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Determination of Sensitivity and Specificity of In-house Sandwich Elisa for the Detection of Infectious Bursal Disease Viruses

P K Saha
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M H Ali
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M B Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M A Islam
School of Sustainable Agriculture, University Malaysia Sabah, 88999, Kota Kinabalu, Sabah, Malaysia

Abstract/ Executive Summary:

The study was designed for the development of an In-House sandwich ELISA as a suitable serological method for the rapid detection of infectious bursal disease virus (IBDV). The test was also designed to compare and evaluate its sensitivity and specificity with other traditional methods used for the detection of IBDV from field outbreak cases prevalent among the poultry population of Bangladesh. To develop the In-House sandwich ELISA, hyper-immune serum was raised against live IBDV vaccine in rabbit which was used to coat each of the 96-well flat bottomed polystyrene microtitre plate whereas, hyperimmune sera raised in chickens against IBDV used as secondary antibody. The newly developed In-House sandwich ELISA was standardized by dispensing different dilutions (10-1 up to 10-4) of rabbit serum. Among them, the 10-2 dilution of serum showed most suitable reading for the detection of IBD virus and used to coat the plate to evaluate its sensitivity and specificity. Sensitivity test was done by different dilutions (10-0 to 10-4) of reference IBD virus. The virus dilution, 10-3 was the highest dilution having lowest capacity to bind with coated antibody of the ELISA plate which indicated that IBD viruses was absent in the dilutions of above 10-3. The cut-off value of negative control samples was determined as 0.937 which indicated titer of tested samples >0.937 was positive and <0.937 was negative. Specificity test was performed using different known viruses (IBDV and NDV) using different dilutions (10-1 up to 10-4). Only the IBDV showed positive result which indicated high specificity of newly developed ELISA plate. A total of 26 samples (feces, cloacal swab, spleen and bursa) from control group, experimental and natural IBDV outbreaks were used as field viral antigen for the evaluation of sensitivity and specificity of the newly developed In-House sandwich ELISA. In case of experimental infection, 5 (62.5%) of 8 feces sample but none of cloacal swab were positive for IBDV whereas, all bursa and spleen samples were positive by both In–House sandwich ELISA and AGIDT. In case of natural outbreak cases, 6 of 6 bursal samples and 4 of 6 spleen samples were positive by In-House sandwich ELISA whereas, AGIDT detected all bursal and 3 spleen samples. No virus was detected from the samples of control group. The result showed 92.85% specificity of the developed sandwich ELISA for detection of IBDV with AGIDT which indicated that the developed ELISA is a sensitive, specific, cost effective and reliable tool for the detection of IBDV antigen from a large number of field samples.

Keywords: Sandwich ELISA, AGIDT, Virus

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Virus

Objectives:

This paper describes the development of an In-House sandwich ELISA for rapid detection of IBDV antigens from large number of field samples and comparison of sensitivity with the agar gel immunodiffusion test (AGIDT) in detecting IBDV antigen using field and laboratory samples.

Methodology:

The New Zealand white rabbits (n=4) were vaccinated with live IBD vaccine (BAL-IBD EM from BESTAR) on day 7, 14 and 21 through S/C route @ 0.5 ml/rabbit and blood was collected prior to first and during each vaccination. The separated serum was checked with reference IBD virus by AGIDT for IBDV antibodies and positive sera were used as coating antibody and positive control. The serum collected from one non-vaccinated control rabbit was screened for the antibodies against IBDV and used as negative control.ISA Brown chickens (n=4) of two months old were vaccinated followed by blood collection thrice at day 7, 14 and 21 with live IBDV vaccine through ocular route. The separated sera samples were checked for anti-IBDV antibodies by AGIDT against reference IBDV antigen and positive sera were pooled to use as secondary antibody and positive control. For negative control serum blood was collected at day 28 from two non-vaccinated birds and checked by AGIDT to confirm the absence of antibody. All the sera samples were preserved at -20⁰C in the screw-capped vial until used.The statistical analysis to compare the specificity between the newly developed In-House sandwich ELISA and agar gel immunodiffusion tests was done according to the statistical formula given by Samad et al. (1994). The statistical formula was used as described below.

Source of Information: Bangl. J. Vet. Med. (2010). 8(2): 97 – 106

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

From the above findings the present study may be concluded that studies of the molecular epidemiology of IBDV are important and the In-House sandwich ELISA could be used as an alternative technique for screening a large number of samples before testing and also for the confirmation of the IBDV quickly from a large number of IBD suspected field samples. If it is produced commercially in a country it can be a valuable tool for the detection of IBDV virus with minimum cost and it is highly reliable like other procedures of IBDV isolation and detection such as AGIDT, molecular detection.

Knowledge Management: Journal