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

J Immunol Methods. 2010 Feb 28;353(1-2):49-61. doi: 10.1016/j.jim.2009.11.017. Epub 2009 Dec 1.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM Proteins / blood
  • ADAM Proteins / immunology
  • ADAM17 Protein
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / isolation & purification*
  • Antibody Specificity
  • Automation, Laboratory*
  • Biomarkers / blood
  • C-Reactive Protein / analysis
  • C-Reactive Protein / immunology
  • Chromatography, Liquid*
  • Enzyme-Linked Immunosorbent Assay*
  • Humans
  • Hybridomas
  • Immunomagnetic Separation*
  • Indicator Dilution Techniques*
  • Mass Spectrometry*
  • Mice
  • Peptide Fragments / blood
  • Peptide Fragments / immunology


  • Antibodies, Monoclonal
  • Biomarkers
  • Peptide Fragments
  • C-Reactive Protein
  • ADAM Proteins
  • ADAM17 Protein