抗蛋白酶3抗体检测及影响因素的研究

Research for influence factors on detection of antibodies to proteinase-3

目的研究血清样本稀释度、封闭液、孵育条件、洗涤条件及酶标板包被后保存时间对血清抗蛋白酶3(PR3)抗体检测结果的影响。方法采用酶联免疫吸附试验(ELISA)对159份血清进行抗PR3抗体检测,并用受试者工作特征曲线(ROC曲线)分析确定阳性界限(cut-off)值。结果血清样本最佳稀释度为1∶50;5%脱脂奶粉封闭酶标板能有效地降低本底值;孵育缓冲液中加入牛血清白蛋白及脱脂奶粉比单用磷酸盐缓冲液(PBS)对测定值有明显影响。经ROC曲线分析实验的阳性临界值为正常阴性对照吸光度(A)值的2.5倍,灵敏度和特异性分别为84.62%和98.46%。结论血清抗PR3检测结果受血清样品稀释、封闭液、孵育条件及阳性临界值的选择等多因素的影响。

Objective To investigate the influence factors on the detective level of serum anti-proteinase 3 (anti-PR3) antibody by analyzing the absorptance at various conditions, including sample dilutions, blocking materials, incubation condition and interval time between blocking and detection. Methods Anti-PR3 from 159 serum samples (13 patients and 146 controls) was assayed by ELISA, and the cut-off values were defined by ROC curve obtained. Results Serum dilution at 1∶50 was suitable for the detection; absorptance of background blocked by 5% defatted milk powder was decreaced significantly; the incubation buffer containing bovine serum albumin or 5% defatted milk powder influenced the detection more obviously than that containing PBS only; the area under curve (AUC) was 0.928 (95% CI 0.834~1.021); cut-off value of absorptance was 0.235; sensitivity and specificity were 84.62% and 98.46% respectively. Conclusion The detection of anti-PR3 can be influenced by serum dilution, blocking materials, incubation condition and the choice of cut-off point.