Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus Thuringensis for the development of a capillary electrophoresis competitive immunoassay

Abstract

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable-(5.0 +/- 1.2) x 10(6) M(-1) and (9.06 +/- 5.7) x 10(6) M(-1)-reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 mug L(-1)). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.