Sm^(3+)标记抗心磷脂抗体IgM时间分辨荧光免疫分析法的建立

Detection of anticardiolipin antibody IgM by Sm^(3+) -labeled time-resolved fluoroimmunoassay

目的建立高灵敏度、宽量程的定量检测患者血清中的抗心磷脂抗体(anticardiolipin antibody,ACA)IgG的Sm3+标记时间分辨荧光免疫分析方法。方法采用ACA抗原(心磷脂+β2糖蛋白I)和兔抗人IgM抗体分别作为固相抗原和钐标记抗体,如果样本中存在ACA抗体,则形成ACA抗原-ACA抗体-钐标记兔抗人IgM抗体复合物,加入解离增强液解离铕离子,检测荧光强度,样本中ACA-IgM抗体含量与荧光强度成正比;对建立的时间分辨荧光免疫(time-resolved fluoroimmunoassay,TRFIA)法检测ACA抗体的线性范围、精密度、检测范围进行分析;对25例ACA-IgM抗体阳性血清标本分别利用TRFIA及ELISA法检测ACA-IgM抗体,分析其相关性;收集50例健康献血者,利用TRFIA检测其血清中的ACA-Ig抗体,计算临床特异度。结果利用SPSS 13.0统计软件进行统计学分析,TRFIA法检测高、中、低3种浓度混合血清的批内(n=20)精密度分别为2.39%、3.26%和3.94%,批间(n=8)精密度分别为2.92%、3.73%和4.35%;方法的灵敏度为0.1 MPL U/ml;TRFIA法检测健康献血者,临床特异度为98%;TRFIA与ELISA 2种方法检测结果的一致性检验采用相关性分析,相关系数为0.956;将1份ACA-IgM抗体强阳性的血清标本从2483 MPL U/L倍比稀释至0.151 MPL U/L,ELISA方法较好的线性范围在4.85-310.4MPL U/L,而TRFIA方法在0.151-2483MPL U/L都有较好的线性。结论首次建立稳定的高灵敏度和宽检测范围的TRFIA法检测人血清中的ACA-IgM抗体,对早期诊断自身免疫性疾病及监测疗效具有重要意义,该方法以其优势有望在各检验科室得以普遍应用。

Objective In an effort to improve the quantitative detection of aCL IgM,we develop a new immunoassay to improve aCL IgM detection based on TRFIA. Methods Using the complex of cardiolipin plus bovine 2GpI as anti gen and $m3＋-labeled rabbit anti-human IgM as conjugate. The precision, sensitivity, specificity, coefficient of recovery and stability of the assay were evaluated and comparison with the classical ELISA Was also made. Results The aCL IgM TRFIA kit we established had a wider detectable range than commercial ELiSA ones when diluted a specimen with strong positive from 0. 151 2483 MPL U/L. We observed that for the established TRFIA kit there was a good liner range within 0. 151-2483 MPL U/L,whereas it was within 4.85-310.4 MPL U/L when using ELISA kits. The intraas- say precision rate and the interassay precision rate were 5 % for 3 different concentrations. The sensitivity was 0. 1 MPL U/ml and the clinical diagnostic specificity was 98%. Average recovery rate was 101.13%. The established assay kit also behaved better in stability. Additionally, the immunoassay we established correlated well with the ELISA and the correlation coefficient was 0. 956. Conclusion We thus conclude that the TRFIA we developed for aCL IgM detec- tion gives promise to a more sensitivity and reable diagnosis of APS and has potential value for large-scale screening programs.