Veterinary Research Communications

, Volume 31, Issue 5, pp 631–641 | Cite as

Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus

  • R. Wu
  • S. Hu
  • Y. Xiao
  • Z. Li
  • D. Shi
  • D. BiEmail author


Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.


avian influenza virus full-length nucleoprotein NP-ELISA detection and quantification of antibodies 



agar gel propagation


avian influenza


avian influenza virus


bovine serum albumin


enzyme-linked immunosorbent assay


ELISA titre


glutathione S-transferase




haemagglutination inhibition


immunoglobulin G








optical density


phosphate-buffered saline




sodium dodecyl sulfate-polyacrylamide gel electrophoresis


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Copyright information

© Springer Science + Business Media, Inc. 2007

Authors and Affiliations

  1. 1.Laboratory of Animal Microbiology and Immunology, State Key Laboratory of Agricultural MicrobiologyCollege of Veterinary Medicine, Huazhong Agricultural UniversityWuhanPeople’s Republic of China

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