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Assay Design and Quality Control in HTS and Lead Optimisation.

Assay Design and Quality Control in HTS and Lead Optimisation. Criteria for assay development and acceptance. Factors affecting assay variability. Statistical analysis in assay development and screening. So what?. “To knowledge by measurement” Kammerlingh.

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Assay Design and Quality Control in HTS and Lead Optimisation.

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  1. Assay Design and Quality Control in HTS and Lead Optimisation.

  2. Criteria for assay development and • acceptance. • Factors affecting assay variability. • Statistical analysis in assay development • and screening. • So what?

  3. “To knowledge by measurement” Kammerlingh “Science is Measurement” Lord Kelvin “An assay is only worth doing if it generates reliable SAR. “

  4. HTS Technologies Scintillation Proximity Assay Homogenous Time Resolved Fluorescence Fluorescence Polarisation Fluorescence Correlation Spectroscopy Fluorescence Intensity Distribution Assay Alphascreen

  5. Assay Acceptance Criteria Reproducible. Predictive

  6. Physical Causes of Assay Variation • Liquid Handling Errors • Reagent adhesion • Evaporation • Mixing • Incubation conditions • Human Error

  7. Biological Causes of Assay Variation • Biochemical Assays: • pH • Temperature • Ion concentration • Reagent Stability • Reagent Aggregation • Reagent Solubility • Order of reagent addition • Solvent effects

  8. Biological Causes of Assay Variation • 2. Cell Based Assays • As for Biochemical assays, plus: • Cell culture plastics • Culture media • Serum • Cell cycle • Passage number • Solvent effects • Infection

  9. Optimisation of Assay Parameters • Hi tech: Automated Assay Development. • JMP • Statistica • Design Expert • Low Tech: Trial and Error

  10. Statistical Analysis

  11. 7000 6000 5000 4000 cpm 3000 2000 1000 0 E1 E4 F10 E7 G1 G4 G7 F1 F4 F7 A1 A4 A7 B1 B4 B7 C1 C4 C7 D1 D4 D7 H1 H4 H7 G10 E10 C10 A10 B10 D10 H10 Sample Number Statistical Analysis

  12. Statistical Analysis Data variability Band Separation Band Data variability Band 3ss 3sc Frequency ms mc Assay Signal

  13. 3SD of sample + 3SD of control mean of sample – mean of control Z = 1 – Zhang, Chung and Oldenburgh, 1999 Statistical Analysis: Z Factor

  14. Statistical Analysis: Z Factor Z FactorAssayScreening 1 SD = 0 or dynamic range Þ¥Ideal assay 1 > Z >= 0.5 Separation band is large Excellent assay 0.5 > Z > 0 Separation band is small Double assay 0 No separation band, sample signal Yes / No assay and control signal variation touch < 0 No separation band, sample signal Unusable assay and control signal variation overlap

  15. Statistical Analysis Z = 1 SD = 0 or Dynamic rangeÞ¥ Ideal 1> Z >= 0.5 Separation band is large Excellent Data variability Band Separation Band Data variability Band 3ss 3sc Frequency ms mc Assay Signal

  16. Statistical Analysis 0.5 > Z > 0 Separation band is small Double assay Z = 0 No separation band Yes / No assay Data variability Band Data variability Band 3ss 3sc Frequency ms mc Assay Signal

  17. Statistical Analysis Z < 0 No separation band Unusable assay 3ss 3sc Frequency ms mc Assay Signal

  18. Quality Control of Screening Data • Standards • Inter Assay Variability • Appropriate plate layouts • Plate Drift • Intra Assay Variability

  19. Assay Plate Layout (Rogue’s Gallery)

  20. Assay Plate Layout

  21. Usable Assay Variability • HTS • Low false positive rate • Lead Optimisation • Ki inter assay variability < 3 fold • (Preferably < 2 fold)

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