1 ASSAY FOR IMMUNOSUPPRESSANT DRUGS
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of PCT International Application PCT/US2007/ 088098 filed Dec. 19, 2007, and claims the priority of U.S. Provisional Application Ser. No. 60/882,863 filed Dec. 29, 2006, the disclosures of Which are incorporated herein by reference in their entireties.
This invention relates to diagnostic immunoassays to determine the concentration levels of an immuno suppressant drug in a test sample, and in particular relates to use of high salt conditions to improve assay sensitivity.
Immunosuppressant drugs such as tacrolimus, everolimus, temsorolimus and cyclo sporine are effective for the treatment of organ or tissue rejection following transplant surgery, of graft versus host disease and of autoimmune diseases in humans. During immunosuppressant drug therapy, monitoring the blood concentration levels of the immunosuppressant is an important aspect of clinical care because insufiicient drug levels lead to graft (organ or tissue) rejection and excessive levels lead to undesired side effects and toxicities. Blood levels of immunosuppressant are therefore measured so drug dosages can be adjusted to maintain the drug level at the appropriate concentration. Diagnostic assays for determination of immuno suppressant blood levels have thus found wide clinical use.
A commonly used competitive format involves the binding of a first antibody to the immunosuppressant and the binding of labeled immunosuppressant (e.g., acridinium-tacrolimus) to the remaining free antibody binding sites, followed by quantitation by detection of the label.
The invention provides a method for assessing the presence or concentration of an immunosuppressant drug in a test sample. The method entails contacting a test sample or test sample extract with an antibody specific for an immunosuppressant drug under conditions suitable for binding of the antibody to the immunosuppressant drug, if present, to form an assay mixture. Such conditions include a salt concentration of greater than about 0.4 M. Binding of the antibody to the immunosuppressant drug is then detected. In particular embodiments, the immunosuppressant drug is tacrolimus, everolimus, zotarolimus, cyclosporine, or an analog of any of these compounds. In preferred embodiments, tacrolimus or cyclosporine. Conveniently, the test sample includes a human blood sample.
In certain embodiments, the immunoassay is carried out in the presence of salt at a concentration less than or equal to about 4.0 M, e.g., at a concentration of about 2.0 M. In particular, embodiments, the salt includes a monovalent anion, such as chloride. In exemplary embodiments, the salt includes a chloride salt of an alkali metal, such as sodium chloride.
The assay mixture can additionally includes a glycol, such as ethylene glycol, propylene glycol, or an analog thereof, and at least one alcohol having five or fewer carbons. Suitable alcohols include, for example, methanol, ethanol, and propanol. The ratio of glycol to alcohol in the assay mixture can be in the range of about 4:1 to about 1:4, or more particularly, in the range of about 4:1 to about 1:2.
Another aspect of the invention is a test kit, which includes: (a) at least one antibody capable of binding specifically to at least one immunosuppressant drug; (b) an assay diluent including a salt at a concentration of at least about 1.0 M; and (c) a lysis reagent including: a glycol selected from ethylene glycol, propylene glycol, and an analog thereof; and at least one alcohol having five or fewer carbons. In particular embodiments, the immunosuppressant drug is tacrolimus, everolimus, zotarolimus, cyclosporine, or an analog of any of these compounds. In preferred embodiments, tacrolimus or cyclosporine.
In certain embodiments, the concentration of salt in the assay diluent is less than or equal to about 10.0 M, e.g., about 4.5 M. In particular, embodiments, the salt includes a monovalent anion, such as chloride. In exemplary embodiments, the salt includes a chloride salt of an alkali metal, such as sodium chloride.
The test kit can also contain a control composition including the at least one immunosuppressant drug recognized by the antibody provided in the kit.
The alcohol in the lysis reagent can include, for example, methanol, ethanol, and/or propanol. The ratio of glycol to alcohol in the lysis reagent can be in the range of about 4: 1 to about 1:4, or more particularly, in the range of about 4:1 to about 1:2.
In an exemplary embodiment, the test kit of the invention includes: (a) at least one antibody or protein capable of binding specifically to at least one immunosuppressant drug selected from the group consisting of tacrolimus and cyclosporine; (b) an assay diluent including a salt at a concentration of at least about 1.0 M; and (c) a lysis reagent comprising: (i) propylene glycol and ethanol at a ratio in the range of about 4:1 to about 1 :2; and (ii) a control composition including the at least one immunosuppressant drug of (a).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph obtained using an ARCHITECT® Tacrolimus Assay (Abbott Laboratories, Abbott Park, Ill.) tested with different concentrations of NaCl in the reaction. Abscissa: tacrolimus amount (ng/mL). Ordinate: signal (Relative light units, or RLUs). Symbols: (solid diamond), 0 M NaCl; (solid triangle), 1 M NaCl; (solid square), 2 M NaCl.
FIG. 2 is a graph obtained using an ARCHITECT® Tacrolimus Assay (Abbott Laboratories, Abbott Park, Ill.) tested with different concentrations of NaCl in the reaction. Abscissa: tacrolimus amount (ng/mL). Ordinate: X/A. Symbols: (solid diamond), 0 M NaCl; (solid triangle), 1 M NaCl; (solid square), 2 M NaCl. It can be seen that curve shape was improved (decreased B/A) by increasing NaCl concentrations in the reaction.
FIG. 3 is a graph obtained using an IMx® Tacrolimus II Assay (Abbott Laboratories, Abbott Park, Ill.) in which rates of blood matrix calibrators were tested with different concentrations of NaCl present in the microparticles. Abscissa: tacrolimus amount (ng/mL). Ordinate: Rate. Symbols: (solid diamond), 1 M NaCl; (solid square), 2 M NaCl; (open circle), IMx® Tacrolimus II marketed assay with no salt addition.
FIG. 4 is a graph obtained using an IMx® Tacrolimus II Assay (Abbott Laboratories, Abbott Park, Ill.). Abscissa: tacrolimus amount (ng/mL). Ordinate: X/A. Symbols: (solid diamond), 0 M NaCl; (solid square), 2 M NaCl; (open circle), IMx® Tacrolimus II marketed assay with no salt addition. It can be seen that curve shape was improved (decreased B/A) by 2 M NaCl concentration in the microparticles.
FIG. 5 is a bar chart obtained using an ARCHITECT® Tacrolimus Assay (Abbott Laboratories, Abbott Park, Ill.) and a variety of different salts at 0.44 M in each reaction. Abscissa: no salt, lithium chloride (LiCl), sodium chloride (N aCl), potassium chloride (KCI), rubidium chloride (RbCl), cesium chloride (CsCl). Ordinate: signal (RLUs). Symbols: (bar with diagonal lines), 0 ng/mL tacrolimus; (stippled bar),
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