Pfizer BioTherapeutics Research and Development, 4300 Oak Park, Sanford, North Carolina 27330, USA.
Anal Chem. 2010 Mar 1;82(5):1786-92. doi: 10.1021/ac902664x.
Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.
本文介绍了一种手动多糖检测方法的改进，该方法适用于结合疫苗（如 Prevenar），可应用于自动化液体处理系统（LHS），以提高性能。蒽酮法用于测定碳水化合物浓度，并通过适当的混合、分配和测量操作扩展到微量滴定板格式。对 LHS 平台进行了改进和开发，包括不同大小的葡聚糖多糖和肺炎球菌 6A 型多糖（PnPs 6A）。标准板配置编程使 LHS 对校准标准品和测试样品进行多次稀释，每个稀释倍数有六个重复制备。这种重复程度最大限度地减少了任何单一偏差或输送误差的影响。对大小从 214 kDa 到 3.755 MDa 的葡聚糖聚合物进行分析表明，无论聚合物链的长度如何，水解都是完全的，这从均匀的浓度测量中可以明显看出。未观察到板位置吸光度偏差；在分析了 12 个板以检查位置偏差的情况下，观察到的最大偏差为 0.02%相对标准偏差（%RSD）。高纯度的葡聚糖还提供了评估 LHS 准确性的机会；对葡聚糖进行了九次重复分析，得出的平均回收率为 101%。至于精密度，在单个批次的 PnPs 6A 上总共进行了 22 次独特的分析，结果的变异性为 2.5% RSD。这项工作证明了 LHS 能够一致地进行蒽酮法，并缩短了检测周期，提高了实验室的容量。
