Patent ID: 7943327

Claim:
A method of measuring circulating free drug present in the serum or plasma of a patient, comprising: (a) collecting blood from a patient who is taking a drug, and purifying serum or plasma containing the circulating free drug from the blood; (b) reacting the serum or plasma containing the circulating free drug with a target cell, wherein the target cell expresses a target that reacts with the drug to bind at least a portion of the drug present in the serum or plasma; (c) reacting the target cell of step b that has been reacted with the circulating free drug present in the patient serum or plasma with a labeled anti-Free-Site antibody, wherein the anti-Free site antibody is a monoclonal antibody that recognizes an epitope on the target when the drug is not bound to the target (drug-free target), but is competitively inhibited by binding of the drug to the target; (d) washing away unreacted 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; (g) comparing the result in step f) against a standard curve wherein the standard curve is prepared by: (i) exposing separate aliquots of unreacted target cells to known amounts of the drug; (ii) reacting the aliquots of the target cells of (i) with the labeled anti-Free-Site antibody; (iii) washing away the unreacted anti-Free-Site antibody from the aliquots of the target cells of (ii); (iv) fixing the aliquots of the target cells of step (iii); (v) quantifying the amount of anti-Free-Site antibody binding to the aliquots of the target cells of step (iv); and (vi) preparing a standard curve showing the relationship between the drug concentration and the quantified binding of the anti-Free-Site antibody to the aliquots of target cells of step (v).