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Development of an Enzyme-Linked Immunosorbent Assay for Measuring

Antibodies in Chicken Serum against Infectious Bursal Disease Virus

Article · January 2003

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Bangladesh Vet. J. (2003) 37(1-4):29-36

Development of an Enzyme-Linked Immunosorbent Assay

for Measuring Antibodies in Chicken Serum
against Infectious Bursal Disease Virus

K.M. Islam1, M.N. Islam2, A.S.M. Bari and M.R. Islam

Department of Pathology, Faculty of Veterinary Science
Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

ABSTRACT
An indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring
antibodies against infectious bursal disease virus (IBDV). For the preparation of ELISA antigen, a
molecularly engineered tissue culture adapted IBDV (BD-3tc) was grown in primary chicken
embryo fibroblast cell culture. The virus from the cell culture supernatant was concentrated (one
hundred fold) with polyethylene glycol and used as the antigen without further purification.
Pools of positive and negative control sera were used for standardization of the test. The
optimum dilutions of the antigen, antibody and conjugate were determined empirically. A
standard curve equation was derived to predict antibody titre from the ELISA absorbance value
of a single dilution (1:1000) of the serum. The developed ELISA was tested on 46 serum samples
collected from chickens infected experimentally with different strains of IBDV. The results
obtained with the developed ELISA (BAU ELISA) were compared with those obtained with a
commercial IBDV ELISA kit (IDEXX ELISA). A good correlation (r = 0.87, n = 46, P < 0.01) was
observed between the titres predicted by these two tests. The test can be used for rapid
seroprofiling of chicken flocks for IBDV antibody before and after vaccination.

INTRODUCTION

Infectious bursal disease (IBD) is a contagious disease of young chickens caused by

a virus belonging to the family Birnaviridae (for a review see Lukert and Saif, 1997). In
the recent years a very virulent (vv) pathotype of IBD virus (IBDV) has emerged which
can cause high mortality in chicks (Chettle et al., 1989; van den Berg et al., 1991). The first
outbreak of IBD occurred in Bangladesh at the end of 1992. The virus was isolated and
identified (Chowdhury et al., 1996; Rahman et al., 1996) and pathogenicity of a local
isolate was studied (Islam et al., 1997). Recently, several Bangladeshi strains of vvIBDV
have been characterized at antigenic and molecular level (Islam et al., 2001a, 2001b). One

Present address: 1Department of Pathology, Sylhet Govt. Veterinary College, Sylhet; 2Department of
Pathology, Dinajpur Govt. Veterinary College, Dinajpur

Copyright © Bangladesh Veterinary Association All rights reserved 0030-9915/03

ELISA for measuring antibodies to IBDV

of the strains (BD 3/99) has also been adapted to grow in chicken embryo fibroblast
(CEF) cell culture by molecular engineering (Islam et al., 2001c; Raue et al., 2004).
Vaccination along with strict biosecurity is the only means of preventing outbreaks
of IBD. However, vaccination failure has been a common problem in Bangladesh. This
failure may be due to inadequate immune response following vaccination or
neutralization of vaccine virus by maternal antibodies present in the chicks. To
overcome this problem it is essential to measure the antibody level prior to and after
vaccination. Enzyme-linked immunosorbent assay (ELISA) is commonly used to
measure antibodies against IBDV (Marquardt et al., 1980; Tsukamoto et al., 1990). ELISA
kits are available commercially, but these are highly expensive. Therefore, the present
study was undertaken with the objective of developing an ELISA locally for measuring
antibody against IBDV.

MATERIALS AND METHODS

Preparation of ELISA Antigen

Confluent monolayers of primary chicken embryo fibroblast (CEF) cell culture
grown in 75 sq. cm. tissue culture flasks were infected with a molecularly engineered
tissue culture adapted vvIBDV (BD-3tc) (Islam et al., 2001c; Raue et al., 2004). When
maximum cytopathic effects (CPE) had developed, the infected culture supernatant was
harvested after five cycles of freezing and thawing and clarified by centrifugation at
3000 rpm for 20 minutes. The virus was concentrated from the culture supernatant by
polyethylene glycol precipitation method (Gould and Clegg, 1985). Briefly, the clarified
culture fluid was taken in a conical flask and placed in an ice-bath on a magnetic stirrer.
Sodium chloride was added to the culture fluid to a final concentration of 0.5M and
dissolved by stirring. Then polyethylene glycol 6000 was added @ 7% and stirred for 4
hours on ice. The suspension was left overnight at 40C. To recover the precipitated
antigen, the suspension was centrifuged at 3000 rpm for 2 hours. The supernatant was
discarded and the pellet was resuspended thoroughly to 1% of the original volume in
TNE buffer (40 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.6). The concentrated antigen
was centrifuged again at 3000 rpm for 10 minutes to remove undissolved particles, if
any. The supernatant was collected, divided into small aliquots and stored frozen at –
200C until used.

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 K.M. Islam et al., 2003

Positive and negative serum samples

A total of 46 IBDV-specific positive serum samples were collected at different time
intervals from chickens infected experimentally at 4 weeks of age with three different
strains of IBDV for a pathogenicity trial (Raue et al., 2004). Five negative sera were
obtained from 35 days old unvaccinated and uninfected control birds from the same
experiment.

Development of ELISA protocol

Development of the ELISA protocol was based on a standard procedure of indirect
ELISA. Briefly, the IBDV antigen was diluted in carbonate/ bicarbonate buffer (pH 9.6)
and dispensed to each well of a 96-well ELISA plate. After incubation at 370C for 60 min
followed by overnight at 40C, the excess antigen was removed and the plate was washed
5 times with a washing buffer (PBS containing 0.05% Tween 20). Uncoated sites of the
wells, if any, were blocked with 1% bovine serum albumin (BSA). The plate was
incubated for 30 min at 370C. Excess BSA was removed and the serum samples, diluted
appropriately in a diluent solution (PBS containing 0.1% Tween 20), were added to the
wells. The plate was incubated for 60 min at 370C. Excess sample was removed and the
plate was washed as before. Then alkaline phosphatase conjugated goat anti-chicken
IgG (Sigma, USA) diluted optimally in the diluent solution was added to each well. The
plate was incubated for 60 minutes at 370C. After washing as before, substrate solution
(0.1% p-nitrophenyl phosphate in diethanolamine buffer) was added to each well and
incubated for 30 minutes at room temperature in dark place. The reaction was stopped
with 3M NaOH solution added to each well. The intensity of color development was
measured at 405 nm with an ELISA reader (SPECRA max 340, Molecular Devices Inc.,
USA). All reagents were added in 100 µl volume per well except the stop solution, which
was added at 50 µl volume. The optimum dilutions of the antigen, test sera and the
conjugate were determined empirically by checkerboard titration.

Prediction of antibody titre

A standard curve equation was derived to predict the antibody titre of a serum from
the absorbance values obtained at a single (1:1000) dilution of the serum (Snyder et al.,
1984; Islam and Jones, 1988). For this, six serum samples having a range of absorbance
values (0.19 to 1.04) were selected. The end-point ELISA titre of each of the six sera was
determined by serial 10-fold dilution. The end-point titres of these six sera and the
corresponding absorbance values at 1:1000 dilutions were subjected to correlation-

31
ELISA for measuring antibodies to IBDV

regression analysis to derive a standard curve equation. This equation was used for
predicting titre of a serum from the absorbance value at a 1:1000 dilution.

Validation of test
To validate the newly developed ELISA (BAU ELISA), 46 positive sera were tested
by BAU ELISA as well as with a commercial ELISA kit (IDEXX ELISA, IDEXX
Laboratory, Inc. Westbrook, Maine 04092, USA). The IDEXX ELISA was performed
according to the manufacturer’s instruction and the antibody titre was determined using
the formula supplied with the kit. The correlation between the titres predicted by the
two systems was examined by correlation-regression analysis.

RESULTS

Optimum dilutions of reagents

The optimum dilutions for the present lot of antigen and the conjugate, as
determined by repeated chequerboard titration, were found to be 10-6 and 10-3,
respectively. The test sera could be used at 1:100 dilution, but the best discrimination
between the ELISA absorbance values of the positive and negative sera were obtained at
a dilution of 1:000 or above (data not shown).

Prediction of antibody titre from single dilution of serum

The six selected sera were titrated in ten-fold serial dilutions. The positive-negative
threshold (PNT) baseline was drawn as the mean plus thrice the standard deviation of
the absorbance values of the five negative serum samples measured separately at 1:100
and 1:1000 dilutions. To determine the end-point titre, serial absorbances of each serum
were plotted against log10 corresponding serum dilution. The point at which absorbance
curve intersected the PNT baseline was considered as the end-point titre.
Correlation regression analysis was performed using the absorbance values of six
sera in the single dilution (1:1000) and the log-transformed end-point titre. The results
are shown in Fig. 1. A good positive correlation was observed (r = 0.995, n=6, P < 0.01).
The slope was 1.37 and the Y-intercept was 2.23. So, the standard curve equation for
prediction of antibody titre from the absorbance value at a 1:1000 dilution was as
follows:
Log 10 titre = 1.37 (Absorbance 1:1000) + 2.23

32
 K.M. Islam et al., 2003

ELISA titre (Log 10) 4.0

3.5

3.0

2.5

2.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
ELISA Absorbance at 1:1000 dilution

Fig. 1. Standard curve for predicting ELISA antibody titre from the absorbance value
measured at a single dilution (1:1000) of a serum (r = 0.995, n = 6, P<0.01)

The original end-point titres and the predicted titres of the 6 selected sera are shown
in Table 1. Both the titres were very close for each serum, not exceeding log10 0.06.

Table 1. IBDV antibody titres of six different sera determined by different methods from
absorbance values measured in a single dilution (1:1000)
Sample No. Absorbancevalues at End-point titre Predicted titre
1:1000 dilution (Log10) (Log10)
1 0.56 3.05 2.99
2 0.19 2.45 2.49
3 1.04 3.65 3.66
4 0.35 2.70 2.71
5 0.78 3.25 3.30
6 0.44 2.85 2.83

33
ELISA for measuring antibodies to IBDV

Validation of the test

The ELSA titres of 46 positive sera determined by the newly developed BAU ELISA
and the commercial IDEXX ELISA kit were subjected to correlation-regression analysis.
The results are shown in Fig. 2. There was a significant positive correlation (r = 0.87, n =
46, P < 0.01) between titres determined by the two methods.

DISCUSSION
An indirect ELISA was developed for the detection of antibody against IBDV in
chicken serum. A computational method was also developed for predicting antibody
titre from the absorbance value at a single dilution (1:1000) of the serum. The ELISA titre
of 46 chicken sera predicted by the developed ELISA system (BAU ELISA) correlated
very well with that predicted by a commercial ELISA kit (IDEXX ELISA).

4
"IDEXX" ELISA titre (Log 10)

1
1 2 3 4 5
"BAU" ELISA titre (Log 10)

Fig. 2. Correlation between the titres determined by “IDEXX” and “BAU” ELISA
methods (r = 0.87, n = 46, P < 0.01)

Highly purified IBDV antigen, prepared by density gradient ultra centrifugation, is

commonly used in ELISA (Marquardt et al., 1980; Tsukamoto et al., 1990). Recombinant

34
 K.M. Islam et al., 2003

subunit IBDV antigen also has been used in ELISA (Martinez-Torrecuadrada et al., 2000).
In the present study, the ELISA antigen was prepared from IBDV-infected tissue culture
fluid by polyethylene glycol precipitation, after removal of the cell debris by low-speed
centrifugation. The concentrated antigen was used without any further purification. This
procedure resulted in very good recovery of the antigen, as the antigen can be used at as
high as 10-6 dilution. However, the non-specific reaction with this crude antigen was
relatively high. Nevertheless, a good discrimination between the positive and negative
serum could be achieved. When ten-fold serial dilutions of the positive and negative
serum pools were tested, a dilution of 1:1000 appeared to be the most suitable for the
present ELISA system. At this dilution the discrimination between the positive and
negative serum was the highest.
A standard curve equation was derived for predicting antibody titre from the
absorbance determined at a single dilution (1:1000) of the serum. A strong correlation
was observed (r = 0.995, P < 0.01) between the absorbance values and the end-point
titres. The titres predicted with the standard curve equation were very close to the
actual end-point titre. Similar computational methods have been described earlier
(Snyder et al., 1984; Islam and Jones, 1988).
The ELISA titres predicted by BAU ELISA were in general slightly higher than that
predicted by IDEXX ELISA. This was probably the reflection of higher background
reaction in the BAU ELISA because of the use of crude antigen. However, a very good
correlation (r = 0.87, n = 46, P < 0.01) was observed between the ELISA titres determined
by these two systems.
The ELISA system developed in this study offers an important tool for seroprofiling
of chicken flocks for IBDV antibodies before and after vaccination, which is essential to
ensure the success of vaccination programmes. However, still there are scopes for
further fine-tuning of the ELISA system, such as better preparation of antigen and
further optimisation of the test conditions.

REFERENCES
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disease in East Anglia. Vet. Rec. 125:271-272.
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Acute infectious bursal disease in chickens; pathological observation and virus
isolation. Asian-Aus. J. Anim. Sci. 9:465-469.
3. Gould, E.A. and Clegg, J.C.S. 1985. Growth, titration and purification of togaviruses.
In: Virology: A Practical Approach. Edited by B. W. J. Mahy. IRL Press, Oxford, pp. 43-
78.

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 ELISA for measuring antibodies to IBDV

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