Development of an Immunochromatographic Strip for Rapid Detection of Canine Adenovirus

Abstract

Although canine adenovirus (CAdV) is highly prevalent in dogs, there is currently a lack of a quick diagnostic method. In this study, we developed a rapid immunochromatographic strip (ICS) assay using colloidal gold coupled to CAdV-2-specific monoclonal antibodies (mAbs). BALB/c mice were immunized with a purified CAdV-2 suspension, and four mAbs (belonging to two different epitopes) were generated and designated as 2C1, 7D7, 10D1, and 4G1. Western blot and protein spectral analysis indicated that the hexon protein of CAdV-2 recognized all four mAbs. The colloidal gold-coupled 7D7 and 2C1 mAbs were chosen for inclusion in the rapid ICS assay. The optimal concentrations of the coating antibody (2C1), the capture antibody (7D7), and the goat anti-mouse antibody were 1.0 mg/ml, 10 µg/ml, and 2.0 mg/ml, respectively. The limit of detection was approximately 2.0 × 10 2 tissue culture infective dose (TCID 50 )/ml. Other common canine viruses were tested to evaluate the specificity of the ICS, and positive results were observed for only CAdV-1 and CAdV-2. The ICS test was conducted on 360 samples to detect CAdV, and the results were compared with those of polymerase chain reaction (PCR) tests. The ICS test was found to be a sufficiently sensitive and specific detection method for the convenient and rapid detection of CAdV.

Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies

“…The CAdV-2 strain used in this study was stored at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Science. Hybridoma lines secreting CAdV-2-specific mAbs were generated in our lab previously [ 15 ]. The pET-30a (+) vector was purchased from Tiangen, China.…”

“…Hybridoma lines secreting CAdV-2-specific mAbs were cultured, and the isotype was determined using the SBA Clonotyping TM System/HRP Kit (Southern Biotech, USA). Specificity characterization of CAdV-2-specific mAbs 2C1 and 7D7 was carried out by indirect immunofluorescence assay (IFA) and WB according to standard procedures [ 15 ], but WB using denaturing SDS-PAGE and native PAGE, respectively. Denaturing SDS-PAGE was carried out according to standard procedures.…”

“…The truncated Hex protein was detected by SDS-PAGE and Western blotting (WB) analysis by anti-His mAb. Immunoreactive bands were verified by an enhanced chemiluminescence system (ECL; PerkinElmer Life Sciences, Fremont, CA, USA) [ 15 ]. The three-dimensional (3D) structure of the expressed protein was predicted with PyMOL software based on data from the SWISS-MODEL online server.…”

“…The expression plasmid was transformed into E. coli BL 21 competent cells (Tiangen); the truncated Hex protein was produced via isopropyl-β-D-thiogalactopyranoside (IPTG, GE Healthcare, Chicago, IL, USA) induction using pET30a-Hex E. coli at 37 • C. The truncated Hex protein was detected by SDS-PAGE and Western blotting (WB) analysis by anti-His mAb. Immunoreactive bands were verified by an enhanced chemiluminescence system (ECL; PerkinElmer Life Sciences, Fremont, CA, USA) [15]. The threedimensional (3D) structure of the expressed protein was predicted with PyMOL software based on data from the SWISS-MODEL online server.…”

Development of indirect ELISA for the detection of canine adenovirus type 2 antibodies in dog sera

“…There are several methods for detecting canine adenovirus (CAV) antigen, including virus isolation, polymerase chain reaction, histopathology, rapid immunochromatographic strip assay and immunohistochemistry [6][7][8][9]. CAV antibodies induced by inoculation of vaccine or natural infection can also be measured by several methods, such as hemagglutination inhibition (HI), virus neutralization (VN), enzyme-linked immunosorbent assay (ELISA), and the indirect immunofluorescence assay in animal serum samples [10,11].…”