Development Of Ochratoxin A Competitive Direct ELISA Kit

Ochratoxins are toxic metabolites which are secreted by fungi such as Aspergillus and Penicillium.Fungi are abundant in agricultural products,and its metabolites are found in various agricultural raw materials and commodities.Mycotoxins have more than 400 different chemical structures,of which OTA is the main component.OTA has nephrotoxicity,immunotoxicity and teratogenic effects and IARC categorizes it as a carcinogen.Instrumental analysis and immunoassay are the most commonly used methods.Instrumental analysis methods often require expensive instruments,cumbersome operations,and require specialized technicians.Immunoassays have the advantages of simple operation,high sensitivity and strong specificity.It can quickly detect a large number of samples to be tested.In this experiment,OTA monoclonal antibodies were prepared and a competitive direct ELISA kit was developed,thereby establishing a rapid,sensitive detection method for OTA.In this study,OTA was coupled to BSA to synthesize the immunogen OTA-BSA,and OTA was coupled OVA to synthesize detecting antigen OTA-OVA.The synthesized antigens were identified by polyacrylamide gel electrophoresis and UV scanning spectrum.The results showed that the synthesis was successful.The OTA-BSA and OTA-OVA coupling ratios were 7.5:1 and 5:1,respectively.The immunization results showed that mice immunized with OTA-BSA produced an immune response.The OTA-OVA was used as coating antigen for detection.Among them,mouse 1 had the best immune effect.The titer of multi-antibody serum was up to 1:51200,and the value of IC50 was 7.98 ng/mL.Mouse 1 spleen cells fused with mouse myeloma cells SP2/0.After fusion,the supernatant of the hybridoma cells was taken and the potency was measured by indirect ELISA.The inhibition was measured by indirect competitive ELISA and the positive clones were screened.After three rounds of subcloning,six positive clone cell lines named 1F5,2C5,2D7,2G3,3D7 and 3F6 were obtained.The 2G3 with the highest positive value was selected to prepare ascites,after which ascites was purified.Polyacrylamide gel electrophoresis was used for identification.The purified monoclonal antibody contained only two clear bands with molecular weights of 50kDa and 25 kDa,indicating that the monoclonal antibody had higher purity.By ELISA,the titer before and after 2G3 purification reached 1:8.192×10⁷.The IC₅₀ was1.22 ng/mL and the affinity constant was 2.11×10¹¹ mol/L.2G3 was detected using a subtype identification kit,and its subtypes were IgG1 and?light chain type.The cell line has high specificity and sensitivity and can be used to develop a kit.Using sodium periodate method,2G3 was attached to HRP to prepare enzyme-labeled antibody.According to the principle of enzyme-linked immunosorbent assay,the enzyme-labeled antibody was used to develop a competitive direct ELISA kit to rapidly detect OTA residues.After optimizing the conditions,the OTA concentration and the binding rate showed a good linear relationship?y=-32.94x+61.00,R²=0.981?.The IC₅₀ was 2.16 ng/mL and the sample recovery rate range was 80.3%¹08%.The cross-reactivity rates of the kit with ochratoxin B and C were 3.5%and 0.28%,respectively,and there was no cross-reaction with other biotoxins.The inter-panel and intra-board average coefficients of variation were 6.41%and 7.07%,respectively.The detection limits of OTA in corn,wheat and peanut were 0.134,0.203 and 0.292 ng/mL,respectively.It can be concluded that the competitive direct ELISA kit prepared in this study can be used for rapid and quantitative detection of OTA in food and feed.