Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry.

Publication Type:

Journal Article

Source:

Journal of immunological methods, Volume 353, Issue 1-2, p.49-61 (2010)

Keywords:

2010, ADAM Proteins, Animals, Antibodies, Monoclonal, Antibody Specificity, Automation, Laboratory, Biological Markers, C-Reactive Protein, Center-Authored Paper, Chromatography, Liquid, Clinical Research Division, Enzyme-Linked Immunosorbent Assay, Humans, Hybridomas, Immunomagnetic Separation, Indicator Dilution Techniques, mass spectrometry, MICE, Peptide Fragments

Abstract:

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) utilizes antibodies to enrich peptides from complex matrices for quantitation by stable isotope dilution mass spectrometry. SISCAPA improves sensitivity and limits the sample handling required for plasma-based analysis. Thus far, SISCAPA assays have been performed using polyclonal antibodies, yet monoclonal antibodies are an attractive alternative since they provide exquisite specificity, a renewable resource, and the potential for isolation of clones with very high affinities (10(-9) M or better). The selection of a good monoclonal antibody out of hundreds-to-thousands of clones presents a challenge, since the screening assay should ideally be in the format of the final SISCAPA assay, but performing the assays manually is labor- and time-intensive. In this manuscript, we demonstrate that monoclonal antibodies can be used in SISCAPA assays, and we describe an automated high-throughput SISCAPA method that makes screening of large numbers of hybridomas feasible while conserving time and resources.