本文選題：抗體 + 抗獨特型抗體　； 參考：《南京農(nóng)業(yè)大學》2009年碩士論文

【摘要】：微囊藻毒素-LR(Microcystin-LR,簡稱MC-LR)是藍藻產(chǎn)生的一類天然毒素�？茖W家的研究表明,被微囊藻毒素(MC)污染的飲用水和水產(chǎn)品,給人類健康帶來了巨大威脅。目前已知最普遍的、含量相對較多、毒性較大的主要是MC-LR,MC-RR,MC-YR等。人們在洗澡、游泳及其他水上休閑和運動時,皮膚接觸含藻毒素水體可引起敏感部位(如眼睛)和皮膚過敏；少量喝入可引起急性腸胃炎；長期飲用則可能引發(fā)肝癌。為了檢測水體及食品中微囊藻毒素的含量,利用毒素標品對水體及食品中毒素的檢測效果顯著,但檢測成本高且操作危險,尋求一種毒素檢測的替代品成為食品安全檢測的首要問題。 利用多克隆抗體技術(shù)制備抗獨特型抗體方法相對單抗生產(chǎn)技術(shù)簡單,容易操作,但如果需要量大,需要多次制備,同時需要除去抗同種型抗體。主要方法是將已生產(chǎn)出的單抗(Ab1)直接免疫試驗動物,然后提取血清,純化血清即可。本實驗首先利用飽和硫酸銨二步沉淀法初步純化兔抗微囊藻毒素多克隆抗體,再用G蛋白親和純化柱進一步純化抗體,通過SDS-PAGE電泳實驗鑒定純化效果顯著,回收率87.57%。 在溫和條件下采用木瓜蛋白酶酶切純化后的兔抗微囊藻毒素多克隆抗體,利用DEAE-52離子交換樹脂柱分離抗體酶切片段,獲得其抗體的決定簇F(ab')2片段,并對該片段的免疫活性及產(chǎn)率進行測定。結(jié)果顯示該片段免疫活性降低50%為1：12000但仍然可以作為抗原制備抗獨特型抗體。然后利用該抗體片段作為新的免疫原,按照常規(guī)免疫方法免疫異源受體BalB/c小鼠,獲得微囊藻毒素抗獨特型抗體。 同樣利用飽和硫酸銨二步沉淀法和蛋白G親和純化柱對新合成抗體進行純化,并檢測抗體純化效果及濃度。通過SDS-PAGE電泳實驗鑒定純化效果顯著,利用ELISA檢測技術(shù)分別對新合成抗體進行效價測定、特異性測定及最佳工作濃度測定。結(jié)果顯示：IgG濃度為27.1mg/ml,最佳工作濃度測定顯示抗原包被濃度為0.25PPM,抗體工作濃度為1：1600。
[Abstract]:-LR (Microcystin-LR, MC-LR) is a kind of natural toxin produced by cyanobacteria. Scientists have shown that drinking water and aquatic products contaminated by microcystin (MC) have brought great threat to human health. At present, the most commonly known, relatively more toxic, mainly MC-LR, MC-RR, MC-YR and so on. People are washing. The skin contact with algal toxin water can cause sensitive areas (such as eyes) and skin allergies during swimming and other water leisure and exercise. A small amount of drinking can cause acute gastroenteritis; long-term drinking may lead to liver cancer. In order to detect the content of microcystins in water and food, toxins are used to detect toxins in water and food. The detection effect is significant, but the detection cost is high and the operation is dangerous. Searching for a substitute for toxin detection becomes the primary problem of food safety detection.
The method of producing anti idiotypic antibody by polyclonal antibody technique is simple and easy to operate, but it needs to be prepared many times and need to remove anti homoantibody. The main method is to immunization the produced monoclonal antibody (Ab1) directly to the animal, then extract the serum and purify the serum. First, the polyclonal antibody of Rabbit anti microcystin was purified by two step precipitation method with saturated ammonium sulfate, then the antibody was further purified by the affinity purification column of G protein. The purification effect of the purified antibody was identified by SDS-PAGE electrophoresis, and the recovery rate was 87.57%.
The Rabbit anti microcystin polyclonal antibody was purified with papain under mild conditions. The antibody fragment was isolated by DEAE-52 ion exchange resin column and the antibody determinant F (ab') 2 fragment was obtained. The immunological activity and yield of the fragment were determined. The results showed that the immune activity of the fragment was 50% 1:1200. 0 but still can be used as an antigen to prepare anti idiotypic antibodies, and then use the antibody fragment as a new immunogen to immunization of the heterologous receptor BalB/c mice in accordance with the routine immunization method and obtain the anti idiotypic antibody of microcystin.
The two step precipitation method of saturated ammonium sulfate and protein G affinity purification column were used to purify the new synthetic antibody, and the effect and concentration of the antibody were detected. The effect of purification was identified by SDS-PAGE electrophoresis. The titer, specificity and optimum working concentration of the new synthetic antibody were measured by ELISA detection technique. The results showed that the concentration of IgG was 27.1mg/ml, and the best concentration of work showed that the antigen concentration was 0.25PPM and the antibody concentration was 1:1600.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 何鳳田,陳寶軍,喬太東,韓者藝,聶勇戰(zhàn),宋保華,樊代明;噬菌體呈現(xiàn)技術(shù)制備結(jié)腸癌抗獨特型抗體[J];中國生物化學與分子生物學報;2001年06期

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