Application Of QD-FLISA For Detection Of Cry1Ab In Genetically Modified Maize

Protein-based detection approaches of GMOs such as ELISA, lateral flow strip and Western blot due to fast and sensitive quality were widely developed. In this study, the quantum dots (QDs) were used as the fluorescence label coupled with the goat-anti-rabbit antibody, then the mixture was detected by fluorospectrophotometer. The polyclonal antibody against purified Cry1Ab protein was obtained by immunizing a New Zealand White rabbit. The quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) using the endotoxin Cry1Ab monoclonal antibody present in genetically modified (GM) maize MON810 was developed.Firstly, Cry1Ab protein was extracted from (Bacillus thuringiensis) HD-1strain, and the concentration of Cry1Ab protein is 2mg/mL which was detected by Bradford method. The Cry1Ab dissolved in 0.9% NaCl was emulsified with an equal volume of Freund's complete adjuvant (then Freund's incomplete adjuvant) to immunize a New Zealand White rabbit (3kg). The titer of purified antiserum (least detectable dose) for indirect ELISA was estimated by purified Cry1Ab protein.The optional concentration of mixture with QDs conjugate antibody is one 25μL solution of MPA-coated CdTe QDs (1 mg/mL), 20μL of EDC (10 mg/mL), 10μL of NHS (1.5 mg/mL) and 0.2 mg of goat anti-rabbit antibodies (Abs) in PBS were reacted together for 4 h at room temperature with gentle agitation. After the reaction finished, the mixture was purified using ultra-filtration membrane according to the instruction of the manufacture.Finally, QD-FLISA and the assay optimization were developed. Then in the established QD-FLISA, the capture antibody concentration of 10μg/mL was selected, and the anti-Cry1Ab detection antibody and the QDs-labeled goat anti-rabbit tracer antibody, and the optimal diluted ratio of the anti-Cry1Ab detection antibody and the QDs labeled goat anti-rabbit tracer antibody were 1:1000 and 1:500, respectively. This assay was highly specific for Cry1Ab protein and has no cross-reactivity with the nontarget proteins tested such as Cry2Ab, Cry1F and Cry3Bb etc. Test linearity was achieved in the range of values from 0.05-5% validated with the extracted protein of MON810 GM maize. The limits of detection (LOD) and quantification (LOQ) were with the value of 2.956pg/mL and 9.854pg/mL, respectively, which were much higher than conventional sandwich ELISA method. The assay fulfilled all the requirements of high sensitivity, high accuracy, high precision and low cross-reactivity. In particularly, all these indicated that this fluoroimmunoassay is high specific and sensitive for detection of Cry1Ab in GMOs.