Quality Control of Coated Antibodies: New, Rapid Determination of Binding Affinity

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.

Ficus sycomorus latex: An efficient alternative Egyptian source for horseradish peroxidase in labeling with antibodies for immunodiagnostic kits

“…The reactivity of the prepared conjugates was examined in 96-well microtiter plates using the ELISA test described by Ricoux et al . [ 16 ]. Briefly, each well was coated overnight with 100 µl of monoclonal mouse anti-albumin IgG (0.5 µg protein/well) dissolved in coating buffer (50 mM sodium carbonate buffer, pH 9.6).…”

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus (Boophilus) annulatus salivary gland

“…Direct ELISA was performed according to the method of Ricoux et al [26]. ELISA plate was coated with 1.5 g/well of the purified rRa-sHSP II in 100 l of coating buffer (50 mM carbonate buffer, pH 9.6).…”