Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten

In this study, by use of synthesized polypeptides as haptens, a monoclonal antibody with broad recognition against seven major Cry1 toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) has been produced and characterized. First, by comparing the three-dimensional structures of seven Cry1 toxins, analyzing the conserved sequences, and considering the antigenicity and hydrophilicity, three polypeptides (T1, T2, and T3) have been chosen and coupled to keyhole limpet hemocyanin as immunogens for the generic monoclonal antibody (Mab) generation. Thereafter, a double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) was developed for simultaneous determination of seven Cry1 toxins. The results revealed that the haptens T1, T2, and T3 had different effects in the production of antibodies. Among them, the obtained Mab (strain 2D3) generated by T2 can recognize seven Cry1 toxins simultaneously. Equilibrium dissociation constant (K D) values for seven Cry1 toxins with Mab 2D3 were 1.198 × 10–8 M for Cry1Aa, 2.197 × 10–8 M for Cry1Ab, 1.367 × 10–8 M for Cry1Ac, 2.092 × 10–8 M for Cry1B, 5.177 × 10–8 M for Cry1C, 4.016 × 10–8 M for Cry1E, and 3.497 × 10–8 M for Cry1F. For 2D3-based DAS-ELISA, the limits of detection (LOD) and limits of quantification (LOQ) can reach 15 and 30 ng·mL–1 for each Cry1 toxin, respectively. Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cry1 toxins, and it will provide a new idea and technical routes for development of multidetermination immunoassays.