| Assay Method Information | |
| | Multi-Point Chaser SPR Assay Variation |
| Description: | Table 4: Chaser assay utilize a single cycle kinetics SPR experiment with a contact time of 120 seconds, a flow rate of 50 μl/min and a dissociation time of 450 seconds. Single cycle kinetics titration utilized an initial blank injection and 5 concentrations with 2 fold serial dilution with a maximum concentration of 500 nM, blanked to a preceding 6 point blank single cycle kinetics injection for double referencing.In the case of potent compounds, the protein-compound half-life cannot be accurately measured using routine fitting of the single cycle titration data. The kd is measured independently by determining the percentage unoccupied compound binding site over time by measuring the binding of a chaser compound measured by SPR.Chaser binding was measured by a multicycle kinetics SPR experiment using a contact time of 20 seconds, a flow rate of 30 μl/min, and a dissociation time of 120 seconds. 7 injections of a single chaser concentration of 15 μM with a preceding blank injection, were recorded spaced out between 674 and 30,263 seconds after the last single cycle kinetics titration injection. The % compound bound at a given time was determined by comparison to a single injection of chaser preceding the single cycle kinetics titration defined below.% Compound Bound=(1−(RUT/RUT0))*100 |
| Affinity data for this assay | |
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