Antibody Characterization for use in Clinical Mass Spectrometry

Abstract

Many biomarkers in human serum are in low abundance. Immunoaffinity enrichment via anti-peptide antibody and analysis via liquid chromatography tandem mass spectrometry (LC-MS/MS) is an attractive workflow that depends on the production of new antibody reagents. Although there are different methods available to characterize antibodies such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and LC-MS/MS, using the same platform for screening and assay development is likely beneficial. Antibody apparent dissociation constant (KD) and on rate and off rate (kon and koff) may be equally as critical as recovery efficiency for screening. A high throughput, manual, in-house antibody LC-MS/MS screen was developed to screen 846 supernatants with antibodies to specific peptides of KLOTHO and Procollagen type III C-terminal Pro-peptide (P3CP) to find the best 15 antibodies and characterize them for future assay development.