Study Of Ochratoxin A Non-toxin Immunological Rapid Detection Method

Ochratoxin is a kind of mycotoxin, which is the secondary metabolites generatedby some kind of Aspergillus and Penicillium. It contains seven structural analogues,but Ochratoxin A （OTA） is the most toxic and the most widely distributed of all andhas the highest toxin production. OTA is immunosuppressive, neurotoxic, renal toxic,hepatotoxic, and has the ability to induce carcinogenesis, teratogenicity andmutagenesis. OTA is difficult to metabolize both in humans and animals; because of itslong half-life, it can accumulate in tissues and blood easily. As OTA is so closelyrelated to the human health, the rapid and accurate detection of OTA is of greatsignificance. Featuring high specificity and sensitivity, ELISA has been widely usedfor quantitative detection of OTA. However, currently-used immunological detectionmethods are mainly dependent on antigens labeled with probes or coupled tomacromolecular protein. As toxin material is expensive and harmful to researchers, theuse of immune-detection method has been limited to a large degree. Recent years haveseen the advent of phage displayed random peptide library in mimicking epitopes. Theuse of phage-displayed toxin epitopes to replace toxin promotes the development ofOTA detection method. In this study, we successfully obtained the OTA mimickingepitopes from the phage-displayed random peptide library, and established a non-toxinimmunological method for OTA detection.1. The affinity panning of Ochratoxin A mimicking epitopesThe purified anti-Ochratoxin A monoclonal antibody has been used as the targetin affinity panning of the phage-displayed random peptide library. After four rounds ofpanning, with the concentration of Tween-20in washing liquid increasing and theconcentration of target molecules decreasing, the amount of eluted phages increasedfrom7.3×10⁵pfu/mL in the first round to7.8×10⁹pfu/mL in the fourth round, andcorrespondingly the recovery rate increased from7.3×10^-4%to7.8%. So positivebinders have been effectively enriched, which laid a good foundation for future experiment.2. The immunological identification of the OTA mimicking epitopesIndirect phage-ELISA indicated that the52phage particles selected from thefourth eluent are positive phages. Then we selected22clones for further directcompetitive ELISA identification. The result showed that the binding of these phageparticles with anti-OTA monoclonal antibody could all be blocked by OTA standardproducts, with the IC₅₀below8ng. Specificity test showed that22phage particles canonly bind to the anti-OTA monoclonal antibody and not with other unrelatedmonoclonal antibody. After sequence analysis, we can obtained the peptide isMPLWXDL （X for any amino acid）.3. Establishment of non-toxic ELISA method for detecting OTAFollowing the successful selection of OTA mimicking epitopes, direct, non-toxicELISA method for detecting OTA was established. After optimizing ELISA workingconditions, the method showed good linearity of standard curve, with R²of0.9921.Results are highly repetitive. The linear detection range is200¹400pg/mL, the IC₅₀is974.67pg/mL. The detection limit is201.33pg/mL.The average of recovery rate ismore than89%.