Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A

Anti-idiotypic nanobodies (AId-Nbs) are novel antigens that can replace the conventional hapten-protein conjugates of small molecule toxins, serving the same function in the competitive immunoassay. Furthermore, nanobodies show increased sensitivity from naive (non immunized) phage display libraries because those obtained have a lower affinity than the binding affinity of the target analytes. Here, we demonstrated a new approach for the development of sensitive and environmentally friendly immuno-PCR (IPCR) for OTA based on phage displayed AId-Nbs against anti-OTA monoclonal antibodies. In this study, a phage displayed AId-Nb was selected from a naive nanobody library. After four cycles of panning, a phage displayed AId-Nb was isolated using a competition-binding biopanning strategy. The half-inhibition concentration (IC50) of the phage-ELISA was 300 pg mL(-1), which was 8.83-fold better than that of a conventional ELISA. Furthermore, a non-toxin quantitative IPCR assay was also developed for the detection of ochratoxin A in agri-products based on phage displayed AId-Nbs. The limit of detection (LOD) of the assay is 4.17 pg mL(-1), which exhibits a 9-fold improvement over the phage-ELISA. The developed method was successfully validated using OTA contaminated agri-products. Furthermore, novel and environmentally friendly IPCR might have potential applications in a general method for the immunoassay of various toxic small molecules.