抗Ⅱ型胶原短肽(260-272)抗体ELISA测定方法的建立与初步应用

Development of ELISA method for anti-typeⅡcollagen peptide(260-272) antibody and its preliminary application

目的了解类风湿关节炎(RA),其他自身免疫病和健康对照组血清抗Ⅱ型胶原短肽,即CⅡ(260-272)抗体水平,探讨其在RA疾病诊断和病情监测中的价值。方法将合成的CⅡ(260-272)短肽与牛血清白蛋白(BSA)耦联得CⅡ(260-272)-BSA复合物,用其作为抗原包被建立抗CⅡ(260-272)抗体的ELISA测定方法,经方法学考核后,对临床病例标本检测结果进行评价。结果建立的抗CⅡ(260-272)抗体的ELISA法实验结果具有较好的稳定性和特异性,其批内和批间变异系数分别为8.4%和9.6%,特异阻断抑制率最高达76.3%。临床检测结果发现抗CⅡ(260-272)抗体在RA中的阳性率为75.0%(30/40),显著高于其他自身免疫病和健康对照组(P<0.01)。以测定吸光度(A)0.213为临界值时,抗CⅡ260-272抗体对RA诊断特异性91.0%,阳性预测值63.8%,阴性预测值94.5%,阳性似然比8.33,阴性似然比0.275,Youden指数0.66。ROC曲线分析结果显示该抗体对RA诊断准确度为中等偏上水平(AUCROC值=0.890)。未发现抗CⅡ(260-272)抗体与类风湿因子(RF)水平相关(在13例RF阴性RA患者中,该抗体阳性10例,阳性率76.9%),但抗CⅡ(260-272)抗体与抗环瓜氨酸肽(CCP)抗体显著相关(P<0.01),且该抗体水平随患者临床症状的改善有逐渐降低趋势。结论成功建立抗CⅡ(260-272)抗体ELISA测定方法,用其检测抗CⅡ(260-272)抗体水平可作为RA疾病诊断和病情监测的一种新的参考指标。

Objective To understand serum levels of anti-type Ⅱ collagen peptide,namely CⅡ（260-272） antibody in rheumatoid arthritis（RA）,other autoimmune diseases and healthy control,and explore the value of this antibody in diagnosis and monitoring of RA disease.Methods Synthetic peptide of CⅡ（260-272） was coupled to bovine serum albumin（CⅡ（260-272）-BSA）,using this conjugate as coated antigen,ELISA method was established for anti-CⅡ（260-272） antibody.After its methodology assessed,results of clinical cases tested by this method were evaluated.Results The establishment of ELISA method for anti-CⅡ（260-272） antibody has the better results of stability and specificity,and its intra-and inter-assay coefficients of variation were 8.4% and 9.6% respectively.The specific inhibitory rate of this method was up to 76.3%.Clinical test results found that the positive rate of anti-CⅡ（260-272） antibody was 75.0%（30/40） in RA,this result was significantly higher than that of other autoimmune diseases and healthy control group（P〈0.01）.When absorbance of 0.213 was considered cut-off value,the anti-CⅡ（260-272） antibody diagnostic specificity,positive/negative predictive value,positive/negative likelihood ratio and Youden＇s index was 91.0%,63.8%/94.5%,8.33/0.275 and 0.66 in RA patients,respectively.ROC curve analysis revealed that this antibody on the accuracy of RA diagnosis was at middle level（AUCROC value= 0.890）.No correlations was found between level of anti-CⅡ（260-272） antibody and rheumatoid factor（RF）（in 13 cases of RF-negative RA patients,10 cases of anti-CⅡ（260-272） antibody were positive,positive rate was 76.9%）,but significantly correlated with anti-cyclic citrullinated peptide（CCP） antibody（P〈0.01）,furthermore,serum levels of anti-CⅡ（260-272） antibody decreased with the improvement of clinical symptoms of RA cases.Conclusion ELISA method for anti-CⅡ（260-272） antibody was successful established,serum levels of this antibody to be tested could be used as a new reference for diagnosis and condition monitoring of RA disease.