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

Kang-Seuk Choi; Jin-Won Kim; Eun-Kyoung Lee; Woo-Jin Jeon; Mi-Ja Park; Yeh-Na Lyoo; Jun-Hun Kwon

doi:10.4167/jbv.2010.40.3.131

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Choi, Kim, Lee, Jeon, Park, Lyoo, and Kwon: Development of Competitive ELISA for Detection of Avian Metapneumovirus Antibodies in Chicken

Original Article

J Bacteriol Virol 2010;40(3):131-143.

Published online: 18 January 2010

DOI: https://doi.org/10.4167/jbv.2010.40.3.131

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

Kang-Seuk Choi^∗, Jin-Won Kim, Eun-Kyoung Lee, Woo-Jin Jeon, Mi-Ja Park, Yeh-Na Lyoo, Jun-Hun Kwon

Avian Diseases Division, National Veterinary Research and Quarantine Service, Anyang, Korea

^∗Corresponding author: Kang-Seuk Choi, DVM, PhD. 175 Anyangro, Manan-gu, Anyang, Gyeonggi 430-757, Korea. Phone: +82-31-467-1821, Fax: +82-31-467-1814 e-mail: kchoi0608@korea.kr

^∗∗ This work was supported by a grant of the National Veterinary Research and Quarantine Service (B-AD15-2008-07-01), Korea.

Revised 30 March 20109 July 2010 Accepted 15 July 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Avian metapneumovirus, ELISA, N protein, Antibody detection

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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.

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.

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.

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.

Table 1.

Detection of antibodies to aMPV in clinical samples of infected chicken farms in Korea by competitive ELISA

Farm^a	SHS^b	Age (w.o)	Serum		Yolk
Farm^a	SHS^b	Age (w.o)	C-ELISA	IDEXX-ELISA	C-ELISA	IDEXX-ELISA
KS (B)	Yes	32	38/40 (95%)^c	39/40 (98%)	NT	NT
SJ (B)	Yes	30	NT	NT	9/15 (60%)	10/15 (67%)
SD (A)	Yes	35	NT	NT	12/30 (40%)	11/30 (37%)
HM (A)^d	No	16	9/10 (90%)^∗	4/10 (40%)^∗	NT	NT
		20	10/10 (100%)	10/10 (100%)	NT	NT
PA (B)	Yes	30	NT	NT	21/30 (70%)	23/30 (77%)
CS (B)	No	65	18/20 (90%)	19/20 (95%)	29/30 (97%)	28/30 (93%)

^a Letter in parenthesis represents subtype of aMPV at the time of diagnosis. aMPV subtype was determined by phylogenetic analysis based on the sequence of G protein gene of aMPV (7).

^b SHS represents swollen head syndrome observed in affected birds at the time of diagnosis.

^c No. positive/No. tested (percent positive). NT, not tested.

^d HN farm was diagnosed as aMPV infection at the 16 weeks old and 4 weeks later re-sampled from the same chicken flock.

^∗ Values between C-ELISA and IDEXX-ELISA differ significantly (Duncan test, p < 0.05)

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