小鼠脾内免疫法制备重组弓形虫SAG1抗原单克隆抗体

Preparation of mouse anti-recombinant SAG1 antigen monoclonal antibody of Toxoplasma gondii by intrasplenic immunization

目的制备小鼠抗重组弓形虫SAG1抗原单克隆抗体,用于早期弓形虫感染的抗原检测。方法用弓形虫RH株重组抗原SAG1进行常规免疫结合小鼠脾内免疫,杂交瘤技术制备单克隆抗体。ELISA法筛选阳性克隆,经亚克隆建株。诱生腹水法制备抗体,protein-G亲合层析法进行纯化,测定其亚类、效价;Westernblot法分析其特异性;夹心-ELISA法检测抗体的敏感性及特异性;检测弓形虫感染小鼠血液循环抗原,同时用PCR法检测弓形虫B1基因并比较结果。结果获得2株抗重组弓形虫SAG1抗原单克隆抗体3B6、10C4,抗体均为IgG1,轻链均为κ链,Western blot显示2株单抗均能识别弓形虫天然的和重组的SAG1抗原。3B6、10C4敏感度分别为31.3ng和62.5ng,与血吸虫病、钩虫病及疟疾患者血清均无交叉反应。弓形虫早期感染检测PCR及ELISA法阳性检出率分别为63.2%、47.4%。结论成功制备小鼠抗重组弓形虫SAG1抗原单克隆抗体,初步用于早期弓形虫感染诊断。

Objective To prepare monoclonal antibody in mice so as to develop an ELISA method for diagnosis of Toxoplasma gondii infection during the initial stage. Methods The mice were immunized by combining routine and intrasplenic immunization with recombinant SGA1 antigen. B lymphocyte hybridization technique was applied to prepare the anti-SAG1 McAbs. Positive clones were screened using ELISA and subcloned to establish cell lines. Ascites was induced to produce the McAbs. Then the McAbs were purified by protein G chromatograph column. The specificity of McAbs was identified by Western blot and sandwich-ELISA. Sensitivity of the McAbs was determined using sandwich-ELISA. Comparasion was carried out between PCR and sandwich-ELISA method. Results Two positive clones were obtained and named as 3B6, 10C4, both could identify the native and recombinant SAG1 antigens. The sensitivity of 3B6, 10C4 was 31.3 ng and 62.5 ng, respectively. There was no cross reaction between the McAbs and positive sera from patients with schistosomiasis, ancylostomiasis or malaria. By using PCR and ELISA, the positive infection rate of T. gondii was 63.2% and 47.4%, respectively. Conclusions Therefore, mouse anti-rSAG1 antigen McAbs have been prepared successfully and primarily applied to early stage diagnosis of T. gondii infection.