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目的制备甲型副伤寒沙门菌菌体抗原单克隆抗体,并对其进行鉴定及初步应用。方法用甲醛灭活的甲型副伤寒沙门菌免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合,筛选出稳定分泌甲型副伤寒沙门菌特异性单克隆抗体的杂交瘤细胞株,对克隆纯化后的阳性细胞扩大培养后,免疫BALB/c小鼠,收获腹水,间接ELISA法检测抗体效价,并进行Ig类和亚类鉴定;腹水经辛酸-硫酸铵盐析法纯化后,采用间接ELISA法、玻片凝集试验、SDSPAGE法、免疫扩散试验进行检测。将纯化后的2株单克隆抗体用于双抗体夹心ELISA法的建立,并用自制配对抗体检测甲型副伤寒患者血浆、乙型副伤寒患者血浆、伤寒患者血浆和正常人血浆。结果共筛选出2株持续分泌单克隆抗体的杂交瘤细胞株,命名为1H4和2A9,腹水抗体效价为1∶10~7和1∶10~6,分别为IgG_1和Ig G_3亚类,轻链均为κ型。纯化后的单克隆抗体1H4、2A9效价分别为1∶10~7和1∶10~3,1H4纯度大于85%,2A9纯度小于60%。2株单克隆抗体与甲型副伤寒沙门菌50503、50973可产生凝集反应,与伤寒沙门菌50096不产生反应;1H4在稀释度为1∶8时可与甲型副伤寒沙门菌菌体特异多糖(organism specific polysaccharide,OSP)产生凝集反应,而2A9在每个稀释度与OSP均不发生反应。将1H4和2A9用于双抗体夹心ELISA检测法建立,确定1H4/HRP-2A9的组合为最佳配对,用该配对抗体检测阳性样本检出率为100%,正常人血浆样本检出率为0。结论获得2株甲型副伤寒沙门菌菌体抗原单克隆抗体,可用于甲型副伤寒沙门菌的快速鉴定。
Objective To prepare the monoclonal antibody against Salmonella paratyphi A, and to identify and apply it. Methods BALB / c mice were immunized with formaldehyde-inactivated Salmonella paratyphi A. The spleen cells were fused with Sp2 / 0 myeloma cells and the hybridoma cells secreting monoclonal antibodies against Salmonella paratyphi A BALB / c mice were immunized with BALB / c mice, and the antibody titer was detected by indirect ELISA. Ig and subclasses were also identified. Ascites was purified by octanoic acid-ammonium sulfate salting-out After indirect ELISA method, slide agglutination test, SDSPAGE method, immune spread test. The purified monoclonal antibodies were used to establish double-antibody sandwich ELISA and plasma of patients with Paratyphus A and PaCO2, plasma of patients with typhoid and plasma of normal people were detected by self-made paired antibodies. Results Two hybridoma cell lines secreting monoclonal antibodies were screened out and named as 1H4 and 2A9. The antibody titers of ascites were 1:10 ~ 7 and 1:10 ~ 6, which were IgG_1 and Ig G_3 subtypes, respectively, which were light The chains are all κ. The purified monoclonal antibodies 1H4, 2A9 titers were 1:10 ~ 7 and 1:10 ~ 3, respectively. The purity of 4H4 was more than 85% and the purity of 2A9 was less than 60%. Two monoclonal antibodies and Salmonella paratyphi A 50503,50973 can produce agglutination reaction, and Salmonella typhimurium 50096 does not produce response; 1H4 at a dilution of 1: 8 with Salmonella paratyphi A-specific polysaccharide (OSP) produced agglutination, whereas 2A9 did not react with OSP at each dilution. 1H4 and 2A9 were used to establish a double-antibody sandwich ELISA assay, and the optimal combination of 1H4 / HRP-2A9 was determined. The detection rate of the positive sample with this paired antibody was 100%, and the detection rate of the normal plasma sample was 0 . Conclusion Two monoclonal antibodies against Salmonella paratyphi A were obtained and could be used for the rapid identification of Salmonella paratyphi A.