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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