抗弓形虫单克隆抗体的制备及夹心ELISA方法的建立

发布时间：2018-05-25 18:41

本文选题：弓形虫 + 单克隆抗体　；参考：《河南农业大学》2009年硕士论文

【摘要】：为建立弓形虫病标准、准确、快速的免疫诊断体系及亚单位疫苗的研制,为弓形虫抗原的分离和纯化及探索弓形虫的保护性免疫等研究提供适宜的工具,本研究利用经淋巴细胞分离液密度梯度离心、微孔滤膜过滤、多次低速离心提纯的弓形虫速殖子,再通过反复冻融和超声波处理后作为抗原免疫Balb/c小鼠6次,获得针对弓形虫速殖子的抗体。间接ELISA检测结果显示,6免后抗体效价均达到1：6400以上。本次建立的间接ELISA方法,其主要条件如下：0.5μg·mL-1的弓形虫速殖子为包被抗原,1%牛血清白蛋白为封闭剂,1：5000稀释HRP标记羊抗鼠IgG作为二抗,TMB(3,3,5,5-四甲基联苯)作为底物。实验证明,该方法特异性强,有较好的重复性。用该法对弓形虫速殖子免疫小鼠的血清抗体及其杂交瘤细胞单抗上清进行检测,获得满意的效果。以PEG2000为促融剂,取效价高的免疫小鼠脾细胞与NSO骨髓瘤细胞融合。经间接ELISA方法筛选阳性孔,有限稀释法进行3-4次克隆和亚克隆,获得4株能稳定分泌抗弓形虫单克隆抗体的杂交瘤细胞株(3A3、3G3、4F4、4G3),1株单抗腹水(3A3)。经鉴定,4株杂交瘤细胞株的上清效价分别在1：800-12800之间,1株腹水(3A3)效价在1：51200,且稳定性良好,单抗亚类为IgG1,与猪等孢子球虫、猪隐孢子虫、猪旋毛虫、猪蛔虫等无特异性。采用免疫荧光法对腹水进行初步鉴定,发现均为抗速殖子单抗。单抗的腹水提纯后进行SDS-PAGE和Westen-blotting分析,单抗的分子量均约为45kDa。用3A3A5单抗包板酶标板与猪抗弓形虫抗体建立夹心ELISA方法,单抗的最佳稀释度为1：800,猪抗弓形虫高免血清的最佳稀释度为1：100-200。特异性试验表明：该方法对于同属于孢子虫属的猪等孢子球虫、猪隐孢子虫、猪旋毛虫、猪蛔虫有较高的识别力,其OD450nm值则显著偏低。该方法不受其它外界因素的影响,灵敏度高,特异性强,对于临床诊断和公共卫生学将是一种简便、可靠的检测方法,为下一步建立弓形虫病快速诊断体系奠定了基础。
[Abstract]:In order to establish the standard of Toxoplasma gondii disease, accurate and rapid immune diagnosis system and the development of subunit vaccine, to provide a suitable tool for the isolation and purification of Toxoplasma antigen and the exploration of protective immunity of Toxoplasma gondii. In this study, Toxoplasma gondii Tachyzoites were purified by density gradient centrifugation, microporous membrane filtration and low speed centrifugation. Balb/c mice were immunized with Toxoplasma gondii Tachyzoites as antigens after repeated freezing and thawing and ultrasonic wave treatment. Antibody against Toxoplasma gondii tachyzoites was obtained. The results of indirect ELISA showed that the titers of antibodies were above 1: 6400. The main conditions of the indirect ELISA method were as follows: Toxoplasma gondii Tachyzoites of 0.5 渭 g / mL-1 were coated with 1% bovine serum albumin (BSA) as an encapsulated antigen, and 1: 5000 diluted HRP labeled sheep anti-mouse IgG as the second antibody TMB3 / 3 (5 / 5) -tetramethyl biphenyls (TMB) as the substrate of Toxoplasma gondii Toxoplasma gondii Tachyzoites (Toxoplasma gondii). Experimental results show that the method is specific and reproducible. The serum antibody and its monoclonal antibody supernatant of mice immunized with Toxoplasma gondii tachyzoites were determined by this method and satisfactory results were obtained. The spleen cells of immunized mice with high titer were fused with NSO myeloma cells using PEG2000 as a fusion agent. Four hybridoma cell lines capable of stably secreting monoclonal antibodies against Toxoplasma gondii were obtained by indirect ELISA screening and limited dilution method for 3-4 times and subclones, and 4 hybridoma cell lines with stable secretion of monoclonal antibodies against Toxoplasma gondii (Toxoplasma gondii) were obtained. The supernatant titers of four hybridoma cell lines were from 1: 800-12800 to 1: 800-12800, respectively. The titer of ascitic cell strain 3A3 was 1: 51200.The Monoclonal subclass was IgG1, which had no specificity with Isosporidium, Cryptosporidium suis, Trichinella spiralis and Ascaris suis. Immunofluorescence assay was used to identify ascites and all of them were identified as anti-tachyzoite monoclonal antibodies. SDS-PAGE and Westen-blotting analysis showed that the molecular weight of McAb was about 45kDa. The sandwich ELISA method was established by using 3A3A5 McAb envelope plate and porcine anti-toxoplasma antibody. The optimal dilution of the McAb was 1: 800, and the optimal dilution of porcine anti-Toxoplasma high immunity serum was 1: 100-200. The specificity test showed that the method had high recognition ability for isosporidium, cryptosporidium, trichinella and Ascaris suis belonging to the same genus of sporidium, and its OD450nm value was significantly lower. This method is not affected by other external factors and has high sensitivity and specificity. It will be a simple and reliable method for clinical diagnosis and public health. It will lay a foundation for establishing a rapid diagnosis system for Toxoplasma gondii in the next step.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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