Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Woo-Jin Jeon; Byung-Sik Chang; Eun-Kyoung Lee; Mi-Ja Park; Hoo-Don Joo; Jun-Hun Kwon; Kang-Seuk Choi

doi:10.4167/jbv.2008.38.3.149

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Jeon, Chang, Lee, Park, Joo, Kwon, and Choi: Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Original Article

J Bacteriol Virol 2008;38(3):149-159.

Published online: 18 January 2008

DOI: https://doi.org/10.4167/jbv.2008.38.3.149

Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Woo-Jin Jeon¹, Byung-Sik Chang², Eun-Kyoung Lee¹, Mi-Ja Park¹, Hoo-Don Joo², Jun-Hun Kwon¹, Kang-Seuk Choi^1,^∗

¹National Veterinary Research Institute, National Veterinary Research and Quarantine ServiceAnyang, Gyeonggi, Republic of Korea

²JenoBiotech Inc, Chuncheon, Gangwon, Republic of Korea

^∗Corresponding author: Kang-Seuk Choi, DVM, MS, PhD. Veterinary Research Institute, National Veterinary Research & Quarantine Service 335 Jungang-ro, Manan-gu, Anyang-si, Gyeonggi 430-824, Republic of Korea. Phone: +82-31-467-1821, Fax: +82-31-467-1814 e-mail: choiks@nvrqs.go.kr

^∗∗ This work was supported by a grant (M-AD15-2007-07-01) from National Veterinary Research & Quarantine Service.

Revised 16 June 200825 August 2008 Accepted 2 September 2008

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

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease of chicken. Agar gel immunodiffusion using IBDV antigen extracted from bursa of Fabricius of infected chicken has been used officially for diagnosis of IBDV in Korea. In this study, in order to replace the IBDV whole virus antigen with non-infectious antigen, recombinant VP2 protein (rVP2) of IBDV was produced using recombinant baculovirus expression system. Purified baculovirus-expressed rVP2 was used as an antigen in an agar gel immunodiffusion (AGID). rVP2 antigen precipitated specifically IBDV antibodies. AGID using rVP2 antigen detected anti-IBDV antibodies from 6 dpi to 28 dpi (termination of the experiment) when specific pathogen free chickens were experimentally infected with IBDV 52/70 strain. This was consistent with result by AGID using IBDV antigen, virus neutralization test (VNT) and a commercial ELISA kit (except for one serum). The sensitivity of rVP2 was the same with that of IBDV antigen when field sera (n=324) were tested by AGID. However, AGID using rVP2 antigen detected maternal antibodies from broiler chickens (n=20) on a broiler farm up to 15 days old, although the detection rate of the AGID was relatively low compared to a commercial ELISA kit. Our results indicate that IBDV whole virus antigen from IBDV infected chickens would be replaced with recombinant VP2 protein as an antigen for AGID.

Keywords: AGID, Antibody detection, Infectious bursal disease, VP2

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Figure 1.

Immunofluorescence staining of IBDV VP2 protein expression. The Sf9 cells infected with recombinant baculovirus (Bac/IBDVP2) were stained using chicken anti-IBDV antiserum (A), IBDV-specific monoclonal antibody R63 (B) and IBDV antibody negative chicken serum (C).

Figure 2.

Analysis of purified IBDV VP2 protein expressed from Sf9 cells by recombinant baculovirus (Bac/IBDVP2). A: SDS-PAGE analysis of purified IBDV VP2 protein fractions F1 (103 μg/ml), F2 (665 μg/ml), F3 (275 μg/ml) and F4 (98 μg/ml); B: Western blot analysis of purified IBDV VP2 protein fraction F2 using IBDV VP2-specific monoclonal antibody R63 (lane 1) and VP3-specific monoclonal antibody B29 (lane 2).

Figure 3.

Titration of IBDV (A) or recombinant IBDV VP2 protein (B) by sandwich ELISA using monoclonal antibody R63 and anti-IBDV chicken serum.

Figure 4.

Detection of anti-IBDV maternal antibodies in broiler measured by AGID test using recombinant IBDV VP2 protein.

Table 1.

Detection of anti-IBDV antibodies in SPF chickens following experimental infection by AGID assays

DPI	ELISA^a	VNT^b	AGID
DPI	ELISA^a	VNT^b	IBDV	rVP2
0	0/10^c	0/10	0/10	0/10
3	0/4	0/4	0/4	0/4
6	6/6	5/6	5/6	5/6
9	6/6	6/6	6/6	6/6
14	7/7	7/7	7/7	7/7
28	7/7	7/7	7/7	7/7

^a Serum with ELISA titers of ≥396 was considered positive.

^b Serum with VN titers of ≥8 was considered positive.

^c No. Positive/No. tested

Table 2.

Detection of anti-IBDV antibodies in SPF chickens following experimental infection by AGID assays

Group	Age	Serum no. tested	AGID
Group	Age	Serum no. tested	IBDV	rVP2
Broiler	1 day old	155 (3)^a	155/155^b	155/155
Boiler	25 to 34 days old	119 (6)	119/119	119/119
Broiler breeder	18 to 75 months old	50 (5)	49/50^c	49/50^c

^a Number in parenthesis represents number of farm tested.

^b No. Positive/No. tested.

^c One serum with negative result in AGID using rVP2 antigen was also negative for AGID using IBDV antigen.

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