巨噬细胞移动抑制因子ELISA方法的建立和应用研究

Development and Application of ELISA Method for Detecting Macrophage Migration Inhibitory Factor

目的建立检测巨噬细胞移动抑制因子含量的ELISA检测法。方法采用方阵滴定法,比较4种封闭液、不同稀释度的捕获抗体、检测抗体和辣根过氧化酶标记链菌素对检测结果的影响,确定ELISA法的最佳实验条件。结果包被用捕获抗体的最适浓度为4mg/L,检测抗体的最适浓度为200mg/L,Streptavidin-HRP的最适稀释度为1∶5000。该方法的检测灵敏度为0.156μg/L,变异系数为7.3%～9.6%,回收率为91.2%～113.6%,与VEGF、TNF-α、IL-6、ICAM-1、胆红素、血红蛋白及甘油三酯均无交叉反应。结论建立的ELISA检测法简便、特异且灵敏,适用于检测血清中的MIF水平。

Objective To develop a new double antibody sandwich ELISA kit for detecting macmphage migration inhibitory factor. Method 4 kinds of block buffer, the different concentration of anti-MIF and biotin-anti-MIF,the different dilution of streptavidin-HRP are used for titration of ELISA test. Result The optimal concentration of anti-MIF was 4 mg/L and biotin-anti-MIF was 200 mg/L. The optimal dilutions of streptavidin-HRP were 1：5000. The sensitivity of this ELISA method was 0.156 μg/L. The variation coefficient was about 7.3%～9.6%, and the recovery rate was 91.2%～113.6%. Furthermore, there was no cross-reaction with VEGF, TNF-α, IL-6, ICAM-1, bilirubin, hemoglobin and triglyceride. Conclusion The ELISA method is a highly sensitive, specific and useful method to detect MIF in serum.