J Bacteriol Virol.  2017 Sep;47(3):148-155. 10.4167/jbv.2017.47.3.148.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

• Yang DK
• Kim HH
• Lee SH
• Ji M
• Cho IS

• KMID: 2392963
• DOI: http://doi.org/10.4167/jbv.2017.47.3.148

Abstract

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.

Keyword

Dog; Indirect ELISA; Rabies virus; Sero-surveillance; Rabies virus nucleoprotein; Recombinant baculovirus system

MeSH Terms

Animals
Antibodies*
Asian Continental Ancestry Group
Baculoviridae
Cell Culture Techniques
Chromatography
Dogs*
Enzyme-Linked Immunosorbent Assay*
Humans
Nucleoproteins
Rabies virus*
Rabies*
Sensitivity and Specificity
Vaccination
Antibodies
Nucleoproteins

Figure

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  Figure 1. The confirmation of recombinant pBlueRVN plasmid construction. RABV nucleoprotein (RVN) gene was inserted into pBluebac4.5/V5-His vector to generate recombinant pBlueRVN plasmid. Insertion of RVN gene into pBluebac4.5/V5 His vector was confirmed by double-digestion with restriction enzyme Bam HI and HindIII followed by agarose electrophoresis. Lane M: 1 kb DNA ladder, lane 1: pBlueRVN.

• 

  Figure 2. Identification of recombinant RVN baculovirus. Cytopathic effect in Sf9 insect cells infected with recombinant RVN baculovirus (A) and normal Sf9 insect cells (B). Immuno-fluorescence in Sf9 insect cells infected with recombinant baculovirus expressing RVN protein using anti-6 histidine antibody (C) and mouse monoclonal antibody against RVN protein (D).

• 

  Figure 3. Identification of the recombinant RVN protein. Recombinant RVN protein expressed using recombinant baculovirus system was separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) and stained with Coomassie blue (A). RVN protein was identified using monoclonal antibody against RVN protein by Western blotting (B). The molecular weight of recombinant RVN protein was 55 kDa. M; protein ladder, lane 1 and 2; purified RVN protein.

• 

  Figure 4. Optimization of Indirect-ELISA (I-ELISA). Concentration of the recombinant RVN antigen (A) and serum dilution factor (B) for indirect enzyme-linked immunosorbent assay (I-ELISA) were determined by a checkerboard titration test. The antigen was diluted to 1:300 (1.0 μg/ml) in carbonate buffer (pH 9.6) and coated wells in a 96-well microplate. The number of remarks indicates RABV antibody titer from fluorescent antibody virus neutralization (FAVN) test.

• 

  Figure 5. Correlation between RABV antibody titers obtained by FAVN test and I-ELISA in 122 dog serum samples.

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