ANTIBODY-BINDING TO A PEPTIDE BUT NOT THE WHOLE PROTEIN BY RECOGNITION OF THE C-TERMINAL CARBOXY GROUP

Antipeptide antibodies have become indispensable tools in modern bioch emistry and molecular biology. Unfortunately, not all antipeptide anti bodies react with their target proteins. The reasons why certain antip eptide antibodies fail to do so are not always clear, although it is c ommonly assumed that conformational difference between the peptide ant igens and the corresponding sequences in proteins accounts for most fa ilures. Here, we report detailed characterization of an antipeptide mA b which reacted avidly with the peptide antigen but did not react with the same sequence in a protein. ELISA analysis using analogs of the a ntigen peptide revealed that this mAb did not react with a C-terminus- extended analog of the antigen peptide and reacted poorly with a pepti de amide analog of the antigen peptide. These results suggest that the mAb recognizes an epitope including the C-terminal-free carboxyl grou p of the peptide. This analysis also revealed that the epitope recogni zed by this mAb was located in the C-terminal pentapeptide, RY-IRS. Fo ur amino acid side chains (R, I, R, and S) in this pentapeptide were s hown by alanine-scanning to be critical for antibody recognition. Anal ysis of the polyclonal antisera raised against this peptide revealed t hat antibodies reacting with this unique carboxyl-containing epitope a re most abundant. This unexpected finding has since been shown in seve ral other cases in this laboratory, suggesting that generation of anti bodies that recognize carboxyl-containing artificial epitopes may be r ather common. We also found that the use of a peptide amide (instead o f peptide acid) antigen did not prevent a similar problem; in this cas e, the C-terminal amide became part of the epitope. Based on these fin dings, we suggest a method for enhancing the probability of isolating protein-reactive mAbS. (C) 1996 Academic Press, Inc.