title: Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA.
abstract: Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID.
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: Devyani
authors.initials: D
authors.lastname: Sengupta
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: Asma
authors.initials: A
authors.lastname: Shaikh
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India. Electronic address: sandeep.bhatia@hsadl.in.
authors.forename: S
authors.initials: S
authors.lastname: Bhatia
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Pateriya
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: R
authors.initials: R
authors.lastname: Khandia
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: R
authors.initials: R
authors.lastname: Sood
authors.affiliation: Department of Biotechnology and Bioinformatics, Barkatullah University, Bhopal, Madhya Pradesh 462021, India.
authors.forename: A
authors.initials: A
authors.lastname: Prakash
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: B
authors.initials: B
authors.lastname: Pattnaik
authors.affiliation: High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: H K
authors.initials: HK
authors.lastname: Pradhan
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D061505:Cell Surface Display Techniques; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology; D005585:Influenza in Birds / Q000175:diagnosis*; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014758:Viral Core Proteins
keywords: AIV; ELISA; Nucleoprotein; Recombinant antibody; ScFv