A quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.

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TitleA quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.
Publication TypeJournal Article
Year of Publication2011
AuthorsLowenthal, MS, Gasca-Aragon, H, Schiel, JE, Dodder, NG, Bunk, DM
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
Date Published2011 Sep 15
KeywordsAmino Acid Sequence, Antibodies, Monoclonal, Antibody Affinity, Chromatography, Liquid, Humans, Immunoprecipitation, Isotope Labeling, Microspheres, Molecular Sequence Data, Nonlinear Dynamics, Peptides, Protein Binding, Reference Standards, Reproducibility of Results, Tandem Mass Spectrometry, Troponin I

A mass spectrometry-based antibody selection procedure was developed to evaluate optimal 'capture' monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K(d)) were determined for 'bound mAb-antigen' vs. 'unbound antigen' using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal 'capture' mAbs were determined through evaluation of relative K(d) constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K(d) constants as determined using NIST Standard Reference Material (SRM) 2921 - Human Cardiac Troponin Complex. This ID LC-MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification.

Alternate JournalJ. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PubMed ID21856254