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.

中文翻译：

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时间分辨荧光免疫分析法测定疫苗中的细小病毒抗原

犬细小病毒2型（CPV-2）作为犬的一种高度传染性和潜在的致命疾病，通常会引起严重的心肌炎和胃肠炎，而疫苗注射大大降低了CPV-2疾病的发生率。然而，目前缺乏简单有效的方法来定量检测疫苗中的CPV-2。因此，本研究旨在制备一种准确测定疫苗中CPV-2抗原（CPV-2-Ag）的方法。在这里，建立并优化了夹心时间分辨荧光免疫分析 (TRFIA)。将抗 CPV-2 抗体固定在 96 孔板上以捕获 CPV-2-Ag，然后与标有铕 (III) (Eu ³⁺) 螯合物；最后，采用时间分辨荧光法测量荧光强度。进行疫苗接种以评估 CPV-2-Ag 浓度与抗体滴度之间的关系。灵敏度为1.15 mEU/mL（Log Y  = 1.524 + 0.8667 × Log X，R ²  = 0.9933），平均回收率在91.00%至106.39%之间，与其他犬病毒抗原无交叉反应。ELISA检测与该方法的相关性高达0.9861。而且，CPV-2-Ag 浓度与抗体滴度之间存在高度相关性 ( R ² = 0.9234）。该免疫测定方法具有较高的灵敏度、准确度和特异性，表明该方法可适用于疫苗评价中CPV-2-Ag的定量检测。