Specific Antibodies against alpha-Melanotropin for Radioimmunoassay. Parallelism between the Immunochemical Cross-Reactions of Melanotropin Structural Analogues and Their Biological Activity

Abstract

The purpose of this experimental investigation was to provide antibodies against α-melanotropin with a high serum titer and with great specificity. To this end, rabbits were immunised with a complex-antigen consisting of human serum albumin containing a rather low number of α-melanotropin molecules attached uniquely through the γ-carboxyl-group of (glutamic acid)-5 (probably mostly to ɛ-amino groups of albumin lysine residues). In such a complex-antigen the groups chemically characteristic of α-melanotropin (C-terminal valine amide and N-terminal N-acetylserine) are expected to be maximally exposed to the surroundings. The antisera had high antibody titers (1:125000 up to 1:900000). Their specificity was directed towards the C-terminal part of α-melanotropin. The cross-reaction with adrenocorticotropin was small (about 0.1%) and negligible with other peptide hormones, including β-melanotropin. The sensitivity of the assay was 40 pg/ml and the detection limit for α-melanotropin about 3 pg. Radioiodination of α-melanotropin with the lactoperoxidase method yielded a monoiodinated tracer with a specific activity of about 1500 Ci/mmol. The biological activity of structural analogues and fragments of α-melanotropin (assay in vitro with skins of Rana pipiens) paralleled their activity in the radioimmunoassay. This may indicate similar structural requirements of the melanophore receptor and antibody populations.