J Bacteriol Virol.  2008 Sep;38(3):149-159. 10.4167/jbv.2008.38.3.149.

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

• Jeon WJ
• Chang BS
• Lee EK
• Park MJ
• Joo HD
• Kwon JH
• Choi KS

• KMID: 1483973
• DOI: http://doi.org/10.4167/jbv.2008.38.3.149

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.

Keyword

AGID; Antibody detection; Infectious bursal disease; VP2

MeSH Terms

Agar
Animals
Antibodies
Baculoviridae
Bursa of Fabricius
Chickens
Enzyme-Linked Immunosorbent Assay
Immunodiffusion
Infectious bursal disease virus
Korea
Neutralization Tests
Specific Pathogen-Free Organisms
Sprains and Strains
Staphylococcal Protein A
Viruses
Agar
Antibodies
Staphylococcal Protein A

Figure

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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).

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  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).

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  Figure 3. Titration of IBDV (A) or recombinant IBDV VP2 protein (B) by sandwich ELISA using monoclonal antibody R63 and anti-IBDV chicken serum.

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  Figure 4. Detection of anti-IBDV maternal antibodies in broiler measured by AGID test using recombinant IBDV VP2 protein.

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