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 (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea 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 lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea 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 lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.
题目: 羊驼噬菌体展示纳米抗体库中lea模拟抗原表位筛选与鉴定的初步研究.
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 (lea)抗体亲和淘选羊驼噬菌体展示纳米抗体文库来选择lea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lea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lea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lea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lea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lea+，15例lea-），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lea模拟抗原的方法，有望实现lea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.
Keywords: alpaca phage display nanobody library; lea antigen; mimotope.