摘要
目的:构建抗人妊娠相关血浆蛋白A(PAPP-A)单抗细胞株,成功筛选配对抗体后建立PAPP-A双抗体夹心化学发光(CLIA)及免疫胶体金(ICG)检测方法。方法:采用杂交瘤技术获得5株稳定分泌抗PAPP-A单抗的细胞株,将细胞株扩大培养并亲和纯化上清液获得抗体,测定抗体纯度、效价、特异性、表位,建立双抗体夹心CLIA及胶体金体系,并对各体系进行灵敏度、特异性等分析性能评估。结果:获得抗人PAPP-A的杂交瘤细胞株(#13-2-2、#20-1-1、#21-1-1、#44-1-1和#53-2-1)。其分泌的抗体效价均大于10-8 g/ml。化学发光体系在0~10000 ng/ml范围内线性关系良好,其准确度的回收率为107.2%;灵敏度为9.56 ng/ml;线性相关系数为0.9966;低高值的变异系数(CV)均小于10%;胶体金体系灵敏度不小于50 ng/ml。两种检测方法与人甲胎蛋白(AFP)、人泌乳素(PRL)、人胎盘催乳素(hPL)和人绒毛膜促性腺激素(HCG)均不存在交叉。结论:本研究成功制备了抗人PAPP-A单抗,建立了检测PAPP-A化学发光及免疫胶体金体系。该体系为PAPP-A的定量检测和产前诊断等临床应用提供了基础。
Objective:Construction of monoclonal antibody cell lines against human pregnancy-associated plasma protein A(PAPP-A)and establish double antibody sandwich CLIA detecting method and a immune colloidal gold strip for human PAPP-A.Methods:Five hybridoma cell lines secreting anti-PAPP-A were obtained by hybridoma technique.The cell lines were expanded and purified by affinity purification,from which the antibodies were tested for antibody purity,titer,specificity,epitope.The establishment,analysis and performance evaluation of a double mAbs sandwich CLIA system and the ICG strip.Results:Five hybridomas named#13-2-2,#20-1-1,#21-1-1,#44-1-1 and#53-2-1 were obtained respectively,which are strong positive signal and high secretion.The titers of the mAbs secreted by these hydridomas were above 10-8 g/ml.The double-antibody sandwich CLIA system had a good linear relationship between 0 ng/ml and 10000 ng/ml.The recovery rate for accuracy was 107.2%and the sensitivity was 9.56 ng/ml.The linear correlation coefficient was 0.9966 and the CV of low and high values were all less than 10%.The sensitivity of the ICG strip was not less than 50 ng/ml.The two system system were also highly specific without cross-reactivity to the tumor marker AFP,PRL,hPL and HCG.Conclusion:The mAbs against human PAPP-A have been prepared.The CLIA system and ICG system for detecting PAPP-A in the human serum have been established successfully,which provides a basis for PAPP-A quantification and clinical application of prenatal diagnosis.
作者
张兰兰
于洋
曹领改
魏德强(指导)
ZHANG Lan-Lan;YU Yang;CAO Ling-Gai;WEI De-Qiang(College of Life Science,Northeast Forestry University,Harbin 150040,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第22期2753-2758,共6页
Chinese Journal of Immunology
基金
中央高校基本科研业务费专项资金(2572019BD03)
中央高校基本科研业务费专项资金(257214AA19)
大学生创新创业项目(201810225135)。
