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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

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?


Assay design and quality control in hts and lead optimisation

“To knowledge by measurement”

Kammerlingh

“Science is Measurement”

Lord Kelvin

“An assay is only worth doing if it

generates reliable SAR. “


Assay design and quality control in hts and lead optimisation

HTS Technologies

Scintillation Proximity Assay

Homogenous Time Resolved Fluorescence

Fluorescence Polarisation

Fluorescence Correlation Spectroscopy

Fluorescence Intensity Distribution Assay

Alphascreen


Assay design and quality control in hts and lead optimisation

Assay Acceptance Criteria

Reproducible.

Predictive


Assay design and quality control in hts and lead optimisation

Physical Causes of Assay Variation

  • Liquid Handling Errors

  • Reagent adhesion

  • Evaporation

  • Mixing

  • Incubation conditions

  • Human Error


Assay design and quality control in hts and lead optimisation

Biological Causes of Assay Variation

  • Biochemical Assays:

    • pH

    • Temperature

    • Ion concentration

    • Reagent Stability

    • Reagent Aggregation

    • Reagent Solubility

    • Order of reagent addition

    • Solvent effects


Assay design and quality control in hts and lead optimisation

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


Assay design and quality control in hts and lead optimisation

Optimisation of Assay Parameters

  • Hi tech:Automated Assay Development.

  • JMP

  • Statistica

  • Design Expert

  • Low Tech: Trial and Error


Assay design and quality control in hts and lead optimisation

Statistical Analysis


Assay design and quality control in hts and lead optimisation

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


Assay design and quality control in hts and lead optimisation

Statistical Analysis

Data variability

Band

Separation

Band

Data variability

Band

3ss

3sc

Frequency

ms

mc

Assay Signal


Assay design and quality control in hts and lead optimisation

3SD of sample + 3SD of control

mean of sample – mean of control

Z = 1 –

Zhang, Chung and Oldenburgh, 1999

Statistical Analysis: Z Factor


Assay design and quality control in hts and lead optimisation

Statistical Analysis: Z Factor

Z FactorAssayScreening

1 SD = 0 or dynamic range Þ¥Ideal assay

1 > Z >= 0.5 Separation band is largeExcellent assay

0.5 > Z > 0 Separation band is smallDouble assay

0 No separation band, sample signalYes / No assay

and control signal variation touch

< 0 No separation band, sample signalUnusable assay

and control signal variation overlap


Assay design and quality control in hts and lead optimisation

Statistical Analysis

Z = 1SD = 0 or Dynamic rangeÞ¥ Ideal

1> Z >= 0.5Separation band is largeExcellent

Data variability

Band

Separation

Band

Data variability

Band

3ss

3sc

Frequency

ms

mc

Assay Signal


Assay design and quality control in hts and lead optimisation

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


Assay design and quality control in hts and lead optimisation

Statistical Analysis

Z < 0 No separation band Unusable assay

3ss

3sc

Frequency

ms

mc

Assay Signal


Assay design and quality control in hts and lead optimisation

Quality Control of Screening Data

  • Standards

  • Inter Assay Variability

  • Appropriate plate layouts

  • Plate Drift

  • Intra Assay Variability


Assay design and quality control in hts and lead optimisation

Assay Plate Layout (Rogue’s Gallery)


Assay design and quality control in hts and lead optimisation

Assay Plate Layout


Assay design and quality control in hts and lead optimisation

Usable Assay Variability

  • HTS

  • Low false positive rate

  • Lead Optimisation

  • Ki inter assay variability < 3 fold

  • (Preferably < 2 fold)


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