Development of ELISA for the determination of transgenic Bt-cottons using antibodies against Cry1Ac protein from Bacillus thuringiensis HD-73

The area cultivated with Bt-cottons expressing Cry1Ac gene increases year by year in China and other countries. To evaluate any potential adverse impacts on the environment from the release of Bt (Bacillus thuringiensis) technology, the development of a method for easily detecting the activity of the CrylAc toxins is of particular interest. The aim of this study was to develop sandwich-ELISA for the detection of CrylAc protein in Bt-cotton tissues. A specific antibody was obtained from rabbits inoculated with CrylAc protein derived from Bt strain HD-73 and a secondary antibody conjugated to HRP could combine the Bt CrylAc protein specifically. The limit of detection was 5 ng/mL and there were no cross-reactions between the positive control of Cry1Ab/1Ac, Cry1C, Cry2A, Cry3Bb1 and Cry9C. Extracts of proteins from cotton leaves were used to evaluate the suitability of the assay. Tris-borate buffer and sodium carbonate buffer were employed for the extraction of protein, the limit absorbance of detection was 0.134 and 0.449, respectively, and the latter produced a higher background. The results showed that cultivars GK-12, GK-22, insect-resistant cotton, bivalent transgenic cotton and shiyuan 321 assayed positively and NON was the negative sample. The PCR method was used for the validation of the developed assay. Although both methods allowed the same results to be obtained, ELISA needed simple equipment and took less time. The developed immunoassay method is considered reliable for the detection of Bt CrylAc protein.