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)×106M−1 and (9.06±5.7)×106M−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 μg/L). 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
Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus Thuringensis for the development of a capillary electrophoresis competitive immunoassay
GIOVANNOLI, Cristina;ANFOSSI, Laura;BAGGIANI, Claudio;GIRAUDI, Gianfranco
2008-01-01
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)×106M−1 and (9.06±5.7)×106M−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 μg/L). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matricesI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.