德国小蠊变应原Bla g 2单克隆抗体的制备与鉴定

发布时间：2018-01-12 13:10

本文关键词：德国小蠊变应原Bla g 2单克隆抗体的制备与鉴定　出处：《中国寄生虫学与寄生虫病杂志》2017年04期 　论文类型：期刊论文

【摘要】：目的制备德国小蠊(Blattella germanica)变应原Bla g 2的单克隆抗体并鉴定其特异性。方法以纯化的0.2 mg/ml Bla g 2蛋白溶液为抗原免疫雌性BLAB/c小鼠6只,免疫4次,每次间隔1周。免疫后第5天取小鼠尾静脉血,制备小鼠血清,第6天加强免疫1次,第7天处死小鼠取脾脏,制备脾细胞悬液。测定小鼠抗血清滴度,运用杂交瘤技术将免疫脾细胞与骨髓瘤细胞融合,采用有限稀释法对阳性孔杂交瘤细胞进行克隆化。蛋白质印迹(Western blotting)检测单克隆抗体特异性,间接ELISA测定单克隆抗体效价,双抗夹心ELISA鉴定单克隆抗体的亚型。结果在小鼠血清稀释至1∶16 000时A450值仍0.100,判断为免疫成功。通过杂交瘤技术获得2株能稳定分泌Bla g 2单克隆抗体的杂交瘤细胞,标记为Bla g 2-1、Bla g 2-2。Western blotting结果表明,Bla g 2-1和Bla g 2-2单克隆抗体对Bla g 2蛋白均具有高度特异性。间接ELISA测得2株单克隆抗体的效价分别为Bla g 2-1(1∶16 000)、Bla g 2-2(1∶8 000)。双抗夹心ELISA结果表明单克隆抗体蛋白亚类均为免疫球蛋白G1(IgG1)。结论制备的德国小蠊变应原单克隆抗体Bla g 2能与Bla g 2蛋白发生特异性结合。
[Abstract]:Objective to prepare Blattella germanica from Blattella germanica. Methods six female BLAB/c mice were immunized with purified 0.2 mg/ml Bla g2 protein solution as antigen. The mice were immunized for 4 times each time for 1 week. The blood samples were taken from the tail vein of the mice on the 5th day after immunization to prepare the serum of mice. The mice were immunized once on the 6th day and the mice were killed on the 7th day to get the spleen. Spleen cell suspension was prepared. The titer of mouse antiserum was measured and the immune spleen cells were fused with myeloma cells by hybridoma technique. Western blotting was used to detect the specificity of monoclonal antibody. Indirect ELISA was used to determine the titer of monoclonal antibody and double antibody sandwich ELISA was used to identify the subtype of monoclonal antibody. Results the A450 value was still 0.100 when the mouse serum was diluted to 1:16. Two hybridoma cells stably secreting Bla g2 monoclonal antibody were obtained by hybridoma technique and labeled as Bla g2-1. The results of Bla g 2-2.Western blotting showed that. Bla g2-1 and Bla g2-2 monoclonal antibodies were highly specific to Bla g2 protein. The titers of the two monoclonal antibodies detected by indirect ELISA were Bla, respectively. G 2-1 (. 1:16. Bla g _ 2-2: 1: 8 000. The results of double antibody sandwich ELISA showed that monoclonal antibody protein subclasses were immunoglobulin G _ (1) G _ (1) and G _ (1) G _ (1). Conclusion Bla g 2, a monoclonal antibody against Blattella germanica allergen, can bind specifically to Bla g 2 protein.
【作者单位】：浙江省舟山医院检验中心;南京医科大学第一附属医院临床实验室;锦州医科大学变态反应学与临床免疫学研究中心;
【基金】：国家科学自然基金(No.30972714)~~
【分类号】：R384
【正文快照】： 过敏性疾病是临床上的常见病和多发病,并有公司。辣根过氧化物酶化学发光(ECL)试剂盒购逐年增多趋势,引起人们的广泛关注[1]。德国小蠊自美国Pierce公司。其他试剂均为国产分析纯。酶(Blattella germanica)是蜚蠊的一种,属于节肢动标仪(iM ark)、凝胶成像系统(ChemiD oc XRS)

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