Development Of Monoclonal Antibodies And Screening Phage Mimotopes For Ochratoxin A

Ochratoxins are secondary metabolites which are produced by some kinds of the Aspergillus genus and Penicillium genus, containding seven kinds of structurally similar compounds,in which OTA is the most common and most toxic. OTA has kindy toxicity, liver toxicity, immunotoxiciy, teratogenic, carcinogenic and mutagenic characteristicsetc, it has great potential hazards to the health of human and animals, OTA was classed as 2B carcinogen by the International Agency for Research on Cancer in 1993. At present, the main methods of detection OTA are HPLC, LC-MS and TLC and fluorescence detection method etc. However,the shortcoming of these methods are tedious sample preparation, high cost,requirements professional personnel to operate and not suitable for the detection of large quantilies of samples. The enzyme linked immunosorbent, which is one method of the immunoassay, not noly has good sensitivity and specificity, but also the low requirement of the sample purity, simple and feasible of the operation and suitable for detection of large quantities of samples.However, the competitive detection of ELISA is a toxic detection system which is based on the competitive antigen of enzyme labeled toxin or toxin coupled to a carrier, and it also has other disadvantages such as expensive toxin products, importing retricitons, complicated coupling reactions and threatening the health of the operators. The appears of Phage display technology effectively solved this problem. The mimotope, which was was non-toxic, was obtained by panning of Phage display technology, can replace toxic standard for toxin detection. This method effectively solved the problem of OTA toxin and inconvenient operation, and provided a guarantee for operators’ health.The main contents of this paper are as follows: 1. Synthesis and identification of complete antigen for Ochratoxin AThe immunogen OTA-BSA and coating antigen OTA-OVA were pepared with OTA coupled with BSA and OVA using NHS method. UV scanning spectrum and SDS-PAGE identification showed that BSA and OVA were successfully coupled with OTA, and the coupling ratio of them were 7:1 and 4.5:1, respectively.The polyclonal antibody was prepared by immunizing Balb/c mice with OTA-BSA, and the titer was up to 1:51200 by ELISA detection coating ELISA plate with OTA-OVA. The result showed that the prepared immunogen OTA-BSA had a satisfied immunogenicity and provided a basis for the preparation of OTA monoclonal antibody. 2. Preparation and identification of monoclonal antibody for Ochratoxin AThe antiter and sensitivity of anti-OTA monoclonal antibody were prepared by immunizing Balb/c mice, which were six weeks of age, with the artificial antigen OTA-BSA. Choose the mice spleen cells, which immune effect was better, to fuse with myeloma cells SP 2/0, and screened by indirect ELISA and indirect competitive ELISA. Two monoclonal cells, which were named 1B2 and 2G3, respectively, and can stably secrete anti-OTA monoclonal antibody, were obtained after three times subclonde. The ascites was prepared by induced ascites in vivo and purified by acid ammonium sulfate. The titers of two strains of monoclonal antibody were up to 10-6, and the values of IC50 were 0.723ng/mL and 0.683ng/mL, respectively,and the affinity constant were 3.05×1010L/moL and 2.56×1011L/moL, respectively. The subtype of two strains monoclonal,which were identified with the mouse monoclonal antibody subtype identification kit,were IgG1. 3. Affinity panning of Ochratoxin A mimotopeThe anti-OTA monoclonal antibody was purified as target in affinity panning of Phage random 7 peptide library. In the eluate titer course of the 4th round of biopanning, 20 clones were picked from the total number of blue plaques less than 100, then through indirect ELISA and indirect competitive ELISA to determine positive phage clones.With the positive clons were secquced, analysised and translated, the acid secquence of positive clones were obtained. All of the sequences of mimotope were TTQVLEV with the monoclonal anbitody 1B2 as target. Two secquences of mimotope, which were obtained in the panning of monoclonal antibody 2G3 as the target, were VIGNSDP and QQSWLHI, respectively.