High throughput screening of antibodies from crude samples

G.A. CANZIANI. S. KLAKAMP, AND D.G. MYSZKA.

Ventura, CA, USA

Conferencia; Gordon Conference:Reversible Associations in Structural and Molecular Biology; 2002

GORDON RESEARCH CONFERENCES

WHAT YOU WILL LEARN ABOUT ANTIBODY SCREENING 1. Antibodies may be screened on Biacore using a capturing assay in a single-concentration analysis mode. 2. Human mAbs be captured out of crude supernatants using anti-IgG monoclonal antibodies that recognize binding sites in the Fc region. 3. Immobilizing the antibody eliminates avidity effects that occur when the antibodies are screened by binding to antigen surfaces and the need to know antibody concentrations in supernatants. 4. The antibody capture levels and the antigen concentration used in the assay are determined empirically to obtain measurable responses that show curvature in the association phase, permitting the determination of the association and dissociation rate as well as the overall surface capacity. 5. A simple kinetic model was used to interpret the kinetics of antigen binding to multiple antibodies simultaneously. 6. High resolution kinetic studies show that the kinetics were comparable to the higher-throughput kinetics within 10% error.