Determination of the Kinetic Rate Constants and Affinity of a Protein-Antibody Interaction Using the BiOptix 404pi™


Biophysical characterization of antigen-antibody interactions is an important application for
label-free instrumentation. In a previous report, Katsamba et al. (1) used the binding of
prostate-specific antigen (PSA) to a monoclonal antibody (mAb) to characterize antibody-antigen
interactions using surface plasmon resonance technology. In this application note, we used the
same model system to demonstrate that the BiOptix 404pi yields similar results.



PSA monoclonal antibody (Fitzgerald #10-P20A, clone 612166) was amine-coupled to different
surface capacities on two flow cells of a BiOptix CMD200 sensor chip using standard EDC/NHS
coupling chemistry with blocking of the remaining active ester groups by ethanolamine. Two
reference flow cells were activated and blocked with no immobilized protein. The running buffer
was PBS, 0.005% polysorbate 20, 200 μg/mL bovine serum albumin, pH 7.4. Six two-fold dilutions
of PSA (Fitzgerald #30C-CP1017) were injected in triplicate at 100 μL/min at 20oC as indicated in
Table 1. Three additional injections of PSA at 592 nM were performed, but instead of following
dissociation for 90 seconds, dissociation was followed for 3600 seconds to obtain a more accurate
estimate of the slow kd observed for this interaction. Buffer blank injections were performed
throughout the randomized PSA injection sequence for double referencing. At the end of each
injection cycle, a six second pulse of 100 mM HCl was used to regenerate the surface.



Data were collected on the Bioptix 404pi instrument and were processed using Scrubber 2.0c
(Biologic Software). The data were globally fit from two different flow cells with different surface
capacities of the anti-PSA mAb to a 1:1 interaction model as shown in Figure 1. Table 2 shows the
calculated kinetic rate constants and affinity versus those that were reported in Katsamba et al.

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