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

Abstract

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.

FIG. 1

Expression of CDV 2544/Han95 N protein by recombinant baculovirus in H. virescens larvae as demonstrated by Western blot analysis. Larvae were prepared 3 to 5 days following subcuticular infection with 5 × 10⁴ TCID₅₀ of recombinant Bac-CDV-N or with wt AcNPV (c, control). Blots were probed with the N-specific MAb CDRCT-8 (Table 1). In each lane 10 μl of a lysate (prepared with 2% SDS in TBS) was loaded. At least 500 μl of lysate was produced from a single larva. p., post.

FIG. 2

Yields and suitability of recombinant CDV N protein prepared from infected Sf9 in vitro cultures and from H. virescens larvae under different lysing conditions. Results were obtained by indirect ELISA with MAb CDRCT-8. Mock-infected Sf9 cells were used as a negative control. ⧫, 4 M urea in TBS (pH 8.7); ■, 2% SDS in TBS (pH 8.7) with lysate from larvae at a starting dilution of 1/1,000; ◊, 1% Triton X-100 in TBS (pH 8.7); □, 1% SDS in TBS (pH 8.7); +, aqua bidest; ○, beta-OTG in TBS (pH 8.7); ●, beta-OTG in 0.1 M glycine (pH 9.6); ★, negative control.

FIG. 3

Characteristics of the IgG-specific CDV N protein-based capture-sandwich ELISA compared to those of V-NA alone (A) and in combination with other nonneutralizing IgG-relevant test systems (B). Values in the boxes are numbers of samples. Performance parameters were calculated according to equations in reference . Values in parentheses are 95% confidence intervals. Wblot, Western blotting; EIA, enzyme immunoassay; Pos. Predict. Value, positive predictive value; Neg. Predict. Value, negative predictive value.

FIG. 4

Correlation between values obtained by the one-step capture-sandwich ELISA (alpha method) and CDV-neutralizing antibody titers (ND₅₀) in concordantly IgG-positive or -negative canine sera originating from Siberia, Russia (A), or from northern Germany (B). Sera were examined in duplicate by the recombinant CDV N protein-based ELISA at a single dilution of 1/100. Results were recorded as percentages of activity relative to those of a standard positive serum and a negative serum obtained with the following equation: percentage of activity = (OD_sample − OD_neg)/(OD_pos − OD_neg) × 100. Correlation (Spearman’s rank analysis) and regression tests were performed for sera showing ND₅₀ titers of ≥1/10. Inter-rater agreement analysis was extended also to seronegative samples. A threshold value of ≥50% activity indicated the presence of a (protective) ND₅₀ titer of ≥1/100 only in sera of panel B. Dotted horizontal and vertical lines indicate thresholds distinguishing seropositive from seronegative values. Solid horizontal and vertical lines indicate thresholds discriminating protective from nonprotective titers. % activ., percentage of activity; EIA, enzyme immunoassay. Values in boxes are numbers of samples with the indicated ND₅₀ and percentages of activity. Values in parentheses are 95% confidence intervals.