Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal

• Ab2β−Nb−AP has the potential to replace chemically-coupled probes.
• Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly.
• We developed a green and rapid one-step competitive enzyme immunoassay.
• The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL−1, respectively, with a linear range of 0.93–7.73 ng mL−1. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL−1, and the IC50 was 0.89 ± 0.09 ng mL−1 with a linear range of 0.29–2.68 ng mL−1. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.

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