The IPMA for demonstrating antibodies to CHV1 was performed as described [15 (link)] and previously used both in epidemiological [10 (link),15 (link)] and prospective [21 (link)] studies. IPMA is a virus neutralisation test. Briefly, monolayers of CHV infected Madin-Darby canine kidney cells in 96-well microtiter plates were incubated with twofold dilutions 1:10 to 1:1280 of test sera. Following washing and incubation steps, secondary peroxidase-conjugated rabbit anti-dog immunoglobulin G was added (DAKO, Denmark), and subsequently the substrate 3-amino-9-ethylcarbazole (Invitrogen AB, Sweden). Positive and negative serum samples were included. Serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining. Titers equal to or above 80 were considered positive for exposure to CHV1. Further, increasing antibody titers were categorized as; 80 = weakly positive, 160 and 320 = moderately positive, 640 ≥ strongly positive.
The validation of the IPMA showed a sensitivity of about 90% compared to an in-house ELISA test (National Veterinary Institute, Sweden) used in a vaccine trial on sera from dogs vaccinated against CHV. Twenty-seven serum samples were collected from 13 dogs at intervals during the trial. Estimation of the specificity is not available.
The validation of the IPMA showed a sensitivity of about 90% compared to an in-house ELISA test (National Veterinary Institute, Sweden) used in a vaccine trial on sera from dogs vaccinated against CHV. Twenty-seven serum samples were collected from 13 dogs at intervals during the trial. Estimation of the specificity is not available.