FcRn Interaction Analyses By Surface Plasmon Resonance (SPR) And
ELISA
The binding interaction of the BsAbs with recombinant cynomolgus monkey
FcRn was monitored by SPR detection using a BIAcore 3000 instrument
(Biacore Inc.) as described previously.24, 25 Briefly,
cynomolgus monkey FcRn was immobilized directly to a CM5 chip using the
standard amine-coupling kit methodology. BsAbs samples were prepared in
running buffer (PBS, pH 6.0, 0.0005% Tween 20) and evaluated in
duplicate. Samples were injected for 30 seconds at a flow rate of 100
µL/min and a dissociation time of 300 seconds at 25◦C.
BsAbs were dissociated from FcRn using 1x PBS (pH 7.4) dissociation
buffer. Kinetic binding constants were determined through global fits of
the average of two data sets collected on separate days using Biacore
T200 Evaluation, version 1.0. BsAb binding to FcRn was also evaluated at
pH 7.4 (PBS, pH pH 7.4, 0.0005% Tween 20) at single concentration (5 µM
diluted PBS, pH pH 7.4, 0.0005% Tween 20). Data collected at pH 7.4 was
not fit since there was no observable signal.
The evaluation of the pH-dependent dissociation of BsAbs from FcRn was
conducted using an ELISA as previously reported.24, 25Briefly, biotinylated cynomolgus monkey FcRn was produced by reacting
each purified soluble protein with EZ-Link® Sulfo-NHS-Biotin (Pierce)
using the conditions supplied by the vendor, and the FcRn:biotin ratio
was measured as 1.0 and 1.0, respectively, using the EZ™ biotin
quantitation kit (Pierce). The pH-dependent ELISA for the BsAbs was
performed as described in earlier studies at pH 6.0 and pH
7.4.24, 25 Optical density (OD) data at pH 6.0 and pH
7.4 were analyzed and expressed as the total BsAb that remained bound to
FcRn at pH 7.4 [(ODpH7.4/ODpH6.0) x
100%)]. The mean and standard deviation of three independent
experiments were determined.