jbv Journal of Bacteriology and Virology J Bacteriol Virol 1598-2467 2093-0249 Journal of Bacteriology and Virology 10.4167/jbv.2020.50.1.017 jbv-50-17 Original Article Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen YangDong-Kun ∗ KimHa-Hyun LeeSiu JiMiryeon HanBok Hee OhSoobin HyunBang-Hun 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 32020 932020 501 1724 07122019 16022019 18022020 Copyright © 2020 Journal of Bacteriology and Virology 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.

 CAV-1 I-ELISA sero-surveillance REFERENCES Hu RL Huang G Qiu W Zhong ZH Xia XZ Yin Z Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction Vet Res Commun 200125 7784 Timurkan MO Aydin H Alkan F Detection and molecular characterization of canine adenovirus type 2 (CAV-2) in dogs with respiratory tract symptoms in shelters in Turkey Veterinarski arhiv 201888 46779 Liu YC Abouhaidar MG Sira S Campbell JB Characterization of the genome of a vaccine strain of canine adenovirus type 1 Virus Genes 19882 6981 Russell WC Adenoviruses: update on structure and function J Gen Virol 200990 120 Walker D Abbondati E Cox AL Mitchell GB Pizzi R Sharp CP Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the UK Vet Rec 2016178 421 Buonavoglia C Martella V Canine respiratory viruses Vet Res 200738 35573 Choi JW Lee HK Kim SH Kim YH Lee KK Lee MH Canine adenovirus type 1 in a fennec fox (Vulpes zerda) J Zoo Wildl Med 201445 94750 Cabasso VJ Infectious canine hepatitis virus Ann N Y Acad Sci 1962101 498514 Wong M Woolford L Hasan NH Hemmatzadeh F A Novel Recombinant Canine Adenovirus Type 1 Detected from Acute Lethal Cases of Infectious Canine Hepatitis Viral Immunol 201730 25863 Yang DK Kim HH Yoon SS Ji M Cho IS Incidence and Sero-survey of Canine Adenovirus Type 2 in Various Animal Species J Bacteriol Virol 201848 1028 Marusyk RG Yamamoto T Characterization of a canine adenovirus hemagglutinin Can J Microbiol 197117 1515 Noon KF Rogul M Binn LN Keefe TJ Marchwicki RH Thomas R An enzyme-linked immunosorbent assay for the detection of canine antibodies to canine adenoviruses Lab Anim Sci 197929 6039 Segura MM Puig M Monfar M Chilló n M Chromatography purification of canine adenoviral vectors Hum Gene Ther Methods 201223 18297 Coronel J Behrendt I Bü rgin T Anderlei T Sandig V Reichl U Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems Vaccine 201937 70118 Muhamuda K Madhusudana SN Ravi V Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans Int J Infect Dis 200711 4415 Gschwender HH Brummund M Lehmann-Grube F Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus J Virol 197515 131722 Schagen FH Rademaker HJ Rabelink MJ van Ormondt H Fallaux FJ van der Eb AJ Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield Gene Ther 20007 15704 Kanegae Y Makimura M Saito I A simple and efficient method for purification of infectious recombinant adenovirus Jpn J Med Sci Biol 199447 15766 Yang DK Kim HH Lee SH Ji M Cho IS Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera J Bacteriol Virol 201747 14855 Burova E Ioffe E Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications Gene Ther 200512 Suppl 1 S517 Konz JO Livingood RC Bett AJ Goerke AR Laska ME Sagar SL Serotype specificity of adenovirus purification using anion-exchange chromatography Hum Gene Ther 200516 134653 Zhu M Carta G Protein adsorption equilibrium and kinetics in multimodal cation exchange resins Adsorption 201622 16579 He Y Gao D Zhang M Expression of the nucleoprotein gene of rabies virus for use as a diagnostic reagent J Virol Methods 2006138 14751 Figures and Tables

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.

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).

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.

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).

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.