African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 4982

Full Length Research Paper

Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

Mariana Monezi Borzi*
  • Mariana Monezi Borzi*
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Ketherson Rodrigues Silva
  • Ketherson Rodrigues Silva
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Maria de Fatima Silva Montassier
  • Maria de Fatima Silva Montassier
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Filipe Santos Fernando
  • Filipe Santos Fernando
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Maria de Lourdes Feres Tamanine
  • Maria de Lourdes Feres Tamanine
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Romeu Moreira dos Santos
  • Romeu Moreira dos Santos
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Elisabete Schirato de Oliveira
  • Elisabete Schirato de Oliveira
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Viviane Casagrande Mariguela
  • Viviane Casagrande Mariguela
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Priscila Diniz Lopes
  • Priscila Diniz Lopes
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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Dilmara Reischak
  • Dilmara Reischak
  • National Agricultural Laboratory (LANAGRO), Campinas, São Paulo, Brazil.
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Andre Oliveira Mendonca
  • Andre Oliveira Mendonca
  • National Agricultural Laboratory (LANAGRO), Campinas, São Paulo, Brazil.
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Renato Luis Luciano
  • Renato Luis Luciano
  • Advanced Center for Technological Research of Poultry Agribusiness /Biological Institute, Descalvado, São Paulo, Brazil.
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Helio Jose Montassier
  • Helio Jose Montassier
  • Laboratory of Immunology and Virology, Department of Veterinary Pathology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil.
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  •  Received: 06 December 2016
  •  Accepted: 23 March 2017
  •  Published: 14 May 2017

Abstract

Avian influenza (AI) causes significant impact on industrial poultry farming, besides infecting a variety of vertebrates. The detection of antibodies against viral antigens by serological methods is important for the epidemiology, control and prevention of AI because their high simplicity and speed for assaying a large number of samples. Obtaining antigenic preparations used for detection of anti-avian influenza virus (AIV) antibodies usually requires complex and expensive procedures and Escherichia coli system expression may be an alternative. The nucleoprotein (NP) of AIV is an ideal antigen candidate because it is highly conserved across AIV strains, resulting in high cross-reactivity and immunogenicity for avian hosts. The NP gene segment was cloned and expressed from AIV isolate H4N6 in E. coli fused to a small ubiquitin-like modifier (SUMO) polypeptide and a poly-histidine tag, obtaining a soluble recombinant NP (rNP) containing the most important epitopes. After purification, the rNP was used as an antigen to develop an indirect rNP-enzyme-linked immunosorbent assay (ELISA) to effectively detect anti-AIV antibodies in chicken serum samples. This rNP-ELISA had high sensitivity (95%), specificity (97%), accuracy (96.7%) and agreement (k=0.88) in a comparative analysis with a commercial ELISA kit. The results suggest that rNP-ELISA offers a viable alternative to improve immunodiagnosis of AIV infection in chickens.

Key words: Escherichia coli system expression, small ubiquitin-like modifier (SUMO)-peptide, immunodiagnosis, poultry.