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P2078
Development & Validation of a Protein-Protein Interaction HTS Assay Based on AlphaLisa™ Technology
Presenter Cleofe Zapatero, GSK, Spain
Additional Authors: Susan E. Hutchinson, Juan Manuel Domínguez, and María-Jesús Vázquez
Protein-protein interactions (“PPI”) are an essential key in all biological processes, from replication and expression of genes to the morphogenesis of organisms.Signalling cascades integrate extra cellular stimuli primarily through regulated protein-protein interactions. Although various methodologies exist for the detection and quantification of protein-protein interactions, ELISA is still the most widely used. This non-homogeneous technology offers great selectivity, sensitivity and assay versatility, however has certain limitations which impede its use in HTS, such as its limited throughput due to the inclusion of washing steps, a generally narrow dynamic range and the incapacity to use low affinity antibodies. Other proximity-based technologies has been tried to monitor PPI, TR-FRET among these. However its use is precluded by the large size of the PPI complex which prevents from efficient energy transfer among the two fluorophores.We have recently evaluated the use of AlphaLISA™ technology for this purpose and have shown that this technology performs well in a HTS environment. Full biochemical validation has been achieved by using the isolated domain of one of the two proteins which has been described to be responsible for the interaction  such domain causes a decrease of the observed interaction in a dose response manner, yielding an IC50 value consistent with the reported Kd value. The assay is amenable to miniaturization in 1536 wells hence enabling its use in uHTS.