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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2726-2732
• Main Authors: Lowenthal, Mark S., Gasca-Aragon, Hugo, Schiel, John E., Dodder, Nathan G., Bunk, David M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analysis; Analytical, structural and metabolic biochemistry; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - metabolism; Antibody; Antibody Affinity; antigens; binding capacity; Biological and medical sciences; Chromatography, Liquid - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Immunoassay; Immunoprecipitation - methods; Isotope dilution; Isotope Labeling; liquid chromatography; Mass spectrometry; Medical sciences; Microspheres; Molecular Sequence Data; monitoring; monoclonal antibodies; Nonlinear Dynamics; Peptides - analysis; Peptides - chemistry; Peptides - metabolism; Pharmacology. Drug treatments; precipitin tests; Protein Binding; quantitative analysis; Reference Standards; regression analysis; Reproducibility of Results; synthetic peptides; tandem mass spectrometry; Tandem Mass Spectrometry - methods; traceability; Troponin; troponin I; Troponin I - analysis; Troponin I - chemistry; Troponin I - metabolism
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.07.037

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.