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Journal of Clinical Microbiology, April 1999, p. 1049-1056, Vol. 37, No. 4
Institute of Virology,
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 (
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
= 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 (
= 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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