Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronomicas (CICV y A) - Instituto Nacional de Tecnologia Agropecuaria (INTA), Buenos Aires, Argentina; CONICET, Argentina.
J Virol Methods. 2010 Oct;169(1):143-53. doi: 10.1016/j.jviromet.2010.07.014. Epub 2010 Jul 22.
Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections.
两种酶联免疫吸附试验（ELISA）被开发并用于牛血清和免疫豚鼠的 BoHV-1 抗体定量，符合 ISO/IEC 17025 标准。该试验的截断值设定为高阳性对照的 20%阳性，用于牛的筛选。使用这个阈值，该试验正确地对 OIE 牛参考血清 EU1、EU2 和 EU3 进行了分类。对于疫苗效力测试，两种物种都选择了 40%的截断值。使用 40%的阈值，试验的可靠性（由其诊断灵敏度和特异性决定）分别为牛的 89.7%和 100%，以及豚鼠的 94.9%和 100%。ELISA 和病毒中和结果之间几乎完全一致。此外，在接种疫苗后，从同一牛或豚鼠中采集的血清的中和抗体效价和 ELISA 抗体效价之间存在良好的相关性，分别在接种后 60 天和 30 天（R（牛）=0.88，R（豚鼠）=0.92；p<0.0001）。当分析接种动物组的平均抗体效价时，也观察到了类似的相关性（R（牛）=0.95 和 R（豚鼠）=0.97；p<0.0001），表明这些 ELISA 对于目标物种和实验室动物模型中的批间疫苗效力测试具有相关性。在同一家实验室中，3 年内用阳性对照进行检测的相对变异系数（CV）表示，试验的中间精密度为牛的 22.2%和豚鼠的 23.1%。在实验室间试验中获得的两种技术的重现性分别为牛的 CV=12.4%和豚鼠的 CV 约为 0，符合 OIE 的要求（CV<30%）。验证的 ELISA 是疫苗效力测试和控制 BoHV-1 感染的重要方法。
