title: Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach.
abstract: Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the Kon and Koff values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.
authors.affiliation: a Institute of Physics; Chinese Academy of Sciences ; Beijing , China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.forename: Hongyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Dai
authors.forename: Shaohu
authors.initials: S
authors.lastname: Hu
authors.forename: Ignacio
authors.initials: I
authors.lastname: Pino
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Eichinger
authors.forename: Huibin
authors.initials: H
authors.lastname: Lyu
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived
keywords: KD; OIRD; OIRD, oblique-incidence reflectivity difference; mAb, monoclonal antibody; IP, immunoprecipitation; IHC, immunohistochemistry; ICC, immunocytochemistry; ChIP, chromatin immunoprecipitation; HuProt, human proteome microarray; ELISA, enzyme-linked immunosor; affinity; avidity; kinetics; monoclonal antibodies; protein microarrays