EMBL-DKFZ Chemical Biology Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany.
PLoS One. 2013 Oct 4;8(10):e75677. doi: 10.1371/journal.pone.0075677. eCollection 2013.
A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2) = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.
一种高度敏感、自动化、纯附加、高通量基于假病毒的中和测定法（HT-PBNA）已被开发用于人乳头瘤病毒（HPV）16、18、31、45、52、58 和牛乳头瘤病毒 1 型。通过时间将 384 孔板的血清稀释度和实际基于细胞的测定法分开，因此可以连续处理多达一百个测定板的批次。在七个独立运行中，对来自 HPV 16 DNA 阳性宫颈上皮内瘤变 2+病变患者的 35 份血清进行了分析，对于在 58 次重复中单独在各个板上测试的标准血清，抗 HPV 16 和 HPV 18 效价的平均变异系数（CV）为 13%。与之前报道的基于分泌型碱性磷酸酶的手动 PBNA（manPBNA）相比，新的 HT-PBNA 基于增加了灵敏度的高斯荧光素酶。与 manPBNA 相比，HT-PBNA 获得的效价通常更高。对于这 35 份血清，通过 HT-PBNA 测定的效价与通过基于 Luminex 珠的 GST 捕获测定法测定的抗 HPV 16 L1 抗体水平之间存在良好的线性相关性（R²=0.7），并且只有 3 份效价低的血清不一致。除了天然低效价抗体反应外，HT-PBNA 的高灵敏度还允许检测由商业 HPV L1 疫苗和实验性 L2 疫苗诱导的交叉中和抗体。在分析 HPV 16 和 18 的世界卫生组织国际标准时，我们确定了分别为 0.864 和 1.105 mIU 的分析灵敏度。
