Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA

J Virol Methods. 2014 Nov:208:129-37. doi: 10.1016/j.jviromet.2014.08.009. Epub 2014 Aug 22.

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.

Keywords: AIV; ELISA; Nucleoprotein; Recombinant antibody; ScFv.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Viral / blood*
  • Cell Surface Display Techniques
  • Chickens
  • Enzyme-Linked Immunosorbent Assay / methods
  • Escherichia coli / genetics
  • Gene Expression
  • Influenza A Virus, H5N1 Subtype / immunology
  • Influenza in Birds / diagnosis*
  • Mice, Inbred BALB C
  • Nucleocapsid Proteins
  • RNA-Binding Proteins* / genetics
  • RNA-Binding Proteins* / immunology
  • RNA-Binding Proteins* / isolation & purification
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Sensitivity and Specificity
  • Single-Chain Antibodies* / genetics
  • Single-Chain Antibodies* / immunology
  • Single-Chain Antibodies* / isolation & purification
  • Viral Core Proteins* / genetics
  • Viral Core Proteins* / immunology
  • Viral Core Proteins* / isolation & purification

Substances

  • Antibodies, Viral
  • NP protein, Influenza A virus
  • Nucleocapsid Proteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • Viral Core Proteins