Mimic Epitope Of Ochratoxin A And Its Application In Phage-free Peptide ELISA

Ochratoxin A(OTA)is a common mutagenic,teratogenic and carcinogenic mycotoxin mainly produced by several Aspergillus and Penicillium species.It is important to develop a rapid,high sensitive and high-throughput OTA analysis method for the safe monitoring of the global food.Enzyme-linked immunoadsordent assay(ELISA)is the most widely used method for OTA detection,while OTA usually is needed for preparing competitive antigen in this method,which is harmful to the opearators.Thus,it is necessary to develop effective and biocompatible substitution of OTA for the development of OTA immunoassay method.In this study,Mimic epitope peptide sequence of OTA which is specific to the anti-OTA monoclonal antibody(m Ab-2A11)was obtained from a phage display random 7-mer petide library by using a modified panninging method.Then,the biotinylated mimic epitope peptide sequence was used as the competing antigen to establish a phage-free direct competetitive ELISA for OTA detection in corn samples.The detailed procedure is stated as follows:Firstly,the microtiter plate was coated with donkey anti-mouse Ig G antibody,and then the anti-OTA m Abs were direct immobilized onto plate through binding the Fc fragment of the m Abs.Then the phage display random 7-mer petide library was added to screen the sequence specific to anti-OTA m Abs.After three-round acid elutions,the PBST buffer containing 10%(v/v)methanol and 0.1 ng/m L OTA was used for competitive elution.Thirty of randomly selected phage particles were identified as positive phage particles,and 16 of them showed high competitive inhibition rate under 0.1 ng/m L of OTA solution.Six kinds of petide sequences were obtained by conducting DNA sequencing for the 16 selected phage particles,and the phage with sequence of“GMVQTIF”showed the lowest IC50 value in a phage ELISA test.Then,biotinylated 12-mer peptide “GMVQTIF-GGGSK-biotin” was designed as a competing antigen to develop a direct competitive ELISA for OTA detection.The proposed ELISA demonstrated a good linear range from 0.005ng/m L to 0.2ng/m L for OTA quantitative detection with a half-maximal inhibitory concentration of 0.024 ng/m L,which is 5-fold lower than that of commercial ELISA kit.The detection limit of the proposed peptide ELISA was achieved at 0.001ng/m L.Moreover,Biotin-peptide ELISA shows a highly selectivity for OTA detection with a negligible cross reaction with other common mycotoxins including AFB1,FB1,DON,and ZEN.The accuracy and precision of peptide ELISA were validated by analyzing the spiked corn samples.The average recoveries(n=3)of the intra-assay ranged from 98.7~120.3% with variation coefficient(CV)ranging from 3% to 10%,and those for the inter-assay ranged from 97.4% to 105.2% with CV ranging from 8% to 20%,respectively.In addition,the results of petide ELISA showed a high correlation coefficient with those of commercial ELISA.In conclusion,a peptide ELISA with high sensitivity,selectivity and reproductivity was developed and can be used for OTA analysis in corn samples.