Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa

Dissertation (MSc)--University of Pretoria, 2013.

The etiological agent of rabies, rabies virus, is a member of the fatal Lyssavirus genus that accounts for the death of more than 55 000 humans per annum, with the number of infected animals far exceeding that number. The process of post-mortem diagnosis of rabies plays a crucial role in general disease surveillance as well as in the implementing and monitoring of disease control programs in animal populations. Although post mortem diagnostic techniques play a crucial role in impeding disease spread, the routine diagnosis of rabies in resource-limited developing countries remains limited due to a lack of stable infrastructures, power supplies, technical expertise and general resources required to perform the routine gold standard fluorescent antibody test (FAT) diagnosis. Based on the aforementioned facts, the development of diagnostic assays that are suitable for application in the resource-limited developing countries has recently gained a lot of consideration, with numerous novel assays being developed and applied in small-scale investigations. Of all the novel diagnostic assays, the direct rapid immunohistochemical test (dRIT) has, to date, shown the most promise in terms of applicability because of its diagnostic sensitivity and specificity, which has been shown to be equal to that of the FAT in five pilot studies. The main drawback with the current application of the dRIT diagnostic assay is that the Centres for Disease Control and Prevention (CDC) are the only supplier of the required cocktail of two-biotinylated monoclonal antibodies. The singular source of biotinylated antibody thus limits the widespread application of the dRIT diagnostic assay because of the limited availability of the cocktail of biotinylated antibodies. This study endeavoured to ascertain whether an alternative antibody preparation could be biotinylated and applied to the dRIT diagnostic assay in order to act as a routine replacement for the cocktail of biotinylated monoclonal antibodies supplied bythe CDC. In order to gain comparative data pertaining to the diagnostic efficacy and versatility of the dRIT diagnostic test relying on the locally produced biotinylated polyclonal antibody, the research involved a multi-faceted investigation. The investigated facets included the comparison of the dRIT test relying on the locally produced biotinylated polyclonal antibody to the FAT test. Apart from the comparison to the gold standard FAT, the three versions of the dRIT test, each relying on one of threebiotinylated antibodies used in the study was performed. The antibodies involved in the comparison included the locally produced biotinylated polyclonal antibody preparation as well as the two-biotinylated monoclonal antibodies (monoclonal antibody 1 and monoclonal antibody 2) that make up the antibody cocktail supplied by the CDC. Apart from the said investigation into the diagnostic efficacy of the dRIT diagnostic assay, the versatility of the given assay was also investigated by adapting the standard operating procedure to accommodate an acetone fixation step. The sample set used for the study included a significant number of central nervous system (CNS) tissues samples (n=250) derived from five of the major mammalian reservoir species in southern Africa as well as a subset of CNS tissue samples derived from mice inoculated with seven representative African rabies-related lyssavirus isolates. The results indicated that the dRIT diagnostic assay, relying on the biotinylated polyclonal antibody preparation, had a diagnostic sensitivity (100%) and specificity (100%) that was marginally higher than that of the widely recognised gold standard FAT diagnostic test that had produced a single false negative result (diagnostic sensitivity of 99,5%) once applied to the known true positive and negative samples included in the study. The dRIT diagnostic test, relying on either of the two-biotinylated monoclonal antibodies, had reduced levels of diagnostic efficacy compared to the FAT assay in terms of the sensitivity of the given assays (monoclonal antibody 1: 83,08% and monoclonal antibody 2: 90,55%) once applied to the known true positive and negative samples included in the study. Monoclonal antibody 2, once applied to the dRIT assay in this study, was the only antibody to produce a single false positive result (diagnostic specificity of 97,96%). Theadaptation of the dRIT protocol to include the acetone fixation step had no influence on the diagnostic efficacy of the dRIT test, while the results of the study were indicative of the fact that the dRIT diagnostic assay could be used to detect the viral antigen of all the representative rabies-related viruses, irrespective of the biotinylated antibody used. While the data obtained from the study was used to interpret the diagnostic efficacy of the various biotinylated antibodies applied to the dRIT diagnostic assay, a simulation framework was also developed to analyse the costs involved in performing routine rabies diagnosis with either the FAT or dRIT diagnostic tests in order to broaden the scope of the research. The only cost in the simulation framework that resulted in a significant difference between the two assays was the capital expenditure required to set up a new diagnostic facility, with the costs indicating that five dRIT diagnostic facilities could be established for the price of one FAT diagnostic facility. In summary, the work presented in this study has shown that not only is it indeed possible to apply alternative biotinylated antibody preparations to the dRIT diagnostic assay, but that it is also necessary to optimize the concentration of the biotinylated antibody preparation of the dRIT diagnostic assay before routine application can occur. In the case of this specific study, the dRIT diagnostic assay relying on the biotinylated polyclonal antibody preparation was shown to be an ideal complimentary diagnostic assay to the FAT due to its high diagnostic efficacy, adaptability and calculated costs.

Microbiology and Plant Pathology

MSc

Unrestricted