期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 879, 期 26, 页码 2726-2732出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2011.07.037
关键词
Immunoassay; Mass spectrometry; Isotope dilution; Antibody; Troponin
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 isotopedilution 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. Published by Elsevier B.V.
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