大田软海绵酸免疫亲和柱的开发及初步应用

发布时间：2018-08-11 20:11

【摘要】：大田软海绵酸(Okadaic acid,OA)是由一些赤潮甲藻类产生的，其毒性对贝类没有什么影响，但如果人类误食了被OA污染的贝类海鲜，常引起腹泻、恶心、呕吐等腹泻性症状，OA虽无强烈的急性毒性，但却有致癌和免疫毒性作用，人类的健康问题受到严重威胁，因此，对该毒素的预防和控制具有重要的公共卫生学意义。在毒素的液相色谱、免疫学等检测方法中，都需要对毒素进行有效的提取，进而真实反应海产食品中的毒素的含量，从而确保食用者的安全，因此建立一种高效、方便、易于操作的样品前处理手段就显得尤为重要。免疫亲和柱作为一种常用的应用于各种检测分析中的前处理手段，具有高效、方便、特异性强等优点，而ELISA方法具有操作简单、结果相对稳定、成本较低等优点，广泛应用于多种食品污染物的快速检测。本研究在获得OA单克隆抗体杂交瘤细胞株的前期工作基础上，自制免疫亲和柱，用来对加标海产样品进行前处理，通过间接竞争ELISA方法，定量分析层析柱的回收率以及应用效果。本论文复苏特异性分泌抗OA的杂交瘤细胞株，通过体内诱生法大量制备小鼠腹水，并运用Protein G亲和层析柱对腹水进行纯化，成功制备出纯度较高的OA单克隆抗体，抗体的效价为2.56×106。利用制备的单克隆抗体与CNBr活化的Sepharose4B进行偶联，偶联率可达到93.4%，成功制备了OA免疫亲和柱，并对其进行条件优化，最终确定上样缓冲液为含0.2mol/L NaCl的PBS(pH=7.4)，上样缓冲液的体积为1.5mL，洗涤液为甲醇：水（v/v：1/20），洗脱液为甲醇：水（v/v：9/10），洗脱液体积是1mL。利用制备的免疫亲和柱对贝类样品进行加标回收试验，利用间接竞争ELISA方法检测回收率达90.3%，且应用HPLC-MS/MS方法验证，得到了相似的结果。进一步对10种贝类样品初步进行了OA含量的测定，结果显示7种样品OA阳性，其中栉孔扇贝中OA含量最高为33.91ng/g，毛蚶次之，，含量为11.45ng/g，其他样品OA含量均小于10ng/g。免疫亲和柱的成功制备，可有效富集样品中OA含量，减少基质的影响，从而提高检测准确率，为监测OA污染情况以及建立OA检测方法，提供了高效的前处理工具。
[Abstract]:Okadaic acidinic acid (OA) is produced by some red tide algaes, and its toxicity has little effect on shellfish. However, if humans miseat shellfish contaminated by OA, they often cause diarrhea and nausea. Although diarrhea symptoms such as vomiting have no acute toxicity, but they have carcinogenic and immunotoxic effects, human health problems are seriously threatened. Therefore, the prevention and control of the toxin has important public health significance. In the detection of toxins by liquid chromatography, immunology and other methods, it is necessary to extract the toxins effectively, so as to truly reflect the content of toxins in seafood, thus ensuring the safety of food users, so as to establish a high efficiency and convenience. Easy-to-operate sample pretreatment is particularly important. As a common pre-processing method used in various detection and analysis, the immuno-affinity column has the advantages of high efficiency, convenience and specificity, while the ELISA method has the advantages of simple operation, relatively stable results, low cost and so on. Widely used in the rapid detection of various food pollutants. In this study, on the basis of the preliminary work of obtaining hybridoma cell lines of monoclonal antibody to OA, a self-made immuno-affinity column was developed to pretreat the labeled marine samples and to compete indirectly with ELISA method. The recovery rate and application effect of chromatographic column were quantitatively analyzed. In this paper, hybridoma cell lines that specifically secrete anti-OA were resuscitated, mouse ascites were prepared by in vivo induction method, and ascites were purified by Protein G affinity chromatography, and high purity OA monoclonal antibodies were successfully prepared. The titer of antibody was 2.56 脳 106. The coupling rate of the prepared monoclonal antibody to the Sepharose4B activated by CNBr was 93.44.The OA immuno-affinity column was successfully prepared and the conditions were optimized. Finally, the sample buffer was determined to be PBS (pH=7.4) containing 0.2mol/L NaCl, the volume of sample buffer was 1.5 mL, the washing solution was methanol: water (v/v:1/20), the eluant was methanol: water (v/v:9/10), and the volume of eluant was 1 mL. The standard recovery test of shellfish samples was carried out by using the prepared immuno-affinity column. The recovery rate of indirect competitive ELISA method was 90.30.The results were verified by HPLC-MS/MS method and the similar results were obtained. The OA content of 10 kinds of shellfish samples was determined. The results showed that OA was positive in 7 samples. The OA content of chlamys farreri was 33.91 ng / g, the highest OA content was 33.91 ng / g in chlamys farreri, the next was 11.45 ng / g, and the OA content of other samples was less than 10 ng / g. The successful preparation of the immunoaffinity column can effectively enrich the OA content in the sample, reduce the influence of matrix, improve the detection accuracy, and provide an efficient pre-processing tool for monitoring the OA contamination and establishing the OA detection method.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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