甲型副伤寒沙门菌菌体抗原单克隆抗体的制备及其初步应用

Preparation and preliminary application of monoclonal antibody against O-antigen of Salmonella paratyphi A

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, identify and preliminarily monoclonal antibody（Mc Ab） against Salmonella paratyphi A. Methods BALB/c mice were immunized with S. paratyphi A inactivated with formaldehyde, of which the spleen cells were fused with Sp2/0 myelomas cells. The hybridoma cell strains stably secreting Mc Ab against S. paratyphi A were screened and purified, with which BALB/c mice were immunized. The ascites of immunized mice was collected,determined for antibody titer by indirect ELISA, and identified for class and subclass of Ig. The ascites was further purified by salting out with caprylic acid-ammonium sulphate, then subjected to indirect ELISA, slide agglutination test,SDS-PAGE and immunodiffusion test. The purified Mc Abs secreted by two hybridoma cell strains were used for development of a double antibody sandwich ELISA for determination of plasma from patients with paratyphoid A,paratyphoid B and typhoid as well as normal human plasma. Results Two hybridoma cell strains stably secreting Mc Ab were obtained and named as 1H4 and 2A9 respectively. The secreted Mc Abs, at titers of 1 ∶ 10~7 and 1 ∶ 10~6, were Ig G_1 and IgG_3 respectively, both of which were of κ chain. The titers of purified Mc Abs 1H4 and 2A9 were 1 ∶ 10~7 and 1 ∶ 10~3,while the purities were more than 85% and less than 60%, respectively. Both the Mc Abs showed agglutination with S. paratyphi A 50503 and 50973 while no agglutination with S. typhi 50096. Mc Ab 1H4 at a dilution of 1 ∶ 8 showed agglutination with organism specific protein（OSP） of S. paratyphi A. However, Mc Ab 2A9 at any dilution showed no agglutination with the OSP. The combination 1H4/HRP-2A9 was optimal to the development of double antibody sandwich ELISA, by which the positive rate of positive samples was 100% while that of normal human plasma was 0. Conclusion The prepared Mc Abs may be used for the rapid identification of S. paratyphi A.