| Fumonisins (FBs), as a group of mycotoxins, are mainly produced by Fusarium moniliforme, Fusarium proliferatum, etc. There are more than 20 kinds of derivatives of mycotoxins. FB1, FB2 and FB3, are the most toxic and prevalent. FB1 usually be found in maize and maize-based food or animal feed. FBs are one kind of the possible carcinogen. The carcinogenicity of FBs have been proved in many animals, and some surveys have showed that the FBs might relate to esophageal cancer in humans. Because the serious threat of FBs to human and animal, More attention has been paid in the field of food hygiene. Currently, several analytical methods have been reported for FBs determination in food samples, including high performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC-MS) and immunological methods, etc. For the need of expensive equipment and complex sample preparation, the application of these methods by using large instrument were limited. Because the immunological methods were sensitive, specific, cheap and simple (sample processing), they were fit for the rapid screening of large numbers of samples. In this study, a indirect competitive ELISA (ic-ELISA), colloidal gold immunochromatographic assay (GICA) and chemiluminescent enzyme-linked immunosorbent assay (CLEIA) were developed to analyze FB1. These results would be contributed to the progress of rapid screening methods for FBs.In this study, the glutaraldhyde was used to conjugate FB1 to ovalbumin for the detection antigen and bovine serum albumin for the immunogen. The conjugates FB1-OVA and FB1-BSA were analyzed by SDS-polyacrylamide gelelectrophoresis and UV scanning method. Routine immunization protocol and quickly immunization protocol were adopted to immunize BALB/c mice, the hybridomas were screened after the cell fusion. Three hybridomas which stable secrete the antibodies against FBs have been selected and the hybridoma named 4B3. The subclass of the McAb was IgG1, the concentration was 2.19 mg/mL, the ELISA titers was 1: 2.56×106, and the affinity constant was 5.21×109 L/mol of the McAb. The cross-reactivities with FB2 and FB3 was 198% and 59%, respectively, and all cross-reactivities (CRs) with DON, ZEN, T-2, CIT, Och-A and AFB1 were lower than 10%.An ic-ELISA method for detection FB1 using the McAb have been established and optimized. The optimized concentration of antigen added to microtiter plates was 0.5μg /mL at 37℃for 2 h. The working concentration of the McAb in the competition condition was 1:30 000 at 37℃for 45 min. The reaction time of HRP secondary antibody and OPD substrate solution were at 37℃for 1 h and 25 min, respectively. The linear regression equation of the ic-ELISA was y = -41.499 x + 89.715, with the correlation coefficient (R2) of 0.981. The linear range was 1.56 ng/mL - 50 ng/mL with the lowest detection limit of 1.30 ng/mL. Recovery experiment of spiked corn samples showed that the total average recovery, coefficient of variation, and inter-assay coefficient of the method were (94.73±4.54)%, 8.31% and 8.54%, respectively. 14 corn samples was tested for FB1 using the ic-ELISA. In these samples, the FB1 was not detected in 5 samples, less than 1 mg/kg in 6 samples, between 1 mg/kg and 2 mg/kg in 3 samples.The nanoparticle-monoclonal antibody probes for FB1 have been synthesized using the 20 nm colloidal gold particles prepared by sodium citrate reduction and the simple colloidal gold immunochromatographic test strip have been assembled. The visible detection limit of the test strip was 2.5 ng/mL and optimal decidable concentration was 30 ng/mL (the test line was disappeared). The stability of the test strip was up to 6 months. When the spiked corn samples were extracted by methanol: water (70:30, v/v) and diluted for 5 times, the results were consistent with the standard curve. The corn samples were detected with the test strip. The results show, the concentration of FB1 was higher than 0.75 mg/kg in 3 samples, was less than 0.25 mg/kg in 10 samples and was between 0.25 mg/kg - 0.75 mg/kg in 1 sample.An ic-CLEIA method for detection FB1 using the McAb have been established and optimized. The optimized concentration of antigen added to polystyrene white flat bottom micro-plate was 25 ng /mL at 4℃overnight. The concentration of the McAb in ic-CLEIA was 1:100 000 at 37℃for 45 min. The working concentration and time of goat anti-mouse IgG-HRP were 1:10 000, at 37℃for 60 min. The chemiluminescent substrate solution was at room temperature away from light for 10 min. The standard curve equation of the ic-CLEIA was logit (y) = 1.079 5 - 2.299 8 log (x) with the correlation coefficient (r) of -0.998 2. The linear range of 0.32 ng/mL - 25 ng/mL and the lowest detection limit of 0.32 ng/mL. The total average recovery, coefficient of variation, and inter-assay coefficient of the method were (100.15±9.55)%, 7.90% and 8.12% in detection of spiked corn samples, respectively. Of the 14 corn samples, the FB1 was not detected in 5 samples, was less than 1 mg/kg in 6 samples and was between 1 mg/kg and 2 mg/kg in 3 samples.After extracted with acetonitrile/water and purified by TC-F120 SPE columns, the FB1 was detected by LC-MS/MS method in corn samples. The results from the immunoassays were compared with those of LC-MS/MS method, which indicated that the results between the immunological detection methods and LC-MS/MS method were consistent. The developed three immunological detection methods including GICA, ic-ELISA and ic-CLEIA can be used for rapid screening FBs in corn.
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