微囊藻毒素-LR抗独特型抗体的制备与鉴定

发布时间：2018-06-28 09:49

本文选题：抗体 + 抗独特型抗体　；参考：《南京农业大学》2009年硕士论文

【摘要】：微囊藻毒素-LR(Microcystin-LR,简称MC-LR)是蓝藻产生的一类天然毒素。科学家的研究表明,被微囊藻毒素(MC)污染的饮用水和水产品,给人类健康带来了巨大威胁。目前已知最普遍的、含量相对较多、毒性较大的主要是MC-LR,MC-RR,MC-YR等。人们在洗澡、游泳及其他水上休闲和运动时,皮肤接触含藻毒素水体可引起敏感部位(如眼睛)和皮肤过敏；少量喝入可引起急性肠胃炎；长期饮用则可能引发肝癌。为了检测水体及食品中微囊藻毒素的含量,利用毒素标品对水体及食品中毒素的检测效果显著,但检测成本高且操作危险,寻求一种毒素检测的替代品成为食品安全检测的首要问题。利用多克隆抗体技术制备抗独特型抗体方法相对单抗生产技术简单,容易操作,但如果需要量大,需要多次制备,同时需要除去抗同种型抗体。主要方法是将已生产出的单抗(Ab1)直接免疫试验动物,然后提取血清,纯化血清即可。本实验首先利用饱和硫酸铵二步沉淀法初步纯化兔抗微囊藻毒素多克隆抗体,再用G蛋白亲和纯化柱进一步纯化抗体,通过SDS-PAGE电泳实验鉴定纯化效果显著,回收率87.57%。在温和条件下采用木瓜蛋白酶酶切纯化后的兔抗微囊藻毒素多克隆抗体,利用DEAE-52离子交换树脂柱分离抗体酶切片段,获得其抗体的决定簇F(ab')2片段,并对该片段的免疫活性及产率进行测定。结果显示该片段免疫活性降低50%为1：12000但仍然可以作为抗原制备抗独特型抗体。然后利用该抗体片段作为新的免疫原,按照常规免疫方法免疫异源受体BalB/c小鼠,获得微囊藻毒素抗独特型抗体。同样利用饱和硫酸铵二步沉淀法和蛋白G亲和纯化柱对新合成抗体进行纯化,并检测抗体纯化效果及浓度。通过SDS-PAGE电泳实验鉴定纯化效果显著,利用ELISA检测技术分别对新合成抗体进行效价测定、特异性测定及最佳工作浓度测定。结果显示：IgG浓度为27.1mg/ml,最佳工作浓度测定显示抗原包被浓度为0.25PPM,抗体工作浓度为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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】