Section2:Stability and Process Studies

From Assay Guidance Wiki

Contents 1 Reagent Stability and Storage Requirements 2 Reaction Stability Over Projected Assay Time 3 Reagent Stability During Daily Operations; Use Of Daily Leftover Reagents 4 DMSO Compatability

Reagent Stability and Storage Requirements

It is important to determine the stability of reagents under storage and assay conditions.

• Use the manufacturer’s specifications if the reagent is a commercial product.
• Identify conditions under which aliquots of the reagent can be stored without loss of activity.
• If the proposed assay will require that the reagent be frozen and thawed repeatedly, test its stability after similar numbers of freeze-thaw cycles.
• If possible, determine the storage-stability of the reagent.
• If reagents are combined and aliquoted together, examine the storage-stability of the mixtures.

Reaction Stability Over Projected Assay Time

Conduct time-course experiments to determine the range of acceptable times for each incubation step in the assay. This information will greatly aid in addressing logistic and timing issues.

Reagent Stability During Daily Operations; Use Of Daily Leftover Reagents

The stability studies will require running assays under standard conditions, but with one of the reagents held for various times before addition to the reaction. The results will be useful in generating a convenient protocol and understand the tolerance of the assay to potential delays encountered during screening.

If possible, reagents should be stored in aliquots suitable for daily needs. However, some information pertinent to saving leftover reagents (particularly expensive ones) for future assays should be obtained.

New lots of critical reagents should be validated using the bridging studies

DMSO Compatability

Test compounds are delivered at fixed concentrations in 100% DMSO, thus solvent-compatibility of assays should be determined. Typically, the uninhibited or fully stimulated assay may be performed in the presence of DMSO concentrations spanning the expected final concentration. Typically, DMSO concentrations from 0 to 10% are tested. Note that this study should be done relatively early in development of the assay because other studies, such as the variability studies, should be performed with the concentration of DMSO that will be used in screening. For cell based assays, it is recommended that the final %DMSO be kept under 1%.