0013-7227/90/1276-2977$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 6 Printed in U.S.A.
The Antigenic Structure of the Human Glycoprotein Hormone a-Subunit. I. Characterization of Anti-a Monoclonal Antibodies* M. C. CHARLESWORTH, D. J. McCORMICK, E. R. BERGERT, T. VUTYAVANICHf, H. HOJO$, AND R. J. RYAN Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905
mAbs A101, A102, and E512 were specific for the a-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free a. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using a, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on a which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated a. (Endocrinology 127: 2977-2984, 1990)
ABSTRACT. The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, a and /3. The /3-subunit confers hormone specificity, while the a-subunit is homologous within a species. To help in determining the antigenic structure of the common a-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric a-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to a- and /3-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free a-subunit, hCG or hFSH.
T
show considerable diversity in their recognition of asubunit or native hormone, and in companion papers using these antibodies, the amino acid residues of overlapping, discontinuous antigenic determinants recognized by these mAbs will be tentatively defined.
HE glycoprotein hormones FSH, LH, CG, and TSH share a common a subunit which is homologous within a species. Hormone specificity is conferred by the unique 0-subunit of each heterodimer. Chemical and enzymatic modifications of the intact hormones and the subunits have yielded some information on the relevant structural features for receptor binding, biological and immunological activity (1, 2), but the antigenic determinants are still largely undefined. Antigenic regions have been demonstrated on the individual subunits and the heterodimer (3-9), indicating that at least some of the epitopes are topographically located on both subunit chains. Identifying the sites of antigenicity on the human a-subunit would prove valuable in determining the surface orientation of amino acid residues, those residues associated with the /3-subunit, and studying any conformational changes that occur upon subunit combination and hormone receptor binding. Eight anti-a monoclonal antibodies (mAb) have been extensively characterized in this study. These mAbs
Materials and Methods Hormones
Received May 21,1990. Address correspondence and requests for reprints to: Dr. Robert J. Ryan, Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905. * Supported by funds from NIH Grant HD-9140, the Mellon Foundation, and the Mayo Foundation.
Human (h)FSH (AFP4161B), hLH (AFP4991), hTSH (AFP 4197C), hCGa (CR123a; > 99% pure), hLH/3 (AFP 3282B), hFSH/3 (AFP 4058C), and hTSH/? (AFP 785B) were provided by the National Pituitary Agency, NIDDKD (Baltimore, MD). hCG and its a- and /?-subunits were prepared in this laboratory using approximately 3000 IU/mg hCG (Diosynth, Chicago, IL) as starting material. The procedures were essentially those described by Morgan et al. (10). The purified native hCG had a potency of >10,000 IU/mg by RRA and RIA. The purified a and |8 subunits had less than 1% cross-contamination as assessed by RIA. Rabbit polyclonal antibody R101 raised against reduced and carboxymethylated (RCXM) human a-subunit was obtained from Dr. Henry Keutmann, Massachusetts General Hospital (Boston, MA) and has been characterized previously (11). Preparation of monoclonal antibodies (mAb) The A series of mAb (A101 and A102) was made using hFSH as the immunogen as described previously (12). The E series
2977
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2978
CHARACTERIZATION OF ANTI-hCGa ANTIBODIES
(E501, E502, E511 and E512) was made using free a-subunit (prepared from hCG) as the immunogen using the same techniques (12). In brief, 8- to 10-week-old BALB/c mice were immunized sc with 50 ng antigen in complete Freund's adjuvent. After 4 weeks, they were bled and antibody titers were tested by RIA. The animal with the highest titer was given 25 ng antigen in saline ip and 3 days later, the spleen was removed for fusion. Fusion was performed using the protocol of Kennett et al. (13) with a 2:1 ratio of spleen and FO mouse myeloma cells (14) and 2-min exposure to 50% polyethylene glycol 4000. Cells were aliquoted into ten 96-well microtiter plates having a feeder layer of BALB/c macrophages. They were fed every 3 days with a hypoxanthine-aminopterin-thymidine medium. After 10-14 days, the spent media from wells containing clones were screened for reaction with free a-subunit by enzyme linked immunosorbent assay (ELISA). Positive clones were further tested in a double antibody RIA, and selected anti-a clones were subcloned twice by using limiting dilution (15). Ascities fluid was produced by ip injection of 0.5-1 x 106 hybridoma cells into BALB/c mice primed 3 weeks earlier with 0.5 ml pristine. Ascites fluid was obtained after 1-2 weeks and the immunoglobulin purified by affinity chromatography using agarose-protein A columns (MAPS kit, Bio-Rad, Richmond, CA). The K series of mAbs (K2.18 and K94.6) was obtained from Dr. George Klee, Department of Laboratory Medicine, Mayo Medical School. These antibodies were made using approximately 3000 IU/mg preparation of hCG (Ayerst, Chicago, IL) as the antigen. Positive clones were identified by immunoradiometric assay using sheep antimouse IgG-coated tubes and incubation with 125I- hCG, hCGa and hCG/3. Antibodies that bound free a subunit were selected, amplified in ascites fluid and purified as above. ELISA The basic procedure was that described by Douillard and Hoffman (16). One hundred-microliter aliquots of antigen (1 Mg/ml) in carbonate buffer (pH 9.8) were pipetted into each well of 96-well microtiter plates and incubated overnight at 4 C. After washing with buffer (150 mM NaCl2, 20 mM Tris, 0.05% Tween 20, 0.01% ovalbumin; pH 7.4), 100 fd of diluted mouse serum or hybridoma culture medium were allowed to react for 1 h at 22 C. The wells were washed again with buffer and then incubated for 1 h with goat anti-mouse immunoglobulin G (IgG) coupled with alkaline phosphatase (PelFreeze, Brown Deer, WI). After an additional wash with buffer, they were incubated for 1 h with substrate (p-nitrophenyl phosphate, Sigma, St. Louis, MO) and absorbance was measured at 410 nm using a microtiter plate reader (Dynatech, Cambridge, MA). IgG subclasses were determined in a similar ELISA procedure using mouse isotype-specific rabbit antibodies and goat antirabbit IgG-peroxidase (HyClone, Logan, UT) RIA Hormones, subunits, and mAbs were iodinated by a modification (17) of the chloramine-T method of Greenwood et al. (18), yielding specific activities of 100-200 nCi/fig for the hormones and subunits, and 9-28 /xCi/ng for the mAbs.
Endo • 1990 Voll27^No6
Antibodies of the A, K, and E series were titered against 125Ilabeled subunits and native hormones indicated in Table 2. One tenth ng of 125I-«- or /?-subunit or hormone was added to graded concentrations of purified mAb (0.15-1000 ng/tube) in 0.5 ml 40 mM Tris/0.1% BSA (Tris/BSA buffer, pH 7.4). After overnight incubation (~16h) at 22 C, assays were terminated by double antibody (goat antimouse IgG)-polyethylene glycol precipitation, and 125I was measured in the pellets. The affinities of the mAbs were determined by the method of Scatchard (19) from RIAs employing monoclonal antibodies and increasing concentrations of unlabeled a in the presence of a fixed amount of labeled a. Cross-reactivities of the mAbs with native glycoprotein hormones and the subunits were determined by displacement of 0.1 ng 125I-a or 125I-hFSH from the E and K mAb series or the A series, respectively, with increasing doses of ligand (hFSH, hLH, hTSH, hCG, a-subunit, and the corresponding /3-subunits) under conditions described in the previous paragraph. All RIAs were repeated at least twice. RRA The ability of the eight monoclonal antibodies to inhibit binding of radiolabeled hCG to ovarian receptor was determined by a competitive RRA using a 2000 X g membrane-rich fraction from superovulated rat ovaries (17). Graded concentrations of mAb were added to tubes containing 1 ng 125I-hCG and 1.25 mg (wet weight) of receptor preparation in a total volume of 1 ml Tris/BSA buffer. Tubes were incubated overnight at 22 C, then centrifuged to precipitate pellet, washed, and counted. Nonspecific binding was assessed by adding an excess of unlabeled hCG (2000 IU/ml) in the presence of tracer and receptor. Data reflect specific binding obtained by subtracting the nonspecific component from total binding. To determine if any of the antibodies recognized receptorbound hormone, unlabeled hCG (50 ng) was incubated with receptor as described above. After centrifuging to precipitate membrane receptor-hormone complex, the pellet was washed three times with fresh buffer, and approximately 100,000 cpm each 125I-mAb was added in the original assay volume. An excess of 100 ng appropriate unlabeled antibody was added to control tubes to correct for nonspecific binding. After an 8-h incubation at 22 C, tubes were centrifuged, and the pellet was washed and counted. Sandwich radioimmunometric assays To determine their specificity for different epitopes on asubunit, native hCG and hFSH, two-site binding assays were performed with all the mAbs used as either capture antibodies (solid phase-bound) or detection antibodies (125I-mAbs). mAbs (1 Mg/50 fii carbonate buffer, pH 9.8) were incubated in triplicate in 96-well microtiter plates (Immulon 1 Removawell strips; Dynatech, Alexandria, VA) overnight at 4 C. After washing with PBS (100 mM phosphate buffer, 150 mM NaCl2; pH 7.4), plates were blocked with 1% BSA/0.25% ovalbumin in PBS for 1 h at 22 C. After a PBS wash, antigens a, hCG, or hFSH (50 Ml/well in 0.1% BSA/0.025% ovalbumin/PBS) were added to the plates. Concentrations of antigen were varied to reflect the
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CHARACTERIZATION OF ANTI-hCGa ANTIBODIES differing affinities among the antibodies: 5 ng a and FSH for K2.18, K94.6, E502, and E511; 100 ng for E501; 500 ng for A101, A102, and E512. hCG was added at a concentration of 500 ng for all the mAb. Capture antibodies reacted with antigen for 8 h at room temperature, after which 100,000 cpm (~2 ng/ 50 /il) of each detection 125I-mAb were added to triplicate wells for determination of binding to antigen. After overnight incubation, wells were washed with PBS, separated, and counted. Nonspecific binding was determined from wells not containing antigen. Assays were repeated twice for each antibody and antigen. Differences in binding of 125I-mAbs to each of the eight capture antibodies were deemed significant at the P < 0.01 level if the total specific counts bound to the well were greater than the mean of the control wells (no antigen) + 3 SD. Reduction and carboxymethylation (RCXM) of a-subunit RCXM a was prepared by the method of Crestfield (20) as previously used in this laboratory (21).
Results RIA
The general properties of the monoclonal antibodies characterized in this report are given in Table 1. Despite the differences in antigen used as immunogen, all mAbs are specific for the a-subunit, although A101 and A102 recognize native hFSH with a potency about 300-fold greater than that for free a-subunit (12). The affinities of five of the antibodies as estimated by the ability of unlabeled a-subunit to compete with iodinated a were very high (10~u to 10~10 M), whereas E512, A102, and A101 had affinity constants (Kds) of 1.2, 2.4, and 4.7 x 10~7 M, respectively. The slopes of the Scatchard plots (not shown) indicated linearity as would be expected for mAbs. The titers of the mAbs to subunits or hormones are shown in Table 2. For the a-subunit, E502 and E511 had the highest titer, followed by K2.18, E501, K94.6, and E512. E502 and E511 also bound hCG, hLH, and hTSH, and E501 bound hFSH, hTSH, and hLH, albeit at a much higher concentration of mAb. None of these antiTABLE 1. Specificities and affinities of mAbs mAb K2.18 E511 E502 K94.6 E501 E512 A102 A101
Species and Class MIgG, MIgG2b MIgG2 MIgG2 MIgG2b MIgG2a MIgG, MIgG,
Immunogen hCG a(hCG) a(hCG) hCG a(hCG) a(hCG) hFSH hFSH
Specificity a a a a a a a a
Kd (1(T10 M)° 0.24 0.34 1.70 1.80 3.70 1200.00 2400.00 4700.00
0 Affinities for the E and K series are for 125I-a subunit; affinities for the A series are for a-subunit using 125I-hFSH as tracer.
2979
TABLE 2. Titers of mAbs against radiolabeled ligand 5
MAba 320 A101 A102 >1000 K2.18 2.6 4.2 K94.6 4.5 E501 E502 0.3 0.4 E511 E512 170.0
hCG
I-Ligand
hLH
330
hFSH
hTSH 0 subunit
2.5 15
>1000 240
>1000 >1000 780 920
700 2.5 7.7
>1000 >1000
22
>1000 >1000 >1000
326 150 170
1000
>1000* >1000* >1000c >1000c >1000" >1000d >1000d >1000d
" Amount of mAb (nanograms of wt/vol) necessary to bind 30% of I-labeled human subunit or hormone (0.15 ng/tube); data shown are means of two assays. b /3-Subunits of hCG and FSH were tested. c /3-Subunits of hCG was tested. d 0-Subunits of hCG, LH, FSH, and TSH were tested. 125
bodies bound any of the 125I-/3 subunits at concentrations of 1 /zg or more. A previous report (12) indicated that A101 at 100 ng bound 45% of hFSH, 8% of a, and no FSH/3, A102 at 300 ng bound 42% of hFSH, 6% of a and no FSH/3. Cross-reactivities of the antibodies with native hormones and jS-subunits of human CG, LH, FSH, and TSH are shown in Table 3. The apparent high potency of FSH/3 in competition with 125I-a for binding to the E and K series mAbs is due to 11.8% contamination of the fi preparation with free a-subunit as determined by RIA (data not shown). Upon correcting for this contamination, most of the j8-subunits were only 0.1-1.5% as potent as free a in competing for binding of a to mAbs E501, E502, E511, K2.18, and K94.6. TSH/3 was 5% as potent as a for binding to K2.18 and K94.6. E512 recognized hLH, FSH, and TSH /3-subunits, but not hCG/3. hCG and hTSH were relatively ineffective in competing with a for binding to E511, E502, K2.18, and K94.6 (1-3%). These antibodies do cross-react with hLH and FSH, as does E501, which also binds hTSH. Interestingly, E512 recognizes the dimeric a-subunit of hLH better than free a. Cross-reaction of E512 with the native hormones was also high. A101 and A102 recognized asubunit in combination with /3 in the glycohormone dimers better than free a. RRA All eight mAbs inhibited binding of 125I-hCG to ovarian receptor with varying potencies as shown in Table 4. Except for the E series mAbs, these potencies didn't correlate with the antibodies' affinities for a-subunit. In addition, none of the antibodies were able to recognize hCG bound to receptor (Fig. 1).
Reactions with RCXM-a Figure 2 indicates the reactions of this panel of monoclonal antibodies with RCXM-a, a denatured and linear-
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2980
C H A R A C T E R I Z A T I O N OF A N T I - h C G a A N T I B O D I E S
Endo. 1990 Vol 127 • No 6
TABLE 3. Cross-reactivities of anti-a mAbs with human glycoprotein hormones and /3-subunits IC50 = nM° mAb
a Subunit
A101 A102 K2.18 K94.6 E501 E502 E511 E512
470.00 240.00 0.02 0.18 0.37 0.17 0.03 121.00
/3 Subunit hCG (%)"
LH (%)
FSH (%)
2100.0 11000.0 3.3 (0.6) 17.0 (1.1) >83.3 >83.3 26.6 (0.1) 16600.0 (0.7)
9100.0 13000.0 1.5 (1.3) 11.0 (1.6) 22.3 (1.7) 16.5 (1.0) 2.4 (1.3) 405.0 (30.0)
1030.0 ' 420.0 0.1 (17.0) 0.9 (20.0) 1.7 (21.0) 0.6 (27.0) 0.4 (7.5) 238.0 (51.0)
Hormone TSH (%)
hCG (%)
LH (%)
0.5 (4.3) 3.9 (4.6) 18.3 (2.0) 10.7 (1.6) 2.5 (1.2) 1330.0 (9.1)
10.6 50.0 0.4 (5.0) 2.4 ( 7.5) 3.6 (10.0) 7.3 (2.3) 1.4 (2.1) 352.0 (34.0)
52.5 200.0 0.1 (20) 0.7 (26) 0.6 (62) 0.4 (43) 0.1 (30) 43.5 (278)
0 Concentration of ligand needed to displace 50% of the binding of 0.1 ng antibody; data shown are means of at least two assays. * Values in parentheses represent percent cross-reactivity with a-subunit.
5
FSH (%) 0.6 1.2
0.1 (20) 0.5 (36) 0.5 (74) 0.6 (28) 0.1 (30) 110.0 (110)
I-a subunit (for E and K series) or
TSH (%) 14.0 26.0 0.4 (5.0) 2.2 ( 8.2) 1.0 (37.0) 3.0 (5.7) 0.7 (4.3) 315.0 (38.0)
125
I-hFSH (for A series) to
TABLE 4. Inhibition of 125I-hCG binding to receptor by anti-a mAbs mAb
IC50 (10-8 M)"
A101 A102 K2.18 K94.6 E501 E502 E511 E512
36.0 14.0 214.0 850.0 16.5
_o
2.1 4.7
c
Ol
1
c/5 CO
Vol. 127, No. 6 Printed in U.S.A.
The Antigenic Structure of the Human Glycoprotein Hormone a-Subunit. I. Characterization of Anti-a Monoclonal Antibodies* M. C. CHARLESWORTH, D. J. McCORMICK, E. R. BERGERT, T. VUTYAVANICHf, H. HOJO$, AND R. J. RYAN Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905
mAbs A101, A102, and E512 were specific for the a-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free a. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using a, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on a which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated a. (Endocrinology 127: 2977-2984, 1990)
ABSTRACT. The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, a and /3. The /3-subunit confers hormone specificity, while the a-subunit is homologous within a species. To help in determining the antigenic structure of the common a-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric a-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to a- and /3-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free a-subunit, hCG or hFSH.
T
show considerable diversity in their recognition of asubunit or native hormone, and in companion papers using these antibodies, the amino acid residues of overlapping, discontinuous antigenic determinants recognized by these mAbs will be tentatively defined.
HE glycoprotein hormones FSH, LH, CG, and TSH share a common a subunit which is homologous within a species. Hormone specificity is conferred by the unique 0-subunit of each heterodimer. Chemical and enzymatic modifications of the intact hormones and the subunits have yielded some information on the relevant structural features for receptor binding, biological and immunological activity (1, 2), but the antigenic determinants are still largely undefined. Antigenic regions have been demonstrated on the individual subunits and the heterodimer (3-9), indicating that at least some of the epitopes are topographically located on both subunit chains. Identifying the sites of antigenicity on the human a-subunit would prove valuable in determining the surface orientation of amino acid residues, those residues associated with the /3-subunit, and studying any conformational changes that occur upon subunit combination and hormone receptor binding. Eight anti-a monoclonal antibodies (mAb) have been extensively characterized in this study. These mAbs
Materials and Methods Hormones
Received May 21,1990. Address correspondence and requests for reprints to: Dr. Robert J. Ryan, Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905. * Supported by funds from NIH Grant HD-9140, the Mellon Foundation, and the Mayo Foundation.
Human (h)FSH (AFP4161B), hLH (AFP4991), hTSH (AFP 4197C), hCGa (CR123a; > 99% pure), hLH/3 (AFP 3282B), hFSH/3 (AFP 4058C), and hTSH/? (AFP 785B) were provided by the National Pituitary Agency, NIDDKD (Baltimore, MD). hCG and its a- and /?-subunits were prepared in this laboratory using approximately 3000 IU/mg hCG (Diosynth, Chicago, IL) as starting material. The procedures were essentially those described by Morgan et al. (10). The purified native hCG had a potency of >10,000 IU/mg by RRA and RIA. The purified a and |8 subunits had less than 1% cross-contamination as assessed by RIA. Rabbit polyclonal antibody R101 raised against reduced and carboxymethylated (RCXM) human a-subunit was obtained from Dr. Henry Keutmann, Massachusetts General Hospital (Boston, MA) and has been characterized previously (11). Preparation of monoclonal antibodies (mAb) The A series of mAb (A101 and A102) was made using hFSH as the immunogen as described previously (12). The E series
2977
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2978
CHARACTERIZATION OF ANTI-hCGa ANTIBODIES
(E501, E502, E511 and E512) was made using free a-subunit (prepared from hCG) as the immunogen using the same techniques (12). In brief, 8- to 10-week-old BALB/c mice were immunized sc with 50 ng antigen in complete Freund's adjuvent. After 4 weeks, they were bled and antibody titers were tested by RIA. The animal with the highest titer was given 25 ng antigen in saline ip and 3 days later, the spleen was removed for fusion. Fusion was performed using the protocol of Kennett et al. (13) with a 2:1 ratio of spleen and FO mouse myeloma cells (14) and 2-min exposure to 50% polyethylene glycol 4000. Cells were aliquoted into ten 96-well microtiter plates having a feeder layer of BALB/c macrophages. They were fed every 3 days with a hypoxanthine-aminopterin-thymidine medium. After 10-14 days, the spent media from wells containing clones were screened for reaction with free a-subunit by enzyme linked immunosorbent assay (ELISA). Positive clones were further tested in a double antibody RIA, and selected anti-a clones were subcloned twice by using limiting dilution (15). Ascities fluid was produced by ip injection of 0.5-1 x 106 hybridoma cells into BALB/c mice primed 3 weeks earlier with 0.5 ml pristine. Ascites fluid was obtained after 1-2 weeks and the immunoglobulin purified by affinity chromatography using agarose-protein A columns (MAPS kit, Bio-Rad, Richmond, CA). The K series of mAbs (K2.18 and K94.6) was obtained from Dr. George Klee, Department of Laboratory Medicine, Mayo Medical School. These antibodies were made using approximately 3000 IU/mg preparation of hCG (Ayerst, Chicago, IL) as the antigen. Positive clones were identified by immunoradiometric assay using sheep antimouse IgG-coated tubes and incubation with 125I- hCG, hCGa and hCG/3. Antibodies that bound free a subunit were selected, amplified in ascites fluid and purified as above. ELISA The basic procedure was that described by Douillard and Hoffman (16). One hundred-microliter aliquots of antigen (1 Mg/ml) in carbonate buffer (pH 9.8) were pipetted into each well of 96-well microtiter plates and incubated overnight at 4 C. After washing with buffer (150 mM NaCl2, 20 mM Tris, 0.05% Tween 20, 0.01% ovalbumin; pH 7.4), 100 fd of diluted mouse serum or hybridoma culture medium were allowed to react for 1 h at 22 C. The wells were washed again with buffer and then incubated for 1 h with goat anti-mouse immunoglobulin G (IgG) coupled with alkaline phosphatase (PelFreeze, Brown Deer, WI). After an additional wash with buffer, they were incubated for 1 h with substrate (p-nitrophenyl phosphate, Sigma, St. Louis, MO) and absorbance was measured at 410 nm using a microtiter plate reader (Dynatech, Cambridge, MA). IgG subclasses were determined in a similar ELISA procedure using mouse isotype-specific rabbit antibodies and goat antirabbit IgG-peroxidase (HyClone, Logan, UT) RIA Hormones, subunits, and mAbs were iodinated by a modification (17) of the chloramine-T method of Greenwood et al. (18), yielding specific activities of 100-200 nCi/fig for the hormones and subunits, and 9-28 /xCi/ng for the mAbs.
Endo • 1990 Voll27^No6
Antibodies of the A, K, and E series were titered against 125Ilabeled subunits and native hormones indicated in Table 2. One tenth ng of 125I-«- or /?-subunit or hormone was added to graded concentrations of purified mAb (0.15-1000 ng/tube) in 0.5 ml 40 mM Tris/0.1% BSA (Tris/BSA buffer, pH 7.4). After overnight incubation (~16h) at 22 C, assays were terminated by double antibody (goat antimouse IgG)-polyethylene glycol precipitation, and 125I was measured in the pellets. The affinities of the mAbs were determined by the method of Scatchard (19) from RIAs employing monoclonal antibodies and increasing concentrations of unlabeled a in the presence of a fixed amount of labeled a. Cross-reactivities of the mAbs with native glycoprotein hormones and the subunits were determined by displacement of 0.1 ng 125I-a or 125I-hFSH from the E and K mAb series or the A series, respectively, with increasing doses of ligand (hFSH, hLH, hTSH, hCG, a-subunit, and the corresponding /3-subunits) under conditions described in the previous paragraph. All RIAs were repeated at least twice. RRA The ability of the eight monoclonal antibodies to inhibit binding of radiolabeled hCG to ovarian receptor was determined by a competitive RRA using a 2000 X g membrane-rich fraction from superovulated rat ovaries (17). Graded concentrations of mAb were added to tubes containing 1 ng 125I-hCG and 1.25 mg (wet weight) of receptor preparation in a total volume of 1 ml Tris/BSA buffer. Tubes were incubated overnight at 22 C, then centrifuged to precipitate pellet, washed, and counted. Nonspecific binding was assessed by adding an excess of unlabeled hCG (2000 IU/ml) in the presence of tracer and receptor. Data reflect specific binding obtained by subtracting the nonspecific component from total binding. To determine if any of the antibodies recognized receptorbound hormone, unlabeled hCG (50 ng) was incubated with receptor as described above. After centrifuging to precipitate membrane receptor-hormone complex, the pellet was washed three times with fresh buffer, and approximately 100,000 cpm each 125I-mAb was added in the original assay volume. An excess of 100 ng appropriate unlabeled antibody was added to control tubes to correct for nonspecific binding. After an 8-h incubation at 22 C, tubes were centrifuged, and the pellet was washed and counted. Sandwich radioimmunometric assays To determine their specificity for different epitopes on asubunit, native hCG and hFSH, two-site binding assays were performed with all the mAbs used as either capture antibodies (solid phase-bound) or detection antibodies (125I-mAbs). mAbs (1 Mg/50 fii carbonate buffer, pH 9.8) were incubated in triplicate in 96-well microtiter plates (Immulon 1 Removawell strips; Dynatech, Alexandria, VA) overnight at 4 C. After washing with PBS (100 mM phosphate buffer, 150 mM NaCl2; pH 7.4), plates were blocked with 1% BSA/0.25% ovalbumin in PBS for 1 h at 22 C. After a PBS wash, antigens a, hCG, or hFSH (50 Ml/well in 0.1% BSA/0.025% ovalbumin/PBS) were added to the plates. Concentrations of antigen were varied to reflect the
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CHARACTERIZATION OF ANTI-hCGa ANTIBODIES differing affinities among the antibodies: 5 ng a and FSH for K2.18, K94.6, E502, and E511; 100 ng for E501; 500 ng for A101, A102, and E512. hCG was added at a concentration of 500 ng for all the mAb. Capture antibodies reacted with antigen for 8 h at room temperature, after which 100,000 cpm (~2 ng/ 50 /il) of each detection 125I-mAb were added to triplicate wells for determination of binding to antigen. After overnight incubation, wells were washed with PBS, separated, and counted. Nonspecific binding was determined from wells not containing antigen. Assays were repeated twice for each antibody and antigen. Differences in binding of 125I-mAbs to each of the eight capture antibodies were deemed significant at the P < 0.01 level if the total specific counts bound to the well were greater than the mean of the control wells (no antigen) + 3 SD. Reduction and carboxymethylation (RCXM) of a-subunit RCXM a was prepared by the method of Crestfield (20) as previously used in this laboratory (21).
Results RIA
The general properties of the monoclonal antibodies characterized in this report are given in Table 1. Despite the differences in antigen used as immunogen, all mAbs are specific for the a-subunit, although A101 and A102 recognize native hFSH with a potency about 300-fold greater than that for free a-subunit (12). The affinities of five of the antibodies as estimated by the ability of unlabeled a-subunit to compete with iodinated a were very high (10~u to 10~10 M), whereas E512, A102, and A101 had affinity constants (Kds) of 1.2, 2.4, and 4.7 x 10~7 M, respectively. The slopes of the Scatchard plots (not shown) indicated linearity as would be expected for mAbs. The titers of the mAbs to subunits or hormones are shown in Table 2. For the a-subunit, E502 and E511 had the highest titer, followed by K2.18, E501, K94.6, and E512. E502 and E511 also bound hCG, hLH, and hTSH, and E501 bound hFSH, hTSH, and hLH, albeit at a much higher concentration of mAb. None of these antiTABLE 1. Specificities and affinities of mAbs mAb K2.18 E511 E502 K94.6 E501 E512 A102 A101
Species and Class MIgG, MIgG2b MIgG2 MIgG2 MIgG2b MIgG2a MIgG, MIgG,
Immunogen hCG a(hCG) a(hCG) hCG a(hCG) a(hCG) hFSH hFSH
Specificity a a a a a a a a
Kd (1(T10 M)° 0.24 0.34 1.70 1.80 3.70 1200.00 2400.00 4700.00
0 Affinities for the E and K series are for 125I-a subunit; affinities for the A series are for a-subunit using 125I-hFSH as tracer.
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TABLE 2. Titers of mAbs against radiolabeled ligand 5
MAba 320 A101 A102 >1000 K2.18 2.6 4.2 K94.6 4.5 E501 E502 0.3 0.4 E511 E512 170.0
hCG
I-Ligand
hLH
330
hFSH
hTSH 0 subunit
2.5 15
>1000 240
>1000 >1000 780 920
700 2.5 7.7
>1000 >1000
22
>1000 >1000 >1000
326 150 170
1000
>1000* >1000* >1000c >1000c >1000" >1000d >1000d >1000d
" Amount of mAb (nanograms of wt/vol) necessary to bind 30% of I-labeled human subunit or hormone (0.15 ng/tube); data shown are means of two assays. b /3-Subunits of hCG and FSH were tested. c /3-Subunits of hCG was tested. d 0-Subunits of hCG, LH, FSH, and TSH were tested. 125
bodies bound any of the 125I-/3 subunits at concentrations of 1 /zg or more. A previous report (12) indicated that A101 at 100 ng bound 45% of hFSH, 8% of a, and no FSH/3, A102 at 300 ng bound 42% of hFSH, 6% of a and no FSH/3. Cross-reactivities of the antibodies with native hormones and jS-subunits of human CG, LH, FSH, and TSH are shown in Table 3. The apparent high potency of FSH/3 in competition with 125I-a for binding to the E and K series mAbs is due to 11.8% contamination of the fi preparation with free a-subunit as determined by RIA (data not shown). Upon correcting for this contamination, most of the j8-subunits were only 0.1-1.5% as potent as free a in competing for binding of a to mAbs E501, E502, E511, K2.18, and K94.6. TSH/3 was 5% as potent as a for binding to K2.18 and K94.6. E512 recognized hLH, FSH, and TSH /3-subunits, but not hCG/3. hCG and hTSH were relatively ineffective in competing with a for binding to E511, E502, K2.18, and K94.6 (1-3%). These antibodies do cross-react with hLH and FSH, as does E501, which also binds hTSH. Interestingly, E512 recognizes the dimeric a-subunit of hLH better than free a. Cross-reaction of E512 with the native hormones was also high. A101 and A102 recognized asubunit in combination with /3 in the glycohormone dimers better than free a. RRA All eight mAbs inhibited binding of 125I-hCG to ovarian receptor with varying potencies as shown in Table 4. Except for the E series mAbs, these potencies didn't correlate with the antibodies' affinities for a-subunit. In addition, none of the antibodies were able to recognize hCG bound to receptor (Fig. 1).
Reactions with RCXM-a Figure 2 indicates the reactions of this panel of monoclonal antibodies with RCXM-a, a denatured and linear-
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C H A R A C T E R I Z A T I O N OF A N T I - h C G a A N T I B O D I E S
Endo. 1990 Vol 127 • No 6
TABLE 3. Cross-reactivities of anti-a mAbs with human glycoprotein hormones and /3-subunits IC50 = nM° mAb
a Subunit
A101 A102 K2.18 K94.6 E501 E502 E511 E512
470.00 240.00 0.02 0.18 0.37 0.17 0.03 121.00
/3 Subunit hCG (%)"
LH (%)
FSH (%)
2100.0 11000.0 3.3 (0.6) 17.0 (1.1) >83.3 >83.3 26.6 (0.1) 16600.0 (0.7)
9100.0 13000.0 1.5 (1.3) 11.0 (1.6) 22.3 (1.7) 16.5 (1.0) 2.4 (1.3) 405.0 (30.0)
1030.0 ' 420.0 0.1 (17.0) 0.9 (20.0) 1.7 (21.0) 0.6 (27.0) 0.4 (7.5) 238.0 (51.0)
Hormone TSH (%)
hCG (%)
LH (%)
0.5 (4.3) 3.9 (4.6) 18.3 (2.0) 10.7 (1.6) 2.5 (1.2) 1330.0 (9.1)
10.6 50.0 0.4 (5.0) 2.4 ( 7.5) 3.6 (10.0) 7.3 (2.3) 1.4 (2.1) 352.0 (34.0)
52.5 200.0 0.1 (20) 0.7 (26) 0.6 (62) 0.4 (43) 0.1 (30) 43.5 (278)
0 Concentration of ligand needed to displace 50% of the binding of 0.1 ng antibody; data shown are means of at least two assays. * Values in parentheses represent percent cross-reactivity with a-subunit.
5
FSH (%) 0.6 1.2
0.1 (20) 0.5 (36) 0.5 (74) 0.6 (28) 0.1 (30) 110.0 (110)
I-a subunit (for E and K series) or
TSH (%) 14.0 26.0 0.4 (5.0) 2.2 ( 8.2) 1.0 (37.0) 3.0 (5.7) 0.7 (4.3) 315.0 (38.0)
125
I-hFSH (for A series) to
TABLE 4. Inhibition of 125I-hCG binding to receptor by anti-a mAbs mAb
IC50 (10-8 M)"
A101 A102 K2.18 K94.6 E501 E502 E511 E512
36.0 14.0 214.0 850.0 16.5
_o
2.1 4.7
c
Ol
1
c/5 CO
