Development and Comparison of Capture Enzyme Linked Immunosorbent Assay and Indirect Immunofluorescent Test in Determination of Nairobi Sheep Disease Virus.

Abstract

The diagnosis of Nairobi Sheep Disease relies on the inoculation of tissue culture, Baby Hamster kidney (BHK-21), with suspensions of infected samples followed by identification of the virus using indirect Immuno-fluorescent assay. These tests have a number of drawbacks including low specificity; visual reading of results which requires highly skilled expertise and tissue culture facilities therefore development of capture Enzyme linked Immuno Sorbent assay (ELISA) would improve the diagnosis of NSD in infected sheep. Nairobi Sheep Disease Virus (NSDV) was isolated, purified and titrated to determine the best working titer for immunization of animals. The purified virus subjected to IIFA test and fluorescence indicated the presence of NSDV. The animals immunized were rabbits and goat which were used for production of antibodies for C-ELISA test. C-ELISA was set-up using anti-goat sera as the primary antibody, purified NSDV as antigen and anti-rabbit sera as the secondary antibody. A 1:400 dilution was established as best dilution for true positive and negative samples.  The diagnostic specificity and specificity of the developed C-ELISA was estimated. False positive samples were picked by IIFA, which was confirmed by tissue culture technique. The level of agreement between developed C-ELISA and IIFA used as a gold test was 95%, and the Kappa index was 0.86. The perfect agreement indicated by Kappa values is a sign that both tests can be used. However, C-ELISA is a better test in that it is more flexible and less subjective. The sensitivity and specificity of C-ELISA was estimated at 80% and 100% respectively. The results showed high diagnostic specificity of developed C- ELISA which could be adapted to test a large number of samples over short periods of time. The test is useful during outbreaks of NSD without need for tissue culture facilities.

Keywords: Tissue culture, Capture Enzyme linked Immuno Sorbent assay, Nairobi Sheep Disease Virus, Kappa index.