Mimotope peptides for nanobodies: A nontoxic alternative to ochratoxin A and its application in chemiluminescence immunoassays for analysis of pepper samples.

Ochratoxin A (OTA) is a common food contaminant and poses a significant threat to human health, which requires rigorous monitoring. Mimotope peptides (MPs) are commonly used as non-toxic alternatives to toxic small molecules in eco-friendly immunoassays. Herein, with an anti-OTA nanobody as the target protein, cyclic 7-mer MPs of OTA were screened using phage display and immunomagnetic separation. The phage MPs (PMP) with the highest sensitivity and its alkaline phosphatase-tagged MP fusion (ALP-MP) were used to develop a PMP-based chemiluminescent immunoassay (PMP-CLIA) and an ALP-MP-based CLIA (AMP-CLIA). After optimization, PMP-CLIA and AMP-CLIA exhibited a limit of detection of 0.128 ng/mL and 0.232 ng/mL. Good accuracy and selectivity were confirmed for both CLIAs by recovery experiments and cross-reactions. Moreover, they were validated by high performance liquid chromatography in detecting real pepper samples. Thus, two CLIAs based on the nanobody and MPs were demonstrated as reliable tools for monitoring OTA in pepper.