Abstract
Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores.Key points• Three CDV mAbs that recognized different epitopes and bound to virion were generated.• The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed.• The sandwich ELISA is an ideal method for detecting CDV infections in the field.
Highlights
Canine distemper virus (CDV) can infect a broad range of carnivores, including Canidae, Procyonidae, Felidae, Mustelidae, Mephitidae, Ailuridae, Viverridae, Hyaenidae, and Phocidae (Beineke et al 2015; Loots et al 2017)
The F protein was purified using a Ni-NTA resin column; the SDS-PAGE analysis was conducted and demonstrated the presence of highly pure recombinant F protein (Fig. 1a). This protein was subsequently shown via Western blot analysis to possess antigenic epitopes that bound to antiCDV antibody from dogs possessing strong immunity to CDV (Fig. 1b)
The antibody titers of serum samples collected from four immunized mice were all 1:105, suggesting that an immune response to recombinant CDV-F protein had been induced in each mouse (Fig. 2a)
Summary
Canine distemper virus (CDV) can infect a broad range of carnivores, including Canidae, Procyonidae, Felidae, Mustelidae, Mephitidae, Ailuridae, Viverridae, Hyaenidae, and Phocidae (Beineke et al 2015; Loots et al 2017). Its 15,690-nt viral genome encodes six open reading frames (ORFs) corresponding to six proteins: nuclear protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L) (Martinez-Gutierrez and Ruiz-Saenz 2016; Sidhu et al 1993). Of these proteins, CDV H and F glycoproteins are located on viral particle surfaces and are responsible for virus attachment to host cells and fusion with the host cell membrane (Rendon-Marin et al 2019). Several previous studies have shown that the F protein is a highly conserved immunogenic protein and an important antigen for inducing neutralizing antibodies against CDV (David et al 2019)
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