Development of new chromatographic tools based on A2A adenosine receptor subtype for ligand characterization and screening by FAC-MS

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A_2A adenosine receptor (A_2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A_2AAR vector (A_2A(+)) and from the same cell line transfected with the corresponding empty vector (A_2A(?)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A_2A(+)-IAM and A_2A(?)-IAM stationary phases. Frontal chromatography experiments on both A_2A(+)-IAM and A_2A(?)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A_2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K _d values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm?×?100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A_2A(+)-OT and A_2A(?)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.