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)
目的 建立检测血清抗 H2A 抗体的间接 ELISA 技术,探讨抗 H2A 抗体对类风湿关节炎( RA) 的临床意义。 方法 RT?PCR扩增 H2A 基因,将其克隆到原核表达载体 pET28a,对重组质粒 H2A / pET28a 进行原核表达,经 Western blot 检测后,利 用 H2A 重组蛋白建立检测人血清中抗 H2A 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 H2A 抗体水平。 结果 表达的 H2A 蛋白相对分子量约为 15 000,与预期大小相符,western blot 结果表明组 蛋白 H2A 与抗 H2A 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 H2A 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 H2A 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(χ2 = 41.026, P<0.01)。 结论 成功建立了检测 血清抗 H2A 抗体的间接 ELISA 技术。 RA 患者血清中抗 H2A 抗体水平明显高于健康人对照组。 抗 H2A 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?H2A protein antibody,and investigate its clinical significance in rheumatoid arthritis ( RA) patients. Methods The H2A segments amplified by RT?PCR were cloned into vector pET28a to perform prokaryotic expression. The indirect ELISA method was established for detection of anti?H2A protein antibody in ser? um by using the recombinant H2A protein demonstrated with western blot. The levels of serum anti?H2A protein antibody were detected in 50 RA patients and 50 healthy controls by the established method. Results The expressed H2A protein was about 15 000 Da, which was consistent with the expected size. Western blot test indicated that the recombinant protein had good reactivity with the anti?H2A 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 2A 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?H2A protein antibody in RA patients(84%, 42 / 50) was significantly higher than that in healthy controls(20%, 10 / 50) with statistically significant difference (χ2 = 41.026, P<0.01). Conclu? sion We developed an indirect ELISA method for detecting serum anti?H2A protein antibody. Serum anti?H2A protein antibody levels in RA patients were significantly elevated.Anti?H2A protein antibody in serum of RA patients may play important pathogenic signifi? cance
王海永,徐敏,李晓军.抗组蛋白 H2 A 抗体间接 ELISA 法的建立和临床初步 应用[J].临床检验杂志,2021,39(10):744-747