Validation of Accuracy of Enzyme-Linked Immunosorbent Assay in Hybridoma Screening and Proposal of an Improved Screening Method

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• 作者：Kazuhiro Sasaki ; Thomas R. Glass ; and Naoya Ohmura
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：April 1, 2005
• 年：2005
• 卷：77
• 期：7
• 页码：1933 - 1939
• 全文大小：408K
• 年卷期：v.77,no.7(April 1, 2005)
• ISSN：1520-6882

文摘

The 96-well plate format of enzyme-linked immunosorbentassay (ELISA) is the de facto standard in screeninghybridomas for active antibody. Despite its widespreaduse, there have been few or no systematic attempts tovalidate its accuracy and answer the fundamental question, is it finding all the positives? We report here on acomparison between ELISA and a semiautomated flow-based kinetic exclusion assay (KinExA), both used inscreening the same hybridoma cell line. Our finding isthat ELISA is both overreporting (false positives) andunderreporting (false negatives) compared to the KinExAsystem. The large number of hybridoma cells (e.g.,cultured in six 96-well plates) that must be checked isdaunting in considering any method other than ELISA forroutine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specifiedpattern, allowing a significant reduction in the totalnumber of measurements required.