title: Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library.
abstract: The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10(9). Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin-streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS-ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.
authors.affiliation: Institute of Life Sciences, Southeast University, Nanjing 210096, People's Republic of China.
authors.forename: Xue
authors.initials: X
authors.lastname: Gong
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Li
authors.affiliation: National Center for International Research Targeting Diagnosis and Therapy, Guangxi Medical University, Nanning, Guangxi 530021, People's Republic of China; Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research, Guangxi Medical University, Nanning, Guangxi 530021, People's Republic of China; Department of Immunology, Guangxi Medical University, Nanning 530021, People's Republic of China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Lu
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China. Electronic address: ykwan@simm.ac.cn.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001710:Biotin / Q000737:chemistry*; D002162:Camelus; D004797:Enzyme-Linked Immunosorbent Assay; D057917:Influenza A Virus, H7N2 Subtype / Q000276:immunology / Q000302:isolation & purification*; D061905:Single-Domain Antibodies / Q000276:immunology*
chemical_list: D061905:Single-Domain Antibodies; D001710:Biotin
keywords: Influenza H7N2 virus; Phage library; Sandwich ELISA; VHH antibody