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

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 (Kd) 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 Kd 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 Kd 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.