title: A quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.
abstract: 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.
authors.affiliation: Analytical Chemistry Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8392, Gaithersburg, MD 20899-8392, USA. mark.lowenthal@nist.gov
authors.forename: Mark S
authors.initials: MS
authors.lastname: Lowenthal
authors.forename: Hugo
authors.initials: H
authors.lastname: Gasca-Aragon
authors.forename: John E
authors.initials: JE
authors.lastname: Schiel
authors.forename: Nathan G
authors.initials: NG
authors.lastname: Dodder
authors.forename: David M
authors.initials: DM
authors.lastname: Bunk
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000915:Antibody Affinity; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D007553:Isotope Labeling; D008863:Microspheres; D008969:Molecular Sequence Data; D017711:Nonlinear Dynamics; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019210:Troponin I / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D019210:Troponin I