It is anticipated that all projects will be run as full collaborations between the research group sponsoring the screen and the staff of the HTCS Facility. Outlined below is an overview of commitments that will be required from the sponsoring group.
Step 1 - Developing a High Capacity Assay for the Primary Screen
Currently, the WEHI/Bio21 Lead Discovery library contains approximately 100,000 compounds and screening the entire library requires the execution of at least 110,000 assays. Therefore, above all, the primary screening assay must be quantitative, robust, reproducible, and simple. Many of the assay methods routinely used in biomedical research labs do not meet the criteria for simplicity and robustness, and therefore a common starting point for a collaborative project with the HTCS Facility is to design and produce the reagents necessary for the construction of a high capacity primary screening assay.
As part of the HTCS tool kit there are many specialised technologies designed specifically for constructing high capacity assays, and we can offer advice on the technologies that are best suited to your target protein. The HTCS Facility has only a limited capacity to produce screen specific reagents such as recombinant proteins and genetically modified cell lines. In most cases, therefore, it is the responsibility of the sponsoring research group to produce the target specific reagents required for the project.
The target specific reagents will depend largely on the technology format of the proposed screen, but broadly they are as follows.
Purified recombinant proteins - for many HTCS assay technologies the target protein must be expressed as a fusion with one of the following common affinity tags; Hexa-His, GST, FLAG, Myc. One to two milligrams of target protein is typically required to run a primary screen, and ideally for quality control purposes the entire amount should be prepared in a single batch. The HTCS Facility can assist in labelling proteins with many of the specialised fluorophores used for formatting HTCS assays.
Antibodies – many screening assays make use of antibodies to provide specificity, for example it is possible to modify the standard ELISA for HTCS. A good source of the appropriate antibodies is required as up to several milligrams can be used in a typical screening campaign. In addition, if development of the high capacity assay requires that the antibody is labelled with a specific fluorophore then the antibody must also be purified. Typically it is difficult to carry out labelling procedures on less than 100 μg of antibody. Peptides - labelled with a specific fluorophore, or biotin. In most cases these types of reagents can be obtained easily from commercial sources.
It is not always possible to construct a high capacity assay using fluorescence-based technologies, and in these cases we often have to use radioisotope labelled probes. We can obtain many of the common radioisotope labelled compounds, but in rare cases custom labelling might be required to produce the appropriate reagents for the screen.
Specific Cell lines – cell lines used for the primary screening assay must be easily cultured, and must give a robust reproducible assay response over several passages. We have successfully run a large screening campaign using primary cells but this is not common. Genetically modified cell-lines – preferably, the genetic material should be stably integrated. In special circumstances it is possible to run a primary screen using transiently transfected cell-lines, however, this approach will generally increase the cost of the screen, and reduce the quality of the data generated.
Step 2 requires use of the specialised liquid handling systems and plate readers in the HTCS Facility, and therefore this part of the project is managed and run exclusively by our staff.
In many instances a further 10,000-20,000 assays will be required to sort through the hits from the primary screen and identify the compounds that have the optimum biochemical and physiochemical characteristics. The exact strategy for selecting the best compounds from population of hits will vary from target to target, but it is anticipated that a proportion of this work will be carried out in the screen sponsors lab. Notably, the screen sponsor will be involved in the latter part of the selection process when many of the initial hits have been eliminated and more complex cell-based and/or in vivo models are required to make the final selections.
The Screen Sponsor Provides:
• Background knowledge of the target protein and its biology
• Screen specific reagents
• The resources to produce the necessary reagents and participate in the screen follow-up work
• Funding to cover the infrastructure and salary costs associated with running the screen
The HTCS Facility provides:
• Expertise and the resources to develop high capacity assays and to execute screening campaigns
• The necessary hardware
• The Lead Discovery Library
• Data management