人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的评价与应用

Evaluation and application of double antibody sandwich ELISA kit for micro-quantitative soluble complement receptor 1 protein in human serum

目的对研制的人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的重复性、稳定性进行评价,并了解其实际应用效果。方法选取中、晚期肝硬化患者50例和健康体检者50例分别作为肝病组和正常对照组,以鼠抗人CD35单克隆抗体、兔抗人sCR1多克隆抗体和辣根过氧化物酶标记的山羊抗兔IgG为包被抗体、夹心抗体和检测抗体,以纯化后的重组人sCR1蛋白为标准品,建立人血清微量sCR1蛋白双抗体夹心ELISA试剂盒,进行试剂盒重复性和稳定性检验,并应用该试剂盒检测两组血清sCR1蛋白水平。结果双抗体夹心ELISA法检测人血清微量sCR1蛋白的线性范围是15.60~250.00ng/mL;血清sCR1蛋白水平对吸光度值的回归方程为Y=112.10 X2+18.21 X+1.694(r2=0.998);重复性检测中,高、低水平标准品检测值的批内相对标准偏差(RSD)分别为6.20%、7.40%,批间RSD分别为6.70%和7.90%;试剂盒稳定性检测中,RSD均不大于0.01;肝病组血清sCR1表达水平显著高于正常对照组(P<0.01)。结论人血清微量sCR1蛋白双抗体夹心ELISA试剂盒线性范围广、重复性好、易于保存,适合于临床和科研检测工作。

Objective To evaluate the repeatability and stability of double antibody sandwich ELISA kit for micro-quantitative soluble complement receptor 1（sCR1）in human serum and to understand its practical application effect.Methods 50 patients with middle and advanced liver cirrhosis and 50 individuals undergoing physical examination served as the liver disease group and normal control group respectively.The mouse anti-human CD35 monoclone antibody,rabbit anti-human sCR1 polyclonal antibody and goat anti-rabbit IgG labeled by horseradish peroxidase served as the envelope antibody,sandwich antibody and detection antiboby.The purified recombinant human sCR1 protein served as the standard substance.The human serum micro-quantitative double antibody sandwich CR1 ELISA kit was established.Then the repeatability and stability tests were performed.Then the sCR1 protein level of two group of serum was detected by this kit.Results The linear range of double antibody sandwich ELISA for detecting human serum micro-quantitative sCR1 protein was 15.60-250.00 ng/mL;the regression equation of sCR1 protein concentration to absorbance value was Y=112.10 X^2＋18.21 X＋1.694（r^2=0.998）;in the repeatability test,the intra-batch relative standard deviation（RSD）in high and low concentrations of standard substance detection value was 6.20% and 7.40% respectively,the inter-batch RSD was 6.70%and 7.90%respectively;in the stability test,RSD was not more than 0.01;the serum sCR1 expression level in the liver disease group was significantly higher than that in the normal control group（P0.01）.Conclusion The human serum double antibody sandwich ELISA kit for detecting human sCR1 has wide linear range,good repeatability,is easy to be stored and suitable for clinical and scientific research detection work.