Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies

Abstract

Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

FIG. 1.

Phylogenetic analysis of filovirus GP amino acid sequences. The phylogenetic tree was constructed by using the neighbor-joining method. For the construction of this tree, we used 10 GP amino acid sequences, each comprising a whole GP amino acid sequence. Numbers at branch points indicate bootstrap values (1,000 replicates).

FIG. 2.

Identification and characterization of purified His-GPs. (a) His-EBOV-GP and His-MARV-GP were analyzed by 8% SDS-PAGE and stained with Coomassie brilliant blue. (b and c) Immunoblotting of purified His-GPs was performed by using MAbs to EBOV (ZGP42/3.7) and MARV GPs (AGP127-8) (b) and His tags (c). Arrows indicate the locations of the His-GPs. The protein bands represent His-ZEBOV-GP (lane 1), His-SEBOV-GP (lane 2), His-CIEBOV-GP (lane 3), His-BEBOV-GP (lane 4), His-REBOV-GP (lane 5), and His-MARV-GP (lane 6). Lane 7 shows FCS-derived proteins used as a control antigen (see Materials and Methods).

FIG. 3.

Sensitivity of ELISAs. His-GPs (a, b, and c), GP-expressing cell lysates (d, e, and f), and VLP (g, h, and i) were used as antigens. The GP amounts were standardized by Western blotting as described in Materials and Methods. Serial 10-fold dilutions of MAbs to EBOV (a, d, and g) and MARV (b, e, and h) were prepared and tested. S139/1 (specific to influenza virus hemagglutinin) was used as a negative-control antibody (c, f, and i).

FIG. 4.

IgG antibodies detected in mouse antisera. Serial 10-fold dilutions of anti-ZEBOV (a and g), anti-SEBOV (b and h), anti-CIEBOV (c and i), anti-BEBOV (d and j), anti-REBOV (e and k), and anti-MARV (f and l) sera obtained from mice immunized with EBOV and MARV VLPs were tested for IgG antibodies reacting with His-GPs (a, b, c, d, e, and f) and VLPs (g, h, i, j, k, and l). Averages and standard deviations for three mice of each group are shown. Asterisks indicate statistically significant differences in OD values between the homologous antigen and all other antigens (P < 0.05).

FIG. 5.

IgG antibodies detected in experimentally infected monkey plasma by ELISA using His-GPs. Monkeys C105, C332, C508, and C725 were infected with ZEBOV, whereas monkeys C0287 and C0436 were infected with SEBOV. Infected monkey sera were diluted at 1:1,000. Naïve monkey sera were diluted at 1:100. Each bar represents the average and standard deviation of data from three independent experiments. Asterisks indicate statistically significant differences in OD values between the Zaire antigen and all other antigens (P < 0.05). The dagger shows statistically different reactions between His-SEBOV-GP and all the other antigens (P < 0.05) except His-ZEBOV-GP.

FIG. 6.

IgG and IgM antibodies detected in human samples. OD values for specific IgG (a) and IgM (b) antibodies in the patient sera are shown. Sera from 21 individuals were analyzed at 1:1,000 dilutions. Naïve human sera (1:100 dilution) were used as a negative control. Each bar represents the average and standard deviation of data from independent experiments. Asterisks indicate statistically significant differences in OD values between the homologous antigen and all other antigens (P < 0.05).