Journal List > J Bacteriol Virol > v.38(3) > 1033899

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

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.

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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).
jbv-38-149f1.tif
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).
jbv-38-149f2.tif
Figure 3.
Titration of IBDV (A) or recombinant IBDV VP2 protein (B) by sandwich ELISA using monoclonal antibody R63 and anti-IBDV chicken serum.
jbv-38-149f3.tif
Figure 4.
Detection of anti-IBDV maternal antibodies in broiler measured by AGID test using recombinant IBDV VP2 protein.
jbv-38-149f4.tif
Table 1.
Detection of anti-IBDV antibodies in SPF chickens following experimental infection by AGID assays
DPI ELISAa VNTb AGID
IBDV rVP2
0 0/10c 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
IBDV rVP2
Broiler 1 day old 155 (3)a 155/155b 155/155
Boiler 25 to 34 days old 119 (6) 119/119 119/119
Broiler breeder 18 to 75 months old 50 (5) 49/50c 49/50c

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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