This is evident in the moderate affinity of the serotype-specific antibodies isolated from the naive libraries in this study. monoclonal Acenocoumarol 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 Acenocoumarol (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 usually a valuable methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability. Introduction Dengue virus infections are a significant public health burden. Dengue computer virus belongs to the genus flavivirus and is transmitted by the mosquito vectors and affinity maturation aspect that occurs in natural humoral immune responses by somatic hypermutation [36]. This is evident in the moderate affinity of the serotype-specific antibodies isolated from the naive libraries in this study. Detection of DENV-2 and DENV-3 rNS1 in this work was markedly poorer than that of DENV-1 and DENV-4 rNS1 in the sandwich ELISA format, despite the conversation kinetics of the anti-DENV-3 antibody being similar to the anti-DENV-1 antibody. The limit of detection is usually governed by the cooperative kinetics of both the capture and detection antibodies, and both should be considered when attempting to improve the sensitivity of the assay. Paramount to the affinity of the antibody for the antigen is the availability of sites around the antigen to which antibodies can bind. This is important in the background of a complex test matrix such as serum, especially during anamnestic infections where DENV NS1 may be sequestered in immune complexes [37]. Hypothetically, the Rabbit polyclonal to HDAC5.HDAC9 a transcriptional regulator of the histone deacetylase family, subfamily 2.Deacetylates lysine residues on the N-terminal part of the core histones H2A, H2B, H3 AND H4. non-immune driven, serotype-specific antibodies would still be able to participate in DENV NS1 capture or detection, if paired with a pan-reactive antibody against a non-immune epitope. To conclude, interrogation of naive phage antibody libraries with a subtractive panning strategy has shown to be effective in isolating serotype-specific antibodies which could add a serotyping capability to existing NS1 assays, and we show that these have potential in a multiplexed Acenocoumarol immunoassay. Acknowledgments We thank Dr Sumukh Kumble for the graphical representation in Fig 1. Elements of this research utilised gear and support provided by the Queensland node Acenocoumarol of the National Biologics Facility (www.nationalbiologicsfacility.com), an initiative of the Australian Government being conducted as part of the NCRIS National Research Infrastructure for Australia. Funding Statement This work was supported by an Australian Research Acenocoumarol Council Linkage Grant with project partner Australian Red Cross Blood Support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are within the paper..