快速斑点酶标法检测抗核抗体

Detection of antinuclear antibody by Rapid-Dot-ELISA

目的建立一种快速、简便的抗核抗体（ANA）检测方法。方法采用快速斑点酶标法（Rapid-DotELISA,R-Dot-ELISA）检测20份ANA阳性系统性红斑狼疮（SLE）患者血清和20份阴性正常健康者血清的ANA。检测中采用不同浓度的核抗原、不同的封闭液、不同浓度的酶标抗体、不同显色时间,摸索最佳检测条件。确定检测条件后,采用R-Dot-ELISA法、ELISA法和免疫荧光法检测190例系统性自身免疫性疾病患者和50例健康者血清的ANA,比较R-Dot-ELISA法与ELISA法和免疫荧光法检测的一致性。结果核抗原浓度为40～160mg/L、封闭液为含1%牛血清白蛋白和10%～20%小牛血清的PBS,酶标抗体稀释度为1∶80～1∶320,显色时间3～5min。R-Dot-ELISA与ELISA法相比,检测结果一致率为95.4%（r=0.907,P〈0.05）,与免疫荧光法相比,检测结果一致率为96.7%（r=0.932,P〈0.05）。结论 R-Dot-ELISA检测ANA具有较高的敏感性及特异性,且快速、简便,适用于基层流行病学调查和临床快速诊断。

Objective To set up a simple and convenient immunoassay for antinuclear antibody （ANA）. Methods The ANA positive sera from 20 systemic lupus erythematosus（SLE） cases and the ANA negative sera from 20 healthy were collected and the 40 sera were tested the ANA with Rapid-Dot-ELISA. In the detection we used different concentrations of antigen, different blocking buffer, different concentrations of enzyme-labeled antibodies and different coloration time to choose the optimum testing conditions. The Rapid-Dot-ELISA, ELISA and immunofluorescence methods were used to test the serum ANA in 190 patients with systemic autoimmune diseases and 50 healthy people. Results The concentration of antigen was 40mg/L to 160rag/L, the blocking buffer containing 1% BSA and 10% to 20% bovine serum, the enzyme labeled antibody concentration were 1：80 to 1： 320, the coloration time in 3 to 5min were the appropriate testing conditions. Compared R-Dot-ELISA with ELISA, the concordance rate of the result was 95.4% （ r = 0. 907, P 〈 0.05 ）. Compared R-Dot-ELISA with immunofluorescence method,the concordance rate of the result was 96.7% （ r = 0. 932, P 〈 0. 05）. Conclusion The sensitivity and specificity of R-Dot-ELISA are high, and the assay is rapid and simple, so R- Dot-ELISA used to detect serum ANA is valuable for application in clinical diagnosis.