In the present study, an easy to use field-deployable methodology was developed for onsite detection of pesticidal crystal protein Cry2Ab from transgenic cotton crops to reduce seed adulteration. Anti Cry2Ab IgG and IgY antibodies were developed against recombinant Cry2Ab protein in New Zealand white rabbits and in white leg horn chickens, respectively. Carboxyl-functionalized CdTe quantum dots (QDs) were used as revealing probes, and nitrocellulose paper was used as an assay matrix. Recombinant Cry2Ab was generated in the lab and used for immunization of chicken and rabbits. After successful immunization and attaining the desired titer values (1:32 000 for IgY and 1:64 000 for IgG), eggs and hyperimmune sera were collected. Anti Cry2Ab IgY was purified as per the standardized protocols, and anti Cry2Ab IgG was purified using protein A affinity chromatography. Sensitivity of the generated antibodies was examined using indirect ELISA methods against recombinant Cr2Ab protein. Specificity evaluation was carried out against other Cry proteins including Cry2Ab, Cry4b, Cry4a, Cry1Ec, and Cry1Ac. Functionalized CdTe QDs were characterized for structure and shape as well as fluorescence properties using standard laboratory techniques. A field-deployable paper-based detection methodology was developed where IgG acted as the capturing antibody and IgY-linked CdTe QDs were used as revealing probes. The limit of detection (LOD) and quantification (LOQ) were found to be 2.91 ng/mL and 9.71 ng/mL, respectively. The effect of matrix interference was assessed on the different plant crude extracts of cottonseed materials.
Keywords: Bt cotton; Cry2Ab; IgY antibody; dot blot assay; quantum dots; visual detection.