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Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera

View Article: PubMed Central - PubMed

ABSTRACT

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle.

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The reactivity of rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) against the NP in Western blot and immunofluorescence assay (IFA). (A) Western blot analysis of one polyclonal antibody and three mAbs in Vero cells before and after SFTSV infection. NP expression was assessed at 6 days post infection (dpi = 6), and mock-infected cells were included. Each cell lysate was separated on an 8–16% gradient SDS-PAGE gel and transferred to membranes for Western blot analysis. The α-GAPDH antibody was used as a loading control. (B) Rabbit anti-NP and mouse anti-SFTSV polyclonal antibodies were diluted 1 : 1,000 for immunoblotting and 1:200 for IFA. Three mAbs were diluted 1 : 1,000 for immunoblotting and 1 : 50 for IFA, and the secondary antibodies for rabbit and mouse IgG were diluted 1 : 1,000 for immunoblotting and 1 : 200 for IFA. DAPI staining (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.
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Figure 2: The reactivity of rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) against the NP in Western blot and immunofluorescence assay (IFA). (A) Western blot analysis of one polyclonal antibody and three mAbs in Vero cells before and after SFTSV infection. NP expression was assessed at 6 days post infection (dpi = 6), and mock-infected cells were included. Each cell lysate was separated on an 8–16% gradient SDS-PAGE gel and transferred to membranes for Western blot analysis. The α-GAPDH antibody was used as a loading control. (B) Rabbit anti-NP and mouse anti-SFTSV polyclonal antibodies were diluted 1 : 1,000 for immunoblotting and 1:200 for IFA. Three mAbs were diluted 1 : 1,000 for immunoblotting and 1 : 50 for IFA, and the secondary antibodies for rabbit and mouse IgG were diluted 1 : 1,000 for immunoblotting and 1 : 200 for IFA. DAPI staining (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.

Mentions: The gene for the recombinant NP was amplified from the commercially synthesized S fragment of SFTSV (GenBank accession No. HQ141612) and cloned into a protein expression vector. A 34 kDa recombinant protein band was well maintained after all purification procedures (Fig. 1). This recombinant NP was used as the antigen to generate polyclonal and mAbs. NP-specific polyclonal antibody was obtained in rabbits after four antigen immunizations, and the titer of the antibody was tested by IFA and Western blotting (Fig. 2). NP-specific mAbs were produced from the spleen hybridoma cells of immunized mice, and the three clones (6D55, 8F31, and 10G7) showed strong interactions in the IFA and Western blot tests; therefore, these clones were used as competitive antibodies to develop the cELISA. A clear band corresponding to the size of NP, which appeared only after SFTSV infection, was observed upon Western blot analysis with all generated antibodies (panel A in Fig. 2). Moreover, IFA using these antibodies showed intense cytoplasmic staining, specifically in SFTSV-infected Vero cells (panel B in Fig. 2).


Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera
The reactivity of rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) against the NP in Western blot and immunofluorescence assay (IFA). (A) Western blot analysis of one polyclonal antibody and three mAbs in Vero cells before and after SFTSV infection. NP expression was assessed at 6 days post infection (dpi = 6), and mock-infected cells were included. Each cell lysate was separated on an 8–16% gradient SDS-PAGE gel and transferred to membranes for Western blot analysis. The α-GAPDH antibody was used as a loading control. (B) Rabbit anti-NP and mouse anti-SFTSV polyclonal antibodies were diluted 1 : 1,000 for immunoblotting and 1:200 for IFA. Three mAbs were diluted 1 : 1,000 for immunoblotting and 1 : 50 for IFA, and the secondary antibodies for rabbit and mouse IgG were diluted 1 : 1,000 for immunoblotting and 1 : 200 for IFA. DAPI staining (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037297&req=5

Figure 2: The reactivity of rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) against the NP in Western blot and immunofluorescence assay (IFA). (A) Western blot analysis of one polyclonal antibody and three mAbs in Vero cells before and after SFTSV infection. NP expression was assessed at 6 days post infection (dpi = 6), and mock-infected cells were included. Each cell lysate was separated on an 8–16% gradient SDS-PAGE gel and transferred to membranes for Western blot analysis. The α-GAPDH antibody was used as a loading control. (B) Rabbit anti-NP and mouse anti-SFTSV polyclonal antibodies were diluted 1 : 1,000 for immunoblotting and 1:200 for IFA. Three mAbs were diluted 1 : 1,000 for immunoblotting and 1 : 50 for IFA, and the secondary antibodies for rabbit and mouse IgG were diluted 1 : 1,000 for immunoblotting and 1 : 200 for IFA. DAPI staining (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.
Mentions: The gene for the recombinant NP was amplified from the commercially synthesized S fragment of SFTSV (GenBank accession No. HQ141612) and cloned into a protein expression vector. A 34 kDa recombinant protein band was well maintained after all purification procedures (Fig. 1). This recombinant NP was used as the antigen to generate polyclonal and mAbs. NP-specific polyclonal antibody was obtained in rabbits after four antigen immunizations, and the titer of the antibody was tested by IFA and Western blotting (Fig. 2). NP-specific mAbs were produced from the spleen hybridoma cells of immunized mice, and the three clones (6D55, 8F31, and 10G7) showed strong interactions in the IFA and Western blot tests; therefore, these clones were used as competitive antibodies to develop the cELISA. A clear band corresponding to the size of NP, which appeared only after SFTSV infection, was observed upon Western blot analysis with all generated antibodies (panel A in Fig. 2). Moreover, IFA using these antibodies showed intense cytoplasmic staining, specifically in SFTSV-infected Vero cells (panel B in Fig. 2).

View Article: PubMed Central - PubMed

ABSTRACT

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle.

No MeSH data available.


Related in: MedlinePlus