Chemiluminescent Enzyme Immunoassay and Bioluminescent Enzyme Immunoassay for Tenuazonic Acid Mycotoxin by Exploitation of Nanobody and Nanobody–Nanoluciferase Fusion

The isolation of nanobodies (Nbs) from phage display libraries is an increasingly effective approach for the generation of new biorecognition elements, which can be used to develop immunoassays. In this study, highly specific Nbs against the Alternaria mycotoxin tenuazonic acid (TeA) were isolated from an immune nanobody phage display library using a stringent biopanning strategy. The obtained Nbs were characterized by classical enzyme-linked immunosorbent assay (ELISA), and the best one Nb-3F9 was fused with nanoluciferase to prepare an advanced bifunctional fusion named nanobody-nanoluciferase (Nb-Nluc). In order to improve the sensitivity and reduce the assay time, two different kinds of luminescent strategies including chemiluminescent enzyme immunoassay (CLEIA) and bioluminescent enzyme immunoassay (BLEIA) were established, respectively, on the basis of the single Nb and the fusion protein Nb-Nluc for TeA detection. The two-step CLEIA was developed on the basis of the same nanobody as ELISA, only with simple substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol. In contrast with CLEIA, the novel BLEIA was conducted in one-step new strategy on the basis of Nb-Nluc and bioluminescent substrate coelenterazine-h (CTZ-h). Their half maximal inhibitory concentration (IC50) values were similar to 8.6 ng/mL for CLEIA and 9.3 ng/mL for BLEIA, which was a 6-fold improvement in sensitivity compared with that of ELISA (IC50 of 54.8 ng/mL). Both of the two assays provided satisfactory recoveries ranging from 80.1%-113.5% in real samples, which showed better selectivity for TeA analogues and other common mycotoxins. These results suggested that Nbs and Nb-Nluc could be used as useful reagents for immunodetection and that the developed CLEIA/BLEIA have great potential for TeA analysis