Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

ABSTRACT In this study, we developed a double antibody sandwich chemiluminescent immunoassay (DAS-CLIA) based on phage-displayed nanobodies for the determination of Cry2A toxin. Nine phage-displayed nanobodies specifically to Cry2A were obtained from a naive nanobody library after four rounds of biopanning. The phage-displayed nanobody P2 with high affinity binding was selected as the detection antibody for the sensitive DAS-CLIA development. The proposed method exhibited high sensitivity with a limit of detection of 0.09？ng/mL, has a wide linear range for Cry2A toxin detection, in the range of 0.1–1000？ng/mL and negligible cross-reactivities to the other Bt toxins. The recoveries of Cry2A toxin spiked in cereal samples (rice, wheat, and corn) ranged from 82.7% to 118%, with a coefficient of variation of less than 10%. This study demonstrated that the DAS-CLIA procedure based on phage-displayed nanobodies may provide an alternative sensitive method for the detection of Bt toxins in agri-products. GRAPHICAL ABSTRAC