羊驼噬菌体展示纳米抗体库中 Le^(a) 模拟抗原表位筛选与鉴定的初步研究  

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library

作　　者：钟晓龙 杨璐 张洁 孙莉萍 马铭梓 樊斌 尚玮 黄远帅 汪德清 ZHONG Xiao-Long;YANG Lu;ZHANG Jie;SUN Li-Ping;MA Ming-Zi;FAN Bin;SHANG Wei;HUANG Yuan-Shuai;WANG De-Qing(Department of Blood Transfusion,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Transfusion Medicine,The First Medical Center,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]西南医科大学附属医院输血科,四川泸州646000 [2]中国人民解放军总医院第一医学中心输血医学科,北京100853

出　　处：《中国实验血液学杂志》2022年第3期877-883,共7页Journal of Experimental Hematology

基　　金：中国人民解放军总医院创新计划项目(NO.CX19006)。

摘　　要：目的:建立一种新型Lewis血型抗原的合成方法,即利用羊驼噬菌体展示纳米抗体库筛选Lewis血型抗原的模拟表位,并将模拟表位序列进行生物合成。方法:用单克隆抗Lewis a(Le^(a))抗体亲和淘选羊驼噬菌体展示纳米抗体文库来选择Le^(a)抗原的模拟表位。利用酶联免疫吸附试验(ELISA)对阳性克隆菌与筛选原Le^(a)单克隆抗体的亲和力进行检测,选择高亲和力的阳性克隆菌进行测序,并对最终确定的序列进行表达合成。最后通过ELISA法对合成后的Le^(a)模拟抗原进行灵敏度、特异度以及临床样本的验证。结果:随机挑取96个噬菌体克隆,有24个阳性克隆,并选取亲和力最强的前14个噬菌体克隆进行测序,结果显示有5个不同序列,选取出现频率最高、差异最大、亲和力最高的3条序列进行表达合成。利用ELISA法检测筛选得到的Le^(a)模拟抗原的灵敏度及特异性,发现微柱凝胶法与ELISA法的最低检出限相差25倍,且Le^(a)模拟抗原与其他5种单抗(anti-A,anti-B,anti-P1,anti-M,anti-N)没有交叉反应(P<0.001)。最后,检测了30例临床血浆样本(15例Le^(a+),15例Le^(a-)),结果显示,15例阳性血浆样本的吸光度的均值高于阴性血浆样本,且具有统计学差异(P=0.02),但临床样本的阳性信号值远低于单抗的阳性信号值。结论:初步建立了利用羊驼噬菌体纳米抗体库筛选Le^(a)模拟抗原的方法,有望实现Le^(a)模拟抗原表位的筛选,从而实现ELISA、化学发光等高灵敏度检测方法在血型抗体鉴定方面的应用。Objective:To establish a new method for synthesizing Lewis blood group antigens,that is,the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.Methods:We selected mimotopes of the Lewis a(Le^(a))antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-Le^(a) antibody.Enzyme-linked immunosorbent assay(ELISA)was used to test the affinity of the positive clones for the monoclonal anti-Le^(a) antibody,and the high-affinity positive clones were selected for sequencing and synthesis.Finally,the sensitivity,specificity and reactivity of the synthesized Le^(a) mimotope in clinical samples were verified by ELISA.Results:A total of 96 phage clones were randomly selected,and 24 were positive.Fourteen positive clones with the highest affinity were selected for sequencing.The result showed that there were 5 different sequences,among which 3 sequences with the highest frequency,largest difference and highest affinity were selected for expression and synthesis.The sensitivity and specificity of Le^(a) mimic antigen by ELISA showed that,the minimum detection limit of gel microcolumn assay(GMA)and ELISA method were 25 times different,and the Le^(a) mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001).Finally,30 clinical plasma samples were analyzed.The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples(P=0.02).However,the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies. Conclusion: A new method of screening Le^(a) mimic antigen by using alpaca phage nanoantibody library has been established,which is expected to realize the screening of Le^(a) mimotopes,thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

关 键 词：模拟表位 羊驼噬菌体纳米抗体库 Le^(a)抗原 

分 类 号：R457.11[医药卫生—治疗学]