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A Quantitative LC-MS/MS Method for Comparative Analysis of Capture-Antibody Affinity Toward Protein Antigens



Mark S. Lowenthal, Hugo H. Gasca-Aragon, John E. Schiel, Nathan Dodder, David M. Bunk


A mass spectrometry-based antibody selection procedure targeting human cardiac troponin I (cTnI) was established for the purpose of developing a well-characterized secondary reference measurement procedure. A panel of six monoclonal antibodies (mAb) was evaluated based on relative binding affinity to cTnI as determined by isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) quantification following immunoprecipitation using a magnetic bead solid support. Dissociation constants (Kd) were determined for each mAb-cTnI pair using non-linear regression curve fitting over the diagnostic range 0.2-0.8 for bound [mAb-cTnI] / total [mAb]. Quantification of all three cTn subunits, and of each antibody isotype, was based on the use of stable isotope-labeled peptide analogs as internal standards. The mAb, 19C7, was determined to have the lowest Kd constant among the pre-screened antibody panel using the NIST Standard Reference Material (SRM) 2921 – Human Cardiac Troponin Complex. ID LC-MS2 is shown to be capable of quantitatively differentiating certain mAbs based on their relative binding affinities. Selection of the optimal ‘capture’ mAb for cTnI will be applied towards the development of a well-characterized assay based on an ELISA measurement procedure.
Clinical Chemistry


troponin I, mass spectrometry, immunoassay, ELISA


Lowenthal, M. , Gasca-Aragon, H. , Schiel, J. , Dodder, N. and Bunk, D. (2011), A Quantitative LC-MS/MS Method for Comparative Analysis of Capture-Antibody Affinity Toward Protein Antigens, Clinical Chemistry, [online], (Accessed June 14, 2024)


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Created September 15, 2011, Updated January 27, 2020