抗瓜氨酸化聚合兔IgG抗体ELISA法的建立及初步应用  

Establishment and preliminary application of an ELISA method for detecting serum anti-citrullinated aggregated rabbit IgG antibody

作　　者：李利平[1,2] 王洁[1,2] 张敏杰[2] 虞伟[2] 李晓军[2] 

机构地区：[1]南京师范大学生命科学学院,南京210046 [2]南京军区南京总医院解放军临床检验医学研究所,南京210002

出　　处：《临床检验杂志》2014年第5期345-349,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81071419);江苏省科技计划-社会发展项目(BE2011786);江苏省"六大人才高峰"项目(内科卫生D203);江苏省生物材料与器件重点实验室开放研究基金(2010LBMD01)

摘　　要：目的建立检测血清抗瓜氨酸化聚合兔IgG(citrullinated aggregated rabbit IgG,Cit-AgRIgG)抗体的ELISA方法,探讨其对类风湿关节炎(RA)的诊断价值。方法将兔IgG于62℃加热聚合变性,用肽酰精氨酸脱亚胺酶(PAD)将内含精氨酸催化生成瓜氨酸,制成瓜氨酸化聚合兔IgG(Cit-AgRIgG)。以此作为抗原,包被反应板微孔,建立检测抗Cit-AgRIgG抗体的ELISA方法。经方法学考核后,检测临床标本并对结果进行评价。结果 Cit-AgRIgG显示出与抗环瓜氨酸肽(CCP)抗体有较高的结合活性。以其为包被抗原建立的检测抗Cit-AgRIgG抗体的ELISA法批内和批间平均变异系数分别为4.5%和9.5%。特异性阻断试验显示,Cit-AgRIgG、AgRIgG与CCP抗原对该抗体的最高抑制率分别为80.0%、65.9%和25.0%。抗Cit-AgRIgG与抗AgRIgG抗体的cut off值分别为0.565和0.459,此时RA患者抗Cit-AgRIgG抗体阳性率显著高于其他疾病和健康人对照组(P均<0.01),其诊断RA的敏感性为79.3%,特异性为96.5%,阳性、阴性预测值分别为84.7%和95.1%,阳性、阴性似然比分别为22.66、0.215,Youden指数为0.758,ROC曲线下面积(AUCROC)为0.890。对RA和健康人对照者检查结果显示,抗CitAgRIgG与抗AgRIgG抗体(RF)总符合率97.7%,与抗CCP抗体总符合率91.7%。结论建立的检测抗Cit-AgRIgG抗体的ELISA法有良好的稳定性和特异性。抗Cit-AgRIgG抗体兼具RF与抗CCP抗体的双重特性,有可能发展成为RA疾病诊断和病情监测的一种新的参考指标。Objective To establish an ELISA method for detecting serum anti-citrullinated aggregated rabbit IgG（ Cit-AgRIgG） antibody,and evaluate its value in the diagnosis of rheumatoid arthritis（ RA）. Methods First,the rabbit IgG solution was warmed up to 62 ℃ for aggregated denaturation to form AgRIgG,and then the arginine in AgRIgG was catalyzed into citrulline by peptidyl arginine deiminase（ PAD） to form Cit-AgRIgG. Using Cit-AgRIgG as coated antigen,an ELISA method was established for detecting anti-CitAgRIgG antibody. Last,the established method was evaluated and used to detect clinical serum samples,and the obtained results were analyzed. Results The Cit-AgRIgG could bind highly with anti-cyclocitrulline peptide（ CCP） antibody. The mean intra-assay and inter-assay coefficients of variation of the established ELISA method for detecting anti-Cit-AgRIgG antibody were 4. 5% and 9. 5%,respectively. The specific inhibitory test showed that the maximal inhibitory rates of Cit-AgRIgG,AgRIgG and CCP antigen on the antiCit-AgRIgG antibody were 80. 0%,65. 9% and 25. 0%,respectively. The cut off values for detecting anti-Cit-AgRIgG and antiAgRIgG antibodies were 0. 565 and 0. 459,respectively. When the established ELISA method was used to detect clinical serum samples from the patients with RA or other diseases and healthy controls,its sensitivity and specificity for the diagnosis of RA were 79. 3% and 96. 5%,respectively,its positive and negative predictive values were 84. 7% and 95. 1%,respectively,its positive and negative likelihood ratios were 22. 66 and 0. 215,respectively,and the Youden index and the area under the curve（ AUCROC） were 0. 758 and 0. 890,respectively. Moreover,the positive rate of anti-Cit-AgRIgG antibody in RA patients was significantly higher than those in the patients with other diseases and healthy controls（ P〈 0. 01）. The total coincidence rates between anti-Cit-AgRIgG antibody and antiAgRIgG antibody and between anti-Cit-AgRIgG antibody and anti-CCP antibody were 97. 7% an

关 键 词：类风湿因子 聚合兔IgG 瓜氨酸化 类风湿关节炎 酶联免疫吸附试验 

分 类 号：R563.1[医药卫生—呼吸系统]