Authors

R Nielsen¹; Y Nossin¹; M Nelson¹; C Manning¹; 
¹ Cell Signaling Technology, Netherlands

Discussion

Cell Signaling Technology (CST) uses a rigorous set of validation strategies, the Hallmarks of Validation, to ensure that antibodies developed are highly specific and sensitive, reducing ambiguity when interpreting results. To discover and develop specific antibodies with utility in multiple applications, it is critical to assess samples throughout the development process using these desired applications. High Content Imaging (HCI) is a powerful tool in this arsenal of assays because it enables antibody screening and validation using a high-throughput immunofluorescence protocol. Antibodies intended for use in cell-based immunofluorescence assays (IF) are evaluated using HCI across multiple stages of development, from primary screens through final product formulation. Another commonly used tool for high-throughput antibody screening is ELISA, however it is not a good measure for prediction of utility in IF. Additionally, HCI is often more predictive of specificity across multiple applications than ELISA, as confirmed by Western Blot assay. As such, at CST HCI is performed alongside ELISA during antibody discovery. Additionally, CST leverages expertise in antibody conjugation to provide directly conjugated antibodies for use in HCI, reducing the staining workflow required for immunofluorescence protocols. Using HCI as an antibody discovery and validation tool has enabled the generation of hundreds of highly specific antibodies and led to the development of a broad portfolio of antibodies that can be used in HCI.