J. Piehler et G. Schreiber, Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy, ANALYT BIOC, 289(2), 2001, pp. 173-186
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.