基于二氧四氢喋啶合酶多聚纳米抗体的血清中可溶性PD-L1蛋白检测方法  

Lumazine synthase displayed poly-nanobodies for detection of soluble PD-L1 in human serum

作　　者：谢媛 刘畅 徐新兰 张鑫 胡倩倩 李江伟[1] Xie Yuan;Liu Chang;Xu Xinlan;Zhang Xin;Hu Qianqian;Li Jiangwei(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《国际生物医学工程杂志》2022年第3期213-219,共7页International Journal of Biomedical Engineering

摘　　要：目的:基于二氧四氢喋啶合酶(LS)多聚纳米抗体,建立一种检测血清中可溶性细胞程序性死亡-配体1(PD-L1)蛋白的方法。方法:采用竞争ELISA筛选识别PD-L1不同表位的纳米抗体作为配对抗体,建立双纳米抗体夹心ELISA检测方法(Nbs-ELISA)。为了进一步提高敏感性,将纳米抗体与LS进行融合,获得多聚形式的纳米抗体作为sPD-L1蛋白的捕获分子,建立基于LS的多聚纳米抗体ELISA检测方法(LSNbs-ELISA)。结果:与Nbs-ELISA方法相比,LSNbs-ELISA方法对sPD-L1的检测敏感性提高了约11倍,Nbs-ELISA对血清中sPD-L1的检出限为2.87 ng/ml,而LSNbs-ELISA对PD-L1的检出限为0.255 ng/ml。2种检测方法对sPD-L1的检测均具有很高的特异性,与掺入血液中的PD-1、CD27、CD70、CD137和CD147蛋白均不结合。结论:建立的基于LS多聚纳米抗体检测血清中PD-L1的检测方法有较高的敏感性和特异性,对肺癌患者血清中可溶性PD-L1的检测具有潜在的临床应用价值。Objective To established a method for the detection of soluble programmed death ligand 1(PD-L1)protein in serum based on the poly nanoantibody of lumazine synthase(LS).Methods A dual nanobody-based sandwich ELISA was established with a competitive ELISA to screen nanobodies recognizing different epitopes of PD-L1 as paired antibodies.To improve sensitivity,PD-L1 nanobody P3C8 and lumazine synthase(LS)were fused,and nanobodies were obtained in polymeric forms as sPD-L1 protein captures,so as to develop an LS-displayed polymeric nanobody-based sandwich ELISA(LSNbs-ELISA)method to detect sPD-L1.Results Compared with the Nbs-ELISA method,the LSNbs-ELISA method is approximately 11-fold more sensitive for sPD-L1 detection.The limit of detections(LODs)of Nbs-ELISA and LSNbs-ELISA for sPD-L1 in serum were 2.87 ng/ml and 0.255 ng/ml,respectively.Both assays were highly specific for the detection of sPD-L1 and did not react with structure-related proteins PD-1,CD27,CD70,CD137,and CD147 when spiked into the human serum.Conclusions The Nbs-ELISA and LSNbs-ELISA assays both have high sensitivity and specificity for detecting sPD-L1 in serum and could have potential clinical applications.

关 键 词：二氧四氢喋啶合酶 多聚纳米抗体 夹心ELISA 可溶性PD-L1 

分 类 号：R446.61[医药卫生—诊断学]