Immuno-analytical method development for detection of transgenic Cry1Ac protein and its validation.

ABSTRACT

BACKGROUND: Bacillus thuringiensis (Bt) synthesizes Cry1Ac protein, which is toxic to many lepidopteran pests, and the cry1ac gene has been expressed in several transgenic crop plants. The Cry1Ac protein has been isolated from Bt kurstaki HD73 and purified to homogeneity. Polyclonal antibodies were raised against purified Cry1Ac in rabbits and goat. Sandwich ELISA was developed for Cry1Ac using goat IgG as a coating antibody, and affinity-purified rabbit IgG as the primary antibody. RESULTS: The sensitivity of the assay was in the range of 0.47-1000 ng. It was subsequently employed in validating biological samples. Fifteen different cotton-seed samples were screened 12 were found to be Bt positive and 3 Bt negative. The CS7 seeds showed the highest Bt content of 8.51 ± 0.45 µg g-1 , followed by CS8 (6.0 ± 0.02 µg g-1 ), CS15 (5.9 ± 0.03 µg g-1 ), CS9 (5.5 ± 0.05 µg g-1 ), and CS10 (4.83 ± 0.013 µg g-1 ). The CS5 seeds showed Bt content of 3.6 ± 0.21 µg g-1 . The F2 generation, CS6 (Kaveri seeds) showed lower Bt content (2.9 ± 0.06 µg g-1 ). The CL5 samples showed Cry1Ac content of 0.99 ± 0.009 µg g-1 . The amount of Cry1Ac protein in leaves, stem, and roots of germinated Bt cotton plants (CS10 and CS4) were 1.76 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , 2.0 ± 0.1 µg g-1 , and 1.6 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , and 2.0 ± 0.01 µg g-1 dry tissue, respectively. CONCLUSION: The method developed can be used for screening the expression levels of Cry1Ac in different transgenic Bt cultivars and also spurious Bt cotton seeds procured by farmers. © 2019 Society of Chemical Industry.