J Vet Sci.  2016 Sep;17(3):307-314. 10.4142/jvs.2016.17.3.307.

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

• Lee H
• Kim EJ
• Song JY
• Choi JS
• Lee JY
• Cho IS
• Shin YK

• KMID: 2413129
• DOI: http://doi.org/10.4142/jvs.2016.17.3.307

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.

Keyword

cattle; enzyme-linked immunosorbent assay; immunofluorescence assay; severe fever with thrombocytopenia syndrome

MeSH Terms

Animals
Antibodies, Monoclonal/*blood
Antibodies, Viral/*blood
Bunyaviridae Infections/diagnosis/*veterinary/virology
Cattle
Cattle Diseases/blood/*diagnosis/virology
Enzyme-Linked Immunosorbent Assay/*veterinary
Fluorescent Antibody Technique/veterinary
Phlebovirus/*immunology/isolation & purification
Antibodies, Monoclonal
Antibodies, Viral

Figure

• 

  Fig. 1 Purified recombinant full-length nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) stained with Coomassie brilliant blue after SDS-PAGE. A recombinant protein band of approximately 34 kDa was expressed after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. A single band remained after the affinity-specific purification. Lane 1, Escherichia (E.) coli BL21 (DE3) of the cell lysate before induction; Lane 2, E. coli BL21 (DE3) cell lysate after 20 h of induction with 0.2 mM IPTG; Lane 3, 100 ng of purified protein.

• 

  Fig. 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.

• 

  Fig. 3 Antibody responses from laboratory immunized cattle. The SFTSV-positive bovine serum was tested in the IFA. The optimized dilution rate of the positive serum samples was 1/80, while less diluted sera (1/10 and 1/50) showed strong background signals in the IFA.

• 

  Fig. 4 The percent inhibition (PI) value and optical density (OD) of laboratory immunized bovine sera in a competitive enzyme-linked immunosorbent assay (cELISA). Serum samples collected from two immunized cattle and one negative control cattle were diluted from 1/2 to 1/300 and tested for (A) PI value and (B) OD value (at 450–630 nm) in cELISA. Error bars represent the standard error of the mean of independent experiments repeated at least three times.

• 

  Fig. 5 Establishment of cut-off PI value in cELISA. Frequency distributions of the PI values for 407 bovine serum samples determined as SFTSV-negative in IFA (1 : 80 dilution). The cut-off of 49.5% (mean ± 3SD) is indicated with the arrow.

• 

  Fig. 6 Scatter dot plot indicating individual and mean values of PI value in cELISA. The scatter dot plot represents the distribution of antibody titers to the NP of SFTSV in IFA-negative (n = 407) and IFA-positive (n = 11) samples. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, with outliers represented as dots; and the data points are plotted as open circles. n = 407 and 11 sample points.

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  Fig. 7 Receiver operating characteristic (ROC) for analysis of the cELISA. ROC curves for cELISA compared with four different IFA cut-off points. AUC, area under the curve.

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