高特异性人血清白蛋白化学发光免疫分析方法的建立

Establishment of a High-specificity Chemiluminescence Immunoassay Assay for Human Serum Albumin

人血清白蛋白（HSA）是人类肝脏功能的重要生物标志物.本研究旨在发展一种具有高度种属特异性和灵敏度的HSA化学发光免疫分析（HSA CLIA）定量检测方法.通过HSA免疫小鼠,制备了6株HSA鼠单克隆抗体;使用间接化学发光检测（Indirect-CLIA）和West blotting检测方法评价单抗对HSA的反应活性;使用免疫组织化学检测（IHC）和West blotting检测方法评价单抗的种属特异性;选择最佳的包被抗体和检测抗体配对;确定HSA-CLIA的定量线性和范围.West bloting和IHC方法中,4F5和5D2抗体反应性最强,且不与小鼠、大鼠、牛、兔、山羊、猴、鸡等常见实验动物血清白蛋白交叉反应.以单抗5D2配合单抗4F5建立的HSA CLIA,线性范围达到122～31 250 pg/mL,可适用于多种样本的HSA定量检测.本研究发展的单克隆抗体和检测方法为HSA的特异性检测提供了重要工具和手段.

Human serum albumin （HSA） is an important bio-marker for liver function.This study aimed to develop a quantitative species specific chemiluminescence immunoassay （CLIA）with high sensitivity for detection of HSA.A total of 6 strains of monoclonal antibodies （mAbs） against HSA were obtained by mouse immunization.Subsequently,the reactivity of mAbs against HSA was evaluated by indirect CLIA and western blotting,and the species specificity of mAbs was performed by western blotting and immunohistochemistry （IHC）.And then,the best capture antibody and the detection antibody pairs were selected.Finally,the quantitative linearity and range of HSA CLIA had been determined.Among these antibodies,the mAb 4F5 and 5D2 had the strongest reactivity against HSA,and did not present cross reactivity with serum albumin from common experimental animals,such as mouse,rat,bovine,rabbit,goat,monkey,and chicken.Based on the mAb pair of 5D2 and 4F5,we developed an HSA chemilumi nescence immunoassay with a linear range from 122 pg/mL to 31 250 pg/mL,which was suitable for HSA quantification of various specimens.The monoclonal antibodies and the detection methods we developed in this report would provide important tools for the specific detection of HSA.