In this study we report on the development of a capture-sandwich ELISA suitable for the rapid and sensitive detection of canine IgG and IgM antibodies specific for CDV. The assay is based on a recombinant wt CDV N protein produced in the baculovirus expression system.
In a previous study we showed by immunological and biochemical analyses that CDV N protein expressed by recombinant baculoviruses in Sf9 insect cell cultures appears to be indistinguishable from authentic N protein expressed by CDV-infected Vero cells (19
). In the framework of the present investigation, however, MAbs which were raised against both the authentic and the recombinant CDV N protein revealed a lowered affinity for baculovirus-expressed N protein. The basis of this phenonemon was not further investigated since it apparently did not create a significant impact on the suitability of the antigen for use in an ELISA. We have further demonstrated that the recombinant CDV N protein can be produced in H. viriscens
larvae at a quality similar to that of infected Sf9 cell cultures. With respect to ease, efficacy, and costs of production, the larval system was found to be clearly superior to insect cell cultures. This result is in line with studies of the recombinant expression of N proteins of other morbilliviruses (12
In a first attempt to use the recombinant antigen for the detection of canine CDV-specific antibodies, direct coating of the antigen failed to give acceptable results due to high background signals. A capture-sandwich application, using a capture antibody in combination with an antigen solution prepared in the presence of SDS, provided improved conditions. The capture antibody served to enrich for the specific antigen and, thus, reduced nonspecific reactivity due to the presence of cellular or baculoviral antigens (11
). The SDS was expected to break up intermolecular N protein aggregrations as well as intramolecular secondary structures. Obviously, these conditions made (nonconformational) B-cell epitopes along the N protein accessible to canine antibodies.
Among a total of 196 sera, 11 false-positive and 9 false-negative samples were initially detected with the capture-sandwich ELISA, when the V-NA was regarded as the gold standard. In 10 of the 11 false-positive samples, all of Siberian origin, however, CDV-specific antibodies were also detected by IPMA. Western blotting revealed these antibodies to be specific for CDV N as well as, in some sera, phosphoprotein. These samples, therefore, must be regarded as CDV seropositive, although antibodies with neutralizing capacities were undetectable. The N protein is the immunodominant morbillivirus protein and elicits the most vigorous antibody response in all morbillivirus infections. Therefore, in individuals with waning morbillivirus-specific humoral immunity, N-specific antibodies may still be detectable when H- and F-specific (neutralizing) antibodies are below detection levels. The use of a N protein-based assay may be more sensitive than V-NA when the qualitative CDV serostatus is evaluated. In addition, it has been shown that in certain cases of fulminant acute distemper infections, as well as in CDV-induced chronic demyelinating central nervous system disease, there is a restricted immune response to the hemagglutinin (H) and fusion (F) glycoproteins carrying neutralization sites but that the humoral response to nucleocapsid proteins appears to be comparatively unimpaired (26
All nine samples which tested negative in the IgG ELISA but proved to harbor CDV-neutralizing antibodies were consecutively shown by IPMA, Western blotting, and an IgM-specific capture-sandwich ELISA to carry CDV-specific antibodies of the IgM class exclusively and, thus, probably reflect an early stage of a CDV infection. Indeed, these sera had been obtained from dogs presenting with clinical signs consistent with acute virulent CDV infection, and in five animals a cell-associated CDV viremia was demonstrated by RT-PCR. As such, the CDV N protein-based IgM-specific ELISA may also be helpful in the serological diagnosis of acute distemper infections.
Having resolved these discrepant results, the performance characteristics of the IgG sandwich ELISA are promising and, in terms of sensitivity, even superior to those of the V-NA as far as qualitative results are concerned. The V-NA, however, provide additional information. Although T-cell-mediated immunity is essential in clearing morbillivirus infections, the ND50
antibody titer can be used as a surrogate marker of the resilience of a CDV-specific immunity. Dogs presenting with titers of ≥1/100 are considered to be protected from distemper (26
). To test for a correlation between N-specific antibody titers and the ND50
of a serum, a one-step-dilution (alpha method) ELISA was implemented. When we analyzed these results statistically, a bias related to the sera’s geographic origins (Hannover, Germany, versus Siberia, Russia) was revealed. Results with sera obtained from German dogs showed a good correlation (r
= 0.835, Spearman’s rank analysis) compared to results with sera obtained from Russian dogs (r
= 0.291). The Russian sera displayed comparatively higher alpha values in relation to the ND50
. Alpha values of ≥50%, therefore, could be taken as surrogate markers for ND50
titers of ≥1/100, which indicate the presence of a resilient CDV-specific immunity, for sera sampled in Germany but, at present, not for sera sampled in Siberia. Three reasons may account for this discrepancy: (i) sampling and storage conditions, (ii) neutralizability of wt CDV virus used for V-NA, and (iii) phylogenetic differences of viruses used for V-NA and for immunization of dogs. (i) While the sera from Germany were sampled and stored under optimal conditions, the Russian sera were obtained and processed under semisterile circumstances and had experienced several cycles of freezing and thawing before examination. This might have affected the stability of neutralizing antibodies more than of (the presumably more abundant) N-specific antibodies. (ii) The CDV wt isolate 2544/Han95 was used in all V-NA. This isolate originated from the same area from which the German canine sera were obtained. By phylogenetic analysis, this isolate has been found to belong to a currently circulating central European CDV genotype (17
). wt isolates from Russia (Lake Baikal, Siberia), in contrast, were shown to belong to a different genotype (7
). The genotypic differences are reflected phenotypically at the amino acid level of the hemagglutinin protein, which elicits the majority of neutralizing antibodies. The nucleocapsid protein, however, displays an overall higher degree of nucleotide sequence and antigen conservation (19
). As such, the less significant correlation between N-specific alpha values and ND50
titers of the Siberian sera might have been due to the use of a heterologous CDV isolate from Germany displaying a slightly different antigenic makeup in the hemagglutinin protein. Unfortunately, no Siberian isolate was available for use in complementing V-NA. (iii) Most of the 63 German dogs had a vaccination record, but all 110 Siberian dogs did not. This suggests that all positive Siberian sera reflect wt CDV infections but that most of the positive German sera probably reflect vaccine-induced immunity. CDV vaccine strains, however, have been shown to form yet another genotype that is distinct from both central European and Siberian wt CDV (20
On the basis of these data it cannot be determined with certainty whether the weak correlation between ND50
titers and N-specific ELISA titers observed with the Siberian sera is due to the antigenic makeup of the CDV isolate used for V-NA or the N protein of the capture-sandwich ELISA. However, minor strain-specific influences on virus-neutralizing antibody titers have been demonstrated for rinderpest (31
), measles (34
), and distemper (18
) viruses, although commonly the morbillivirus species are considered serologically monotypic. In CDV serology, minor strain-specific antigenic variation causes us to have reservations about the validity of the ND50
threshold titer of 1/100 since it is likely to vary with the CDV strain used in V-NA and it also challenges the role of V-NA as the gold standard. In view of our data on the Siberian sera, use of the V-NA for seroprevalence studies may lead to an underestimation of prevalence rates when no local (i.e., homologous) CDV isolate is at hand. So, at least for screening purposes, the N-specific capture-sandwich IgG ELISA may prove to be a first-choice option.
In conclusion, results of the recombinant one-step-dilution capture-sandwich ELISA introduced here showed very good agreement in an inter-rater analysis with results of V-NA, and it may therefore be a practicable and cost-effective substitute for CDV V-NA in an evaluation of qualitative CDV-specific IgG serostatus. This ELISA format likewise appears to be suitable for serologic diagnosis of acute distemper infections by detection of specific IgM. Provisional results from ongoing studies in our lab indicate that the ELISA can be easily adapted for detection of CDV-specific antibodies also in sera of noncanine species, such as the exotic felids (unpublished data).