J Bacteriol Virol.  2020 Mar;50(1):17-24. 10.4167/jbv.2020.50.1.017.

Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen

• Yang DK
• Kim HH
• Lee S
• Ji M
• Han BH
• Oh S
• Hyun BH

• KMID: 2471834
• DOI: http://doi.org/10.4167/jbv.2020.50.1.017

Abstract

Canine adenovirus type 1 (CAV-1) causes infectious hepatitis in members of the family Canidae, including dogs. An indirect enzyme-linked immunosorbent assay (I-ELISA) that detects CAV-1 antibodies is required for large-throughput tests of dog sera. We collected 165 serum samples from dogs of Chungbuk and Gyeongbuk provinces between February 2016 and October 2018. The Korean CAV-1 vaccine strain CAV1V was propagated in Madin-Darby canine kidney (MDCK) cells and purified via Nuvia cPrime anion-exchange chromatography; the virus served as an I-ELISA antigen. Virus-neutralizing anti-CAV-1 titers in dog sera were measured using the virus neutralization (VN) method. The I-ELISA was optimized using purified CAV-1 antigen and serum samples. This kit was used to evaluate dog sera. The VN and I-ELISA data were compared. The sensitivity, specificity, and accuracy of the I-ELISA were 97.0%, 74.2%, and 92.7% compared to the VN assay, respectively. The I-ELISA data significantly correlated with those of VN (r = 0.88). These results suggest that the I-ELISA is useful for serosurveillance of CAV-1 in dog sera.

Keyword

CAV-1; I-ELISA; sero-surveillance

MeSH Terms

Adenoviruses, Canine*
Animals
Antibodies
Canidae
Chromatography
Chungcheongbuk-do
Dogs
Enzyme-Linked Immunosorbent Assay*
Gyeongsangbuk-do
Hepatitis A
Humans
Kidney
Methods
Sensitivity and Specificity
Antibodies

Figure

• 

  Fig. 1. Optimal harvesting time determined via analyses of the CAV1V growth kinetics (A). The titer of CAV1V propagated in MDCK cells was measured two times and expressed as mean viral titer. Infected MDCK cells (staining with monoclonal antibody against CAV-1 and the anti-mouse IgG FITC conjugate) show specific nuclear fluorescence (B), Scale bars, 100 μm.

• 

  Fig. 2. CAV-1 antigen loaded onto a Nuvia cPrime column connected to a peristaltic pump (P-1) (A). The concentrations of proteins eluted from the Nuvia cPrime column and their hemagglutinating activities as measured using NanoDrop 1000 UV/Vis spectrophotometry and admixture with 0.6% (v/v) guinea pig erythrocytes, respectively (B). Canine adenovirus type 1 particles were evident via electron microscopy of the seventh eluate, scale bar, 100 nm (C).

• 

  Fig. 3. Determination of the concentration of the purified CAV-1 antigen (A) and serum dilution (B) by indirect enzyme-linked immunosorbent assay (I-ELISA). The concentrations of antigen and dilutions of serum were analyzed according to cut-off values (> 0.4). The numbers in the legend are the virus-neutralizing antibody (VNA) titers (0-512) of CAV-1 in dog serum.

• 

  Fig. 4. Correlation between the VNA titer and absorbance of l-ELISA for detecting CAV-1 antibodies in 165 dog serum samples. The correlation is indicated by the linear regression line and r-value (0.88).

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