免疫金银法检测抗角质蛋白抗体的影响因素

Influence factors in detection of anti-keratin by immunogold silver method

目的：探讨免疫金银法检测抗角质蛋白抗体（AKA）的影响因素.方法：收集AKA阳性的类风湿性关节炎患者血清20份,同时收集20份正常健康者血清作为对照,采用免疫金银法检测上述血清AKA,检测中采用不同的封闭液、不同浓度的金标抗体、不同的显色时间,比较分析不同条件下免疫金银法检测AKA的阳性率及结果易判断程度,以确定较合适的检测条件.同时采用免疫荧光法检测AKA,比较2种方法检测AKA的敏感性.结果：以含10％～20％的羊血清及1％BSA的封闭液、1：80～1：320的金标抗体、10～15 min的显色时间为较合适的检测条件.免疫金银法检测AKA较免疫荧光法敏感性高,且差别有统计学意义.结论：免疫金银法检测AKA对类风湿性关节炎具有较高的敏感性和特异性,临床上用于类风湿性关节炎的辅助诊断具有较高的诊断价值.

Objective： To study the influence factors in detection of anti-keratin（AKA） antibody by immunogold silver method. Methods： The AKA positive sera from 20 rheumatoid arthritis cases and the negative sera from 20 healthy controls were collected and sera AKA was tested by immunogold-silver method. Different blocking buffer, different concentrations of gold labeled antibodies and different coloration time were used to compare the positive rate of AKA under different testing conditions in order to establish a suitable detecting condition. The AKA was tested by immunofluoreseenee as control. The positive rate of AKA by the two methods were compared. Results： The blocking buffer containing the 1NBSA and 10 %-20% sheep serum, the gold labeled antibody concentration in 1 ： 80-1 = 320 and the ehromogenic time in 10-15 minutes was the appropriate testing conditions. The AKA positive rate tested by immunogold-silver staining was higher than that of immumofluorescence. Conclusion： The sensitivity and specificity to AKA of immunogold-silver staining are much more higher than immumofluoreseence, so the immunogold-silver staining is more valuable for rheumatoid arthritis auxiliary diagnosis.