血管内皮生长因子定量检测试剂盒的研制

 目的   研制血管内皮生长因子（vascular endothelial growth factor，VEGF）检测试剂盒（酶标法）。方法  以一株特异性抗VEGF单克隆抗体作为捕获抗体，另一株单克隆抗体作为检测抗体，通过棋盘滴定法确定两抗体的最佳浓度组合。制备冻干VEGF165参考品，并对样本稀释液、封闭液等实验条件进行优化。检测自制试剂盒的特异性并对其进行初步应用。结果  棋盘滴定法确定捕获抗体的质量浓度为10 μg/ml，检测抗体的稀释倍数为1:20 000。冻干VEGF165的质量浓度为720~880 pg/ml，样本间变异系数＜10%。以5%牛血清白蛋白作为封闭液和样本稀释液最为合适。自制试剂盒与10种细胞因子均无交叉反应，具有较高的特异性和灵敏度。结论  成功研制出VEGF检测试剂盒。

Development of quantitative detection kit for vascular endothelial growth factor

Objective To develop a vascular endothelial growth factor (VECF) assay kit. Methods Two anti-VEGF monoclonal antibodies were used as capture and detection antibodies, respectively, and optimum concentration combination of the antibodies was determined by chessboard titration. Freeze-dried VEGF165 reference material was prepared. Sample diluent and blocking solution were screened. The kit was tested for specificity and applied preliminarily. Results The concentration and dilution multiple of the capture antibody and detection antibody were identified as 10 μg/ml and 1:20 000, respectively. The concentration of freeze-dried VEGF165 reference material was 720- 880 pg/ml and the variation coefficient was < 10%. 5% bovine serum albumin was chosen both as sample diluent and as blocking solution. No cross-reactions between the kit and 10 other cytokines were found. The kit had high sensitivity and specificity. Conclusion The VECF assay kit was successfully developed.