title: Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1.
abstract: BACKGROUND
  Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus.
  
  
  METHODS
  Nanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay.
  
  
  RESULTS
  We have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50-1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%.
  
  
  CONCLUSIONS
  Collectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the influenza virus.
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, P.R. China. zhumin555111@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, P.R. China. gongxue0715@163.com.
authors.forename: Xue
authors.initials: X
authors.lastname: Gong
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 210009, P.R. China. hyh@njtech.edu.cn.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Nanobody Engineering and Research Center, Nantong, 226010, P.R. China. ywansystemsbiology@gmail.com.
authors.forename: Weijun
authors.initials: W
authors.lastname: Ou
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, P.R. China. ywansystemsbiology@gmail.com.|Jiangsu Nanobody Engineering and Research Center, Nantong, 226010, P.R. China. ywansystemsbiology@gmail.com.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000276:immunology; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D061905:Single-Domain Antibodies; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D061905:Single-Domain Antibodies; D001710:Biotin; D019809:Streptavidin