title: Demonstration of complimentarity between monoclonal antibodies (MAbs) to human chorionic gonadotropin (hCG) and polyclonal antibodies to luteinizing hormone/hCG receptor (LH-R) and their use in better understanding hormone-receptor interaction.
abstract: We have earlier reported that polyclonal antisera raised in rabbits to a luteinizing hormone/chorionic gonadotropin receptor (LH-R) purified from sheep luteal tissue has antibodies exhibiting hormone agonistic and antagonistic activities. Western blot analysis showed this antibody (LHR-anti IgG) to be highly specific to sheep luteal LH receptor (LH-R) (Jeyakumar and Moudgal, 1991). Using this, along with a battery of mouse monoclonal antibodies (MAbs) to hCG, an attempt has been made to better understand the interaction of LH/hCG with its receptor. Of the eight hCG MAbs screened, three (B14/B7, B52/18 and A7/G4) were specific to the beta-subunit; while a second set of three (G10/F7, H9/E9 and B52/21) were specific to the alpha-subunit. Two additional MAbs (B52/28 and F9/G8) did not recognize individual subunits, but bound like the rest intact hCG. Both 125I hCG and 125I anti LHR-IgG bound specifically to ovine luteal membrane LH-R. Assuming that a certain degree of similarity should exist between hCG and LHR-anti IgG, different hCG MAbs were tested for their ability to block the binding of either 125I hCG or 125I LHR-anti IgG to sheep luteal LH-R. It appears that hCG and LH-R share a minimum of four sites that are complementary to each other and these are recognized by the hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9. Whereas two of the MAbs B14/B7 and G10/F7 blocked the binding of both 125I labeled hCG and LHR-anti IgG to the receptor, MAbs A7/G4 and H9/E9 only inhibited the binding of 125I LHR-anti IgG to the LH-R. Although individually B14/B7 and G10/F7 blocked the binding of 125I LHR-anti IgG to LH-R to a maximum extent of 43%, together they inhibited binding by as much as 80%. The ability of B14/B7 to inhibit binding of 125I LHR-anti IgG to the receptor was also significantly increased by the addition of A7/G4. Finally, by demonstrating direct binding of the immobilized hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9 to LHR-anti IgG, we have been able to establish that the receptor binding sites of hCG and LHR-anti IgG are complementary and that a set of four sites are recognizable by the hCG MAbs. From the degree of interaction, it appears that two sites recognized by MAbs B14/B7 and G10/F7 (representing a site each in the beta- and alpha-subunit of hCG) have a prominent role in the interaction of hCG with its receptor. Thus, this study has provided us with an opportunity to investigate the interaction of LH/hCG with its receptor by an indirect approach of monitoring the binding of their respective antibodies with each other.
authors.affiliation: Department of Biochemistry and Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore.
authors.forename: M
authors.initials: M
authors.lastname: Jeyakumar
authors.forename: H N
authors.initials: HN
authors.lastname: Krishnamurthy
authors.forename: R R
authors.initials: RR
authors.lastname: Dighe
authors.forename: N R
authors.initials: NR
authors.lastname: Moudgal
mesh_terms: D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001667:Binding, Competitive; D002462:Cell Membrane / Q000502:physiology; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003338:Corpus Luteum / Q000502:physiology; D005260:Female; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D011817:Rabbits; D011974:Receptors, LH / Q000276:immunology* / Q000502:physiology*; D012756:Sheep
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D011974:Receptors, LH; D007986:Luteinizing Hormone