US 7,320,879 B2 | ||
Method of determining site-specificity and kit therefor | ||
Robert Karlsson, Uppsala (Sweden); Helena Nordin, Uppsala (Sweden); and Susanna Nyberg, Ramlösa (Sweden) | ||
Assigned to Biacore AB, Uppsala (Sweden) | ||
Filed on Nov. 25, 2003, as Appl. No. 10/722,061. | ||
Claims priority of provisional application 60/430244, filed on Dec. 02, 2002. | ||
Claims priority of application No. 0203548 (SE), filed on Dec. 02, 2002. | ||
Prior Publication US 2004/0166549 A1, Aug. 26, 2004 | ||
Int. Cl. G01N 1/30 (2006.01) |
U.S. Cl. 435—40.5 | 16 Claims |
1. A method of determining the binding site specificity of a first analyte that binds to a ligand having at least two different
binding sites, comprising:
immobilizing the ligand to a sensing surface of a biosensor,
providing a second, reference analyte which binds reversibly to the ligand at a binding site thereof and which is selected
to have a dissociation phase, after interaction with the ligand has ceased, that is either substantially faster or substantially
slower than that of the first analyte,
contacting a mixture of the first analyte and the second, reference analyte with the immobilized ligand to permit association
to the ligand,
stopping the contacting of the first analyte and the second, reference analyte with the ligand, and subjecting the immobilized
ligand to conditions which permit dissociation of bound first analyte and second reference analyte therefrom,
monitoring in real time by a label-free detection technique at least the dissociation phase of the interaction of the mixture
of the first analyte and the second, reference analyte with the immobilized ligand to obtain a dissociation phase binding
curve,
successively increasing the concentration in the mixture of the one of the first analyte and the second, reference analyte
that has the faster dissociation phase, and
determining, from a label-free detection technique, the influence of the increased concentration on the dissociation phase
binding curve profile of the mixture to determine therefrom if the first analyte and the second, reference analyte bind to
the same or different binding sites on the ligand,
wherein an influence in which a substantially reduced contribution to the dissociation phase binding curve profile for the
mixture from the one of the first analyte and the second, reference analyte that has the slower dissociation phase indicates
that the first analyte and the second, reference analyte bind to the same binding site.
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