Structural requirement for the recognition of luteinizing hormone releasing hormone (LHRH) by monoclonal and conventional anti-LHRH antibodies

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Abstract
Immunochemical studies on monoclonal and conventional anti-LHRH antibodies have been reported. The association constants (Ka) were in the order of 109–1010 L/mol when calculated from Scatchard and Steward–Petty plots. Heterogeneity indices (a) calculated from Sips plot were nearly 1.0, indicating the homogeneous nature of monoclonals. Most of the conventional anti-LHRH produced by monkey, baboon, rabbit, and dog, by immunization using LHRH linked to tetanus toxoid by the carbodiimide condensation method, showed a single slope in Scatchard analysis (except two dog antisera) and a values were nearly 1.0. Monoclonals and conventionals reacted most strongly with native LHRH(NH2). Monoclonals showed poor reactivity with LHRH free acid and LHRH fragments containing free acid. The C-terminus tetrapeptide 7–10 showed 10 times more reactivity than tripeptide 4–6. The heptapeptide 4–10 showed 100 and 1000 times more reactivity than the tetra- and tri-peptide, respectively. Introduction of the tripeptide pGlu-His-Trp-OH to heptapeptide 4–10 caused five times more inhibition in reactivity than the heptapeptide. Conventional anti-LHRH antibodies manifested specificity to the C-terminus of LHRH. These sera did not react with LHRH free acid and LHRH fragments of sequence 4–6, 7–10, and 4–10. The complete loss of reactivity of conventional antibodies and poor reactivity of monoclonal antibodies was partly regained when LHRH free acid was coupled to Lys, Lys-MDP, or (Ala)2-tuftsin, suggesting that for monoclonals and conventionals the antigenic determinant was confined to the conformation involving the C-terminus amide of LHRH.