【摘要】：大田軟海綿酸(Okadaic acid,OA)是由一些赤潮甲藻類產(chǎn)生的，其毒性對(duì)貝類沒(méi)有什么影響，但如果人類誤食了被OA污染的貝類海鮮，常引起腹瀉、惡心、嘔吐等腹瀉性癥狀，OA雖無(wú)強(qiáng)烈的急性毒性，但卻有致癌和免疫毒性作用，人類的健康問(wèn)題受到嚴(yán)重威脅，因此，對(duì)該毒素的預(yù)防和控制具有重要的公共衛(wèi)生學(xué)意義。在毒素的液相色譜、免疫學(xué)等檢測(cè)方法中，都需要對(duì)毒素進(jìn)行有效的提取，進(jìn)而真實(shí)反應(yīng)海產(chǎn)食品中的毒素的含量，從而確保食用者的安全，因此建立一種高效、方便、易于操作的樣品前處理手段就顯得尤為重要。 免疫親和柱作為一種常用的應(yīng)用于各種檢測(cè)分析中的前處理手段，具有高效、方便、特異性強(qiáng)等優(yōu)點(diǎn)，而ELISA方法具有操作簡(jiǎn)單、結(jié)果相對(duì)穩(wěn)定、成本較低等優(yōu)點(diǎn)，廣泛應(yīng)用于多種食品污染物的快速檢測(cè)。本研究在獲得OA單克隆抗體雜交瘤細(xì)胞株的前期工作基礎(chǔ)上，自制免疫親和柱，用來(lái)對(duì)加標(biāo)海產(chǎn)樣品進(jìn)行前處理，通過(guò)間接競(jìng)爭(zhēng)ELISA方法，定量分析層析柱的回收率以及應(yīng)用效果。 本論文復(fù)蘇特異性分泌抗OA的雜交瘤細(xì)胞株，通過(guò)體內(nèi)誘生法大量制備小鼠腹水，并運(yùn)用Protein G親和層析柱對(duì)腹水進(jìn)行純化，成功制備出純度較高的OA單克隆抗體，抗體的效價(jià)為2.56×106。利用制備的單克隆抗體與CNBr活化的Sepharose4B進(jìn)行偶聯(lián)，偶聯(lián)率可達(dá)到93.4%，成功制備了OA免疫親和柱，并對(duì)其進(jìn)行條件優(yōu)化，最終確定上樣緩沖液為含0.2mol/L NaCl的PBS(pH=7.4)，上樣緩沖液的體積為1.5mL，洗滌液為甲醇：水（v/v：1/20），洗脫液為甲醇：水（v/v：9/10），洗脫液體積是1mL。利用制備的免疫親和柱對(duì)貝類樣品進(jìn)行加標(biāo)回收試驗(yàn)，利用間接競(jìng)爭(zhēng)ELISA方法檢測(cè)回收率達(dá)90.3%，且應(yīng)用HPLC-MS/MS方法驗(yàn)證，得到了相似的結(jié)果。進(jìn)一步對(duì)10種貝類樣品初步進(jìn)行了OA含量的測(cè)定，結(jié)果顯示7種樣品OA陽(yáng)性，其中櫛孔扇貝中OA含量最高為33.91ng/g，毛蚶次之，，含量為11.45ng/g，其他樣品OA含量均小于10ng/g。免疫親和柱的成功制備，可有效富集樣品中OA含量，減少基質(zhì)的影響，從而提高檢測(cè)準(zhǔn)確率，為監(jiān)測(cè)OA污染情況以及建立OA檢測(cè)方法，提供了高效的前處理工具。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.6

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