Rapid and sensitive detection of immunoglobulin M (IgM) and IgC antibodiesagainst canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

Canine distemper morbillivirus (CDV) infection causes a frequently fatal sy stemic disease in a broad range of carnivore species, including domestic do gs. In CDV infection, classical serology provides data of diagnostic and pr ognostic values (kinetics of seroconversion) and is also used to predict th e optimal vaccination age of pups, Routine CDV serology is still based on t ime- and cost-intensive virus neutralization assays (V-NA). Here, we descri be a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that us es recombinant baculovirus-expressed nucleocapsid (N) protein of a recent C DV wild-type isolate (2544/Han95) for the detection of CDV-specific antibod ies 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 s erostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement ana lysis (kappa = 0.988) indicated that the ELISA can be used unrestrictedly a s a substitute for the V-NA for the qualitative determination of CDV-specif ic IgG serostatus. In an attempt to semiquantify N-specific antibodies, a o ne-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha v alues of greater than or equal to 50% showed very good inter-rater agreemen t (kappa = 0.968) with V-NA titers of greater than or equal to 1/100 50% ne utralizing dose (ND50) as measured against the central European CDV wild-ty pe isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of greater than o r equal to 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 leucocy tes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was d etected by reverse transcription (RT)-PCR, The recombinant capture-sandwich ELISA detecting N-specific antibodies of the Ige class provided superior s ensitivity and specificity and thus represents a rapid and cost-effective a lternative 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 acut e distemper infections.