Elsevier

Analytical Biochemistry

Volume 325, Issue 2, 15 February 2004, Pages 301-307
Analytical Biochemistry

Kinetic screening of antibodies from crude hybridoma samples using Biacore

https://doi.org/10.1016/j.ab.2003.11.004Get rights and content

Abstract

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (ka and kd) and affinity (KD) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to ∼200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.

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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