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Journal of Clinical Microbiology, April 1999, p. 1049-1056, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

Veronika von Messling,1 Timm C. Harder,2,* Volker Moennig,1 Peter Rautenberg,2 Ingo Nolte,3 and Ludwig Haas1

Institute of Virology, Hannover Veterinary School, D-30559 Hannover,1 Institute of Medical Microbiology and Virology, Christian-Albrechts-University, D-24105 Kiel,2 and Small Animal Clinic, Hannover Veterinary School, D-30519 Hannover,3 Germany

Received 28 September 1998/Returned for modification 11 December 1998/Accepted 11 January 1999

Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (kappa  = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of >= 50% showed very good inter-rater agreement (kappa  = 0.968) with V-NA titers of >= 1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of >= 1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.


* Corresponding author. Mailing address: Institute of Medical Microbiology and Virology, Christian-Albrechts-University, Brunswiker Str. 4, D-24105 Kiel, Germany. Phone: 49-431-597-3331. Fax: 49-431-597-3296. E-mail: harder{at}medmicrobio.uni-kiel.de.


Journal of Clinical Microbiology, April 1999, p. 1049-1056, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.



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Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 1999 by the American Society for Microbiology. All rights reserved.