title: Epitope mapping of human follicle stimulating hormone-alpha using monoclonal antibody 3A identifies a potential receptor binding sequence.
abstract: Five monoclonal antibodies (mabs) were generated (3A, 4B, 5F, 2E, 1E) by immunizing BALB/c mice with human (h) FSH. The mabs were used to relate antigenic structures (epitopes) to function (receptor binding). All five mabs could immunoneutralize (inhibit binding to receptor) hFSH and could be placed into two groups based on potency (degree of neutralization). Group I mabs (5F, 2E, 1E) were less potent than group II mabs (3A, 4B) even though group I mabs had a 2-fold higher average affinity constant than group II. Those data suggested that group II mabs recognize an epitope near or in the receptor binding site of hFSH. Immunoradiometric epitope cross-matching demonstrated that group I and group II mabs recognize different epitopes. Further characterization of 5F and 3A (representative of group I and group II, respectively) utilized an enzyme-linked immunosorbent assay (ELISA) and a RIA. In the ELISA, both mabs bound hFSH and hFSH alpha but not hFSH beta. In the RIA, 3A bound [125I]hFSH and [125I]hFSH alpha but not [125I]hFSH beta. In contrast, 5F bound only [125I]hFSH. hFSH effectively competed with [125I]hFSH for 5F and 3A. In contrast, hFSH alpha competed with [125I]hFSH for 5F but not for 3A even though 3A could bind hFSH alpha in the ELISA and the RIA. These results suggest that 3A and 5F recognize different epitopes. The epitope recognized by 3A is unique in that its conformation appears to be dependent on association with hFSH beta. Since 3A was a more potent inhibitor of receptor binding than 5F, its epitope specificity was characterized further by epitope mapping. This was accomplished utilizing a peptide ELISA and by affinity chromatography. The results from epitope mapping demonstrated that 3A recognizes sequences 61-78 and 73-92 with binding to 73-92 being 4-fold greater than to 61-78. Thus, the epitope comprised of sequence 73-92 (and to a lesser extent 61-78) appears to be important for receptor binding.
authors.affiliation: Department of Biochemistry, Albany Medical College, New York 12208.
authors.forename: R S
authors.initials: RS
authors.lastname: Weiner
authors.forename: J A
authors.initials: JA
authors.lastname: Dias
authors.forename: T T
authors.initials: TT
authors.lastname: Andersen
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005640:Follicle Stimulating Hormone / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification; D015592:Immunoradiometric Assay; D009500:Neutralization Tests; D009994:Osmolar Concentration; D010455:Peptides / Q000276:immunology; D011962:Receptors, FSH / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D010455:Peptides; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone