A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton.
A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.
Shan
Guomin
G
Dow AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, Indiana 46268, USA.
Embrey
Shawna K
SK
Schafer
Barry W
BW
D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis*; D001427:Bacterial Toxins / Q000032:analysis*; D004731:Endotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D003368:Gossypium / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000032:analysis*; D007306:Insecticides / Q000032:analysis*; D030821:Plants, Genetically Modified / Q000737:chemistry*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis