| Mycotoxins are secondary metabolites of fungi.Ochratoxin A(OTA)as a secondary metabolite was first isolated from a culture of Aspergillus ochraceus in 1965.The major producers of OTA are Aspergillus and Penicillium.OTA is one of the highest significant mycotoxins,and are capable of having toxic effects on human and animals.It has a great public health and agro economic significance,due to the nephrotoxic,genotoxic,neurotoxic,immunotoxic,embryotoxic and teratogenic effects and its suspected carcinogenicity.OTA has been found in agricultural products such as barley,wheat,sorghum,oats and maize in the fields or during storage.Therefore,it is particularly important to study the rapid and highly sensitive detection method for OTA.The purpose of this study was to develop a monoclonal antibody against OTA and its application in an indirect competitive ELISA assay(ic ELISA),colloidal gold nanoparticles(CGNs)and nanoflowers gold strip(AuNFs)which are sensitive,specific,rapid and reproducible.In this study,Ochratoxin A-BSA conjugate(OTA-BSA)was used as an immunogen to immunize four female BALB/c mice.Antigen was emulsified in Freund’s complete adjuvant for the first immunization while emulsification for subsequent immunization was carried out in Freund’s incomplete adjuvant.The mice serum was tested by ELISA using OTA-KLH as coated antigen.High titers of immunized mice were obtained.Splenocytes were collected from the mouse with the highest titres,and then were fused with Sp2/0myeloma cells to generate hybridoma cells by polyethylene glycol(PEG)-mediated method.After sub-cloning,three positive cells(6E5,10D3 and8F5)were obtained by indirect ELISA.Positive cells were injected into BALB/c mice abdomen,and then antibody was purified with two-step precipitation methods(Caprylic acid and saturated ammonium sulphate).The isotyping result showed that all three anti-OTA mAb was the Ig G1 subclass and the concentrations of mAb were 1.5,1.43 and 2.25 mg/m L,respectively.The 6E5 mAb was selected and chosen for further study,because it reacted strongly to its antigen(OTA-KLH)and was stable to freeze-thawing.The average affinity constant(Kaff)of 6E5 anti-OTA mAb was3.7x10~8L/mol.To measure the cross-reactivity of the 6E5 mAb,ic-ELISA was performed towards structurally related,sharing common epitopes antigens.The results showed that this anti-OTA mAb was capable of binding Ochratoxin B(OTB)with cross-reactivity,whereas no cross-reactivity to other mycotoxins including Zearalenone(ZEN),Citrinin(CTN),Deoxynivalenol(DON)and Fumonsins B1(FB1)was observed.For investigation of the competitive effect between OTA and OTB,further optimization of the indirect competitive ELISA conditions was carried out.A competitive binding curve of OTA and OTB was obtained,and the result showed that this anti-OTA mAb can detect OTA specifically,with low cross-reactivity to OTB(13.4%).A stable 6E5 anti-OTA mAb was employed for the establishment of ic-ELISA,CGNs and AuNFs for detection for OTA toxin in real samples.The optimum parameters to maintain the stable results for the detection of OTA toxin in real samples were adjusted.The optimized ic-ELISA for OTA detection resulted in a linear range of 0.06-0.6 ng/m L.The 50%inhibitory concentration(IC50)was reached at 0.2 ng/m L,and the lower detection limit(LOD)was 0.03 ng/m L.The mean recovery rate from the spiked samples was obtained as(89.315±2.257)%with average coefficient of variation(CV)of 2.182%(lower than 5%difference),which means systematic error was low.By using the same anti-OTA mAb,sensitive and rapid CGNs were also established.Several mycotoxins other than OTA were used to determine the cross-reactivity of the strip assay.The formation of one red line occurs on the test line,indicating that no cross-reactivity(high specificity)was observed.For sensitivity assay,different concentrations(0-10μg/m L)of OTA standard solution were used.The LOD determination for OTA was5μg/m L.For examination of the robustness of these CGNs,real food matrices(corn samples)were collected and used in this evaluation,and the results indicated that these samples were free of OTA contamination,and could be detected out within 5 min.Therefore,the robustness of CGNs was validated and could be used successfully.For AuNFs preparation,result demonstrated that solution was successfully prepared and were conjugated successfully with anti-OTA mAb.The specificity of the strip assay was analyzed by different toxins including OTB,CTN,FB1,DON and ZEN.The result showed that no cross-reactivity(high specificity)was observed among these tested toxins with exception of low cross-reactivity to OTB toxin.In order to determine the limits of strip assay,different concentrations of OTA standard solution were subjected to the AuNFs.The color line disappeared at 1μg/m L,indicating that the limit of detection for OTA was 1μg/m L.AuNFs established in this study were applied to test OTA in real samples,corn and coffee samples.The results showed that two bands appeared on the test strips,and all the reaction could be finished within 5 min.The results indicated that these samples were free of OTA contamination.Therefore,the multi-branched AuNF as probe can be successfully applied to OTA detection.Data obtained from OTA detection in corn and coffee samples revealed that the ic-ELISA,CGNs and AuNFs results were in a good agreement.Therefore,the anti-OTA mAb secreted by 6E5 can be used through these three established assay for robustness detection of OTA in corn and coffee samples. |
