建立非均衡竞争叶酸定量测定的化学发光免疫分析法

Detection of human serum folic acid by non-equilibrium competitive immunoassay using FITC detecting system

目的 制备抗人叶酸（FA）抗血清并应用异硫氰酸荧光素（FITC）系统开发新型非均衡竞争技术,建立可常规应用的定量检测血清 FA的化学发光免疫分析法.方法 FITC-FA类似物、FA抗体-HRP依次加入包被有抗 FITC抗体的化学发光板,形成FITC抗体-FITC-FA类似物-FA抗体-HRP的免疫反应复合物;并进行方法学评价,同时与非 FITC检测系统及罗氏化学发光免疫分析系统检测结果进行比较.结果 成功制备FA抗血清并建立基于FITC系统的非均衡竞争化学发光免疫法;经方法学评价,自研法的线性相关系数绝对值〉0.9900,灵敏度1.21 nmol/L,线性范围1.21~38.80 nmol/L,批内变异系数〈5%,自研法定量检测性能优于非FITC系统;与罗氏检测系统结果有较好相关性（R=0.9081）.结论 建立的非均衡竞争定量检测血清FA化学发光免疫分析法,具有良好的检测灵敏度与特异性,可应用于常规检测.

Objective To prepare anti-folic acid （FA）polyclonal antibody and develop a new non-balanced competing chemilumi-nescence analysis for clinical detection of FA.Methods Established the detection method by added FITC-FA-analogs and FA-HRP-antibody in the light emitting plate,which coated with anti-FITC antibody,to form the immune response complex of FITC/antibod-y-FITC-FA-analogs/FA-antibody-HRP.Then methodology evaluation was performed to evaluate the method performance;and fur-ther compared the detecting results with non-FITC system detection system and Electrochemiluminescence system （Roche Elecsys 2010）.Results The ELISA results showed that the prepared anti-FA antibodies can recognize serum FA specificly.The methodolo-gy evaluation indicated that the linear correlation coefficient of the standard curve was 0.990 0;The analytical sensitivity was 1.21 ng/mL;the range of linear detection was 1.21—38.80 ng/mL;The coefficient variability of intra-assay was 〈5%,which was better than the results of non-FITC detection system;and the correlation coefficient was 0.998 6 compared with the Elecsys-2010 detection system.Conclusion The established chemiluminescence immunoassay for human serum FA has a good sensitivity and specificity, and suitable for clinical serum FA quantitative detecting.