Characterisation, quality control and standardisation
Stringent and correctly applied QC will be a vital element in the AFFINOMICS resource. A fundamental task will be detailed binder characterisation and validation in specific applications. This aspect is all too often sidelined by commercial vendors: recent results in the SH2 pilot project have confirmed impressions that the functionality of a sizeable fraction of commercially available antibodies is at least questionable, and in some cases considerably worse, and that quality rather than cost are the key issues for users. Implementation of standardised quality control procedures must therefore be central to any publicly funded reagent initiative and a major role for AFFINOMICS will be to define and put into practice such protocols for the affinity reagents making up the resource. It is therefore important to consider what attributes are required of affinity reagents for functional proteomics analysis and applications in common use. Their essential properties can be summarised as: diversity of target recognition, high individual specificity (low cross-reactivity), a range of binding affinities (e.g. 10-6 to 10-12 M), utilisation in quantitative as well as qualitative assays, and adaptability to many different assay designs. Generic properties which will be measured for all binder formats are binding constants, target specificity, epitope definition and cross-reactivity; they will be viewed in relation to intended applications using standardised testing by surface plasmon resonance (SPR), Western blotting (WB), immunoprecipitation (IP), protein and peptide arrays, immunohistochemistry (IHC) and immunofluorescence. Evaluation methods will be adapted to appropriately high throughput, e.g using microarray-based systems which allow detailed analyses of binding properties including specificity, affinity and epitope mapping against a large number of antigens in one reaction. The procedures and protocols developed and refined within AFFINOMICS are designed to become a future ‘gold standard’ for binder QC applicable to both the academic and commercial sectors. The results of QC testing will be made publicly available through AFFINOMICS databases.