DETERMINATION OF SENSITIVITY AND SPECIFICITY OF IN-HOUSE SANDWICH ELISA FOR THE DETECTION OF INFECTIOUS BURSAL DISEASE VIRUSES

Abstract

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, hyper-immune 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.DOI = http://dx.doi.org/10.3329/bjvm.v8i2.11185 Bangl. J. Vet. Med. (2010). 8 (2) : 97-106