Quality Control of Coated Antibodies: New, Rapid Determination of Binding Affinity
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Rémy Ricoux
Abstract
A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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- Methods for the Determination of Plasma Total Homocysteine: a Review
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- Interferon β1a Therapy Changes Lipoprotein Metabolism in Patients with Multiple Sclerosis
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