CONTRACEPTION
PROCESSING OF THE PREPARATIONS OF I?-SIJBWIT OF HUMAN CHORIONIC GONADOTROPIN FOR MINIMIZATION OF CROSS-REACTIVITY WITH l!UMAN LUTEINIZILG HORMONE
G.P.
‘Talwar, N.
?1.C. Sharma, Shastri and
S.K. Dubev, M. S. Ramakrishnan
Department of Biochemistry, All India Medical Sciences, New Delhi-IlOOlh,
Salahuddin,
Institute India
of
AHSTRAC’I
B-HCG preparations demonstrate microheterogeneity and are resolved in multiple bands on electrophoresis in polyacrylamide gels. The gels were cut into segments and the immunological reactivity of eluates with anti-B-HCG and anti-JJLH sera was inb-estigated. The hands reacting with the two antigonadotropin sera were identified. Graded ahsorption of II-HCG with anti-heterologous [ovine1 Lll gave preparations with none or minimal cross-reactivity with anti -JILIJ.
,\cccptcd
FEBRUARY
[or
publication
1976
October
VOL. 13 NO. 2
20,
1975
131
CONTRACEPTION
INTRODUCTION -Human chorionic gonadotropin (HCG) is commercially prepared from the urine of pregnant women! who excrete large quantities of this hormone especially in the first trimester of pregnancy (l-3). It is not unexpected that the hormone recovered in the urine represents at least in part the moieties metabolised to variable extent in the kidney and other “target” and “non- target” organs. HCG is composed of two suunits; the a-subunit is largely identical to the a-subunit of TSH, FSH, and LH, the E-subunit in each case ascribes the particular hormonal specificity (4-6). Many preparations of the B-subunit of HCG obtained by chromatography of urea dissociated HCG show microheterogeneity (7) and can be seen to consist of more than one glycopeptides in terms of their physico-chemical properties. Besidesuartiallv metabolized elvcopeotides. it is also possible that undissociated HCG and a-subunit are’present to some extent as contaminants in these preparations. The purpose of this investigation was to assess the immunological reactivity of various components of B-HCG preparations with antisera raised objective of this work was to against HCG and HLH. Another evolve a method for obtaining a preparation of B-HCG with none or minimal cross-reactivity with anti-HLH sera. MATERIALS AND METHODS Analytical grades of acrylaChemicals and Reagents: mide, N,N’-methylene-bis-acrylamide: N,N,N’ -N’-tetramethyl ethylene diamine and other reagents used in polyacrylamide gel electrophoresis were obtained from Canalco, Bethesda, Other chemicals used were either from Sigma Maryland. or of analytical grade of guaranteed Chemical Co., U.S.A. purity from British Drug House (India) Pvt. Ltd. and E. Merck 125I (IMS 30) was obtained from Radio[India) Pvt. Ltd. Anti-C-HCG, anti-HLH, HCG (CR-115) Chemical Center, Amersham. and HLH (LER-960) were from National Institute of Health. Electrophoresis was carried out in 7.5: polyacrylamide gels according to the method of Davis (8) with the modifications that in place of the sample and spacer gels, 5% sucrose was layered on the gel and the sample was applied all solutions for making in 10% sucrose. For urea gels, the buffer and the sample contained urea to a final the gel, BDH grade) was further concentration of 8 M. Urea (Analar purified before use by passing a 10 M solution through a cation-anion exchange resin column prepared by mixing mixed equal quantities of Dowex-1 x 8 and Dowex-50 x 4 of 200 mesh The gels were stained with amido black in case of size. B M urea gels and with Coomassie brilliant blue R-250 (9) for
132
FEBRUARY
1976 VOL. 13 NO. 2
CONTRACEPTION
other runs. The bands were quantitated stained gels in Chromoscan MK II (Joyce England) at 620 nm.
by scanning Loebl & Co.
the Ltd.
Immunological reactivity of the bands: From a set a part were stained and others kept of gels run in parallel, for slicing into 5.0 mm thick segments. The slices were eluted by keeping them immersed for 4 days at 4OC in 3 ml of 10 mM phosphate buffer pH 7.4 containing 0.9% NaCl and 0.1% sodium azide. In some experiments the elution was performed by homogenization of the slices in the same buffer in a Potter-Elvehjem homogenizer. Both procedures gave similar results with duplicate samples. Aliquots of the eluates were taken at different dilutions and tested for reactivity by competition the binding of lz51-HCG and lzsI-HLH to anti-6-HCG anti-HLH sera. The procedures for radioimmunoassays the same as described elsewhere (10).
for and were
The antisera raised against purified Immunoadsorbent: ovine LH In 3 rabbits were pooled and dialyzed against 50 mM phosphate buffer pH 7.4 with 3 changes. The immunoadsorbents were prepared by polymerization with glutaraldehyde according to the method of Avrameas and l‘ernynck (11). RESULTS Electrophoretic pattern of B-HCG preparations: On electrophoresis in 7. 5% polyacrylamide gels containing 8 M urea, three of the B-HCG preparations received from other investigators (Dr. V. Stevens and Dr. O.P. Bahl) were found to resolve into 7 to 8 bands (Figure 1). Immunological reactivity of the bands: The poly acrylamide gels were cut into 5 mm segments. These were eluted with buffer as described in methods section. The immunological reactivities of the eluted fractions with anti-B-HCG and anti-HLH were determined by competition offered by these fractions in the binding of ‘251-IICG and rz51-HLH to anti-B-HCG and anti-HLH, respectively. Figure 2 shows the results of a representative experiment. The antiB-HCG reactivitv was predominantly located in two or three adjacent fractions. The anti-HLH reactivity was partly seen in a fraction comigrating with one of these but was also seen in a fraction migrating slightly slower than the antiHCG reacting fractions. Reduction of anti-HLII reactivity: and B-HCG have large homologies, there are in the primary structure of these peptides.
FEBRUARY
1976
VOL. 13 NO. 2
Although 6-IILH also differences These, as also
133
CONTRACEPTION
+
Figure
134
1:
Electrophoretic patterns of some preparations of beta-HCG in 7.5% polyacrylamide gels in presence Protein samples were kept for 2 hours of 8M urea. in 8M urea at 37OC prior to electrophoresis. Gels 1 to 3: different batches of beta-HCG, 4. alphaHCG.
FEBRUARY
1976
VOL.
13
NO. 2
CONTRACEPTION
70-
6@
_
ORlGlN
Figure
DISTANCE
cm
2: Immunoreactivity of B-HCG fractions resolved by electrophoresis on polyacrylamide gels with antiB-HCG and anti-HLH. ( ) scan of the Coomassie blue stained gel at 620 nm. Reactivity with antiHLH sera (@----+) and with anti- @-HCG sera (-1. 20 ug of @-HCG was loaded. For staining the gels, 50 pg of B-HCG was used.
FEBRLIARY
1976
VOL.
13 NO. 2
135
CONTRACEPTION
the microheterogeneity noted in B-HCG preparation due to endogenous metabolism, could lead to conformations in which antigenic determinants reacting preferentially with anti-HCG or anti-HLll are esposed. It was thus considered whether graded absorption with an immunoadsorbent prepared from an anti-serum against Lll from a heterologous species (say ovine) would not selectively remove material reacting with high avidity with anti-HLII sera. Figure 3 shows the results of an experiment in which the preparation used for the experiment shown in Figure 2 was absorbed with anti-ovine l,II immunoIt may he stated that the time of adsorbent for 30 minutes. adsorption with the immunoadsorbent will vary with the avidity of the antibodies employed for immunoadsorbents and with the The yield of degree of heterogeneity of B-IICG preparations. e-HCG after processing varied from 40 to 65: from preparation to preparation. The reaction with anti-H111 is negligible in Table I summarizes the immunothe processed preparation. logical reactivities against anti-IlLII of the polyacrylamide gel resolved fractions of B-HCG hcfore and after “processing” with the immunosorbent.
80
70
60
50 i
0R;GIN
Figure
136
3:
DISTANCE
cm
Immunoreactivity of the “processed” (anti-ovine LII immunoadsorbent absorbed) B-HCG preparation. scan of the stained gel at 620 nm, t-----* anti-HLH reactivity,uanti2.5 lig of B-HCG after processing B-HCG reactivity. For staining the gels, was loaded on the gel. 25 ng of processed B-HCG was used.
FEBRUARY
1976 VOL. 13 NO. 2
CONTRACEPTION
'Table
I:
Slice No.
Cross-reactivity of processed and native with anti-HLH (Values are ng HLH/ml)
Experiment I Native Processed
Experiment II Native Processed
62
16
108
3 3
54
11
48
106
4
60
L
120
3
11
3
116
6
15
4
274
6
11
3
620
0
8
4
8
353
9
377
4 7
94
4
13
68
3
10
B-HCG
4
11
860
14
136
5
12
720
12
68
5
13
750
8
128
5
14
235
3
1,240
5
15
56
0
1,020
6
16
26
0
1,440
4
17
10
0
1,200
6
18
10
0
0
4
19
17
0
0
4
LO
12
0
0
5
21
0
0
72
5
22
0
0
0
5
23
0
0
0
2
24
0
0
0
4
25
0
0
0
4
26
0
0
0
4
27
0
0
0
4
28
0
0
0
6 (continued)
FEBRUARY
1976
VOL. 13 NO. 2
137
CONTRACEPTION
Table
I:
(continued)
Each slice was eluted in 2 ml in Experiment
in 3 ml huffer II.
in
Slice No. 2 refers to the slice from the acrylamide gel (7.5%) after elctrophoresis before and after processing.
Experiment
I and
origin of polyof B-HCG
The assay procedure was the same as for radioimmunoassay of HCG except that the incubation period with anti-HLH sera was 3 days. Iodination of HLH was carried out for 30 seconds instead of 2 minutes 3s is the case for HCG. DISCUSSION The use of HCG as an antigen is obviously attendant with the disadvantage of evoking cross-reaction with a number of hormones such as LH. FSH and TSH. 3s would be expected by virtue of the largely identical a-subunits of This has been indeed found to be the case these hormones. and immunization of six human female subjects with HCG coupled with sulfanilazo groups was found to give rise to antibodies reacting not only against HCG but also with HLH It is thus clear that the entire molecule of HCG f12)., Immunization with the cannot be used for isoimmunization. B-subunit of HCG can produce sera in some animals reacting with HCG and devoid of cross-reactivity with HLH and other hormones (10, 13). The use of preparations of B-HCG obtained after managed absorption with heterologous anti-LH immunosorbents is helpful in eliminating the undissociated HCG and other moieties reacting with high affinity with HLH and gives anti-sera with none or minimal cross-reactivity with HLH. REFERENCES
138
1.
Chorionic Jones, G.E.S., Delfs, E. and Stran, 1I.M.: gonadotropin and pregnanediol values in normal preg75:359 (1944). Bull. Johns Hopkins Hosp. nancy .
2.
Albert, A. and Berkson, J.: for chorionic gonadotropin. 11:805-820 (1951). Metab.
3.
Lorain, J.A.: in the urine 331 (1950).
of
A clinical bio-assay J. Clin. Endocrinol.
The estimation of pregnancy women.
chorionic gonadotropin J. Endocrinol. 6:319-
FEBRUARY
1976
VOL.
13 NO. 2
CONTRACEPTION
4.
Canfield R.E., Morgan, F.J., Kammerman, S., Bell, J.J. and Agosto, G.M.: Studies on human chorionic gonado27:121-164 (1971). tropin. Rec. Progs. Horm. Res.
5.
Morgan, F.J., Birken, S. and Canfield, R.E.: Chemistry In: Gonadotropin and of human chorionic gonadotropin. Gonads1 Function (ed. N.R. Moudgal), Academic Press, N.Y. 1974, pp 79-92.
6.
Pierce, J.G., Bahl, O.P., Cornell, J.S. and Swaminathan, N.: Biologically active hormone prepared by recombinaand the hormone specific chain tion of n-chain of HCG of bovine thyrotropin or bovine luteinizing hormone. 3. Biol. Chem. 246:2321-2324 (1971).
7.
The Graesslin, D., h'eise, H.C. and Braendle, W.: micro-heterogeneity of human chorionic gonadotropin FEBS Letters 31:214(HCG) reflected in the B-subunit. 216 (1973).
8.
Davis, B.J.: Disc electrophoresis-II. application to human serum proteins. Sci. 121:404-4'7 (1964).
9.
Chrambach, A., Reisfeld, R.A., Ivyckoff, M. and Zaccari, J.: A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electro20:150-154 (1967). phoresis. Anal. Biochem.
Method and Ann. N.Y. Acad.
10.
Salahuddin, M., Ramakrishnan, S., Dubey, S.K. and Talwar, G.P.: Immunological reactivity of antibodies produced by Pr-B-HCG-TT with different hormones. Contraception 13:163 (1976).
11.
Avrameas, S. and Ternynck, T.: The cross-linking proteins with glutaraldehyde and its use for the Immunochemistry preparation of immunoadsorbents. 6:53-66 (1969).
12.
Stevens, V.C. and Crystle, zation with hapten-coupled cycle. Obstet. 8 Gynecol.
13.
Vaitukaitis, J.L., Braunstein, G.D. and Ross, G.T.: A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. 113:751Am. J. Obstet. Gynec. 7.58 (1972).
FEBRUARY
1976
VOL.
13 NO. 2
of
Effects of immuniC.D.: HCG on the human menstrual 42:485-495 f1973).
139
PROCESSING OF THE PREPARATIONS OF I?-SIJBWIT OF HUMAN CHORIONIC GONADOTROPIN FOR MINIMIZATION OF CROSS-REACTIVITY WITH l!UMAN LUTEINIZILG HORMONE
G.P.
‘Talwar, N.
?1.C. Sharma, Shastri and
S.K. Dubev, M. S. Ramakrishnan
Department of Biochemistry, All India Medical Sciences, New Delhi-IlOOlh,
Salahuddin,
Institute India
of
AHSTRAC’I
B-HCG preparations demonstrate microheterogeneity and are resolved in multiple bands on electrophoresis in polyacrylamide gels. The gels were cut into segments and the immunological reactivity of eluates with anti-B-HCG and anti-JJLH sera was inb-estigated. The hands reacting with the two antigonadotropin sera were identified. Graded ahsorption of II-HCG with anti-heterologous [ovine1 Lll gave preparations with none or minimal cross-reactivity with anti -JILIJ.
,\cccptcd
FEBRUARY
[or
publication
1976
October
VOL. 13 NO. 2
20,
1975
131
CONTRACEPTION
INTRODUCTION -Human chorionic gonadotropin (HCG) is commercially prepared from the urine of pregnant women! who excrete large quantities of this hormone especially in the first trimester of pregnancy (l-3). It is not unexpected that the hormone recovered in the urine represents at least in part the moieties metabolised to variable extent in the kidney and other “target” and “non- target” organs. HCG is composed of two suunits; the a-subunit is largely identical to the a-subunit of TSH, FSH, and LH, the E-subunit in each case ascribes the particular hormonal specificity (4-6). Many preparations of the B-subunit of HCG obtained by chromatography of urea dissociated HCG show microheterogeneity (7) and can be seen to consist of more than one glycopeptides in terms of their physico-chemical properties. Besidesuartiallv metabolized elvcopeotides. it is also possible that undissociated HCG and a-subunit are’present to some extent as contaminants in these preparations. The purpose of this investigation was to assess the immunological reactivity of various components of B-HCG preparations with antisera raised objective of this work was to against HCG and HLH. Another evolve a method for obtaining a preparation of B-HCG with none or minimal cross-reactivity with anti-HLH sera. MATERIALS AND METHODS Analytical grades of acrylaChemicals and Reagents: mide, N,N’-methylene-bis-acrylamide: N,N,N’ -N’-tetramethyl ethylene diamine and other reagents used in polyacrylamide gel electrophoresis were obtained from Canalco, Bethesda, Other chemicals used were either from Sigma Maryland. or of analytical grade of guaranteed Chemical Co., U.S.A. purity from British Drug House (India) Pvt. Ltd. and E. Merck 125I (IMS 30) was obtained from Radio[India) Pvt. Ltd. Anti-C-HCG, anti-HLH, HCG (CR-115) Chemical Center, Amersham. and HLH (LER-960) were from National Institute of Health. Electrophoresis was carried out in 7.5: polyacrylamide gels according to the method of Davis (8) with the modifications that in place of the sample and spacer gels, 5% sucrose was layered on the gel and the sample was applied all solutions for making in 10% sucrose. For urea gels, the buffer and the sample contained urea to a final the gel, BDH grade) was further concentration of 8 M. Urea (Analar purified before use by passing a 10 M solution through a cation-anion exchange resin column prepared by mixing mixed equal quantities of Dowex-1 x 8 and Dowex-50 x 4 of 200 mesh The gels were stained with amido black in case of size. B M urea gels and with Coomassie brilliant blue R-250 (9) for
132
FEBRUARY
1976 VOL. 13 NO. 2
CONTRACEPTION
other runs. The bands were quantitated stained gels in Chromoscan MK II (Joyce England) at 620 nm.
by scanning Loebl & Co.
the Ltd.
Immunological reactivity of the bands: From a set a part were stained and others kept of gels run in parallel, for slicing into 5.0 mm thick segments. The slices were eluted by keeping them immersed for 4 days at 4OC in 3 ml of 10 mM phosphate buffer pH 7.4 containing 0.9% NaCl and 0.1% sodium azide. In some experiments the elution was performed by homogenization of the slices in the same buffer in a Potter-Elvehjem homogenizer. Both procedures gave similar results with duplicate samples. Aliquots of the eluates were taken at different dilutions and tested for reactivity by competition the binding of lz51-HCG and lzsI-HLH to anti-6-HCG anti-HLH sera. The procedures for radioimmunoassays the same as described elsewhere (10).
for and were
The antisera raised against purified Immunoadsorbent: ovine LH In 3 rabbits were pooled and dialyzed against 50 mM phosphate buffer pH 7.4 with 3 changes. The immunoadsorbents were prepared by polymerization with glutaraldehyde according to the method of Avrameas and l‘ernynck (11). RESULTS Electrophoretic pattern of B-HCG preparations: On electrophoresis in 7. 5% polyacrylamide gels containing 8 M urea, three of the B-HCG preparations received from other investigators (Dr. V. Stevens and Dr. O.P. Bahl) were found to resolve into 7 to 8 bands (Figure 1). Immunological reactivity of the bands: The poly acrylamide gels were cut into 5 mm segments. These were eluted with buffer as described in methods section. The immunological reactivities of the eluted fractions with anti-B-HCG and anti-HLH were determined by competition offered by these fractions in the binding of ‘251-IICG and rz51-HLH to anti-B-HCG and anti-HLH, respectively. Figure 2 shows the results of a representative experiment. The antiB-HCG reactivitv was predominantly located in two or three adjacent fractions. The anti-HLH reactivity was partly seen in a fraction comigrating with one of these but was also seen in a fraction migrating slightly slower than the antiHCG reacting fractions. Reduction of anti-HLII reactivity: and B-HCG have large homologies, there are in the primary structure of these peptides.
FEBRUARY
1976
VOL. 13 NO. 2
Although 6-IILH also differences These, as also
133
CONTRACEPTION
+
Figure
134
1:
Electrophoretic patterns of some preparations of beta-HCG in 7.5% polyacrylamide gels in presence Protein samples were kept for 2 hours of 8M urea. in 8M urea at 37OC prior to electrophoresis. Gels 1 to 3: different batches of beta-HCG, 4. alphaHCG.
FEBRUARY
1976
VOL.
13
NO. 2
CONTRACEPTION
70-
6@
_
ORlGlN
Figure
DISTANCE
cm
2: Immunoreactivity of B-HCG fractions resolved by electrophoresis on polyacrylamide gels with antiB-HCG and anti-HLH. ( ) scan of the Coomassie blue stained gel at 620 nm. Reactivity with antiHLH sera (@----+) and with anti- @-HCG sera (-1. 20 ug of @-HCG was loaded. For staining the gels, 50 pg of B-HCG was used.
FEBRLIARY
1976
VOL.
13 NO. 2
135
CONTRACEPTION
the microheterogeneity noted in B-HCG preparation due to endogenous metabolism, could lead to conformations in which antigenic determinants reacting preferentially with anti-HCG or anti-HLll are esposed. It was thus considered whether graded absorption with an immunoadsorbent prepared from an anti-serum against Lll from a heterologous species (say ovine) would not selectively remove material reacting with high avidity with anti-HLII sera. Figure 3 shows the results of an experiment in which the preparation used for the experiment shown in Figure 2 was absorbed with anti-ovine l,II immunoIt may he stated that the time of adsorbent for 30 minutes. adsorption with the immunoadsorbent will vary with the avidity of the antibodies employed for immunoadsorbents and with the The yield of degree of heterogeneity of B-IICG preparations. e-HCG after processing varied from 40 to 65: from preparation to preparation. The reaction with anti-H111 is negligible in Table I summarizes the immunothe processed preparation. logical reactivities against anti-IlLII of the polyacrylamide gel resolved fractions of B-HCG hcfore and after “processing” with the immunosorbent.
80
70
60
50 i
0R;GIN
Figure
136
3:
DISTANCE
cm
Immunoreactivity of the “processed” (anti-ovine LII immunoadsorbent absorbed) B-HCG preparation. scan of the stained gel at 620 nm, t-----* anti-HLH reactivity,uanti2.5 lig of B-HCG after processing B-HCG reactivity. For staining the gels, was loaded on the gel. 25 ng of processed B-HCG was used.
FEBRUARY
1976 VOL. 13 NO. 2
CONTRACEPTION
'Table
I:
Slice No.
Cross-reactivity of processed and native with anti-HLH (Values are ng HLH/ml)
Experiment I Native Processed
Experiment II Native Processed
62
16
108
3 3
54
11
48
106
4
60
L
120
3
11
3
116
6
15
4
274
6
11
3
620
0
8
4
8
353
9
377
4 7
94
4
13
68
3
10
B-HCG
4
11
860
14
136
5
12
720
12
68
5
13
750
8
128
5
14
235
3
1,240
5
15
56
0
1,020
6
16
26
0
1,440
4
17
10
0
1,200
6
18
10
0
0
4
19
17
0
0
4
LO
12
0
0
5
21
0
0
72
5
22
0
0
0
5
23
0
0
0
2
24
0
0
0
4
25
0
0
0
4
26
0
0
0
4
27
0
0
0
4
28
0
0
0
6 (continued)
FEBRUARY
1976
VOL. 13 NO. 2
137
CONTRACEPTION
Table
I:
(continued)
Each slice was eluted in 2 ml in Experiment
in 3 ml huffer II.
in
Slice No. 2 refers to the slice from the acrylamide gel (7.5%) after elctrophoresis before and after processing.
Experiment
I and
origin of polyof B-HCG
The assay procedure was the same as for radioimmunoassay of HCG except that the incubation period with anti-HLH sera was 3 days. Iodination of HLH was carried out for 30 seconds instead of 2 minutes 3s is the case for HCG. DISCUSSION The use of HCG as an antigen is obviously attendant with the disadvantage of evoking cross-reaction with a number of hormones such as LH. FSH and TSH. 3s would be expected by virtue of the largely identical a-subunits of This has been indeed found to be the case these hormones. and immunization of six human female subjects with HCG coupled with sulfanilazo groups was found to give rise to antibodies reacting not only against HCG but also with HLH It is thus clear that the entire molecule of HCG f12)., Immunization with the cannot be used for isoimmunization. B-subunit of HCG can produce sera in some animals reacting with HCG and devoid of cross-reactivity with HLH and other hormones (10, 13). The use of preparations of B-HCG obtained after managed absorption with heterologous anti-LH immunosorbents is helpful in eliminating the undissociated HCG and other moieties reacting with high affinity with HLH and gives anti-sera with none or minimal cross-reactivity with HLH. REFERENCES
138
1.
Chorionic Jones, G.E.S., Delfs, E. and Stran, 1I.M.: gonadotropin and pregnanediol values in normal preg75:359 (1944). Bull. Johns Hopkins Hosp. nancy .
2.
Albert, A. and Berkson, J.: for chorionic gonadotropin. 11:805-820 (1951). Metab.
3.
Lorain, J.A.: in the urine 331 (1950).
of
A clinical bio-assay J. Clin. Endocrinol.
The estimation of pregnancy women.
chorionic gonadotropin J. Endocrinol. 6:319-
FEBRUARY
1976
VOL.
13 NO. 2
CONTRACEPTION
4.
Canfield R.E., Morgan, F.J., Kammerman, S., Bell, J.J. and Agosto, G.M.: Studies on human chorionic gonado27:121-164 (1971). tropin. Rec. Progs. Horm. Res.
5.
Morgan, F.J., Birken, S. and Canfield, R.E.: Chemistry In: Gonadotropin and of human chorionic gonadotropin. Gonads1 Function (ed. N.R. Moudgal), Academic Press, N.Y. 1974, pp 79-92.
6.
Pierce, J.G., Bahl, O.P., Cornell, J.S. and Swaminathan, N.: Biologically active hormone prepared by recombinaand the hormone specific chain tion of n-chain of HCG of bovine thyrotropin or bovine luteinizing hormone. 3. Biol. Chem. 246:2321-2324 (1971).
7.
The Graesslin, D., h'eise, H.C. and Braendle, W.: micro-heterogeneity of human chorionic gonadotropin FEBS Letters 31:214(HCG) reflected in the B-subunit. 216 (1973).
8.
Davis, B.J.: Disc electrophoresis-II. application to human serum proteins. Sci. 121:404-4'7 (1964).
9.
Chrambach, A., Reisfeld, R.A., Ivyckoff, M. and Zaccari, J.: A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electro20:150-154 (1967). phoresis. Anal. Biochem.
Method and Ann. N.Y. Acad.
10.
Salahuddin, M., Ramakrishnan, S., Dubey, S.K. and Talwar, G.P.: Immunological reactivity of antibodies produced by Pr-B-HCG-TT with different hormones. Contraception 13:163 (1976).
11.
Avrameas, S. and Ternynck, T.: The cross-linking proteins with glutaraldehyde and its use for the Immunochemistry preparation of immunoadsorbents. 6:53-66 (1969).
12.
Stevens, V.C. and Crystle, zation with hapten-coupled cycle. Obstet. 8 Gynecol.
13.
Vaitukaitis, J.L., Braunstein, G.D. and Ross, G.T.: A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. 113:751Am. J. Obstet. Gynec. 7.58 (1972).
FEBRUARY
1976
VOL.
13 NO. 2
of
Effects of immuniC.D.: HCG on the human menstrual 42:485-495 f1973).
139
