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Guidelines for HTS Assay Methods

I. A. Assay Requirements Detection Modes one of the following 1. Absorbance 2. Luminescence 3. Fluorescence intensity 4. Fluorescence polarization (FP) 5. Fluorescence resonance energy transfer (FRET) 6. Fluorescence for high content microscopic detection a. Up to 4 different fluorescent labels b. Must label cell nuclei Controls 1. Must have 2 controls: 2.

B.

Maximum control Minimum control 4 controls maximum in an assay No dose response curves

C.

96 well microtiter plate format Or 384-well microtiter plate format Reproducibility 1. %CV < 10% for controls in biochemical assay 2. % CV < 12% for controls in cell-based assay %CV = standard deviation (s.d.) mean 3. x 100

D.

Signal of individual wells a. No signal patterns across microtiter plates b. Plate-to-plate variability is low c. Day-to-day variability is low

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E.

Signal / Background > 3.0 Signal / Background > 10 preferred Signal / Background (S/B) = high control signal low control signal

F.

Z > 0.5 Z = 1 3(s.d high control + s.d.low control) (mean high control mean low control)

G.

Assay Performance in the presence of DMSO 1. No significant effect on assay in presence of DMSO concentration required for the assay 2. Data for controls in the presence of 0.05% to 1% DMSO 3. Determination of DMSO concentration required for the assay a. Test compounds in UC Compound Library at 10 mM in 100% DMSO b. Transfer test compounds into assay directly from 100% DMSO solution c. Based on: assay volume (20 70 l in a well) transfer volume (down to approximately 10 nl) test compound concentration d. A common test compound concentration is 10 M

H.

Reagent Stability 1. All reagents added to assay must be stable on the automation system for at least 2 hours 2. Possible to place reagent vessel on ice Stable cell lines for cell-based assays Cells must be mycoplasma free Antibiotics in media with cell-based assays Calculations used for results Desired concentration of test compounds in screen Criteria for HTS hit (examples: level of inhibition, most potent inhibitors, most potent agonists)

I. J. K. L. M. N.

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II. A.

Preferred Assay Characteristics Single measurement by detector 1. Single time point measurement gives best throughput 2. Multiple wavelengths measured is acceptable 3. Cannot measure fluorescence and absorbance in the same assay Minimum number of wash steps zero is best 1. Wash steps decrease assay reproducibility 2. Wash steps decrease throughput Minimum number of reagent addition steps 1. Increasing number of steps decreases reproducibility 2. Increasing number of steps decreases throughput 3. Number of reagent addition steps limited by number of dispensers Shortest assay duration with good assay performance 1. Multiple day assays decrease throughput 2. Multiple day assays increase time to adapt and validate the automated assay method Plate reader detection preferred over Opera detector (high content assays) 1. Significantly faster to develop and validate 2. Significantly faster to run screen

B.

C.

D.

E.

III. A.

Provided by Collaborator Detailed bench level protocol 1. Source of reagents: vendor and catalog number 2. Concentration of all reagents 3. Volumes of all reagents 4. Timing for all assay steps 5. Temperature for all assay steps 6. Detection equipment used and detection parameters, example: wavelength 7. Storage of all reagents a. Freeze-thaw cycle effects b. Light sensitivity c. Solution or solid 8. Preparation of all reagents 9. Reagent stability during assay (assay conditions of temp, time, etc.) 10. Handling of cells a. Media b. How often to split, splitting ratios c. Cell density and appearance for assay

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11. B.

d. Number of cell passages that can be used in assay Information on known inhibitors or stimulators, if appropriate or available

Adequate reagents available for assay adaptation & screening 1. Preferably large batches with single lot numbers for the entire screen 2. Supplier a. commercial supplier b. OR supplied by collaborator (examples: antibody, receptor protein) Additional information available 1. Information on target, desired effect on target 2. Assay performance information a. For enzyme assays Substrate concentration effect Enzyme concentration effect Reaction Time effect Detection reagent effects time & concentration Temperature effects b. For binding assays Ligand concentration effect Receptor concentration effect Time effects, to determine equilibration time for binding Detection reagent effects time and concentration Temperature effects Effect of varying Ligand competitor on assay, especially for a competition assay

C.

For more detailed information on HTS assay methods: Assay Guidance Manual Version 5.0, 2008, Eli Lilly and Company and NIH Chemical Genomics Center. Available online at: http://www.ncgc.nih.gov/guidance/manual_toc.html

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Appendix IV. A. Stand Alone Detector Capabilities- PE Envision Detection Modes 1. Absorbance 2. Luminescence 3. Fluorescence intensity 4. Fluorescence polarization (FP) 5. Fluorescence resonance energy transfer (FRET) 6. Time resolved fluorescence (TRF) 7. Time resolved fluorescence resonance energy transfer (TF-FRET) Additional Capabilities 1. Onboard injector allowing reagent addition with immediate measurement 2. Plate stacker

B.

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