouchterlony serum were kept in the refrigerator to be used as method (Figure 5), despite adjustments in con- controls for each immunoelectrophoretic run. If centration of reactants and changes in buffers. normal male urine extract adsorptions of rabbit Comparative studies using identical samples of HCG antiserum were to remove the precipitating antigen and rabbit HCG antiserum by immuno- antibodies to protein contaminants, then the im- electrophoresis (Figure 4) and immunodiffusion munoelectrophoretic study of HCG with adsorbed (Figure 5) showed a difference of three precipi- and unadsorbed samples of identical rabbit HCG tin lines between the two methods. Similar dis- antiserum would be expected to show a difference crepancies have been reported by others (4, 6). in the number of precipitin lines. This was not Therefore, the proof of immunological unity or the case. In every instance, the number of pre- number of antigen-antibody systems present solely cipitin lines between the central well containing from studies by the Ouchterlony method appears HCG and the troughs on each side of the well, to be incomplete. containing adsorbed or unadsorbed samples of The HCG preparation used in this study con- identical rabbit HCG antiserum, showed an equal tains aproximately 2,500 IU per mg and is roughly number of precipitin lines. The precipitin lines one-fifth as pure as that of Got, Levy, and Bour- formed by reaction between the 1ICG and adsorbed rillon (20) of 12,000 IU per mg. Therefore, ap- HCG antiserum were sometimes of slightly di- proximately 80% of the preparation represents minished intensity, but not consistently so. Im- contaminants. Various investigators have re- munoelectrophoretic studies with adsorbed or un- ported these contaminants to be serum and uri- adsorbed rabbit HCG antiserum that were less nary proteins (3, 6-8). We have searched for than 12 hours old showed five precipitin lines serum proteins in the HCG preparation, using (Figure 4). Adsorbed and unadsorbed rabbit cellulose acetate membrane electrophoresis and HCG antisera stored in the refrigerator for 24 numerous immunoelectrophoretic studies without hours or more show only three precipitin lines. success. Our studies indicate an absence of se- These studies indicate no disappearance of pre- rum proteins in Vitamerican HCG preparation; cipitin lines directly attributable to adsorption if present, they are altered beyond recognition or with normal male urine extract. It also appears are in undetectable quantities. that the limits of the particular antigen-antibody The presence of a "urinary protein" has been reaction have been reached, and factors such as
554 SHINICHI HAMASHIGE AND EDWARD R. ARQUILLA age of antisera and addition of extraneous ma- tion of HCG preparations have been performed and terial could easily cause lines to disappear, and the presence of contaminants has been demon- so lead to false "purification" of antisera. strated. 5) The effect of adsorbing rabbit HCG To study further the effect of normal male urine antiserum with urine extract indicates that it is extract adsorption on rabbit HCG antiserum, he- not possible to make such antisera specific for magglutination titrations were performed with HCG. adsorbed and unadsorbed samples of identical antiserum. Unlike the bioassay method, the he- REFERENCES magglutination method measures a sum of im- 1. Wide, L., and C. A. Gemzell. An immunological munological reactions, since the HCG and its pregnancy test. Acta Endocr. (Kbh.) 1960, 35, 261. homologous antisera have been shown to contain 2. McKean, C. M. Preparation and use of antisera to several protein components. human chorionic gonadotrophin. Amer. J. Obstet. Gynec. 1960, 80, 596. The HCG conjugated to sheep red cells with 3. Brody, S. and G. Carlstrdm. Immuno-assay of hu- bis(diazo)benzidine is a mixture of proteins, and man chorionic gonadotropin in normal and patho- it is not unreasonable to expect all protein moieties logic pregnancy. J. clin. Endocr. 1962, 22, 564. to be attached to the red cells to a greater or lesser 4. Keele, D. K., J. Remple, J. Bean, and J. Webster. degree. In the HCG hemagglutination titration, Immunologic reactions to human chorionic gonado- the HCG-conjugated cells are mixed with the tropin. J. clin. Endocr. 1962, 22, 287. 5. Midgley, A. R., Jr., and G. B. Pierce, Jr. Immuno- HCG antiserum and the result is a summation of histochemical localization of human chorionic go- several antigen-antibody reactions. If normal nadotropin. J. exp. Med. 1962, 115, 289. male urine extract adsorption removes all anti- 6. Midgley, A. R., Jr., G. B. Pierce, Jr., and W. 0. bodies except HCG-specific antibody or antibodies, Weigle. Immunobiological identification of human a change in hemagglutination titre is expected. chorionic gonadotropin. Proc. Soc. exp. Biol. Our study of thrice-adsorbed rabbit HCG anti- (N. Y.) 1961, 108, 85. 7. Rao, S. S., and S. K. Shahani. The antigenicity of serum shows no change in hemagglutination titre, human chorionic gonadotrophin. Immunology which suggests no significant removal of antibod- 1961, 4, 1. ies from the antiserum (Figure 4). This result 8. Lunenfeld, B., C. Isersky, and M. C. Shelesnyak. is consistent with the immunoelectrophoretic stud- Immunologic studies on gonadotropins. I. Im- ies. The results from the immunoelectropho- munogenic properties and immunologic characteri- retic and hemagglutination studies indicate that zation of human chorionic gonadotropin prepara- tions (HCG) and their homologous antisera. J. HCG antiserum with multiple protein antibodies clin. Endocr. 1962, 22, 555. cannot be made HCG-specific by adsorption with 9. Nichol, J. C., and H. F. Deutsch. Biophysical stud- normal male urine extract. The production of ies of blood plasma proteins. VII. Separation of HCG-specific antiserum is probably dependent -y-globulin from the sera of various animals. J. Amer. chem. Soc. 1948, 70, 80. upon use of pure HCG as an antigen. 10. Diczfalusy, E. An improved method for the bioas- say of chorionic gonadotrophin. Acta Endocr. SUM MARY (Kbh.) 1954, 17, 58. 11. Robbins, S. L., and F. Parker, Jr. The use of the 1) With partially purified, commercial, human male North American frog (Rana pipiens) in the chorionic gonadotropin (HCG) as an antigen, diagnosis of pregnancy. Endocrinology 1948, 42, antiHCG antibodies have been consistently pro- 237. 12. Arquilla, E. R., and A. B. Stavitsky. The produc- duced in rabbits and guinea pigs. 2) The anti- tion and identification of antibodies to insulin and body has been demonstrated immunologically by their use in assaying insulin. J. clin. Invest. 1956, hemagglutination and by neutralization of the bio- 35, 458. logical activity of HCG in both the frog and rat. 13. Kabat, E. A., and M. M. Mayer. Experimental Im- 3) An immunological method for the detection of munochemistry, 2nd Ed. Springfield, Ill., Charles C Thomas, 1961. human chorionic gonadotropin by hemagglutina- 14. Chromatographic and Electrophoretic Techniques, tion inhibition has been described. 4) Electro- Zone Electrophoresis, Ivor Smith, Ed. New York, phoretic and immunoelectrophoretic characteriza- Interscience, 1960, vol. 2,
IMMUNOLOGICAL STUDIES WITH HUMAN CHORIONIC GONADOTROPIN 555 15. Frank, R. T., U. J. Salmon, and R. Friedman. De- 18. Schuyler, L. H., K. Anderson, S. Saslaw, and C. C. termination of luteinizing and follicle-stimulating Erickson. A serologic study of chorionic gonado- principles in castrate and menopause urine. Proc. trophin. Proc. Soc. exp. Biol. (N. Y.) 1950, 75, Soc. exp. Biol. (N. Y.) 1935, 32, 1666. 552. 16. Scheidegger, J. J. Une micro-methode de l'immuno- 19. Van Den Ende, M. Precipitins in anti-gonadotrophic electrophorese. Int. Arch. Allergy 1955, 7, 103. sera. Endocrinology 1939, 1, 156. 17. Heller, C. G., and E. J. Heller. Gonadotrophic hor- 20. Got, R., G. Levy, and R. Bourrillon. Analyse im- mone: clinical application of extraction methods munoelectrophoretique de la gonadotropine choriale for assay purposes. Endocrinology 1939, 24, 319. humaine. Experientia (Basel) 1959, 15, 480.
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