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

Dong-Kun Yang; Ha-Hyun Kim; Siu Lee; Miryeon Ji; Bok Hee Han; Soobin Oh; Bang-Hun Hyun

doi:10.4167/jbv.2020.50.1.017

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Yang, Kim, Lee, Ji, Han, Oh, and Hyun: Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen

Original Article

J Bacteriol Virol 2020;50(1):17-24.

Published online: 9 March 2020

DOI: https://doi.org/10.4167/jbv.2020.50.1.017

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

Dong-Kun Yang^∗, Ha-Hyun Kim, Siu Lee, Miryeon Ji, Bok Hee Han, Soobin Oh, Bang-Hun Hyun

Viral Disease Research Division, Animal and Plant Quarantine Agency, MAFRA, Gimcheon, 39660, Republic of Korea

Corresponding Dong-Kun Yang, Ph D, DVM Animal and Plant Quarantine Agency, 177 Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do 39660, Republic of Korea Phone: +82-54-912-0785 Fax: +82-54-912-0812 E-mail: yangdk@korea.kr

Received 7 December 201916 February 2019 Accepted 18 February 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: CAV-1, I-ELISA, sero-surveillance

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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).

Table 1.

The sensitivity, specificity, and accuracy of I-ELISA for the detection of CAV-1 antibodies compared to the VN test

		VN test
		Positive		Negative	Sum
	Positive	130		8	138
I-ELISA	Negative	4		23	27
	Sum	134		31	165
	Sensitivity^∗		97.0%
	Specificity^†		74.2%
	Accuracy^#		92.7%

^∗ Sensitivity (%) = (number of positive results in both tests/total number of positive results in the reference test) × 100,

^† Specificity (%) = (number of negative results in both tests/total number of negative results in the reference test) × 100,

^# Accuracy (%) = (actual number of both positive and negative results/total number of samples) × 100.

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