Journal List > J Bacteriol Virol > v.47(3) > 1034264

Yang, Kim, Lee, Ji, and Cho: Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

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.

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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.
jbv-47-148f1.tif
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).
jbv-47-148f2.tif
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.
jbv-47-148f3.tif
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.
jbv-47-148f4.tif
Figure 5.
Correlation between RABV antibody titers obtained by FAVN test and I-ELISA in 122 dog serum samples.
jbv-47-148f5.tif
Table 1.
Oligonucleotide primers to amplify the RABV N gene and confirm the pure plaque of recombinant RVN baculovirus
Primer Oligo nucleotide sequences (5′ – 3′) Genomic region
NF CCGGATCCGATGGATGCCGACAAGATTGTATTC RABV N
NR CCAAGCTTTGAGTCACTCGAATATGTCTTGTT
Bac F TTT ACT GTT TTC GTA ACA ACA GTT TTG Baculovirus multi-cloning site
Bac R CAA CAA CGC ACA GAA TCT AGC

Underlined sequences indicate restriction enzyme sites (BamH I and Hind III) and start codon. RABV N: rabies virus nucleoprotein.

Table 2.
Determination of the sensitivity, specificity, and accuracy of I-ELISA for the detection of RABV antibodies in comparison with those of FAVN
    No. of samples with FAVN
    Positive Negative Sum
I-ELISA Positive 37 6 43
Negative 5 74 79
Sum 42 80 122
  Sensitivity   88.1%  
  Specificity∗∗   92.5%  
  Accuracy∗∗∗   91.0%  

Sensitivity (%) = [(number of positives in both tests) / (number of positives in the FAVN test)] × 100

∗∗ Specificity (%) = [(number of negatives in both tests) / (number of negatives in the FAVN test)] × 100

∗∗∗ Accuracy (%) = [(number of positives in both tests + number of negatives in both tests) / (total number of samples)] × 100. I-ELISA: indirect enzyme-linked immunosorbent assay; FAVN: fluorescent antibody virus neutralization.

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