| Ochratoxin A (OTA) generated by fungi is a toxic compound that is usuallyfound as a contaminant in a wide range of food and feedstuf, and one of the reasonsthat cause acute or chronic toxicity to humans and animals. Aiming to minimize thepotential risk of OTA to humans and animals, many studies have been performed todevelop diverse methods for OTA determination. The most widely used method isimmunoassay which is known for simplicity, rapidity and specificity.However, most antigens used were synthesized by chemical methods. Itinvolves the operation of toxic mycotoxins which pose threats to human beings, aswell as lot-to-lot variation. Moreover, OTA like many mycotoxins is expensive topurchase that makes antigen not cost-effective. Consequently, the use of alternativeform of antigen such as phage-displayed mimotope would be of great value inimmunoassays.Mimotopes are known to be able to mimic the reaction between epitopes inantigens and their corresponding antibodies, and generally panned from phagedisplay random peptide libraries. However, these mimotopes were limited by thediversities of the peptide libraries and may not exhibit high sensitivities. For thepurpose of improving the sensitivity, the original OTA mimotopes screened from therandom peptide libraries can be evolved in vitro by constructing a second-generationpeptide library. Ultimately, by using genetic engineering and protein engineering anOTA complete antigen with immunoreactivity can be available, and it is utilized todevelop immunoassays. The essential results of this research are shown as follows:1. Selection of OTA mimotopesTo screen OTA mimotopes from two phage display random peptide libraries,anti-OTA monoclonal antibody was employed as a target. Following three cycles ofpanning, seven OTA mimotopes were identified and obtained. A motif sequenceFQLH was found in four mimotopes by sequence alignment. Thus we infer thatFQLH is the conserved region of the OTA mimotope, and it was seeded in asecondary library for evolution.2. Construction and repanning of OTA mimotopes in the second-generation peptide libraryThe motif sequence was incorporated into the second-generation peptide libraryby introducing FQLH flanked by three random amino acid residues. The total titer ofthe library was1.2×108pfu before amplification, and it is sufcient to encode all ofthe6.4×107(206) possible peptides theoretically. The binding between phagedisplay mimotopes and anti-OTA monoclonal antibody can be improved by stringentscreening. The seven phages selected from the second-generation peptide libraryshowed lower half inhibition concentration (IC50) values of0.0520.336ng/mL thanthose selected from the random peptide library (0.50-1.03ng/mL) by phage ELISA.3. Immunoassays set up with the phage-displayed mimotopeThe IC50of the chemiluminescent ELISA developed with the OTA mimotopewas0.04ng/mL, and the linear range was0.0060.245ng/mL. The mimotope wasalso used to develop a qualitative dipstick assay with a cutof level of1ng/mL. Atotal of40samples of foodstufs and feedstufs were analyzed using phage-basedchemiluminescent ELISA, commercial ELISA kit, and phage-based dotimmunoassay, respectively. The results obtained from the three methods were inagreement with each other.4. Biosynthesis and application of OTA antigens in immunoassaysSeven biosynthetic OTA antigens were expressed by E. coli cells after cloningthe gene of OTA mimotope into an expression plasmid pMAL-pIII, respectively. Thechemiluminescent ELISA for OTA set up with the biosynthetic antigen exhibited adetection limit of0.22ng/mL, IC50of0.82ng/mL, working range of0.30-2.17ng/mL. The OTA biosynthetic antigen was also used to develop a dipstick assay witha cut-off level of5ng/mL. We validated the two types of immunoassays bymeasuring OTA recovery using spiked corn, rice and wheat and comparing to thechemosynthetic antigen-based chemiluminescent ELISA. No significant distinctionbetween the three immunoassays was observed. |
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