Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method.
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 semiautomated 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.
Sasaki
Kazuhiro
K
Biotechnology Sector, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko City, Chiba, Japan 270-1194. k-sasaki@criepi.denken.or.jp
Glass
Thomas R
TR
Ohmura
Naoya
N
D000818:Animals; D000915:Antibody Affinity; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004492:Edetic Acid / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005189:False Positive Reactions; D006825:Hybridomas / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
D004492:Edetic Acid