Establishment of multiple highly sensitive immunoassay platforms for detecting Bacillus thuringiensis Cry1Ah insecticidal protein in agricultural and food samples.

Abstract

Genetically modified crops are a hot topic concerning agricultural and food safety. Bt Cry1Ah insecticidal protein has been used in genetically modified crops for pest management, but there have been no immunoassay techniques available for specific-detecting its residues. In this study, a mAb (H10-mAb) and pAbs (287#pAbs) with high activity against Cry1Ah insecticidal protein were prepared, and their affinities (Kaff) were measured as 2.25 × 109 L/mol and 1.36 × 109 L/mol. Thereafter, three immunoassay platforms-DAS-ELISA, AIE-labeled FLISA, and colloidal gold-labeled LFIA-were established for detecting Cry1Ah insecticidal protein. The LODs were 5.53, 3.60, and 7.80 ng/mL, respectively, all reaching a highly sensitive level of ∼ng/mL. The DAS-ELISA and FLISA demonstrated good practicality and applicability for detecting Cry1Ah insecticidal protein spiked into river water, farmland soil, corn leaves, and corn kernels samples. The recoveries were 79.87 %-98.22 % with CVs of 1.70 %-10.78 %. The LFIA not only demonstrates good practicality and applicability for detecting Cry1Ah insecticidal protein spiked into the same samples but is also applicable to the determination of transgenic corn leaves for Cry1Ac (a type of Bt insecticidal protein that exhibits cross-reactivity). The key amino acids of H10-mAb to Cry1Ah and Cry1Ac are Y33, Y47, Y58, and Y98, all of which are in its VH fragment by docking simulation analysis. This study, for the first time, successfully constructed multiple highly sensitive immunoassay platforms suitable for monitoring Cry1Ah insecticidal protein in agricultural and food samples. Meanwhile, the study pinpointed the key active sites of the relevant H10-mAb, thereby indicating the direction for further optimizing the activity modification of this antibody in the future.