To evaluate the MAb based IgG sandwich ELISA and IgM capture ELISA system, 115 serum samples were collected from patients who recovered from suspected SFTS disease in Henan Province, China. The diagnostic criteria for suspected cases were as follows: relevant epidemiological history (i.e., working, living or traveling in the hilly, forest or mountain during SFTS epidemic season or being bitten by ticks within two weeks before disease onset), fever and other clinical manifestations with reduced peripheral blood platelet counts and leukopenia, following “Guideline for the prevention and treatment of fever with thrombocytopenia syndrome (2010 version)”.26

The rSFTSV-N protein expressed and purified as previously reported25 was emulsified with equal volumes of Freund's complete adjuvant (MP Biochemicals, CA, USA) and injected intraperitoneally into three 6-week old female BALB/c mice (Chongqing Tengxin Bill Experimental Animal Sales Co., LTD Chongqing, China), at a dose of 100 μg per injection. On day 14, all mice were boosted with rSFTSV-N protein in equal volumes of Freund's incomplete adjuvant (MP Biochemicals, CA, USA). Ten days after the second immunizations, blood samples were collected for antibody titer measurement via indirect ELISA. On day 28, 29, 30, mice were injected intraperitoneally with 100 μg/day rSFTSV-N protein for three times. On day 31, blood samples were collected and the spleen was taken out for cell fusion.

After immunization, the spleen cells were collected for cell fusion with SP2/0 myeloma cells (from the previous experiment)27 using polyethylene glycol (PEG 1500, Roche, Indiana, USA). The hybridoma cells were grown in a selective medium of hypoxanthine aminopterin - thymidine (HAT) (Gibco, NY, USA) for 10 days and then screened by indirect ELISA to select cells producing antibody against rSFTSV-N protein. The positive clones were diluted to establish single stable clones by limited dilution. In short, hybridoma cell suspension was diluted in growth medium (RPMI 1640, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) and inoculated to 96-well microplates with approximately 1 cell/well for 10 days before performing indirect ELISA to select clones secreting the desired antibody. Subsequently, the positive clones were transferred to culture flasks and propagated in growth media. At last, large-scale production of MAbs were done by propagating positive clones in Hybridoma-SFM medium (Gibco, NY, USA) and injected hybridoma cells intraperitoneally into mouse to produce aseptic fluid. MAb HiTrap protein G chromatograph kit (GE Healthcare, Uppsala, Sweden) was applied to purify MAbs.

The presence of antibodies in the supernatant of hybridoma cells culture media was assessed by ELISA according our previously published protocols27 with the following modification. The 96-well plates were processed as follows: coating of 50 ng/well rSFTSV-N protein at 4°C overnight, then blocking using PBS-T with 5% nonfat milk, at room temperature for 1h followed by adding hybridoma culture supernatant or adding pre-immunization and post-immunization mice serum samples for negative and positive controls, respectively, at 37°C for 1h; followed by the detection of bound IgG with horseradish-peroxidase-conjugated (HRP) goat anti-mouse IgG (American Qualex, San Clemente, CA) diluted 1:10,000 at 37°C for 1h. Then H2O2-ABTS [2,2’ azinobis (3-ethylbenzthiazolinesulfonic acid)] substrate was added and optical density (OD) value was recorded. All clones with OD value twice or higher than that of the negative control were considered positive.

Indirect immunofluorescence test was applied to determine the reactivity of MAbs with SFTSV, SFTSV-infected Vero-E6 (from the previous experiment)27 were collected and centrifuged at 600 x g for five minutes, and then, the cell pellet was washed three times with PBS and spotted onto the Teflon-coated 8-multi-well glass slide (MP Biochemicals, CA, USA). After the slide was air dried, cells were fixed in cold acetone at 4°C for 10 min. After blocking with BlockAce for 30 min at room temperature, the cells were incubated with 15 μl culture media of the hybridoma cells in a wet chamber at 37°C for 1 h. Then, the slides were washed and air-dried once more, followed by detection using 15 μl of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG (Bethyl Laboratories Inc. Montgomery, USA) at a dilution of 1:50 to every test well and reacting in dark at 37°C for 1h. Finally, the slides were washed and sealed with mounting medium for fluorescence (VectorLaboratories, Inc.). The images were acquired using an OLYMPUS IX73 immunofluorescence microscope.

Western blot analysis was performed as described before.25 Briefly, protein molecular weight marker (PageRulerTM Prestained Protein Ladder, Thermo Scientific, Denmark) and the purified rSFTSV-N protein were separated in a 15% polyacrylamide gel. The separated proteins were transferred onto a polyvinylidene fluoride (PVDF) microporous membrane (Immobilon, Millipore, USA). The membrane was first blocked and then incubated at 4°C overnight or 37°C for 1 hour with the supernatant of hybridoma cells, followed by a secondary antibody of goat anti-mouse IgG (American Qualex, Califonia, USA, 1:1000 dilution). Protein bands were visualized using Metal Enhanced dimethyl aminobenzidine (DAB) Substrate Kits (Solarbio, China).

The MAb isotype was determined by the SBA Clonotyping System-HRP kit according to the manufacturer's instructions (Southern Biotech, USA).

To evaluate MAbs for diagnosis of SFTSV infection, we established MAb-based IgG sandwich ELISA and IgM capture ELISA system for human sera. These ELISA systems that have common procedures are described here and the procedures specific for each assay will be described under each assay.

Ninety-six well Nunc immunoplates (Thermo Scientific, Denmark) were used with a sample volume of 100 μL/ well. The coating buffer was 0.01M PBS, pH 7.4, and the plate coating was done at 4°C overnight. After exposure to a specific reagent at each step of the system, except at the last step as described below, plates were washed three times with wash buffer (0.01 M PBS with 0.1% (vol/vol) Tween 20 (PBS-T)). PBS-T with 5% nonfat milk (Difco, Detroit, USA) was used to dilute all serum samples and reagents. In addition to substrate, all incubations were done at 37°C for 1 h. Besides, plates with 100 μL/well H2O2-ABTS substrate (Kirkegaatrd & Perry, Gaithersburg, MD) were incubated at 37°C for 30 min. A spectrophotometer was used to read the plates and record the OD values at 410 nm.

In the MAb-based IgG sandwich ELISA, 96-well plates were processed as follows: coating of 50 ng/well of MAb overnight before blocking using PBS-T with 5% nonfat milk for 1h, followed by 50 ng/well rSFTSV-N protein, then human serum samples diluted at 1:1,000 in 5% nonfat milk in PBS-T were added, afterwards detection of bound IgG with HRP-goat anti-human IgG (American Qualex, Califonia, USA) diluted at 1:30,000, which was made visible after adding H2O2-ABTS substrate. OD values measured at 410 nm using a microplate spectrophotometer. Each serum specimen was tested twice and the average OD value was calculated. Each test used positive and negative control serum samples. The mean OD value of a sample more than twice the mean OD of the negative control serum was considered positive.

The MAb-based IgM capture ELISA followed the steps below. Firstly, for each serum sample to be tested, four wells were coated with 1:500 dilution of anti-human IgM (Cappel, MP Biochemicals) before blocking using PBS-T with 5% nonfat milk for 1h. Secondly, 1:400 diluted human serum was added to the four wells. Afterwards two wells were added with 50 ng of rSFTSV-N protein (positive antigen) and other two wells were added with 50 ng of nonreactive protein (negative antigen). And then, 100 μL/well of purified MAb diluted at 1:10,000 was added to all four wells, after which a 1:10,000 diluted peroxidase conjugated anti-mouse IgG (American Qualex, Califonia, USA) was then added. Finally, the remaining steps for color development were the same as above.

Detection of total antibody against SFTSV28 was done using a commercial ELISA kit (Xinlianxin Biomedical Technology CO., LTD, Wuxi, Jiangsu, China) following the manufacturer's instructions. The kit is a double-antigen sandwich enzyme-linked immunosorbent assay that detects total antibodies. This commercial kit has a sensitivity of 100% and specificity of 99.57% compared with virus neutralization testing.28 Indirect IgG and IgM ELISA were performed as previously described.25

BALB/c mice were immunized with rSFTSV-N protein five times with an immunization regimen as shown in Figure 1A. After subcloning and multiple rounds of indirect ELISA screening, three positive hybridoma clones producing MAbs against rSFTSV-N protein (designated as 5G12, 4A10, 1C3) were obtained.

Fig. 1.

Preparation of monoclonal antibodies against rSFTSV-N protein. (A) BALB/C mice immune schedule. (B) Immunofluorescence analysis to verify the specificity of MAbs. SFTSV infected and mock Vero-E6 cells reacted with three MAbs and then detected by FITC labeled anti-mouse IgG and observed under an immunofluorescence microscope.

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Indirect immunofluorescence test showed strong fluorescence in Vero-E6 cells infected with SFTSV; no fluorescence was observed in mock Vero-E6 cells (Fig. 1B). Western blot assay showed that these three MAbs all reacted with rSFTSV-N protein (Fig. 2A). The classes of three MAbs were determined to be IgG2b and κ chain (Fig. 2B). These results confirmed that the three hybridoma cells secreted MAbs specific for SFTSTV.

Fig. 2.

Western blot ananysis and isotype detection of MAbs. (A) Western-blot analysis of MAbs against rSFTSV-N protein. a: MAb 4A10; b: MAb 1C3; c: MAb 5G12. Lane 1: protein marker; lane 2: purified rSFTSV-N protein. (B) The antibody subclasses of three MAbs. The subclasses of the 3 MAbs were determined to be IgG2b and κ chain using a mouse MAb isotyping kit.

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To evaluate the possibility of using the newly developed MAbs for diagnosis, MAb-based IgG sandwich ELISA for human serum was established. Among the 115 human serum samples from SFTS suspected patients, 85 samples were IgG positive and 30 were IgG negative by the IgG sandwich ELISA system, and the three MAbs showed nearly uniform reactivity. These results perfectly matched the total antibody sandwich ELISA and the rSFTSV-N protein based indirect IgG ELISA results with a 100% concordance to these two ELISA systems (Tables 1 and 2).

IgM capture ELISA for human serum was established using the MAb as detecting antibody. Among the 115 human serum samples from SFTS-suspected patients, 85 samples were IgM positive and 30 were IgM negative by the IgM capture ELISA system which perfectly matched the total antibody sandwich ELISA results with a 100% concordance with the total antibody sandwich ELISA system (Table 3), and similarly, the three MAbs showed nearly uniform reactivity in this method.

Compared with the rSFTSV-N protein based indirect IgM ELISA, among the 85 positive samples detected by IgM capture ELISA, indirect IgM ELISA detected 77 positive, missing eight positive samples. The concordance of the two methods was 93.04% (Table 4).