Lebani Kebaneilwe, Jones Martina L, Watterson Daniel, Ranzoni Andrea, Traves Renee J, Young Paul R, Mahler Stephen M
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
ARC Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Queensland, Australia.
PLoS One. 2017 Jul 6;12(7):e0180669. doi: 10.1371/journal.pone.0180669. eCollection 2017.
The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.
登革病毒（DENV）感染具有多维度特性，该病毒有四种不同血清型，这使得针对感染诊断设计的检测方法的敏感性变得复杂。不同的病毒标志物在感染的不同阶段能得到最佳检测。对感染进行早期识别具有特别重要的临床意义，这对疾病管理和血液筛查检测的开发至关重要。非结构蛋白1（NS1）是感染的早期替代标志物，其在血清中的检测与可检测到的病毒血症同时出现。这项工作的目的是分离并鉴定针对四种DENV血清型中每种血清型与NS1结合的血清型特异性单克隆抗体。这是通过噬菌体展示和消减生物淘选策略实现的，该策略将抗体选择导向血清型特异性表位。这种抗体分离策略比免疫技术具有优势，在免疫技术中很难避免对交叉反应性、免疫显性表位产生抗体反应。通过酶联免疫吸附测定（ELISA）和表面等离子体共振确认了每种抗体对重组抗原的血清型特异性。通过对DENV感染的Vero细胞进行ELISA和免疫荧光测定，实现了与天然DENV NS1结合的确认。未观察到与寨卡病毒或库京病毒的交叉反应。一种先前分离的与免疫显性表位结合的泛反应性抗体能够在夹心ELISA中与每种血清型特异性抗体配对，这表明血清型特异性抗体结合的表位在空间上均与免疫显性表位不同。这些抗体适用于在多重检测中同时检测和血清分型人血清中的DENV NS1。这项工作表明，噬菌体展示与新型生物淘选策略相结合是一种有价值的体外方法，可用于分离能够在具有高同源性的抗原之间进行区分以用于诊断应用的结合物。
