Residue-specific radioimmunoanalysis: a novel analytical tool. Application to the C-terminus of CCK/gastrin peptides

Abstract

Five antisera directed against the common bioactive C-terminal tetrapeptide sequence of cholecystokinin (CCK) and gastrin were examined with respect to the significance of each residue for the antibody binding. Systematic substitutions and/or derivatizations of each of the four residues showed a unique pattern for each antiserum although they were raised against the same antigen and have the same sequence-specificity. The pattern of reactivity towards the related cardioexcitatory FMRF amide peptide, and analogues hereof confirmed the residue specificity of the antisera. While it is well known that even small covalent modifications of the antigen can influence the antibody binding profoundly, the great variations in significance of each residue among randomly selected antisera raised against the same antigen and specific for the same sequence has not been known so far. Hence, by appropriate combination of antisera their different residue specificity can be used for detection of amino acid substitutions or modifications. Such immunochemical sequence analysis requires only femto- or picomolar amounts of peptides, which need not necessarily be purified. Thus, residue-specific immunoanalysis may be a versatile tool in studies of species differences, phylogenesis and synthesis of peptides.