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.

MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics.

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.