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.

Andre Coetzer,Charles E Rupprecht,Claude T Sabeta,Louis H Nel,Wanda Markotter

doi:10.1371/journal.pntd.0003189

Abstract

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.

Highlights

Rabies is a neglected zoonosis that is responsible for the death of tens of thousands of people per annum [1]
We have evaluated a modification of the direct rapid immunohistochemical test (dRIT) in which a polyclonal antibody preparation was biotinylated and compared to the monoclonal antibodies used for the development of all subsequent experimental applications of the dRIT to date
We conclude that the dRIT is a superior test for rabies diagnosis that is adaptable to tolerate the use of different antibody preparations

Summary

Introduction

Rabies is a neglected zoonosis that is responsible for the death of tens of thousands of people per annum [1]. The majority of human rabies deaths are associated with canine rabies in resourcelimited countries. Rabies is caused by multiple lyssaviruses (Genus: Lyssavirus, Family: Rhabdoviridae), of which the prototype is rabies virus (RABV). While RABV is most important from a global disease perspective, there are more than 12 other lyssavirus species, most of which have been associated with infrequent cases of human rabies [2,3]. Classical rabies has the highest known case-fatality rate of any infectious disease, and is preventable by means of effective pre- and post-exposure prophylaxis, the disease is still widespread throughout developing countries on the African and Asian continents [1,4,5,6,7]. The process of post-mortem diagnostic confirmation of rabies plays a crucial role in general disease surveillance and is involved in disease management programs for animal populations (e.g. identifying disease outbreaks within geographical regions where dog vaccination campaigns are being implemented), as well as in risk assessments for consideration of human prophylaxis

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