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

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. streptomycin (100 μ g/mL), and the antimycotic amphotericin B (0.25 μ g/mL) at 37 ° C under 5% (v/v) CO 2 , and used for viral antigen production and serological assay. The CAV1V strain of CAV-1, which is the CAV-1 vaccine used in Korea, was employed as a viral antigen. A total of 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces from 2016 to 2018, and the sera were used in each test. We do not know if the dogs had been inoculated with the CAV vaccine. checked for cytopathic effect (CPE) over 5 days post inoculation (DPI). The virus-neutralizing antibody (VNA) titer of CAV-1 was the reciprocal of the highest serum dilution that completely inhibited the CPE. Each serum sample was diluted from 1:2 to 1:256. A VNA titer ≥ 1:2 was considered positive. serum panel samples at dilutions of 1:20 to 1:40,960, were used to determine the appropriate concentrations at 37 ° C for 1 h. Next, 100 µ L amounts of anti-dog IgG horseradish peroxidase (HRP) conjugate (KPL, Gaithersburg, MD, USA) were added to all well of the microplate, which was incubated for 1 h at the above temperature. After washing, 50 µ L amounts of 2 ’ 2-azino-bis-(3-ethylbenzothiazoline) substrate (ABTS) solution were added to the plate, which was then incubated for 10 min at room temperature. Finally, stop solution (1.0% w/v sodium dodecyl sulfate) were added to stop the reaction. The absorbance of the I-ELISA was measured at 405 nm in a spectrophotometer (Sunrise ELISA reader; Tecan, Switzerland). Under the optimized I-ELISA conditions, 100 µ L serum diluted 100-fold in dilution buffer (1% [w/v] skim milk in PBS) was added to a 96-well microplate coated with CAV1V antigen. After incubation at 37 ° C for 1 h, the plate was washed with PBS containing 0.05% Tween 20 (PBST) and incubated with 100 µ L anti-dog IgG HRP conjugate diluted 4,000-fold in dilution buffer for 1 h at 37 ° C. After washing, 50 µ L ABTS substrate solution and 50 µ L stop solution (1.0% [w/v] sodium dodecyl sulfate) were added to all wells of the microplate. Serum samples exhibiting absorbances greater than the cutoff of 0.4 were evaluated as positive. The specificity, sensitivity, and accuracy of the I-ELISA were determined as previously reported (15).