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 (KD) 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.