抗组蛋白H_(2)A抗体间接ELISA法的建立和临床初步应用  

Establishment and clinical preliminary application of indirect ELISA detection method for anti-H_(2) A protein antibody

作　　者：王海永 徐敏 李晓军 WANG Haiyong;XU Min;LI Xiaojun(Department of Clinical Laboratory,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210002,Jiangsu;Institute of Clinical Laboratory Medicine,Eastem Theater General Hospital,Nanjing 210002,Jiangsu,China)

机构地区：[1]南京中医药大学附属医院检验科,南京210002 [2]东部战区总医院中心实验科,南京210002

出　　处：《临床检验杂志》2021年第10期744-747,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81470071);国家临床重点专科军队建设项目(2014ZDZK003);江苏省临床医学科技专项(BL2014072)。

摘　　要：目的建立检测血清抗H_(2)A抗体的间接ELISA技术,探讨抗H_(2)A抗体对类风湿关节炎(RA)的临床意义。方法RT-PCR扩增H_(2)A基因,将其克隆到原核表达载体pET28a,对重组质粒H_(2)A/pET28a进行原核表达,经Western blot检测后,利用H_(2)A重组蛋白建立检测人血清中抗H_(2)A抗体的间接ELISA方法。用建立的间接ELISA方法检测50例RA患者和50例健康人对照组血清抗H_(2)A抗体水平。结果表达的H_(2)A蛋白相对分子量约为15000,与预期大小相符,western blot结果表明组蛋白H_(2)A与抗H_(2)A抗体阳性血清具有良好的反应原性。经方法学评估,建立的ELISA法在特异性、可重复性方面均达到检测要求。RA组抗H_(2)A抗体水平高于健康人对照组(P<0.01)。当cut-off值设为0.494时,RA患者血清抗H_(2)A抗体的阳性率为84%(42/50),明显高于健康人对照组20%(10/50),差异具有统计学意义(χ^(2)=41.026,P<0.01)。结论成功建立了检测血清抗H_(2)A抗体的间接ELISA技术。RA患者血清中抗H_(2)A抗体水平明显高于健康人对照组。抗H_(2)A抗体在RA患者血清中出现可能具有重要的致病意义。Objective To establish an indirect ELISA method for detecting serum anti-H_(2)A protein antibody,and investigate its clinical significance in rheumatoid arthritis(RA)patients.Methods The H_(2)A segments amplified by RT-PCR were cloned into vector pET28a to perform prokaryotic expression.The indirect ELISA method was established for detection of anti-H_(2)A protein antibody in serum by using the recombinant H_(2)A protein demonstrated with western blot.The levels of serum anti-H_(2)A protein antibody were detected in 50 RA patients and 50 healthy controls by the established method.Results The expressed H_(2)A protein was about 15000 Da,which was consistent with the expected size.Western blot test indicated that the recombinant protein had good reactivity with the anti-H_(2)A protein antibody serum.The specificity and reproducibility of the assay fulfilled the requests of clinical testing by methodological evaluation.The levels of serum anti-H_(2)A protein antibody in RA patients were significant higher than those of healthy controls(P<0.01).When cut-off value for the ELISA was set at 0.494,the positive rate of anti-H_(2)A protein antibody in RA patients(84%,42/50)was significantly higher than that in healthy controls(20%,10/50)with statistically significant difference(χ^(2)=41.026,P<0.01).Conclusion We developed an indirect ELISA method for detecting serum anti-H_(2)A protein antibody.Serum anti-H_(2)A protein antibody levels in RA patients were significantly elevated.Anti-H_(2)A protein antibody in serum of RA patients may play important pathogenic significance.

关 键 词：类风湿关节炎 组蛋白H_(2)A 抗H_(2)A抗体 间接酶联免疫吸附试验 

分 类 号：R446.6[医药卫生—诊断学]