Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method
Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method
复制标题
DOI：
10.1021/ac048823k
复制
发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Ohmura, N
中科院分区：
文献类型：
--
作者：
Sasaki, K;Glass, TR;Ohmura, N
酶联免疫吸附试验（ELISA）的96孔板格式是筛选杂交瘤活性抗体的事实上的标准。尽管它被广泛使用，但很少或根本没有系统的尝试来验证其准确性并回答基本问题，它是否找到了所有的积极因素？我们在这里报告的ELISA和半自动化的流动为基础的动力学排斥试验（KinExA），都用于筛选相同的杂交瘤细胞系之间的比较。我们的发现是，与KinExA系统相比，ELISA既有多报（假阳性），也有漏报（假阴性）。大量的杂交瘤细胞（例如，在六个96孔板中培养），必须检查，这是令人生畏的，在考虑任何方法以外的ELISA常规筛选。为了克服这一点，我们设计了一种采样策略，其中威尔斯在一个指定的模式相结合，允许显着减少所需的测量总数。
The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semi-automated flow-based kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.