Production and characterisation of monoclonal antibodies to Ebolavirus nucleoprotein and their application in development of a rapid antigen detection test

Abstract

Viruses belonging to Filoviridae family, namely ebolaviruses and marburgviruses, cause filoviral haemorrhagic fever (FHF), resulting in case fatality rates of up to 90 percent depending on the virus species and strain. There has been an increase in the incidence of FHF outbreaks in Africa in the last two decades, with some caused by newly found viruses and others occurring in previously unaffected areas. For early diagnosis of Ebola virus disease (EVD), antigen detection tests are best suited. Ebolavirus nucleoprotein (NP) is ideal for antigen detection because it is highly conserved among ebolaviruses, has strong antigenicity and is abundant in ebolavirus particles. The purpose of this study was to produce and characterise monoclonal antibodies (mAbs) to ebolavirus NP, and with these mAbs develop a specific, sensitive, rapid and easy to use, disposable immunochromatographic strip test for the diagnosis of ebolavirus infections. Plasmids expressing Zaire ebolavirus NP, glycoprotein (GP), and viral protein 40 (VP40) were constructed and transfected into 293T cells to produce recombinant NP and virus-like particles (VLPs). The VLPs were inoculated into mice and a panel of mouse mAbs to Zaire ebolavirus NP was produced. These mAbs were grouped according to their specificity and cross-reactivity to the other known Ebolavirus species. Synthetic peptides were used to map the epitopes recognised by these mAbs, which included two regions highly conserved among the currently known Ebolavirus species. A selected mAb, recognising all known Ebolavirus species on ELISA and Western blot, was then used to develop an immunochromatography-based rapid diagnostic test. This test was able to positively identify Zaire ebolavirus, Tai Forest ebolavirus and Bundibugyo ebolavirus VLPs. Further studies need to be conducted to optimise the test and determine its specificity and sensitivity to live ebolavirus in comparison to other established diagnostic tests. Once validated, this test has great potential for use in the field as a rapid diagnostic tool for EVD