J Bacteriol Virol.  2010 Sep;40(3):131-143. 10.4167/jbv.2010.40.3.131.

Development of Competitive ELISA for Detection of Avian Metapneumovirus Antibodies in Chicken

• Choi KS
• Kim JW
• Lee EK
• Jeon WJ
• Park MJ
• Lyoo YN
• Kwon JH

• KMID: 2055050
• DOI: http://doi.org/10.4167/jbv.2010.40.3.131

Abstract

Avian metapneumovirus (aMPV) causes an acute and highly contagious upper respiratory tract infection in turkeys and chickens. In this study, a competitive ELISA (C-ELISA) was developed for the detection of antibodies to aMPV in chicken sera and/or their egg yolks. This assay is based on the competitive binding of monoclonal antibody with serum antibodies to recombinant aMPV N protein expressed by a recombinant baculovirus. The C-ELISA showed specificity and sensitivity of 100% and 98.0%, respectively, when compared to the virus neutralization test. In specific pathogen-free chickens experimentally infected with aMPV SC1509 strain, the C-ELISA started to detect antibodies to aMPV as early as 5 days post infection from birds infected with aMPV, while a commercial ELISA kit detected first 10 days post infection. The C-ELISA was similar or superior to a commercial ELISA kit when serum and egg yolk samples collected from chickens on six outbreak farms were tested for diagnosis. The C-ELISA developed in the present work provides a short turnaround time and can be a useful diagnostic and screening tool for aMPV infection in the field.

Keyword

Avian metapneumovirus; ELISA; N protein; Antibody detection

MeSH Terms

Antibodies
Baculoviridae
Binding, Competitive
Birds
Chickens
Egg Yolk
Enzyme-Linked Immunosorbent Assay
Mass Screening
Metapneumovirus
Neutralization Tests
Respiratory Tract Infections
Sensitivity and Specificity
Sprains and Strains
Turkeys
Viruses
Antibodies

Figure

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  Figure 1. RT-PCR amplification of full-length N gene of aMPV from a nasal turbinate sample of aMPV-infected chicken. (A) Schematic diagram for amplification of full-length N gene of aMPV by RT-PCR using primers NF1 and NR1. (B) RT-PCR result. M, molecular marker; Lane 1, nasal turninate sample.

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  Figure 2. Expression of recombinant aMPV N protein by the rBac/AMPVN. The cell extracts of expressed protein were either fractionated using SDS-PAGE and then visualized by Commassie blue staining (A) and immunoblotting using anti-aMPV antiserum (B) or titrated by ELISA using mAb12.2.1 (C). Lane 1, normal Sf9 cells; lane 2, Sf9 cells infected with rBac/AMPVN; M, molecular marker. Arrow represents recombinant protein having molecular weight of 44 kDa.

• 

  Figure 3. Competitive binding of mAbs and serum antibodies to the recombinant aMPV N protein in a competitive ELISA. Serum panel comprising 10 aMPV antibody-positive and 10 aMPV antibody-negative chicken sera were used for the ELISA. Filled and empty bars represent positive and negative chicken sera, respectively.

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  Figure 4. Sensitivity and specificity of a competitive ELISA using positive (n=102) and negative (n = 200) chicken sera. Arrow indicates the cutoff value of 50 in the C-ELISA.

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  Figure 5. Detection of anti-aMPV antibodies in experimentally aMPV (subtype B)-infected chickens by a competitive ELISA and comparison with IDEXX-ELISA. Cutoff values of C-ELISA (A) and IDEXX-ELISA are PI value of 50 and S/P ratio of 0.2, respectively.

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