Kinetic screening is of paramount importance when it is to select custom-made antibodies, tailored for their respective scientific, diagnostic, or pharmaceutical application. Here a kinetic screening protocol is described, using a Biacore A100 surface plasmon resonance biosensor instrument. The assay is based on an Fc-specific antibody capture system. Antibodies from complex mixtures, like from mouse hybridoma supernatants are captured on the sensor surface in an oriented manner. The method uses a single injection of one antigen concentration for the determination of six relevant screening parameters, which comprehensively describe the antibody’s kinetic rate profile and its valence mode. The method enables the scientist to rank and finally select rare and outstanding antibodies according to their kinetic signatures.
Kinetic screening Molar Ratio Valence Dissociation half-life (t1/2 diss) Binding Late Stability Late Antibody Capture Level kdSurface plasmon resonance
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Canziani GA, Klakamp S, Myszka DG (2004) Kinetic screening of antibodies from crude hybridoma samples using biacore. Anal Biochem 325(2):301–307PubMedCrossRefGoogle Scholar
Wassaf D, Kuang G, Kopacz K et al (2006) High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays. Anal Biochem 351(2):241–253PubMedCrossRefGoogle Scholar
Safsten P, Klakamp SL, Drake AW et al (2006) Screening antibody–antigen interactions in parallel using biacore a100. Anal Biochem 353(2):181–190PubMedCrossRefGoogle Scholar
Johnsson B, Lofas S, Lindquist G (1991) Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem 198(2): 268–277PubMedCrossRefGoogle Scholar
Leonard P, Safsten P, Hearty S et al (2007) High throughput ranking of recombinant avian scfv antibody fragments from crude lysates using the biacore a100. J Immunol Methods 323(2): 172–179PubMedCrossRefGoogle Scholar
Pasqualucci L, Guglielmino R, Houldsworth J et al (2004) Expression of the aid protein in normal and neoplastic B cells. Blood 104(10): 3318–3325PubMedCrossRefGoogle Scholar