Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique.
A refined surface plasmon resonance method was developed to measure the kinetics of peptide binding to rabbit monoclonal antibodies (RabMAbs). Optimized amounts of RabMAbs were captured onto sensor chips from hybridoma supernatants followed by binding of free peptides from solution. This allowed kinetic measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies and determination of affinity constants without complications contributed by avidity considerations. Peptide-binding responses were normalized for the amount of antibody present in each sample and a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants ka and kd, and the affinity constant KD (kd/ka), could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of peptide antigens were performed to validate the reliability of single-concentration measurements. By combining data on affinity, activity and concentration, ranking of the antibody-containing supernatants was performed, allowing selection of high quality RabMAbs for binding of peptides in solution.
Pope
Matthew E
ME
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6.
Soste
Martin V
MV
Eyford
Brett A
BA
Anderson
N Leigh
NL
Pearson
Terry W
TW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity / Q000502:physiology*; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D007700:Kinetics; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D020349:Surface Plasmon Resonance / Q000379:methods*
D000911:Antibodies, Monoclonal; D010455:Peptides