重组日本血吸虫谷胱甘肽-硫-转移酶单克隆抗体在急性血吸虫病临床诊断中的应用  

Immunodiagnosis of acute schistosomiasis using glutathione-S-transferase recombinant protein of Schistosoma japonicum monoclonal antibody

作　　者：王细宏[1] 罗飞[2] 沈继龙[2] 

机构地区：[1]安徽医科大学,安徽合肥230022 [2]教育部重要遗传病基因资源利用重点实验室,安徽合肥230022

出　　处：《检验医学》2008年第6期578-581,共4页Laboratory Medicine

基　　金：国家高技术研究发展计划863项目(2004AA2Z3570)

摘　　要：目的研究重组抗原26 ku日本血吸虫谷胱甘肽-硫-转移酶(SjGST)及其单克隆抗体在血吸虫患者分子诊断中的应用。方法用已纯化的重组SjGST抗原免疫近交系(BALB/c)小鼠,数周后取免疫鼠脾细胞,与SP2/0骨髓瘤细胞进行细胞融合,选择性培养基筛选出杂交瘤细胞,经有限稀释法克隆化,扩大培养制备针对重组SjGST抗原的单克隆抗体细胞株。亲和层析法纯化单克隆抗体。酶联免疫吸附试验(ELISA)检测急性血吸虫病患者和非流行区正常人血清。结果成功获得重组蛋白26 ku SjGST,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示与预期相对分子质量大小相符的蛋白条带。成功获得能够长期分泌抗重组SjGST单克隆抗体的杂交瘤细胞株,ELISA与蛋白质印迹试验均显示杂交瘤细胞培养上清中含有重组SjGST抗体。ELISA检测患者血清结果表明,26 ku SjGST用于急性血吸虫病患者血清中抗体检测的阳性率为90.6%,26 ku SjGST抗体用于急性血吸虫病患者血清中循环抗原检测的阳性率为59.4%。结论该重组蛋白用于急性血吸虫病患者血清中抗体的检测具有良好的免疫反应性,26 ku SjGST抗体对循环抗原的检测可作为抗体检测的有效补充。Objective To evaluate the value of 26 ku glutathione-S-trausferase recombinant protein of Schistosoma japonicum（SjGST） and anti-recombinant SjGST monoclonal antibody in molecular diagnosis for schistosomiasis. Methods BALB/c mice were immunized by the purified antigen of recombinant SjGST for five or six times. HGPRT - SP2/ 0 was fused with mice spleen B lymphoeytes. The hybridoma cell was cultured in the HAT selective medium. The positive clone was screened by indirect enzyme-linked immunosorbent assay （ELISA） and cloning by limiting dilution assay. The serum of patients with acute schistosomiasis and normal controls were defected by ELISA. Results There was a special protein band observed between 20.1 to 31.0 ku in sodium dodecy sulfate-polyacrylamide gel. The hybridoma cell and monoclonal antibody against recombinant SjGST was produced. The results of ELISA and Westen-blot displayed that there was anti-recombinant SjGST antibady in cell culture supernatant. The positive rates were 90.6% in detecting of circulating antibody and 59.4% in detecting of circulating antigen by ELISA. Conclusions The 26 ku recombinant SjGST can be applied as a diagnostic antigen for acute schistosomiasis and detection of circulating antigen by anti-26 ku recombinant SjGST monoclonal antibody will be useful as supplement combination with detecting of circulating antigen.

关 键 词：谷胱甘肽-硫-转移酶 单克隆抗体 日本血吸虫 免疫学诊断 

分 类 号：R446.62[医药卫生—诊断学]