title: Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.
abstract: The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Aneela
authors.initials: A
authors.lastname: Fatima
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Wang
authors.affiliation: Guangzhou Wondfo Biotech Co., Ltd, Scientific City, Guangzhou, PR China.
authors.forename: Keren
authors.initials: K
authors.lastname: Kang
authors.affiliation: Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, PR China.
authors.forename: Liliang
authors.initials: L
authors.lastname: Xia
authors.affiliation: Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Life Science, General Hospital of The People's Liberation Army, Beijing, PR China.
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D002846:Chromatography, Affinity; D003715:Dengue / Q000175:diagnosis / Q000276:immunology* / Q000821:virology; D003716:Dengue Virus / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D055767:Immobilized Proteins / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D007159:Immunologic Tests / Q000379:methods*; D007700:Kinetics; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D017510:Protein Folding; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D055767:Immobilized Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C079340:NS1 protein, Dengue virus type 2; D010455:Peptides; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins
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