Establishment of antibodies-based novel multi-modal highly sensitive immunoassay platforms for detecting Bacillus...

Vip3Aa toxin is a Bacillus thuringiensis (Bt)-derived insecticidal protein widely deployed in transgenic crops, and sensitive residue analysis of Vip3Aa toxin is required for agricultural surveillance. Here, we generated an anti-Vip3Aa toxin monoclonal antibody (mAb, H12-mAb) together with a polyclonal antibody preparation (pAbs, Y26-pAbs) and evaluated their binding affinities (1.34 × 10⁷ and 6.35 × 10⁷ L/mol, respectively). Using this pair of antibodies, we built three complementary assay formats to detect Vip3Aa toxin: a double antibody sandwich ELISA (DAS-ELISA) for routine quantification, an aggregation-induced emission fluorescent microspheres (AIE)-based fluorescent immunoassay (AIE-FLIA) for enhanced sensitivity, and a gold nanoparticle (AuNPs)-based lateral flow immunoassay (AuNPs-LFIA) for rapid screening. The resulting limits of detection (LODs) were 9.68 ng/mL (DAS-ELISA), 1.95 ng/mL (AIE-FLIA), and 31.25 ng/mL (AuNPs-LFIA), which are well below the reported Vip3Aa expression (> μg/g) levels in transgenic tissues. Method performance was verified in representative agricultural matrices, providing intra-assay recoveries of 90.3-99.1% with coefficients of variation (CVs) of 1.4-9.7% and inter-assay recoveries 83.6-99.9% with CVs of 2.9-10.6%. Additionally, the AIE-FLIA and AuNPs-LFIA also exhibited good practicality and applicability on detecting Vip3Aa toxin. Overall, these multi-modal immunoassays enable both laboratory quantification and rapid field-oriented screening of Vip3Aa toxin in agricultural samples.