Simultaneous Assay of Target and Target-Drug Binding
AssigneeLaboratory Corporation of America Holdings
InventorsNorman B. Purvis, Gregory T. Stelzer
Life of this patent
Estimating the term…
Abstract
Whole cell, simultaneous target and drug-target assay using differentially labeled antibodies and flow cytometry. First antibody binds to total target and second antibody binds to the drug binding site of the target, thus drug binding will competitively inhibit the second antibody allowing for a competitive inhibition assay of drug-target binding. The assay allows for whole cell analysis and even analysis of mixed populations of cells, yet provides detailed kinetic assessment of drug activity.
Claims30
A method of measuring circulating free drug present in a biological fluid sample from a patient, comprising:
- (a) obtaining a biological fluid sample from a patient who is taking a drug, wherein the biological fluid sample contains circulating free drug;
- (b) combining the biological sample with a target cell, wherein the target cell expresses a target that interacts with the drug to bind to at least a portion of the drug present in the biological fluid sample,
- (c) mixing the target cell of step (b) that has been combined with the circulating free drug present in the biological fluid sample with a labeled anti-Free-Site antibody, wherein the anti-Free Site antibody is a monoclonal antibody that recognizes and binds to an epitope on the target when the drug is not bound to the target but is competitively inhibited by binding of the drug to the target;
- (d) washing away unbound anti-Free-Site antibody from the target cell of step (c);
- (e) fixing the target cell of step (d);
- (f) quantifying the amount of anti-Free-Site antibody binding to the target cell of step (e) by flow cytometry;
- and
- (g) determining the amount of circulating free drug present in the biological fluid sample by comparing the amount of anti-Free-Site antibody bound to the target cell as quantified in step (f) to a standard curve of pre-determined amounts of Anti-Free-Site antibody bound to target cells previously exposed to known amounts of the drug.
A method of determining drug binding for a drug administered to a subject, comprising:
- (a) administering a drug to a subject, wherein the drug interacts with a target expressed by cells in the subject;
- (b) collecting a biological sample from the subject, wherein the biological sample contains target-expressing cells;
- (c) isolating the target-expressing cells from the biological sample;
- (d) incubating the isolated target-expressing cells with an anti-Total antibody and an anti-Free-Site antibody, wherein the anti-Total antibody and the anti-Free-Site antibody are differentially labeled;
- (e) fixing the isolated target-expressing cells of step (d);
- (f) detecting the amount of the anti-Total antibody bound to the isolated target-expressing cells of step (e), wherein the anti-Total antibody binds to an epitope on the target regardless of whether the target is also interacting with the drug;
- (g) detecting the amount of the anti-Free-Site antibody bound to the isolated target-expressing cells of step (e), wherein the anti-Free-Site antibody recognizes and binds to an epitope on the target when the drug is not interacting with the target but is competitively inhibited by the interaction of the drug with the target,
- wherein the amount of the anti-Total antibody detected in step (f) corresponds to the total amount of target expressed by the isolated target-expressing cells and the amount of the anti-Free-Site antibody detected in step (g) corresponds to the total amount of drug-free target, and
- wherein the amount of drug-bound target is proportional to the amount of the anti-Total antibody detected in step (f) less the amount of the anti-Free-Site antibody detected in step (g).
Dependencies are inferred from the claim text.