How biotinylation can interfere with recognition: a surface plasmon resonance study of peptide-antibody interactions.
Biotinylation is one of the most frequently used labelling procedures in biochemistry and molecular biology. To study the influence of biotinylation on peptide antigenicity, we selected a peptide derived from the second extracellular loop of the beta(2)-adrenergic receptor. Interactions between different biotinylated and nonbiotinylated analogs and a monoclonal antibody directed against an epitope present within the N-terminal end of this peptide were studied in detail. Taking advantage of the BIACORE 3000 surface plasmon resonance equipment, we were able to compare antibody interactions with the immobilised peptides and with the same peptides in solution. While the nonbiotinylated peptide, immobilised by its N-terminus, was not recognised by the antibody, it was recognised either after immobilisation by means of the thiol group of the C-terminal cysteine residue or as a free peptide tested as analyte with the monoclonal antibody immobilised on the chip. The N-terminal biotinylated forms were well recognised when immobilised on streptavidin but poorly (for the aminocaproyl-biotin derivative) or not at all (for the biotinylated derivative) when they were allowed to react with immobilised monoclonal antibody. These results indicate that the biotinyl moiety interacts with residues that are important for antibody recognition in solution but such interactions are abrogated when it is bound to the streptavidin. Molecular modeling confirmed that the N-terminus of the peptide mimicked to some extent the streptavidin binding site.
Peter
Jean-Christophe
JC
UPR 9021 CNRS Immunologie et Chimie Thérapeutiques, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, F-67084, Strasbourg, France.
Briand
Jean-Paul
JP
Hoebeke
Johan
J
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000276:immunology; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D018343:Receptors, Adrenergic, beta-2