Preparation Of Ochratoxin A Artificial Antigen And Anti-Ochratoxin A Polyclonal Antibody

Ochratoxin A (OA) is a virose fungal secondary metabolites, mainly produced by certain Aspergillus and Pencillium strains, which can contaminate food, vegetables, coffee, grapes cococa and other cash crops. It has been widespread around the world, while it could contaminate a large number of species, and the producers can adapte to a extensive environment. It has a strong renal toxicity, liver toxicity, teratogenicity, carcinogenicitu, immune toxicity and embryyo toxicity. Many countries have established limits, so the establishment of low cost, fast and efficient field detection is extremely important. Immunological methods were widely popular due to its rapid, high sensitivity, high specificity, low cost, portable and easy operation.In this study, artificial antigens were obtained by coupling OA to carrier protein through the active ester method. Ultraviolet-visble (UV-Vis) spectroscopy,. fourier transform infrared (FT-IR) spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy were used to identify and analysis the artificial antigens comprehensively, even the response mechanism of the OA and carrier protein was further explored by fluorescence quenching. Then the BALB/c mice were immuned, and studied the characteristics of antiserum, to select the available antiserum for the detection of the recovery of OA in spiked samples and specimen, and the indirect competitive ELISA was established, the specific details are as follows:1 OA artificial antigens construction and its structure analysisDue to the low molecular weight (403.8) of OA, it is a hapten with only reactiongenicity and without immunogenicity. It can stimulate the body to produce antibodies only by the artificial antigens which is coupled OA to carrier protein. In this study, activated ester method was applied to construct the immunogen OA-BSA and coating antigen OA-OVA by conjugating OA with bovine serum blbumin (BSA) and ovalbumin (OVA), respectively, through the active carboxyl belonged to OA. UV-Vis, FT-IR, fluorescence and CD spectroscopies were applied to analyze artificial antigens. In UV-Vis, the maximum absorbent wavelenght (λmax) was red shifted to 380 nm for the lowered pKa in OA due to the combination of OA and carrier protein. The molar ratio of OA with BSA and OVA was 11:1 and 7.6:1, respectively. OA utilization ratio was 50.5% and 38.6%, respectively. In FT-IR, compared with carrier protein, the bands of primary amine at 1545 cm-1 in the artificial antigens was decreased markedly, and there was a small but sharp peak at 1589 cm-1 caused by OA lactone ring in the role of carrier protein. In the study of fluorescence spectroscopy, OA had a strong quenching effect on carrier protein at the 340 nm in the artifical antigens. Further analysis by synchronous fluorescence, OA had a quenching effect on tyrosine residues and tryptophan residues, and the fluorescence peaks of tyrosine residues and tryptophan residues both had a slight blue shift in the artifical antigens, indicating its polarity was decreased and the hydrophobicity was increased after coupling. These results show that OA has been successfully coupled with carrier protein through the active ester method. In addition, to investigate the coupling reaction, fluorescence spectra was adopt to analyse the reaction of different concentration OA and BSA. We found that the quenching effect was gradual strengthening while the concentration of OA was increased, but the trend was slow and could not achieve the level of OA-BSA attained by activity ester. In the synchronal fluorescent, the quenching effect of OA only effected on aminoisocaproic acid residues in direct response. All this demonstrated the effectiveness of ester activity. In the CD of artificial antigen, the positive peak and negative peaks intensity are significantly reduced. The CDSSTR software analysis showed that the a-helical conten of protein was decreased, while theβ-sheet content increased, indicating a more extended structure more and more conducive to OA exposure. In the mixture, with the increasion of OA concentration, the negative peaks intensity are increased gradually. The CDSSTR software analysis showed that the a-helical conten of protein was increased, while the P-sheet content decreased, which means that peptide chain contracted and protein hydrophobicity increased. The significant difference fluorescence spectroscopy and CD spectroscopy between Atificial antigens and mixture proved the success of artificial antigens. 2 Anti-OA antibodies preparation and their characters analysisThree groups of BALB/c mice were immunized by OA-BSA, there mice per group, the dose was set to 80μg OA-BSA. Immunization period of the first group was 21 d, the second was 14 d, the third was 7 d. Mice of three groups had immune response in the antibody production process curves. The antibodies of the first and the second group were produced significantly after the third immune, and mildly after the fourth immune. The third group was produced significantly after the sixth immunization, and became stable after the seventh immunization. The nature of the antiserum of the first and the second group after the third immunization and the third group after the seventh immunization were analyzed, the titers were up to 1.0×105. Indirect competitive ELISA was used to detect the antiserum sensitivity of mice after the best reaction concentration of antigen-antibody was detected by chess-board experiment. The sensitivity of the first group were between 5-7 ng/mL, the linearity range was 1-10 ng/mL,.the sensitivity of the second group were between 13-15 ng/mL, the linearity range was 1-100 ng/mL. While the sensitivity of the third group was between 28-37 ng/mL, the linearity range was 1-100 ng/mL. The specificity of antiserums was good, it had no cross reaction with aflatoxin B1, citrinin and zearalenone in the specific analysis.3 The recovery of OA in spiked samples and the detection of specimenThe ELISA standard curve of OA antiserum which was used to detect OA was obtain by icELISA, the linear equation was y=-0.1397x+1.9539, R2=0.9937. The average recoveries achieved 71.5%-88.2%, with coefficients of variation (CVs) between 0.137%-20.56%. On the basis, some rice and maize samples purchased from the market near Huazhong Agricultural University and samples from Hubei Academy of Agricultural were chose for testing, and some samples had OA which can be detected.