Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Cell culture.P3/Ag568 was used as the parental cell line for hybridomas. The cells were maintained in RPMI 1640 (Gibco BRL, Rockville, Md.) supplemented with 10% fetal bovine serum and antibiotics (streptomycin and penicillin; Gibco BRL). Hypoxanthine-aminopterin-thymidine supplement (HAT) (Gibco BRL) was added to the medium for the selection of hybridomas according to the manufacturer's instructions. Hypoxanthine-thymidine supplement (HT) (Gibco BRL) was added to the medium to switch the medium from HAT to RPMI 1640.

Clinical specimens.Livers (monkey no. 2882 and 2877), spleens (monkey no. 2877, 2882, and 2885), and sera (monkey no. 2866 and 2612), which were collected from EBO-R-infected cynomolgus macaques during the EBO-R outbreak in the Philippines in 1996 (14, 25) and kept frozen at −80C, were used in the study. EBO-R-NP antigens or genomic RNA were detected in these samples by antigen capture ELISA using 3-3D (29), immunohistochemistry (14), or RT-PCR using primers RES-Nf2 (5′-TGAGCTCCGGAAGAAGGACGGTGT-3′) and RES-Nr2 (5′-ACCATCATGTGTCCAACTGATTGC-3′). Five liver specimens and 79 serum specimens from EBO-R-noninfected cynomolgus macaques were used as the negative controls. Liver and spleen tissues were homogenized at approximately 10% (wt/vol) in phosphate-buffered saline (PBS) containing 0.05% Tween 20, 1% Triton X-100, and 5% nonfat milk (Triton-milk-PBS-T). After centrifugation at 15,800 × g for 10 min, the supernatants were used in the experiments. Sera were diluted with Triton-milk-PBS-T and used in the experiments.

Preparation of histidine-tagged Ebola virus rNPs (His-EBO-R-NP and His-EBO-Z-NP) for antigen capture ELISA.Recombinant NPs (rNP) of EBO-R (His-EBO-R-NP) and EBO-Z (His-EBO-Z-NP) were prepared to be the antigens in antigen capture ELISA. The recombinant baculoviruses, which expressed His-EBO-R-NP (referred to as Ac [Autographa californica]-His-EBO-R-NP) or His-EBO-Z-NP (referred to as Ac-His-EBO-Z-NP), were produced as described previously (15, 33). Ac-His-EBO-R-NP or Ac-His-EBO-Z-NP was grown in Tn5 insect cells as reported previously (24). Tn5 cells infected with the recombinant baculoviruses were washed twice with PBS after 3 days of culture at 26°C and then incubated for 15 min in PBS containing 1% NP-40. After sonication for 1 min, the lysates were used in the experiments.

Preparation of MAbs.MAbs were prepared as described previously (29). Briefly, BALB/c mice were subcutaneously immunized with His-EBO-R-NP together with Freund complete adjuvant (Becton Dickinson, Franklin Lakes, N.J.) and boosted subcutaneously three times with IMJECT-ALUM (Pierce, Rockford, Ill.) at 2-week intervals. Three days after the last immunization, the spleens were removed, and the spleen cells were fused with P3/Ag568 cells by using polyethylene glycol 4000 (catalog no. 4030-035; Gibco BRL) according to the manufacturer's instructions. The supernatants of the hybridoma cells were screened in an immunoglobulin G (IgG) ELISA (29) with His-EBO-R-NP as an antigen. Crude hybridomas were isolated from ELISA-positive wells, and the hybridoma clones were established by the limiting dilution method. MAbs were purified from the culture supernatants with a MAb Trap GII antibody purification kit (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) according to the manufacturer's instructions. The isotypes of the MAbs were determined with a mouse MAb isotyping kit from Gibco BRL.

Preparation of polyclonal antibodies.Rabbit antisera to the glutathione S-transferase (GST)-tagged EBO-R NP C-terminal half (amino acids [aa] 360 to 739) (GST-EBO-R-ΔNP) (15) and to His-EBO-Z-NP (32, 33) were prepared as described previously. Briefly, rabbits were immunized four times subcutaneously with GST-EBO-R-ΔNP or His-EBO-Z-NP together with IMJECT-ALUM (Pierce). Serum was obtained 7 days after the last immunization.

IFA.Indirect immunofluorescence assays (IFAs) using HeLa cells expressing the entire NPs of EBO-R or EBO-Z were performed as described previously (15, 32). IFA slides coated with Vero E6 cells infected with EBO-S and gamma irradiated were kindly supplied by the Centers for Disease Control and Prevention, Atlanta, Ga. The well of the slide was spotted with 20 μl of each MAb at a concentration of 100 ng/μl, and the slides were incubated under humidified conditions at 37°C for 1 h. After being washed with PBS, the slides were reacted with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H+L) antiserum (catalog no. 62-6511; Zymed Laboratories, Inc., South San Francisco, Calif.) at a dilution of 1:100. The slides were washed with PBS and examined for the staining pattern under a fluorescent microscope.

Epitope mapping with GST-truncated EBO-R rNP fusions.A series of truncated EBO-R rNPs were prepared to determine the epitopes recognized by Res2-6C8 and Res2-1D8. Table 1 shows the primers used for the construction of the peptides. A truncated EBO-R NP corresponding to aa 631 to 739 was prepared as follows. PCR using the primers RES-N8F and RES-N8R (29) was performed to amplify the corresponding region of EBO-R cDNA. The PCR fragment was subcloned into a pGEX-2T vector (Amersham Pharmacia Biotech) to construct pGEX-ΔNP_631-739. The insert sequences were confirmed to be identical to the originals. Then the GST-tagged truncated EBO-R NP was expressed in Escherichia coli (BL-21 strain) and purified by glutathione Sepharose 4B column chromatography according to the manufacturer's instructions (Amersham Pharmacia Biotech). PCR was also performed with the forward primers R635f, R636f, R637f, and R638f and a reverse primer, RES-N8R. As described above, each of the truncated EBO-R NPs corresponding to aa 635 to 739, 636 to 739, 637 to 739, and 638 to 739 was expressed and purified. The plasmids to express the truncated EBO-R NP corresponding to aa 631 to 638, 631 to 639, 631 to 640, 631 to 641, 631 to 642, and 631 to 643 were constructed by replacing a corresponding codon with a stop codon by site-directed mutagenesis of pGEX-ΔNP_631-739 using the primer pairs Mu639f/Mu639r, Mu640f/Mu640r, Mu641f/Mu641r, Mu642f/Mu642r, Mu643f/Mu643r, and Mu644f/Mu644r according to the manufacturer's instructions (QuikChange site-directed mutagenesis kit; Stratagene, La Jolla, Calif.). Each of the truncated EBO-R NPs was expressed and purified as described above. Then, the reactivity of Res2-6C8 and Res2-1D8 to truncated EBO-R rNPs was examined by IgG ELISA.

Antigen capture ELISA.Antigen capture ELISA was performed as described previously (29). Briefly, a microtiter plate (Becton Dickinson) was coated with purified MAbs (100 ng per well in 100 μl of PBS) overnight at 4°C. The three MAbs Res2-6C8, Res2-1D8, and 3-3D were used as the capture antibodies in the assay. MAb 3-3D was prepared against EBO-Z NP and was previously reported to detect the NPs of EBO-Z, EBO-R, and probably EBO-S in the antigen capture ELISA (29). After washing the plates three times with PBS containing 0.05% Tween 20 (PBS-T), 200 μl of PBS-T containing 5% nonfat milk (milk-PBS-T) was added to the wells for blocking for 1 h at 37°C. The milk-PBS-T was then removed, and 100 μl of the samples was added to the wells, which were then incubated for 1 h at 37°C. Triton-milk-PBS-T was used as the dilution buffer for the samples. After being washed three times with PBS-T, anti-GST-EBO-R-ΔNP rabbit serum and anti-His-EBO-Z-rNP rabbit serum at a dilution of 1:1,000 were added to the wells of the plates coated with the Res2-6C8 or Res2-1D8 and 3-3D, respectively. The plates were incubated for 1 h at 37°C and washed three times with PBS-T. Then, horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) antiserum (catalog no. 62-6120; Zymed Laboratories, Inc.) was added to the wells at a dilution of 1:1,000, and the plates were incubated for 1 h at 37°C. After being washed three times with PBS-T, 2,2′-azinobis[3-ethylbenziazoline-6-sulfonic acid] (ABTS) substrate (ABTS tablet and buffer; Roche Diagnostics, Mannheim, Germany) was added to the wells. The plates were then incubated for 30 min at room temperature, and the optical density at 405 nm (OD) was recorded.

In the antigen capture ELISAs using Res2-6C8, Res2-1D8, and 3-3D, the mean + 3 standard deviations (SDs) of OD of 79 sera from EBO-R-uninfected macaques were 0.11, 0.07, and 0.11, respectively, and those of 5 livers from EBO-R-uninfected macaques were 0.15, 0.08, and 0.15, respectively. Therefore, we defined the cutoff value as 0.2 in the assay.

Development of two MAbs specific to the EBO-R NP.Thirty-two hybridoma clones secreting MAbs to EBO-R rNP were established, and the reactivities of these MAbs to His-EBO-R-NP were examined in the antigen capture ELISA format (data not shown). Two MAbs, Res2-6C8 and Res2-1D8, were reactive in the antigen capture ELISA format and were used in the present study. The isotypes of Res2-6C8 and Res2-1D8 were IgG2b and IgG1, respectively. Ebola virus species specificity of Res2-6C8 and Res2-1D8 was examined by IFA with HeLa cells that expressed the rNP of EBO-R or EBO-Z or by using Vero E6 cells infected with EBO-S. These two MAbs reacted to EBO-R NP (Fig. 1A and B), but not to EBO-Z NP (Fig. 1D and E) or EBO-S NP (Fig. 1G and H).

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FIG. 1.

IFA using HeLa cells expressing rNP of EBO-R (A to C) or EBO-Z (D-F) and using Vero cells infected with EBO-S (G to I). The reactivities of Res2-6C8 (A, D, and G), Res2-1D8 (B, E, and H), and 3-3D (C, F, and I) to NP of EBO-R, EBO-Z, and EBO-S are shown. Res2-6C8 and Res2-1D8 reacted to EBO-R NP, but not to the NPs of EBO-Z and EBO-S. The MAb 3-3D reacts to the NPs of all three Ebola virus species.

Epitopes recognized by Res2-6C8 and Res2-1D8.The epitopes recognized by Res2-6C8 and Res2-1D8 were determined by IgG ELISAs using the truncated EBO-R rNPs (Fig. 2). Res2-6C8 and Res2-1D8 recognized the amino acid residues between aa 631 and 739, and the minimum epitopes were further determined. Res2-6C8 reacted to the truncated EBO-R rNPs corresponding to aa 636 to 739 and 631 to 639, but not to aa 637 to 739 and 631 to 638. On the other hand, Res2-1D8 reacted to the truncated EBO-R NP corresponding to aa 636 to 739 and 631 to 643, but not to aa 637 to 739 and 631 to 642. These results showed that the epitopes recognized by Res2-6C8 and Res2-1D8 were aa 636 to 639 (4 aa residues, ₆₃₆DPDI₆₃₉) and aa 636 to 643 (8 aa residues, ₆₃₆DPDIGQSK₆₄₃) of EBO-R NP, respectively. The amino acid sequences of the epitopes were aligned to that of EBO-R Pennsylvania isolate in 1989 to 1990, EBO-Z, and EBO-S. The 8 aa residues were identical to those of the EBO-R Pennsylvania isolate in 1989 to 1990 (Table 2). The amino acid sequences of EBO-Z and EBO-S NPs corresponding to the amino acids of EBO-R, ₆₃₆DPDIGQSK₆₄₃, were NQDSDNTQ and EALPINSK, respectively (Table 2).

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FIG. 2.

Reactivities of Res2-6C8 and Res2-1D8 to the truncated EBO-R rNPs in IgG ELISA. Res2-6C8 recognizes the amino acid residues between aa 636 and 639 (sequence, DPDI). Res2-1D8 recognizes the amino acid residues between aa 636 and 643 (sequence, DPDIGQSK). The mean OD value ± SD in the IgG ELISA is shown.

Development of the antigen capture ELISAs using these novel MAbs.The sensitivity of the antigen capture ELISAs prepared with these two MAbs was tested. As shown in Fig. 3A, Res2-6C8 and Res2-1D8 detected His-EBO-R-NP up to dilutions of 1:128,000 and 1:64,000 in the antigen capture ELISA, respectively. The ELISA prepared with 3-3D detected His-EBO-R-NP up to the dilution of 1:32,000. As expected, Res2-6C8 and Res2-1D8 did not detect His-EBO-Z-NP in the antigen capture ELISA (Fig. 3B).

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FIG. 3.

Reactivity of MAbs Res2-6C8 (□), Res2-1D8 (▵), and 3-3D (♦) to the rNPs of EBO-R and EBO-Z in the antigen capture ELISA. His-EBO-R-NP (A) and His-EBO-Z-NP (B) were used as antigens. The mean ± SD of four assays is shown.

Detection of EBO-R NP in the specimens from EBO-R-infected monkeys.Serum, liver, and spleen specimens from EBO-R-infected macaques were examined for the presence of EBO-R NP by antigen capture ELISAs using Res2-6C8, Res2-1D8, and 3-3D. Res2-6C8 and Res2-1D8 detected the EBO-R NP antigens in the sera, livers, and spleens (Table 3). Res2-6C8 detected the NP in all of the samples, while Res2-1D8 and 3-3D detected the NP in six of seven and five of seven samples, respectively. The endpoint titers were higher in the ELISAs using Res2-6C8 and Res2-1D8 than in the ELISA using 3-3D.