0013-7227/90/1275-2090$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Monoclonal Antibodies against Luteinizing Hormone Receptor. Immunochemical Characterization of the Receptor* MAI THU VUHAI-LUUTHI, ANDRE JOLIVET, BAHIJA JALLAL, ROLAND SALESSE, JEAN-MICHEL BIDART, ANNE HOULLIER, ANNE GUIOCHON-MANTEL, JEAN GARNIER, AND EDWIN MILGROM Institut National de la Sante et de la Recherche Medicale (M.T.V.-L, A.J., A.H., A.G.-M., E.M.) Unite 135, Hormones et Reproduction, Hopital de Bicetre, 94270 Le Kremlin-Bicetre, France; Institut National de la Recherche Agronomique (B.J., R.S., J.G.), Unite d'Ingenierie des Proteines, Biotechnologies, 78350 Jouy-enJosas, France; Institut Gustave Roussy (J.M.B.), Unite de Biochimie Clinique, 94805 Villejuif, France
ABSTRACT. Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-DiE8 anti/3hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (~15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width
of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 4845,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species. {Endocrinology 127: 2090-2098, 1990)
T
HE structure and the function of the LH/human CG (hCG) receptor still remains the subject of controversy (1, 2). Conflicting results have been reported on the size and the number of receptor subunits as determined by purification, cross-linking, or ligand blotting (1, 2). Some reports have suggested that the receptor consists of a single subunit (3-8) whereas other experiments have revealed the existence of several proteins with varying molecular weights (9,10). In the latter case it is not known if all these receptor species are due to intracellular synthesis or if some result from artifactual proteolysis (11-13). Moreover, the structural relationship between these putative receptor components re-
Received April 25, 1990. Address requests for reprints to: E. Milgrom, Hormones et Reproduction (Institut National de la Sante et de la Recherche Medicale, Unite 135), Hopital de Bicetre, Faculte de Medecine Paris-Sud, Le Kremlin-Bicetre, France. * This work was supported by the Institut de la Sante et de la Recherche Medicale, the Institut National de la Recherche Agronomique, the Centre National de la Recherche Scientifique, the Unite de Formation et de Recherche Kremlin-Bicetre, the Institut Gustave Roussy, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Medicale, and Transbio Company.
mains unclear. Are they derived from the same gene (for instance by premessenger alternative splicing, protein post translational modifications etc...), or are they completely unrelated polypeptides? Their functional role has also been the subject of many unanswered queries. Is hormone binding assumed by a single polypeptide chain, or are several chains necessary (as for instance in the insulin receptor)? Are the same polypeptide chains also responsible for the biological responses which follow hormone binding, i.e. activation of adenylate cyclase (14, 15), possible activation of phospholipase C (16-19) and steroidogenesis (20)? The degree of similarity of receptors in different target cells of a given species has also been questioned (21, 22). Although the recent cloning and sequencing of the receptor cDNA have imparted important information (23, 24), the lack of well characterized immunological tools has impeded its extensive characterization. We report here the preparation and characterization of a panel of monoclonal antibodies against the LH/hCG receptor and their subsequent use for immunochemical studies and immunopurification of the receptor.
2090
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
Materials and Methods Tissue processing and receptor solubilization Porcine testes (Large White strain, age 10-20 days) (25) were kept at —20 C. After thawing they were dissected and homogenized in a Waring blender ( 4 x 3 sec at 4 C) in Tris, 10 mM; NaCl, 100 mM; KC1, 3 mil; MgCl2, 0.5 mM; CaCl2, 0.9 mM; and HC1, pH 7.4, buffer containing benzamidine, 1 mM (Serva, Heidelberg, W. Germany) and phenylmethyl-sulfonyl fluoride, 1 mM (Serva) (1 ml buffer/g tissue). After centrifugation (105,000 X g, 30 min, 4 C), the pellet was resuspended in the same volume of PBS: sodium phosphate, 10 mM; NaCl, 150 mM; pH 7.4, containing 20% glycerol and then frozen at -20 C (the pellets have been kept frozen for up to 6 months without loss of activity). The pellets were thawed and incubated overnight at 4 C with 125 I-hCG, (20 nM; SA, 1 Ci/mmol). After a 2-fold dilution they were washed twice in the same buffer (centrifugation for 30 min at 105,000 x g, and 4 C). The pellets were resuspended in the same volume of PBS, pH 7.4, containing MgCl2, 0.5 mM; CaCl2, 0.9 mM; benzamidine, 1 mM; phenylmethyl-sulfonyl fluoride, 1 mM; bacitracin (100 Mg/nil) (Sigma, St Louis, MO), aprotinin, 38 Mg/ml (Calbiochem, San Diego, CA), leupeptin, 5 mM (Sigma), and pepstatin (1 Mg/ml) (Sigma). Triton X-100 (Packard, Downers Grove, IL) was added (final concentration 0.5%), and the suspension was strongly agitated with a magnetic stirrer overnight at 4 C. Centrifugation for 60 min at 105,000 x g yielded the solubilized membrane preparation. 125 I-FSH-receptor complexes were prepared from the same testes. 125I-TSH receptor complexes were prepared from porcine thyroids. The procedures were similar except that a lower ionic strength buffer was used (Tris, 20 mM; NaCl, 25 mM, pH 7.4). 125
/ labeling of hormone. Measurement of protein-bound hormone Highly purified hCG (a gift from Dr. R. Canfield, NIH, SA, 13,400 U/mg) was labeled with 125I by the Iodo-Gen method (Pierce, Rockford, IL) (26). The specific activity varied between 500-1200 Ci/mmol, and the biological activity, determined as described (27), was 30%. When necessary the specific activity was decreased by dilution with unlabeled hormone (Gonadotrophine Chorionique Endo, Organon, St Denis, France; SA, 5000 U/mg). Protein-bound hormone was measured by precipitation with 15% polyethylene glycol 6000 (Merck, Darmstadt, W. Germany) in presence of 150 mM NaCl for 10 min at 4 C (centrifugation, 3000 X g X 10 min). Similar procedures were used for porcine FSH (a gift from Dr. Y. Cambarnous, Nouzilly, France; SA, 5000 U/mg) and bovine TSH (a gift from Dr. J. Pierce, Los Angeles, CA; SA, 70 U/mg). The biological activity of 125I-labeled porcine FSH and bovine TSH was 10% and 5%, respectively. Immunopurification of hCG-receptor complexes using anti-hCG antibodies DiE8 antihCG monoclonal antibodies (28) were purified and coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA) at a concentration of 10 mg antibody/ml gel. Triton X-100 solubilized membranes containing hCG-recep-
2091
tor complexes were filtered (10 ml/h) through three successive columns containing: inactivated Affi-Gel 10 (15 ml), nonhormone related IDA 10 antibody (29) coupled to Affi-Gel 10 (10 ml) and D^ antibody coupled to Affi-Gel 10 (2 ml). The last column was disconnected and then washed successively, first with PBS containing 0.5% Triton X-100 and various protease inhibitors (see above), then PBS containing 0.1% Triton X100 and 2 M NaCl, PBS containing 0.1% Triton X-100 and 0.1% deoxycholate (Sigma), PBS containing 0.1% Triton X100 and sodium dodecyl sulfate (SDS) 0.01% (BDH, Poole, England), PBS containing 0.1% Triton X-100 and ethylene glycol 30% (Merck), PBS containing 0.5% Triton X-100 and finally ammonium bicarbonate 25 mM, pH 8, buffer containing 0.1% Triton X-100. In each case the volume of the washing buffer was 80 ml. hCG-receptor complexes were eluted with ammonium bicarbonate, 50 mM; pH 10.8, buffer containing 0.2% Triton X-100 (3 ml). Because the buffer was volatile the sample was easily concentrated if necessary. Immunization and preparation of hybridomas A 10-week-old Balb/c mouse was immunized with three sc injections of 200 /A. The first injection contained 960 pmol hCG-receptor complex (assuming a 1:1 ratio of hormone to receptor) in complete Freund's adjuvant. The second injection was performed 15 days later and contained 830 pmol hCGreceptor complexes in incomplete Freund's adjuvant. The third injection, 7 days later, contained 830 pmol in the same adjuvant. One week later 500 pmol hCG-receptor complexes (200 fi\) were injected ip and 250 pmol (100 /A) iv. The mouse was killed 4 days later, and hybridomas were produced by fusion with Sp 2 / O-Ag cells, as previously described (30). The cloning and production of ascites have been previously reported (31). The antibodies were purified on protein A-Sepharose columns following supplier's instructions (Bio-Rad, Richmond, CA). Detection of antireceptor and anti-hCG antibodies by immunoprecipitation Solubilized 125I-hCG-receptor complexes (200 fi\, 20,000 cpm) were incubated overnight at 4 C with either 400 fi\ cell culture supernatant or diluted mouse serum. Carrier mouse immunoglobulins (5 ng in 10 n\) and antimouse immunoglobulin antiserum (corresponding to 100 /x\ serum precipitated by ammonium sulfate at 50% saturation) were then added. After a 4-h incubation at 4 C a centrifugation was performed through a 1 M sucrose cushion in PBS, 0.1% Triton X-100 buffer, as previously described (31). Immunoprecipitated complexes were counted for radioactivity and compared to background values obtained using cell culture media from nonreceptor related hybridomas. A similar experiment was performed with 125I-hCG alone instead of 125I-hCG-receptor complexes. In some experiments 125 I-FSH-receptor and 125I-TSH-receptor complexes were used. Enzyme-linked immunosorbant assay (ELISA) tests for the detection of antibodies against the receptor, hCG, and major membrane proteins Purified hormone-receptor complexes (200 fmol in 20 /ul/ well) were mixed with 20 n\ of methanol in wells of Nunc
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
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(Rockilde, Denmark) ELISA plates. After 1 h at room temperature, the supernatant was discarded and the plate was washed twice. Other plates were coated with 200 fmol hCG or with 20 n\ Triton X-100 membrane extract diluted 10-fold (see above). ELISA tests were performed thereafter as described (31).
Endo • 1990 Vol 127 • No 5
3, buffer and immediately neutralized with Tris, 1 M, pH 10. In some comparative experiments (see Figs. 5 and 6) DiE8Affi-Gel 10 immunomatrix was used instead of LHR 38-AffiGel 10.
Results Protein assays Proteins were assayed either by the micro BCA method (Pierce) or by the amido Schwartz method (32) (if protein concentration was lower than 0.2 mg/ml). Immunoblot study of LH/hCG receptor Testicular membranes were solubilized in PBS containing Triton X-100 (0.7%) and iV-ethylmaleimide, 5 mM. Aliquots (50 n\) containing 100-200 fmol solubilized membrane receptor or 1 pmol purified receptor were electrophoresed on 7.5% polyacrylamide gels as described (33), and the proteins were electrotransferred overnight in a Tris, 25 mM; glycine, 195 mM, pH 8.2, buffer containing 10% methanol. The nitrocellulose membranes were saturated by PBS, pH 7.4, containing 5% BSA and then incubated for 2 h at room temperature with purified monoclonal antibodies (5 Mg/ml) in PBS containing 0.5% BSA, Triton X-100 (0.05%) and polyoxyethylene sorbitan monolaurate (Tween 20) (0.05%) (Sigma, St Louis, MO). After washing in the same buffer containing also SDS (0.02%), detection of bound monoclonal antibodies was performed using either an antimouse immunoglobulin antiserum and 126I-protein A as previously described (33), or, in the case of the experiment reported in Fig. 2, using 125I-antimouse immunoglobulin F (IgF) (ab')2 (Amersham, Arlington Heights, IL). Immunoenzymatic assays of LH/hCG receptor Receptor was detected in the eluate of affinity columns by dispensing 20 ^1 each fraction (previously diluted 200-fold) on ELISA plates (Nunc, Roskilde, Denmark) and then precipitating the proteins with 20 ^1 methanol (see above). Saturation of free plastic sites was done with PBS, pH 7.4, containing Tween 20 (0.1%) and BSA (0.1%) (PBS-Tween-BSA buffer). Biotinylated antibodies (400 ng/100 /xl/well) in the PBS-TweenBSA buffer were incubated 1 h at 20 C. After five washes with PBS-Tween, peroxidase-linked streptavidin (Sigma) was added (50 ng/100 /xl) in PBS-Tween-BSA and incubated for 1 h. Coloration was elicited using O-phenylene diamine (Merck, Darmstadt, W. Germany) as substrate. Optical density was read at 492 nm. Immunopurification of receptor using antireceptor antibodies Monoclonal antireceptor LHR 38 (10 mg/ml gel) was coupled to Affi-Gel 10 at pH 7.5. Deactivated Affi-Gel 10 and IDA 10Affi-Gel 10 (29) served as nonspecific immunoadsorbants. Receptor solubilized in 0.7% Triton X-100 was diluted 2-fold and chromatographed first through the two nonspecific columns (Affi-Gel 10 and IDA 10-Affi-Gel 10) (10 ml/liter extract) and then through the LHR 38-Affi-Gel 10 column (1 ml/liter extract). The flow rate was 10 ml/h. The last column (containing antireceptor antibodies) was disconnected and washed as previously described. Receptor was eluted in glycine, 50 mM; pH
Preparation of monoclonal antibodies The DXE8 antihCG monoclonal antibody (28) was used for immunoaffinity purification of hormone-receptor complexes. Two properties of this antibody dictated its choice: 1) its binding to the hormone did not prevent the interaction of the latter with the receptor; and 2) DxEs antibody-receptor complexes were unstable at alkaline pH whereas receptor-hormone complexes were relatively stable at the same pH (data not shown). Membranes of piglet testes were incubated with 125I-hCG and extensively washed to remove loosely bound proteins and free hormone. Membrane proteins were then solubilized with Triton X-100 and chromatographed through an Affi-Gel 10 immunomatrix containing DiEg antibody preceded by two columns devised to decrease the nonspecific binding of proteins to the immunomatrix (33). The recovery of hCG-receptor complexes from the immunoaffinity column was 37%; yielding 48 ^g protein and 840 pmol hCGreceptor complexes (starting with 750 testes and 13.2 g membrane proteins). The specific activity (1750 pmol/ mg protein) indicated a purity of approximately 15% assuming the receptor had a mol wt of 80,000-100,000. However the frequency of monoclonal antibodies obtained in subsequent experiments indicated that this purity was probably underestimated, or that the receptor is particularly immunogenic. Polyethylene glycol precipitated 50% of the eluted radioactivity showing that a sizable proportion of the receptor-hormone complexes had been preserved [it is known that polyethylene glycol precipitation of receptor-hormone complexes is incomplete at low protein concentrations (34)]. A mouse was immunized according to the schedule described in Materials and Methods, and its immunological response was followed by various tests (see Materials and Methods). After a strong positive response had been observed, the mouse was killed, and its spleen was used to prepare hybridomas. Screening of 909 wells gave 28 positive results (Table 1). Six were clearly antibodies against the hormone (they reacted in ELISA tests with the purified antigen, but also with pure hCG, and they immunoprecipitated both 125I-hCG-receptor complexes and 125I-hCG alone). Two were antibodies against nonreceptor and nonhormone contaminants of the antigen preparation: they reacted in ELISA tests with the antigen but also with crude Triton membrane extract (the latter test showing that they corresponded to relatively abundant membrane proteins). Twenty (71%) of the positive hybridomas secreted antibodies against the receptor. They were positive in
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
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TABLE 1. Detection of hybridomas secreting antibodies against LH/hCG receptor, hCG, and contaminating proteins ELISA Tests 0 Clones
IgG class
Immunoprecipitation0
hCG
. ,. Antigen0b
Membrane extract
125
125
I-hCG
I-hCG-Receptor complexes
IDA 10°
IgGl
Vol. 127, No. 5 Printed in U.S.A.
Monoclonal Antibodies against Luteinizing Hormone Receptor. Immunochemical Characterization of the Receptor* MAI THU VUHAI-LUUTHI, ANDRE JOLIVET, BAHIJA JALLAL, ROLAND SALESSE, JEAN-MICHEL BIDART, ANNE HOULLIER, ANNE GUIOCHON-MANTEL, JEAN GARNIER, AND EDWIN MILGROM Institut National de la Sante et de la Recherche Medicale (M.T.V.-L, A.J., A.H., A.G.-M., E.M.) Unite 135, Hormones et Reproduction, Hopital de Bicetre, 94270 Le Kremlin-Bicetre, France; Institut National de la Recherche Agronomique (B.J., R.S., J.G.), Unite d'Ingenierie des Proteines, Biotechnologies, 78350 Jouy-enJosas, France; Institut Gustave Roussy (J.M.B.), Unite de Biochimie Clinique, 94805 Villejuif, France
ABSTRACT. Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-DiE8 anti/3hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (~15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width
of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 4845,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species. {Endocrinology 127: 2090-2098, 1990)
T
HE structure and the function of the LH/human CG (hCG) receptor still remains the subject of controversy (1, 2). Conflicting results have been reported on the size and the number of receptor subunits as determined by purification, cross-linking, or ligand blotting (1, 2). Some reports have suggested that the receptor consists of a single subunit (3-8) whereas other experiments have revealed the existence of several proteins with varying molecular weights (9,10). In the latter case it is not known if all these receptor species are due to intracellular synthesis or if some result from artifactual proteolysis (11-13). Moreover, the structural relationship between these putative receptor components re-
Received April 25, 1990. Address requests for reprints to: E. Milgrom, Hormones et Reproduction (Institut National de la Sante et de la Recherche Medicale, Unite 135), Hopital de Bicetre, Faculte de Medecine Paris-Sud, Le Kremlin-Bicetre, France. * This work was supported by the Institut de la Sante et de la Recherche Medicale, the Institut National de la Recherche Agronomique, the Centre National de la Recherche Scientifique, the Unite de Formation et de Recherche Kremlin-Bicetre, the Institut Gustave Roussy, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Medicale, and Transbio Company.
mains unclear. Are they derived from the same gene (for instance by premessenger alternative splicing, protein post translational modifications etc...), or are they completely unrelated polypeptides? Their functional role has also been the subject of many unanswered queries. Is hormone binding assumed by a single polypeptide chain, or are several chains necessary (as for instance in the insulin receptor)? Are the same polypeptide chains also responsible for the biological responses which follow hormone binding, i.e. activation of adenylate cyclase (14, 15), possible activation of phospholipase C (16-19) and steroidogenesis (20)? The degree of similarity of receptors in different target cells of a given species has also been questioned (21, 22). Although the recent cloning and sequencing of the receptor cDNA have imparted important information (23, 24), the lack of well characterized immunological tools has impeded its extensive characterization. We report here the preparation and characterization of a panel of monoclonal antibodies against the LH/hCG receptor and their subsequent use for immunochemical studies and immunopurification of the receptor.
2090
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
Materials and Methods Tissue processing and receptor solubilization Porcine testes (Large White strain, age 10-20 days) (25) were kept at —20 C. After thawing they were dissected and homogenized in a Waring blender ( 4 x 3 sec at 4 C) in Tris, 10 mM; NaCl, 100 mM; KC1, 3 mil; MgCl2, 0.5 mM; CaCl2, 0.9 mM; and HC1, pH 7.4, buffer containing benzamidine, 1 mM (Serva, Heidelberg, W. Germany) and phenylmethyl-sulfonyl fluoride, 1 mM (Serva) (1 ml buffer/g tissue). After centrifugation (105,000 X g, 30 min, 4 C), the pellet was resuspended in the same volume of PBS: sodium phosphate, 10 mM; NaCl, 150 mM; pH 7.4, containing 20% glycerol and then frozen at -20 C (the pellets have been kept frozen for up to 6 months without loss of activity). The pellets were thawed and incubated overnight at 4 C with 125 I-hCG, (20 nM; SA, 1 Ci/mmol). After a 2-fold dilution they were washed twice in the same buffer (centrifugation for 30 min at 105,000 x g, and 4 C). The pellets were resuspended in the same volume of PBS, pH 7.4, containing MgCl2, 0.5 mM; CaCl2, 0.9 mM; benzamidine, 1 mM; phenylmethyl-sulfonyl fluoride, 1 mM; bacitracin (100 Mg/nil) (Sigma, St Louis, MO), aprotinin, 38 Mg/ml (Calbiochem, San Diego, CA), leupeptin, 5 mM (Sigma), and pepstatin (1 Mg/ml) (Sigma). Triton X-100 (Packard, Downers Grove, IL) was added (final concentration 0.5%), and the suspension was strongly agitated with a magnetic stirrer overnight at 4 C. Centrifugation for 60 min at 105,000 x g yielded the solubilized membrane preparation. 125 I-FSH-receptor complexes were prepared from the same testes. 125I-TSH receptor complexes were prepared from porcine thyroids. The procedures were similar except that a lower ionic strength buffer was used (Tris, 20 mM; NaCl, 25 mM, pH 7.4). 125
/ labeling of hormone. Measurement of protein-bound hormone Highly purified hCG (a gift from Dr. R. Canfield, NIH, SA, 13,400 U/mg) was labeled with 125I by the Iodo-Gen method (Pierce, Rockford, IL) (26). The specific activity varied between 500-1200 Ci/mmol, and the biological activity, determined as described (27), was 30%. When necessary the specific activity was decreased by dilution with unlabeled hormone (Gonadotrophine Chorionique Endo, Organon, St Denis, France; SA, 5000 U/mg). Protein-bound hormone was measured by precipitation with 15% polyethylene glycol 6000 (Merck, Darmstadt, W. Germany) in presence of 150 mM NaCl for 10 min at 4 C (centrifugation, 3000 X g X 10 min). Similar procedures were used for porcine FSH (a gift from Dr. Y. Cambarnous, Nouzilly, France; SA, 5000 U/mg) and bovine TSH (a gift from Dr. J. Pierce, Los Angeles, CA; SA, 70 U/mg). The biological activity of 125I-labeled porcine FSH and bovine TSH was 10% and 5%, respectively. Immunopurification of hCG-receptor complexes using anti-hCG antibodies DiE8 antihCG monoclonal antibodies (28) were purified and coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA) at a concentration of 10 mg antibody/ml gel. Triton X-100 solubilized membranes containing hCG-recep-
2091
tor complexes were filtered (10 ml/h) through three successive columns containing: inactivated Affi-Gel 10 (15 ml), nonhormone related IDA 10 antibody (29) coupled to Affi-Gel 10 (10 ml) and D^ antibody coupled to Affi-Gel 10 (2 ml). The last column was disconnected and then washed successively, first with PBS containing 0.5% Triton X-100 and various protease inhibitors (see above), then PBS containing 0.1% Triton X100 and 2 M NaCl, PBS containing 0.1% Triton X-100 and 0.1% deoxycholate (Sigma), PBS containing 0.1% Triton X100 and sodium dodecyl sulfate (SDS) 0.01% (BDH, Poole, England), PBS containing 0.1% Triton X-100 and ethylene glycol 30% (Merck), PBS containing 0.5% Triton X-100 and finally ammonium bicarbonate 25 mM, pH 8, buffer containing 0.1% Triton X-100. In each case the volume of the washing buffer was 80 ml. hCG-receptor complexes were eluted with ammonium bicarbonate, 50 mM; pH 10.8, buffer containing 0.2% Triton X-100 (3 ml). Because the buffer was volatile the sample was easily concentrated if necessary. Immunization and preparation of hybridomas A 10-week-old Balb/c mouse was immunized with three sc injections of 200 /A. The first injection contained 960 pmol hCG-receptor complex (assuming a 1:1 ratio of hormone to receptor) in complete Freund's adjuvant. The second injection was performed 15 days later and contained 830 pmol hCGreceptor complexes in incomplete Freund's adjuvant. The third injection, 7 days later, contained 830 pmol in the same adjuvant. One week later 500 pmol hCG-receptor complexes (200 fi\) were injected ip and 250 pmol (100 /A) iv. The mouse was killed 4 days later, and hybridomas were produced by fusion with Sp 2 / O-Ag cells, as previously described (30). The cloning and production of ascites have been previously reported (31). The antibodies were purified on protein A-Sepharose columns following supplier's instructions (Bio-Rad, Richmond, CA). Detection of antireceptor and anti-hCG antibodies by immunoprecipitation Solubilized 125I-hCG-receptor complexes (200 fi\, 20,000 cpm) were incubated overnight at 4 C with either 400 fi\ cell culture supernatant or diluted mouse serum. Carrier mouse immunoglobulins (5 ng in 10 n\) and antimouse immunoglobulin antiserum (corresponding to 100 /x\ serum precipitated by ammonium sulfate at 50% saturation) were then added. After a 4-h incubation at 4 C a centrifugation was performed through a 1 M sucrose cushion in PBS, 0.1% Triton X-100 buffer, as previously described (31). Immunoprecipitated complexes were counted for radioactivity and compared to background values obtained using cell culture media from nonreceptor related hybridomas. A similar experiment was performed with 125I-hCG alone instead of 125I-hCG-receptor complexes. In some experiments 125 I-FSH-receptor and 125I-TSH-receptor complexes were used. Enzyme-linked immunosorbant assay (ELISA) tests for the detection of antibodies against the receptor, hCG, and major membrane proteins Purified hormone-receptor complexes (200 fmol in 20 /ul/ well) were mixed with 20 n\ of methanol in wells of Nunc
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
2092
(Rockilde, Denmark) ELISA plates. After 1 h at room temperature, the supernatant was discarded and the plate was washed twice. Other plates were coated with 200 fmol hCG or with 20 n\ Triton X-100 membrane extract diluted 10-fold (see above). ELISA tests were performed thereafter as described (31).
Endo • 1990 Vol 127 • No 5
3, buffer and immediately neutralized with Tris, 1 M, pH 10. In some comparative experiments (see Figs. 5 and 6) DiE8Affi-Gel 10 immunomatrix was used instead of LHR 38-AffiGel 10.
Results Protein assays Proteins were assayed either by the micro BCA method (Pierce) or by the amido Schwartz method (32) (if protein concentration was lower than 0.2 mg/ml). Immunoblot study of LH/hCG receptor Testicular membranes were solubilized in PBS containing Triton X-100 (0.7%) and iV-ethylmaleimide, 5 mM. Aliquots (50 n\) containing 100-200 fmol solubilized membrane receptor or 1 pmol purified receptor were electrophoresed on 7.5% polyacrylamide gels as described (33), and the proteins were electrotransferred overnight in a Tris, 25 mM; glycine, 195 mM, pH 8.2, buffer containing 10% methanol. The nitrocellulose membranes were saturated by PBS, pH 7.4, containing 5% BSA and then incubated for 2 h at room temperature with purified monoclonal antibodies (5 Mg/ml) in PBS containing 0.5% BSA, Triton X-100 (0.05%) and polyoxyethylene sorbitan monolaurate (Tween 20) (0.05%) (Sigma, St Louis, MO). After washing in the same buffer containing also SDS (0.02%), detection of bound monoclonal antibodies was performed using either an antimouse immunoglobulin antiserum and 126I-protein A as previously described (33), or, in the case of the experiment reported in Fig. 2, using 125I-antimouse immunoglobulin F (IgF) (ab')2 (Amersham, Arlington Heights, IL). Immunoenzymatic assays of LH/hCG receptor Receptor was detected in the eluate of affinity columns by dispensing 20 ^1 each fraction (previously diluted 200-fold) on ELISA plates (Nunc, Roskilde, Denmark) and then precipitating the proteins with 20 ^1 methanol (see above). Saturation of free plastic sites was done with PBS, pH 7.4, containing Tween 20 (0.1%) and BSA (0.1%) (PBS-Tween-BSA buffer). Biotinylated antibodies (400 ng/100 /xl/well) in the PBS-TweenBSA buffer were incubated 1 h at 20 C. After five washes with PBS-Tween, peroxidase-linked streptavidin (Sigma) was added (50 ng/100 /xl) in PBS-Tween-BSA and incubated for 1 h. Coloration was elicited using O-phenylene diamine (Merck, Darmstadt, W. Germany) as substrate. Optical density was read at 492 nm. Immunopurification of receptor using antireceptor antibodies Monoclonal antireceptor LHR 38 (10 mg/ml gel) was coupled to Affi-Gel 10 at pH 7.5. Deactivated Affi-Gel 10 and IDA 10Affi-Gel 10 (29) served as nonspecific immunoadsorbants. Receptor solubilized in 0.7% Triton X-100 was diluted 2-fold and chromatographed first through the two nonspecific columns (Affi-Gel 10 and IDA 10-Affi-Gel 10) (10 ml/liter extract) and then through the LHR 38-Affi-Gel 10 column (1 ml/liter extract). The flow rate was 10 ml/h. The last column (containing antireceptor antibodies) was disconnected and washed as previously described. Receptor was eluted in glycine, 50 mM; pH
Preparation of monoclonal antibodies The DXE8 antihCG monoclonal antibody (28) was used for immunoaffinity purification of hormone-receptor complexes. Two properties of this antibody dictated its choice: 1) its binding to the hormone did not prevent the interaction of the latter with the receptor; and 2) DxEs antibody-receptor complexes were unstable at alkaline pH whereas receptor-hormone complexes were relatively stable at the same pH (data not shown). Membranes of piglet testes were incubated with 125I-hCG and extensively washed to remove loosely bound proteins and free hormone. Membrane proteins were then solubilized with Triton X-100 and chromatographed through an Affi-Gel 10 immunomatrix containing DiEg antibody preceded by two columns devised to decrease the nonspecific binding of proteins to the immunomatrix (33). The recovery of hCG-receptor complexes from the immunoaffinity column was 37%; yielding 48 ^g protein and 840 pmol hCGreceptor complexes (starting with 750 testes and 13.2 g membrane proteins). The specific activity (1750 pmol/ mg protein) indicated a purity of approximately 15% assuming the receptor had a mol wt of 80,000-100,000. However the frequency of monoclonal antibodies obtained in subsequent experiments indicated that this purity was probably underestimated, or that the receptor is particularly immunogenic. Polyethylene glycol precipitated 50% of the eluted radioactivity showing that a sizable proportion of the receptor-hormone complexes had been preserved [it is known that polyethylene glycol precipitation of receptor-hormone complexes is incomplete at low protein concentrations (34)]. A mouse was immunized according to the schedule described in Materials and Methods, and its immunological response was followed by various tests (see Materials and Methods). After a strong positive response had been observed, the mouse was killed, and its spleen was used to prepare hybridomas. Screening of 909 wells gave 28 positive results (Table 1). Six were clearly antibodies against the hormone (they reacted in ELISA tests with the purified antigen, but also with pure hCG, and they immunoprecipitated both 125I-hCG-receptor complexes and 125I-hCG alone). Two were antibodies against nonreceptor and nonhormone contaminants of the antigen preparation: they reacted in ELISA tests with the antigen but also with crude Triton membrane extract (the latter test showing that they corresponded to relatively abundant membrane proteins). Twenty (71%) of the positive hybridomas secreted antibodies against the receptor. They were positive in
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MONOCLONAL ANTIBODIES AGAINST LH RECEPTOR
2093
TABLE 1. Detection of hybridomas secreting antibodies against LH/hCG receptor, hCG, and contaminating proteins ELISA Tests 0 Clones
IgG class
Immunoprecipitation0
hCG
. ,. Antigen0b
Membrane extract
125
125
I-hCG
I-hCG-Receptor complexes
IDA 10°
IgGl
