Chapter 10 Assay Validation in High Throughput Screening – from Concept to Application

1. Introduction

1.1. HTS assay validation

1.2. Instrumentation in HTS

2. Protocol of a typical assay validation

3. Plot interpretation

4. Considerations

4.1. Biological reagents

4.1.1. Reagent consistency

4.1.2. Reagent stability

4.1.3. Sequence of reagent addition

4.2. Consumable composition

4.2.1. Consumable batch/lot variations

4.2.2. Leaching and adsorption behavior of consumables

4.3. Plate type selection

4.4. Incubation conditions

4.5. Proper use of internal assay controls

4.5.1. Non-biologically vs. biologically-relevant controls

4.5.2. Interaction of positive controls with reagent

4.5.3. Hill slope steepness and “medium” signal stability

4.5.4. Stability and solubility of positive controls

4.5.5. Appropriate washing protocols for liquid transfer components when dealing with “persistent-binding” positive controls

4.5.6. Negative control selection

5. Potential limitations

6. Conclusion

Abbreviation

Author details

References