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目的研制血管内皮生长因子(vascular endothelial growth factor,VEGF)化学发光免疫测定试剂盒,并对该试剂盒进行验证。方法以试剂盒的技术指标和血清检测结果为参考依据,建立检测方法,确定检测范围,并对包被抗体(1∶500、1∶1 000和1∶2 000倍稀释)及酶标抗体浓度(1∶3 000、1∶5 000、1∶6 000和1∶8 000倍稀释)、包被温度(室温和4℃)、封闭液成分(1%小牛血清、1%和0.5%牛血清白蛋白)及干燥方法(超净台吹干和使用真空干燥机进行干燥)进行优化;同时对试剂盒的线性、检出限、精密性、准确度、特异性及热稳定性进行验证;采用本试剂盒对139例非肿瘤人群和100例肿瘤患者的血清样本进行初步检测。结果方法的最佳检测条件为:包被抗体浓度为1∶1 000,酶标抗体浓度为1∶3 000,对血清样本的检测范围为6.25~800 pg/ml,最佳包被浓度为4℃,最佳封闭液成分为0.5%牛血清白蛋白,最佳干燥方法为真空干燥法。试剂盒线性相关系数r>0.99,校准曲线各点偏差的绝对值<10%,最低检出限≤3.125 pg/ml,空白检出限≤3.125 pg/ml,批内差异<5%,批间差异<7%,回收率为85%~99%;特异性检验结果为本试剂盒对EGF、FGF-2、PDGF-AA、Pl GF-1、Pl GF-2、HGF、TNF-α、TNF-β检测的交叉反应率≤0.52%;经37℃保存3、7 d后,试剂盒线性相关系数r>0.99,空白检出限≤3.125 pg/ml,批内差异<8%,回收率为80%~82%。139例非肿瘤人群和100例肿瘤患者的血清样本中VEGF的中位水平(M)分别为44.6和83.4 pg/ml。结论本研究开发的试剂盒具有较高的准确性、灵敏性、特异性,适用于血清VEGF的临床检测,具有较好的临床应用前景。
Objective To develop a chemiluminescent immunoassay kit for vascular endothelial growth factor (VEGF) and verify the kit. Methods Based on the technical indicators and serum test results of the kit, a detection method was established to determine the detection range. The coated antibody (1: 500, 1: 1000, and 1: 2000 dilution) and enzyme- (1: 3000, 1: 5000, 1: 6000 and 1: 8000 dilutions), coating temperature (room temperature and 4 ° C), blocking solution Serum albumin) and drying methods (ultra-clean bench drying and drying with a vacuum dryer) were optimized; at the same time, the linearity, detection limit, precision, accuracy, specificity and thermal stability of the kit were verified; Serum samples from 139 non-tumor patients and 100 cancer patients were initially tested using this kit. Results The best detection conditions were as follows: the concentration of coating antibody was 1: 1 000, the concentration of enzyme-labeled antibody was 1: 3000, the detection range of serum sample was 6.25-800 pg / ml, and the optimal coating concentration was 4 ℃, the best blocking solution is 0.5% bovine serum albumin, the best drying method is vacuum drying method. The linear correlation coefficient of the kit was> 0.99. The absolute value of the deviation of each point of the calibration curve was <10%, the minimum detection limit was 3.125 pg / ml, the blank detection limit was 3.125 pg / ml, the difference within the batch was less than 5% The specificity test results were as follows: the difference was less than 7% and the recovery rate was 85% ~ 99% The cross-reaction rate was-0.52%. After 37 ℃ stored for 3 and 7 days, the linear correlation coefficient of the kit was 0.99, the blank detection limit was 3.125 pg / ml, the difference was within 8% 80% ~ 82%. Serum samples of 139 non-tumor and 100 tumor patients had median VEGF levels (M) of 44.6 and 83.4 pg / ml, respectively. Conclusion The kit developed in this study has high accuracy, sensitivity and specificity for the clinical detection of serum VEGF, and has good clinical application prospects.