双抗体夹心ELISA法分析人血APOL1含量与EV71感染程度的相关性

The double antibody sandwich ELISA method to analyze the correlation between the level of APOL1 in human serum and the severity of EV71 infection

目的建立检测载脂蛋白L1(APOL1)的ELISA方法,探讨肠道病毒71型(EV71)感染患儿血液中APOL1的含量与EV71感染程度的关系。方法合成APOL1多肽,采用经典方法制备APOL1单克隆抗体,建立检测APOL1蛋白的双抗体夹心ELISA方法,并检测EV71感染的临床血样68份。结果筛选出5B5和7A9两个特异性的单克隆抗体,据此建立ELISA双抗体夹心检测方法。在78.125~5000 ng/mL范围内,APOL1蛋白浓度有较好的检测线性,批内和批间变异系数不超过10%。在EV71⁃IgM阳性组和弱阳性组感染患儿血清中,APOL1含量分别为(1008.00±18.34)ng/mL和(885.80±15.76)ng/mL,两组血清中的APOL1含量差异有统计学意义(P<0.05)。结论建立了稳定可靠用于检测血清中APOL1蛋白的双抗体夹心ELISA方法。EV71⁃IgM抗体阳性组患儿血清中APOL1含量明显高于弱阳性组,提示APOL1含量和EV71感染程度具有一定相关性。

Objective To establish ELISA method for detecting Apolipoprotein L1(APOL1)content and explore a correlation between the level of APOL1 in serum of Human enterovirus 71(EV71)patients and the severity of EV71 infection.Methods After synthesizing APOL1 antigen polypeptide,the specific monoclonal antibody against APOL1 was prepared and identified by the classical monoclonal antibody preparation technology.The double⁃antibody sandwich ELISA methods for detecting APOL1 protein were established and 68 clinical serum samples with EV71 infection were detected.Results Two monoclonal antibodies specfic for APOL1 polypeptide,5B5 and 7A9 were selected to develope a sandwich ELISA method.In the range of 78.125⁃5000 ng/mL,the concentration of APOL1 protein was linear,and the coefficient of variation within and between batches was not more than 10%.The content of APOL1 in patients′serum of the EV71⁃IgM positive group and the weak positive group were(1008.00±18.34)ng/mL and(885.80±15.76)ng/mL,respectively.The levels of APOL1 in the two groups were significantly different(P<0.05).Conclusion The stable and reliable sandwich ELISA methods was established for detecting APOL1 content in human serum.The content of APOL1 in the serum of the EV71⁃IgM antibody positive group was significantly higher than that of the weak positive group,indicating that the content of APOL1 had a certain correlation to the severity of infection of EV71.