Kinetic screening of antibodies from crude hybridoma samples using Biacore
Section snippets
Materials and methods
Binding experiments were performed using Biacore 2000 and 3000 optical biosensors equipped with research-grade CM5 sensor chips (Biacore AB, Uppsala, Sweden). Amine-coupling reagents (EDC, NHS; and sodium ethanolamine HCl, pH 8.5) were obtained from Biacore AB. Goat anti-human-Fc purified IgG antibody (2 mg/mL, 0.1% sodium azide) was purchased from Caltag Laboratories (No. H10500; Burlingame, CA), carboxymethyl dextran sodium salt (No. 27560) was purchased from Fluka Chemical Corp. (Milwaukee,
Antibody screening assay
A capture method was used to monitor antigen binding to monoclonal antibodies isolated from crude supernatants. First, the Fc-specific anti-human IgG was immobilized in all flow cells. Second, each of three supernatants from the panel was flowed across an IgG surface to capture antibody and create a stable, homogeneous surface. Third, antigen binding and dissociation were monitored over each captured antibody. The antibody/antigen complexes were completely stripped from the surface to assay
Discussion
We demonstrated a method to determine binding kinetics for antibody/antigen interactions in a screening mode on Biacore instruments. A key to the method is the ability to immobilize and quantitate the amount of antibody from crude samples using specific capturing systems. Immobilizing the bivalent antibodies (instead of the antigen) avoided avidity effects, which have led to misinterpretations of antibody/antigen binding kinetics in the past [10], [11]. The use of a specific IgG recognition
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