We produced non-recombinant infectious virus-based slurry (for IgM) and lysate (for IgG) antigen preparations for each of the five EBOV species. In order to confirm the presence of EBOV antigen in each viral lysate and slurry antigen preparations, we performed Western blots, using HMAF poly-clonal antibodies, raised against ZEBOV, SEBOV, REBOV, and CIEBOV () and rabbit poly-clonal antibodies, raised against ZEBOV, SEBOV, and REBOV (), as detector antibodies. Although neither detector antibody mixture contained antibodies specifically raised against BEBOV, the presence of reactive nucleoprotein (NP) bands near the 100 kilodalton weight marker, both in the lysate and slurry preparations, indicates the presence of viral antigen in both of the preparations. While only a faint NP band was detected in the SEBOV lysate preparation using the HMAF detector antibody mixture, the presence of antigen was apparent using the rabbit polyclonal antibody mixture. This may suggest an issue in reactivity of the HMAF antibody mixture against the SEBOV lysate preparation. However, we noted the presence of a strong NP band in the SEBOV slurry preparation, using the HMAF antibody mixture, indicating the utility of the HMAF as a detector antibody for the SEBOV IgM ELISA assay.
Western blots showing lysate and slurry antigen preparations for each EBOV species.
Samples for this study are convalescent specimens collected as part of diagnostic activities for outbreaks due to ZEBOV (Kikwit, Democratic Republic of Congo, 1995 
), SEBOV (Gulu, Uganda, 2000 
), BEBOV (Bundibugyo, Uganda, 2007 
), and REBOV (Philippines 
) (). We quantitatively examined the IgM antibody reactivity to autologous versus heterologous virus antigen by comparing adjusted sum OD values for each of the individual virus slurry antigen preparations, among outbreak samples. While many of the samples, particularly from Kikwit and Bundibugyo, had low IgM titers, overall adjusted sum OD IgM values tended to be higher to autologous than heterologous virus antigen preparations (). For instance, adjusted sum OD values for samples from the Kikwit outbreak were significantly higher against ZEBOV antigen, than against SEBOV, BEBOV, and REBOV. Similar trends are also apparent for samples from the Gulu and Bundibugyo outbreaks. Interestingly we note that samples from the Kikwit outbreak had significantly higher adjusted sum OD values against CIEBOV than against ZEBOV, and additionally samples from Bundibugyo had higher (although not significantly different) values against CIEBOV than BEBOV antigen. All samples from the Philippines were demonstrated to be IgM negative during diagnostic testing and thus adjusted sum OD values were not examined in this study.
IgM adjusted sum OD values for outbreak samples to each of the five EBOV antigens.
We additionally examined IgG antibody reactivity of autologous versus heterologous virus antigen by comparing adjusted sum OD values for each of the individual virus lysate antigen preparations among samples collected from each of the outbreaks. Owing to the convalescent stage at which most samples were collected, overall IgG adjusted sum OD values were mostly higher than IgM values (). Similar to trends observed for IgM responses, samples collected from Gulu and Bundibugyo outbreaks had significantly higher adjusted sum OD IgG values against autologous antigen than against heterologous antigen (with the exception of samples from Bundibugyo having higher values against CIEBOV than against BEBOV). In contrast, adjusted sum OD values for samples from Kikwit did not differ between ZEBOV and SEBOV, BEBOV, or REBOV, and had higher values for CIEBOV in comparison to ZEBOV antigen. Interestingly, samples from the Philippines had higher adjusted sum OD values against ZEBOV, SEBOV, and CIEBOV, than against autologous REBOV antigen.
IgG adjusted sum OD values for outbreak samples to each of the five EBOV antigens.
We examined the kinetics of antibody development, for samples from Kikwit, Gulu, and Bundibugyo, by plotting the adjusted sum OD to autologous antigen for each of the sets of samples, relative to time post symptom onset (). The combined data for samples from these three outbreaks indicated early presence of IgM antibodies (earliest samples for this study were at 14 days post symptom onset). While sample collection dates varied for the Kikwit, Gulu, and Bundibugyo samples, adjusted sum OD values peaked between 30–50 days, and largely declined by 80 days post symptom onset. As with IgM, IgG antibodies were present, even in most early samples, however, adjusted sum OD values remained high over the full course (as long as 117 days) of sample collection post-symptom onset.
IgM and IgG adjusted sum OD values to autologous antigen by day of sample collection relative to symptom onset.
While the adjusted sum OD measure allowed us to quantitatively compare serologic cross-reactivity between autologous and heterologous antigens using a continous variable measure, we additionally wanted to examine the performance of heterologous antigen from a discrete (positive or negative) diagnostic standpoint. In order to assess the utility of heterologous antigen for the serologic diagnosis of EBOV infection by IgM ELISA, we selected all individuals with positive IgM antibody responses to respective autologous antigen from Kikwit, Gulu, and Bundibugyo outbreaks, and examined the sensitivity of the heterologous antigens for serologic diagnosis of EBOV in these samples (). While the overall sensitivity of heterologous pairs varied widely, many heterologous virus combinations had low sensitivity for detection of positive IgM antibody responses. For instance, SEBOV, BEBOV, and REBOV antigen preparations had a sensitivity of less than 40% for all combinations of heterologous outbreak samples.
Sensitivity of IgM ELISA, using heterologous antigen.
We similarly examined the diagnostic utility of heterologous antigen for the serologic diagnosis of EBOV infection by IgG ELISA. In contrast to the above results for the IgM assay, heterologous antigens had a high sensitivity in the detection of IgG antibodies (). With the exception of samples from Gulu, which displayed a diagnostic sensitivity of 74% with ZEBOV and REBOV antigen, all heterologous antigen pairs displayed at least 95% sensitivity for detection of IgG antibodies, and for many combinations, heterologous antigen detected positive results for 100% of samples.
Sensitivity of IgG ELISA, using heterologous antigen.