Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy

Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constan ts over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFN alpha2) and th e extracellular domain of its receptor ifnar2 (ifnar2-EC), We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, nonco mpetitive mAb provided stable, but still reversible, immobilization of ifna r2-EC, We measured kinetics and affinity of numerous of mutants of IFN alph a2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and associati on rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 muM were measured with protein concentra tions up to 50 muM without no background signal or nonspecific binding. The instrument detection limit is similar to 10 pm without the need for temper ature stabilization or referencing channels. The system proved effective fo r large-scale mutational analysis involving alanine scanning mutagenesis an d double mutant cycles. (C) 2001 Academic Press.