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 biochemistry and molecular biology. Unfortunately, not all antipeptide antibodies react with their target proteins. The reasons why certain antipeptide antibodies fail to do so are not always clear, although it is commonly assumed that conformational difference between the peptide antigens and the corresponding sequences in proteins accounts for most failures. Here, we report detailed characterization of an antipeptide mAb which reacted avidly with the peptide antigen but did not react with the same sequence in a protein. ELISA analysis using analogs of the antigen peptide revealed that this mAb did not react with a C-terminus-extended analog of the antigen peptide and reacted poorly with a peptide amide analog of the antigen peptide. These results suggest that the mAb recognizes an epitope including the C-terminal-free carboxyl group of the peptide. This analysis also revealed that the epitope recognized by this mAb was located in the C-terminal pentapeptide, RY-IRS. Four amino acid side chains (R,I,R, and S) in this pentapeptide were shown by alanine-scanning to be critical for antibody recognition. Analysis of the polyclonal antisera raised against this peptide revealed that antibodies reacting with this unique carboxyl-containing epitope are most abundant. This unexpected finding has since been shown in several other cases in this laboratory, suggesting that generation of antibodies that recognize carboxyl-containing artificial epitopes may be rather common. we also found that the use of a peptide amide (instead of peptide acid) antigen did not prevent a similar problem; in this case, the C-terminal amide became part of the epitope. Based on these findings, we suggest a method for enhancing the probability of isolating protein-reactive mAbs.
Liang
T C
TC
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225, USA. tliang@utmmg.med.uth.tmc.edu
Luo
W
W
Hsieh
J T
JT
Lin
S H
SH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology*; D011506:Proteins / Q000276:immunology*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins