【作者】 付云洁；

【导师】 刘志国；

【作者基本信息】 武汉工业学院 ， 微生物与生化药学， 2011， 硕士

【摘要】 本研究通过制备苏丹红I多克隆抗体，建立了两种用于快速测定食品中苏丹红I的免疫学检测方法—竞争酶联免疫吸附法（ELIS A）和胶体金免疫层析试纸条法。本文利用重氮法合成苏丹红I衍生物，再用混合酸酐法将苏丹红I衍生物和明胶偶联，形成的苏丹红I衍生物-明胶复合物作为免疫抗原。按照常规免疫程序免疫新西兰白兔制备抗苏丹红I多克隆抗体。免疫程序结束后采用颈动脉取血法获得抗血清，抗血清经辛酸硫酸铵法和亲和层析法纯化后，可以得到特异性的抗苏丹红I抗体。通过优化ELIS A操作中的各种条件，来测定特异性抗体的效价和亲和力，并建立食品中苏丹红I竞争ELISA的检测方法。利用酶标记的苏丹红I抗原和待测样品中可能存在的苏丹红I竞争性的与固相载体上的苏丹红I抗体结合，待测样品中苏丹红I含量多，则形成的非酶标记复合物多，酶标苏丹红I抗原与抗体则结合少，即显色程度与待测样品中的苏丹红I含量成反比。通过绘制线性回归标准曲线，计算样品中苏丹红I的浓度。结果表明苏丹红I抗体的效价为1: 32 000。抑制率曲线得到竞争ELIS A最低检测限为1.0μg /L，IC 50为15.0μg/L，表明抗体具有较高的亲和力。加标回收率为85.8 8 %-97. 83 %，变异系数为4.9%-7 .4 %。该方法对苏丹红II、III、IV的交叉反应率分别为2.7%、6.5%和4.1%。胶体金免疫层析试纸条的制备，是以硝酸纤维素膜（NC膜）作为载体，将抗苏丹红I抗体和抗BSA抗体包被在NC膜上，分别作为检测线（T）和质控线（C）。用胶体金标记的苏丹红I抗原喷涂在金标垫上，最佳工作条件确定后，组装试纸条。在层析作用下，待测样品中可能存在的苏丹红I与金标苏丹红I竞争结合NC膜上的抗体。待测液中的苏丹红I含量越多，与金标抗原的竞争作用越明显。检测线T的显色程度与待测样品中的苏丹红I含量成反比。通过对NC膜上两种包被抗体的浓度和金标垫上金标抗原含量的优化，确定试纸条的最低检测限和灵敏度。结果表明，本方法对辣椒粉中的苏丹红I最低检测限为10μg/ kg。在苏丹红I浓度为5.0 -20 .0μg /kg范围内可实现定量分析。变异系数≤9.23 %。对其他苏丹红染料有低的交叉反应性。与HPLC方法相比，相关系数R2 =0.9 41，测定结果相一致。本方法高效、简便且耗时短，适用于食品中苏丹红I残留的快速检测及大量苏丹红I待检样品的筛查测定。更多还原

【Abstract】 Two methods based on immunoassays were developed for rapid detection of Sudan I in food by preparing anti-Sudan I polyclonal antibodies. The assays were enzyme-linked immunosorbent assay and colloidal gold immunochromatographic method.Sudan I derivate was synthesized via diazotization measures, and Sudan I-gelatin immunogen was obtained by mixed acid ahhdride method to couple Sudan I derivative with gelatin. The immunoantigen was injected into new Zealand white rabbits to prepare the anti-Sudan I polyclonal antibodies. The hyperimmune serums were collected when the immune programme was finished, and purified via octanoicacid ammoniumsulfate precipitation and affinity chromatography. Finally, the specific anti-Sudan I antibodies were obtained.An indirect ELISA was applied to test the titer and affinity of Sudan I antibodies.Results showed the titer of antibody was 1: 32 000. The limit detection was 1.0 jig/L and IC50 was 15.0 jig/L by the inhibition ratio curve, which proved the antibodies have high affinity. The recoveries were among 85.88%-97.83%, and coefficients of variation were from 4.9% to 7.4%. The cross-reactivity for Sudan II, III, IV were 2.7%, 6.5% and 4.1%, respectively.During the process of immunochromatographic (IC) strip, the reaction was based on the nitrocellulose (NC) membrane, and the Sudan I antibody and anti-BSA antibody were immobilised on the NC membrane as the test reagent and control reagent, respectively. The goId-labled Sudan I antigen was coated on the glassfiber. With the help of chromatograph affect, the possible Sudan I in samples and the go Id-labeled Sudan I-BSA conjugate would competitively capture the immobilised antibody. The more Sudan I present in the samples, the more effective competition reaction with gold-labeled conjugate occurred. The limit detection was found to be 10 fig/kg in chili powder by optimizing the concentrations of anti-Sudan I antibodies and anti-BSA antibodies. Quantitative analysis of Sudan I could be achieved between 5.0 fig/kg and 20.0 jig/kg. Low cross reactivity was found among other Sudan dyes. Compared with HPLC methods, the results showed a good correlation with a square of correlation coefficient of 0.941. The analysis was high performance, concenient and could be completed in shorter time, suitable for rapid detect Sudan I residue in food and preliminary screening detections for abundant samples.更多还原