Determination of parvovirus antigen in the vaccine using time‐resolved fluorescence immunoassay

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV‐2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV‐2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV‐2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV‐2 antigen (CPV‐2‐Ag) in vaccine. Here, a sandwich time‐resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti‐CPV‐2 antibodies were immobilized on 96‐well plates to capture CPV‐2‐Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+) chelates; finally, time‐resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV‐2‐Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2 = 0.9933), and the average recovery is among 91.00% to 106.39% without cross‐reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV‐2‐Ag concentration and antibody titers (R2 = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV‐2‐Ag in vaccine evaluation. The time‐resolved fluorescence immunoassay can be used for detection of the CPV‐2 antigen in vaccine and prediction the vaccination effect.