J Vet Sci.  2014 Dec;15(4):503-509. 10.4142/jvs.2014.15.4.503.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

• Cho KH
• Kim J
• Yoo HA
• Kim DH
• Park SY
• Song CS
• Choi IS
• Lee JB

• KMID: 2070236
• DOI: http://doi.org/10.4142/jvs.2014.15.4.503

Abstract

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Keyword

canine distemper virus; enzyme-linked immunosorbent assay; H gene; hydrophilic extra-viral domain; immunochromatography

MeSH Terms

Animals
Antigens, Viral/*diagnostic use
Distemper/diagnosis/*virology
Distemper Virus, Canine/*immunology
Dog Diseases/*diagnosis/virology
Dogs
Enzyme-Linked Immunosorbent Assay/*veterinary
Escherichia coli/genetics
Genetic Vectors/genetics
Hemagglutinins, Viral/*diagnostic use
Antigens, Viral
Hemagglutinins, Viral

Figure

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  Fig. 1 Diagram of the test strip for detecting anti-CDV antibody and interpretation of the test results. Band shown in (A), (B), and (C) correspond to as SN titers below 1:16, from 1:16 to 1:64, and above 1:128, respectively.

• 

  Fig. 2 (A) HΔ200 (lanes 1 and 2, SDS-PAGE and Western blotting analyses using anti-His6 antibody, respectively). (B) HEVD (lanes 1 and 2, SDS-PAGE and Western blotting analyses using anti-His6 antibody, respectively). HEVD antigenicity was observed with Western blotting (C) using anti-CDV polyclonal antibody (1:1,000) and anti-goat HRP-conjugated antibody (1:2,000).

• 

  Fig. 3 (A) Dot blotting for HEVD and HΔ200 using anti-CDV polyclonal antibody (left) or anti-His6-peroxidase antibody (right) and ELISA. (B) performed for 12 sera samples divided into three categories: ones with low titers (S0, L1~L4), medium titers (S3, M1~M4), and high titers (S7, H1~H4). S units (S0 to S6) were measured by ImmunoComb. For dot blotting, each serum sample was diluted 1:3,000. The anti-CDV polyclonal antibody detected HEVD more easily than HΔ200, while anti-His6-peroxidase antibody was sensitive to both proteins.

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