1. INTRODUCTION

1. Identifying potential assay formats compatible with High Throughput Screen (HTS), and Structure Activity Relationship (SAR)
2. Developing optimal assay reagents
3. Optimizing assay protocol with respect to sensitivity, dynamic range, signal intensity and stability
4. Adopting screening assays to automation and scale up in microtiter plate formats
5. Statistical validation of the assay performance parameters
6. Secondary follow up assays for chemical probe validation and SAR refinement
7. Data standards to be followed in reporting screening and SAR assay results

Assays developed for HTS can be roughly characterized as cell-free or cell-based in nature. The choice of either biochemical or cell-based assay design and the particular assay format is a balancing act between two broad areas. On one side of the fulcrum is the need to ensure that the measured signal is providing relevant data to the desired biological process. For assays that are to be used in HTS, this must be balanced with the ability of these assays to support reagents that yield robust data in microtiter plate formats where typically 105 to 106 samples are screened in the assay.

This process originates during method development and continues throughout the assay life cycle (Figure 1). Successful completion of validation at an earlier stage increases the likelihood of success at later stages. During method development, assay conditions and procedures are selected that minimize the impact of potential sources of invalidity (e.g. so-called false positives or false negatives) on the measurement of analyte or the biological end point (eg. Gene expression, protein phosphorylation ) in targeted sample matrices or test solutions. There are three fundamental general areas in method development and validation: (a) Pre-study (Pre-screen) validation (b) In-study (In-screen) validation, and (c) Cross-validation or method transfer validation. These stages encompass the systematic scientific steps in assay development and validation cycle.

Pre-study validation: The investigator is faced with a number of choices with respect to the assay design and format. For many well characterized target classes there are a number of methods and kits available. At this stage the choice of an assay format is made. Close attention must be paid at this early stage to factors such as the selection of reagents with appropriate specificity and stability. Validation of assay performance at this stage should proceed smoothly if high quality procedures are chosen during method development. This requires the generation and statistical analysis of confirmatory data from planned experiments to document that analytical results satisfy pre-defined acceptance criteria. The choice of detection is made here. If fluorescent labels are chosen, careful attention must be paid to the wavelength to ensure low interference by compounds, compatibility with microtiter plate plastics and that appropriate filters are available on high-throughput plate readers. If available, the assay sensitivity and pharmacology is evaluated using control compounds. Section IV illustrates procedures common to compound evaluation using dose-response curves. Several examples of assay design and optimization are given in the additional sections of this manual for well-studied target classes (Sections V-XI). A complete discussion of design of experiment procedures will be a topic for a future chapter in this manual.

In-study validation: These procedures are needed to verify that a method remains acceptable during its routine use. For assays to be run in HTS the assay must be adapted to microtiter plate volumes. Therefore, plate acceptance testing is required where the assay is run in several microtiter plates (at least 96-well plates). From this data, statistical measures of assay performance such as Z-factors are calculated. Some methods may require additional experiments to validate the automation and scale up of an assay that may not have been addressed in earlier stages. The plates should contain appropriate maximum and minimum control samples to serve as quality controls of each run to check the performance. This will allow the investigator to check for procedural errors and to evaluate stability of the method over time. Assaying a randomly selected subset of test samples at multiple dilution levels monitors parallelism of test and standard curve samples. Sections II and III illustrate the procedures typically used to evaluate assay performance in microtiter plates and some of the common artifacts that are observed.

Cross validation: This portion includes the assay hand-off from the individual investigator’s team to the high-throughput screening center. More broadly, this procedure is used at any stage to verify that an acceptable level of agreement exists in analytical results before and after procedural changes in a method as well as between results from two or more methods or laboratories. Typically, each laboratory assays a subset of compounds and the agreement in results is compared to predefined criteria that specify the allowable performance for HTS. Considerations in adapting assays to automated robotic liquid handling and plate screening protocols are also discussed in the sections of this manual.

1. Findlay JWC, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, Bowsher RR: Validation of Immunoassays for Bioanalysis: A Pharmaceutical Industry Perspective. J. Phamac. Biomed. Analysis, 21, 1249-73, 2000.
2. Smith WC, Sittampalam GS: Conceptual and statistical Issues in the validation of analytical dilution assays for pharmaceutical applications. J. Biopharm. Stat., 8, 509-532, 1998.