快速斑点酶标法检测抗丝聚蛋白抗体

The detection of anti-filaggrin antibody by Rapid-Dot-ELISA

目的建立一种快速、简便的抗丝聚蛋白抗体（anti-filaggrin antibody,AFA）的检测方法。方法采用快速斑点酶标法（Rapid-Dot-ELISA,R-Dot-ELISA）检测15份AFA阳性的类风湿性关节炎（rheumatoid arthritis,RA）患者和15份AFA阴性的正常健康者血清的AFA。检测中采用不同浓度的丝聚蛋白、不同的封闭液、不同浓度的酶标抗体,摸索检测条件。确定检测条件后,采用R-Dot-ELISA和ELISA法检测100例RA患者和150例包括系统性红斑狼疮（SLE）、强直性脊柱炎等非RA的自身免疫病患者和50例健康者血清的AFA,比较两种方法检测AFA的一致性,并判断R-Dot-ELISA检测AFA对RA的诊断价值。结果丝聚蛋白浓度为40-160μg/m L,封闭液为含1%BSA和10%小牛血清的PBS,酶标抗体稀释度为1∶80-1∶320是较合适的检测条件。R-Dot-ELISA与ELISA法检测AFA,结果一致率为98%（r=0.947,P〈0.05）,对RA诊断的敏感度、特异度等统计学指标无差异。结论 R-Dot-ELISA检测AFA快速、简便,且对RA的诊断具有较高的敏感性及特异性,适用于基层流行病学调查和临床快速诊断。

Objective To development a simple and convenient immunoassay method for detection anti-filaggrin antibody（AFA）.Methods The AFA positive sera from 15 rheumatoid arthritis（RA） cases and the AFA negative sera from 15 healthy were collected and tested the AFA by Rapid-Dot-ELISA（R-Dot-ELISA）.The different concentrations of antigen,blocking buffer and enzyme-labeled antibodies were used to optimize the testing conditions.100 patients with RA and 150 patients with other immune disease and 50 healthy people were conducted consistency check of two methods,Rapid-Dot-ELISA and ELISA.Results The appropriate testing conditions were 40 - 160μg /m L of the antigen concentration,the blocking buffer containing the 1% BSA and 10% bovine serum and the enzyme labeled antibody concentration in 1∶ 80 - 1∶ 320.Compared R-Dot-ELISA with ELISA,the result of concordance rate was 98%（r = 0.947,P〈0.05）.Conclusion It is a rapid,simple,sensitivity and specificity method to detect AFA by using of R-Dot-ELISA for RA,and the assay is valuable for application in clinical diagnosis of RA.