DEVELOPMENT OF AN ELISA SYSTEM FOR THE DETECTION AND STRAIN IDENTIFICATION OF LASSA AND EBOLA VIRUSES USING MONOCLONAL ANTIBODIES

An ELISA was developed which was able to detect Lassa and Ebola virus antigens in supernatant fluids from infected Vero cells and in animal sera, using monoclonal antibodies. Plaque infectivity titers in these fluids were as low as 10('3) PFU/ml, a level of virus comparable to that found in the blood of infected patients. By selecting a monoclonal antibody specific for the VP3 protein of Ebola mayinga, and a second monoclonal antibody specific for the VP3 protein of E. boniface, these two strains of Ebola virus could be distinguished.;When the test was applied to blood and tissues from experimental animals infected with Lassa or Ebola virus, Lassa josiah antigen was reliably detected in specimens containing more than 10('7) PFU/ml. Experiments were conducted to explore reasons for this loss of sensitivity, but no definite conclusions could be made. Results suggest that E. mayinga antigen was detected in a formalin-inactivated guinea pig serum originally containing 10('3.17) PFU/ml, but not in a duplicate serum inactivated by gamma irradiation.;Attempts were made to develop a competitive binding ELISA for Ebola antigen in which the antibody in known antibody-containing sera was reacted with the solid-phase Ebola antigen (either purified virus or whole Ebola-infected Vero cells). Monoclonal antibody was then added to determine if the antibody binding sites on the viral antigen had been blocked by serum antibodies. Bound monoclonal antibody was detected by the addition of biotinylated anti-mouse IgG and avidin-biotinylated-HRP complex (ABC), but competition was not observed.;Beads coated with antibody by amination and diazotization, and by a second method using a carboiimide reagent and a commercially prepared kit (Bio-Rad), were found to be less satisfactory in capturing viral antigen than immobilizing antigen in filter paper disks contained in the wells of a specially designed 96-well filtration manifold. No difference in test sensitivity was observed when antigen was immobilized in filter paper compared to antigen bound directly to the walls of 96-well microelisa plates. The use of polyclonal capture antibody to trap test antigen served only to increase test background, not test sensitivity.