| Fumonisins are secondary metabolites mainly produced by Fusarium moniliforme which are common contaminants of food and feed especially in corn, wheat and some agricultural products and bring serious threat to human and animal healthy. So far,15 fumonisins had been found and the mainly poisons are FB1, FB2 and FB3 in the nature. FB1 usually be found in corn and its products, and is also the main cause of the toxicity.FB1 has neurotoxicity, cell toxicity, immune toxicity, liver and kidney toxicity and carcinogenicity etc. Taking into account its toxicity, the International Agency for Research on Cancer (IARC) classifies FB1 into the 2B group of molecule. Many studies found that FB1 was closely related to the esophageal cancer of human, but it had no direct evidences. Nowdays, FB1 is commonly analyzed by high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS) and enzyme- linked immunosorbent assay (ELISA). HPLC, GC and LC-MS methods are expensive and time consuming, and require appropriate instrumentation and trained personnel. ELISA has the advantages of high specificity, sensitivity and simple, and has become the most popular and useful screening tool which are widely used.In this study, the glutaraldehyde was used to conjugate FB1 to ovalbumin (OVA) for the complete antigen (FB1-OVA). The conjugate FB1-OVA was analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectroscopy (UV) and mass spectrum (MS). The results indicated that the coupling method was success. The molecular weights of FB1-OVA and OVA were measured as 51928.612 and 44265.718 by MS and the conjugate ratio was 10.6:1. The hybridoma was expansive incubated and injected in mice, after the ascites was collected and purified, the antibodies against FB1 were gained. The subclass of the McAb was IgGl, the concentration was 2.56 mg/mL and the titer was 1:1× 105 and can be produced stablely.The Ci-ELISA method for detection FB1 using the McAb have been established and optimized. The linear regression equation of the Ci-ELISA was y=-38.524+ 98.816 with the correlation coefficient (R2) of 0.9961. The linear range was 2.5～250 ng/mL and the sensitivity of 12 ng/mL, the lowest detection limit of 2.28 ng/mL. The intra-assay and inter-assay coefficient of variation was 2.35% and 1.96%. Recovery experiment of spiked corn samples showed that the average recovery rate ranged from 102±4% to 90.5±3.2% and from 96.3±7.6% to 86.7±5% during intra-assay and inter-assay, respectively. The mean intra-assay and inter-assay coefficient of variation was 3.84% and 6.22%. The cross reactivity with FB2 was 60.4% and all cross reactivity (CR) with DON, AFB1, OTA were lower than 1%.The established Ci-ELISA method for FB1 was adopted to determine 144 corns originating from Fuyang, Bozhou, Bengbu, Hefei, Anqing and Huaibei city of Anhui province. The results indicated that the positive ratio of FB1 was the lowest and the contamination ratio was 20.8% and the pollution levels were 0.062- 0.237μg/kg and the average level was 0.074μg/kg in Bengbu. The positive ratio of FB1 was the highest and the contamination ratio was 95.8% and the levels were 0.52-1.37 μg/kg and the average level was 0.883 μg/kg in Fuyang.The Ci-ELISA method was developed in our experiments which provide a solid foundation for formulating the local food safety standards of Anhui province, and also provide a safe and reliable technical support for the supervision of food safety and the healthy diets of livestock and poultry. |
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