| SudanI, one of the most widely used synthetic fat-soluble organic colorants, has been proved to be a potential carcinogen and forbitted as an additive used in foods. In May 2003, France gave a warning against chilli powders from India that was found to be contaminated with Sudan I. In China, from February until May 2005, Sudan I was also found in batches of roasted chicken wings and chicken burgers on sale and in some commercial products such as piccalilli and chilli sauce which led to a great panic . Along with technological development and national attention, its detection method has also developed continuously. Diferent analytical methods are currently used:electrochemical analysis, Spectrometry, Chromatography, LC-MS, IAS, etc. Enzyme linked immunoabsorbant assay (ELISA) is a simple, fast, highly sensitive and selective analytical method widly used in clinical detection, biological determination, food analysis and enviornment monitoring. In order to find a rapid and sensitive means of immunological analysis to detect the SudanI residue, in this study, based on the analysis of molecular structure and immunogenicity of Olaquindox, the technology of monoclonal antibodies production was applied to prepare the monoclone antibody against SudanI and then assemble rapid ELISA kit and strip for SudanI residual detection. The main contents and resuLts of this study were as follows:1. Synthesis and identification of the artificial antigen for SudanIA derivative(CSDI)of Sudan I was synthesized by diazotization and CSD I was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) as immunogen or coating antigen respectively. UV and SDS-PAGE were used to identify Sudan I artificial antigen. BALB/c mice were immunized with BSA-Sudan I. The titers of polyclonal antiserum was detected by indirect ELISA, the sensitivity of polyclonal antiserum was identified by blocking ELISA. The animal immunization results showed that three BALB/c mice indirect ELISA titers against SudanI were above 1×1.28-4 and the IC50 of No.3 mice was 67ng/mL.The high-titer, sensitivity and specific anti-SudanI polyclonal antiserum have been generated. The artificial antigen of SudanI and its mice polyclonal antiserum laid a foundation base for product the monoclonal antibodies and rapid test reagent against SudanI.2. Preparation of monoclonal antibodies and immunological characterization of SudanIThe titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. SudanImAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive SudanImAb were induced from in vivo method. There hybridoma cell lines of 6H6B1,6H6E6,6H6G10,6F12E2,6F12E5和6F12G2 were screened for specificity to SudanI, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:6.4×102 ~1: 1.28×103 in supernatant, 1:6.4×105 of 6H6E6 in ascites, and the affinity constant(Ka) was 1.85×1010L/moL and 1.94×1010L/moL, the mAb of 6H6E6 showed good sensitivity with IC50 of 2ng/mL to SudanI. The rate of cross reaction of SudanImAb with carbadox was no cross-reactivity to other compounds. SudanImAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of SudanI residues in animal food.3. Development of rapid determination of SudanI residue methodBased on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of SudanI (SudanI-Kit) was developed with SudanImAb. The calibration curve of SudanI-Kit with standard SudanI inhibitor was typical sigmoid curve fitted to the four parameters logistic equation, the detection limit for SudanI was 0.2ng/mL and IC50 was 2ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The recoveries from pig liver and pork were 90.005%和88.475%, respectively, when 1, 20, 60, 100ng/mL SudanI were spiked. The validity of SudanI-Kit in 4℃was above six months.
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