Monoclonal Antibody scFv 1N8 Anti-N Protein of Canine Distemper Virus

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.

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Quantitation of Canine Distemper Virus and Antibodies by Enzyme-Linked Immunosorbent Assays Using Protein A and Monoclonal Antibody Capture

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100203 ◽

1989 ◽

Vol 1 (2) ◽

pp. 110-115 ◽

Cited By ~ 3

Author(s):

Leon N. D. Potgieter ◽

Peace A. Ajidagba

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Cell Cultures ◽

Canine Distemper Virus ◽

Solid Phase ◽

Sandwich Elisa ◽

Protein A ◽

Canine Distemper ◽

Clinical Specimens ◽

Immunosorbent Assays

Monoclonal antibodies produced from 19 cloned hybridomas were selected for this study. Specific canine distemper virus (CDV) antibodies in medium from cloned hybridomas were detected by direct enzyme-linked immunosorbent assays (ELISA) and by indirect immunofluorescence. Three different sandwich ELISA systems were developed either to detect CDV in cell cultures and clinical specimens or to detect specific antibody in canine sera. Protein A and monoclonal antibodies attached in sequence to a solid phase constituted the capture system in the assays. Viral antigens were detected by sandwiching extracts of clinical specimens (or infected cell cultures), monoclonal antibody, and peroxidase-labeled protein A in sequence onto the capture layer. In 1 procedure, biotin-labeled antibody and peroxidase-labeled avidin were used as the last 2 layers in the assay. The CDV antibodies in dog sera were quantitated in a similar manner, but the sequential sandwiching levels consisted of partially purified CDV, serum specimen, and peroxidase-labeled protein A, respectively. The procedures were specific and highly sensitive.

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Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein

Virus Research ◽

10.1016/j.virusres.2015.10.022 ◽

2016 ◽

Vol 213 ◽

pp. 1-5 ◽

Cited By ~ 5

Author(s):

Li Yi ◽

Yuening Cheng ◽

Miao Zhang ◽

Zhigang Cao ◽

Mingwei Tong ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Nucleocapsid Protein ◽

Canine Distemper Virus ◽

Cell Epitope ◽

Canine Distemper ◽

B Cell Epitope

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Phylogenetic Analysis and Biological Characteristics Identification of a Canine Distemper Virus Strain Isolated from a vaccinated Domestic Dog in the Northeast China

10.21203/rs.3.rs-349122/v1 ◽

2021 ◽

Author(s):

Cao Zhigang ◽

Yi Li ◽

Yuening Cheng ◽

Pengfei Shi ◽

Jianke Wang ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Host Range ◽

Genome Sequence ◽

Canine Distemper Virus ◽

Immunofluorescence Assay ◽

Domestic Dog ◽

Whole Genome Sequence ◽

Canine Distemper ◽

N Protein ◽

H Protein

Abstract Canine distemper virus (CDV) is currently circulating in domestic and wild animals. The host range of CDV comprises all families within the order Carnivora, and it has recently expanded to nonhuman primates, moreover the host range still has the potential for further expansion. In this research, a CDV strain named BT was isolated from a vaccinated domestic dog in Changchun, Northeast of China, and identified by Electron microscope (EM) and Immunofluorescence Assay (IFA). The whole genome sequence of the virus was obtained by RT-PCR and Sanger sequencing. Phylogenetic analysis was performed based on the Coding sequence (CDS) of hemagglutinin (H) protein and the complete genome sequence of the virus respectively, the results showed that the CDV-BT strain still classified into the Asia-1 lineage. This research shows that CDV-BT strain’s antigenicity of the epitope 444GDKYPIHFNDER455 in nucleocapsid (N) protein and the epitope 178ARGDIFPPY186 in H protein were significantly different from vaccine strain by amino acid substitutions, and suggests that the characterization of genetic diversity among the circulating CDVs is essential for future CDV’s research and disease monitoring.

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Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody

Cellular and Molecular Life Sciences ◽

10.1007/bf01922469 ◽

1991 ◽

Vol 47 (8) ◽

pp. 842-845 ◽

Cited By ~ 20

Author(s):

D. Hamburger ◽

C. Griot ◽

A. Zurbriggen ◽

C. Örvell ◽

M. Vandevelde

Keyword(s):

Monoclonal Antibody ◽

Structural Change ◽

Canine Distemper Virus ◽

Canine Distemper

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Evaluation of infection with N protein-specific Immunoglobulin M and G in naturally occurring distemper in dogs

Veterinární Medicína ◽

10.17221/31/2019-vetmed ◽

2020 ◽

Vol 65 (No. 4) ◽

pp. 168-173

Author(s):

HS Saltik ◽

M Kale

Keyword(s):

Canine Distemper Virus ◽

Indirect Elisa ◽

Clinical Symptoms ◽

Clinical Manifestations ◽

Specific Treatment ◽

Immunoglobulin M ◽

High Morbidity ◽

Canine Distemper ◽

Cutaneous Lesions ◽

N Protein

In dogs, canine distemper has a worldwide distribution with high morbidity/mortality, despite the widespread usage of vaccines and has no specific treatment. In susceptible animals with the canine distemper virus, respiratory, gastrointestinal and nervous system disorders, immunosuppression and cutaneous lesions can also be seen. Especially puppies and unvaccinated dogs are prone to get the viral infection. IgM and IgG antibodies constitute the major component of the natural antibodies produced during the primary and secondary antibody response that have long been recognised to inhibit viral infections. In the present study, the presence of the viral N protein-specific IgM and IgG was investigated by indirect ELISA in naturally infected dogs. Moreover, the rate of outbreaks in naturally infected dogs was shown by the detection of new and re-infections. In the Western Mediterranean region, blood serum samples were collected from 50 unvaccinated dogs for the mentioned infection between 2015 and 2017. At 0–12 months, in the dogs with clinical symptoms, the indirect ELISA detected 4% acute, 54% early convalescent, 40% late convalescent and 2% no infections phases. The clinical manifestations were studied in four main groups follow as: respiratory, gastrointestinal, nervous and cutaneous symptoms. The evaluation showed that the canine distemper virus N protein-specific antibodies detection by the indirect ELISA is quick and safe in naturally infected dogs. In conclusion, the method is very useful for the pre-diagnosis of the disease when evaluated together with the clinical symptoms. It helps to distinguish acute and convalescent (early/late) phases. Distinguishing these phases of infection is important for monitoring the spread of the outbreaks and identifying the risk of severe forms of canine distemper.

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Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma

International Journal of Molecular Sciences ◽

10.3390/ijms22073578 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3578

Author(s):

Federico Armando ◽

Adnan Fayyad ◽

Stefanie Arms ◽

Yvonne Barthel ◽

Dirk Schaudien ◽

...

Keyword(s):

Tumor Microenvironment ◽

Virus Infection ◽

Tumor Growth ◽

Canine Distemper Virus ◽

Xenograft Model ◽

Histiocytic Sarcoma ◽

Oncolytic Viruses ◽

Canine Distemper ◽

Murine Xenograft Model ◽

The Impact

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

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