Genomics Research Center, Academia Sinica, 128 Academia Rd., Sec.2, Nankang Dist., Taipei, 115, Taiwan.
Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan.
Sci Rep. 2020 Aug 7;10(1):13318. doi: 10.1038/s41598-020-70135-6.
Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.
基于同时结合靶分析物的捕获和检测抗体的夹心免疫复合物的免疫测定法已成为分子分析中的强大技术。单分子检测技术的最新发展使夹心免疫测定法的检测限接近飞摩尔（10-15M），这推动了开发用于检测和定量生物标志物的越来越广泛应用的敏感和特异性抗体的需求。夹心免疫测定法的基础是基于抗体的亲和试剂，其传统来源是免疫动物的单克隆或多克隆抗体。动物来源的抗体作为亲和试剂的缺点在于需要数月的开发时间，并且由于动物体液免疫反应的免疫优势，抗体候选物的选择有限。因此，开发能够区分高度相关抗原的动物抗体可能具有挑战性。为了克服动物免疫系统带来的限制，我们开发了一种基于噬菌体展示的合成抗体文库的体外方法，用于针对密切相关的流感病毒核蛋白（NP）亚型的多种抗体作为亲和试剂，旨在区分禽流感病毒（H5N1）和季节性流感病毒（H1N1 和 H3N2），NP 之间的氨基酸序列同一性高达 90-94%。我们应用该方法在四周内获得了一组 IgG，它们具有针对一组具有高达 90%以上的氨基酸序列同一性的代表性 NP 的可区分特异性，并且从抗体文库中获得的抗体无需进一步的亲和力精炼，其亲和力与 NP 的小鼠抗体相当，夹心 ELISA 检测裂解病毒中 NP 的检测限低于 1 nM。该 IgG 组能够快速区分由于高致病性禽流感病毒引起的感染与季节性流感的感染，以应对可能的紧急情况，即禽流感病毒在与季节性流感感染重叠的情况下在人群中传播。结果表明，体外抗体开发方法能够开发出否则无法从动物抗体技术获得的诊断抗体。
