Category: Assay Development and Screening
Scientists typically optimize assay parameters without consideration of liquid handler performance. Instead, default liquid handler values are selected during transition to an automated platform. This study illustrates the importance of optimizing both the assay and any associated liquid handling steps with the overall goal of minimizing variability. Assay optimization parameters such as incubation time and temperature, and liquid handler optimization parameters such air gaps, aspirate/dispense speeds, fluid exit rates, and calibration were all studied. Parameters that bridged assay and liquid handling optimization such as mixing, buffer type and labware were also considered. The aim of this study was to optimize the entire assay and process prior to automation. By evaluating the entire assay assembly process, we aim to reduce variability as early as possible, thereby allowing assay transfer to become less troublesome. The study was designed to evaluate assay performance by comparing dose-response (IC50), Hill slope, curve span, and precision across the different parameters. The model assay was based upon the binding protein streptavidin and its strong binding affinity toward biotin and derivatives of biotin. In this assay, biotin-4-fluorescein (B-Fl) competes with biotin and its derivatives for binding to streptavidin. If B-Fl binds, fluorescence is quenched. If it remains free in solution the fluorescence is maintained. By examining various liquid handling parameters, we were able to demonstrate that some affected the outcome of the assay, while others did not. This methodology allows the user to pinpoint which liquid handler variable parameters have the most impact on the particular assay.
Nathaniel Hentz– Assistant Director, Analytical, North Carolina State University, Raleigh, NC
Assistant Director, Analytical
North Carolina State University
Dr. Nathaniel Hentz is Assistant Director of the Analytical Lab at the Golden LEAF Biomanufacturing Training and Education Center (BTEC), where he provides analytical support for the various biomanufacturing processes taught at BTEC. He has nearly 12 years of experience developing high-throughput screening assays, automating and optimizing laboratory equipment and investigating new technologies with Eli Lilly & Co. and Bristol-Myers Squibb. Dr. Hentz has also served as an independent consultant in the automated liquid handling quality control, microfluidic separations and ADME/tox fields. He received his Ph.D. in analytical chemistry from the University of Kentucky in 1996 and B.S. in chemistry from Eastern Michigan University in 1990.