抗CCP抗体斑点免疫印迹试验测定方法的建立与初步应用

Development of Dot-immuno-bloting technique for anti-CCP antibody determination and its preliminary application

目的建立检测抗环瓜氨酸肽(CCP)抗体的斑点免疫印迹试验,探讨其在类风湿关节炎(RA)疾病诊断中的价值。方法通过方阵滴定法选择斑点免疫印迹试验检测抗CCP抗体的最佳实验条件,并用该法检测RA、其他疾病及健康人血清,同时与自行建立的ELISA法及欧蒙试剂抗CCP抗体检测结果作对比分析。结果斑点免疫印迹试验检测抗CCP抗体对RA的诊断敏感性为52%,特异性为94%,并具有较好的方法学稳定性和特异性。在48例被诊断为RA患者中有25例抗CCP抗体阳性,显著高于其他疾病组和正常对照组(P<0.01)。对比研究结果表明,该法与基于RSA-CCP固相模式建立的ELISA法具有高度的相关性(χ2=131.712)和一致性(Kappa=0.771,P<0.001),与欧蒙试剂检测结果亦具有高度的相关性(χ2=24.147)和很好的一致性(Kappa=0.714,P<0.001)。结论成功建立了抗CCP抗体检测的斑点免疫印迹试验,实验结果稳定,特异,可靠,值得进一步推广和研究。

Objective To establish Dot-immuno-bloting（DIB） technique for anti-Cyclic citrullinated peptide （anti-CCP） anti- body, and study its value in diagnosing rheumatoid arthritis（RA）. Methods By optimizing experimental conditions of DIB for anti- CCP antibody determination, serum levels of anti-CCP in patients with RA, other autoimmunity and medicine diseases as well as normal control was investigated,and was also determined by ELISA（Based on our established method and EUROIMMUN assay）. Results The diagnostic sensitivity of anti-CCP antibody for RA was 52%, and specificity was 94% by this DIB technique. In all patients with RA（ n = 48） ,2.5 of them were positive with anfi-CCP antibody, they were significantly increased than other diseases and normal control （P 〈 0.01）.Parallel investigation showed that serum levels of anti-CCP antibody detected by DIB was highly significant association （ X^2 = 131.712） and consistency （ Kappa = 0.771） with of ELISA method established based on RSA-CCP immobilization（ P 〈 0.（301）,and was also significant association （ X^2 = 24.147） and consistency （ Kappa = 0.714 ） with of EUROIMMUN assay （ P 〈 0.001 ）. Conclusion DIB technique used for anti-CCP antibody determination was successfully established,results of this assay was stable, specific and reliable. So,this DIB technique is worth of popularizing and further investigation.