An Extraction Reagent of Immunosuppressant Drug for Immunoassays

Abstract

The disclosure provides a reagent for extracting immunosuppressant drugs from whole blood sample for immunoassay. The extraction reagent comprises protein denaturant, proteolytic enzyme, surfactant and pH buffer. The disclosure also provides a method and an immunoassay kit for detection of the immunosuppressant concentration in a whole blood sample using the extraction reagent. The extraction reagent of the present disclosure doesn't need the use of organic solvent as that in the traditional extraction method, therefore the adverse effects of the organic solvent on the antibody activity in a detection system and the other relative defects associated to its use are obviated. The drug extraction process of the present disclosure doesn't need centrifugation, the processed sample can be directly applied for immunoassay. The operation for drug extraction by the present disclosure is simple, and the detection result based on this extraction method is accurate.

What is claimed is: 
     
         1 . An extraction reagent of the immunosuppressant drug in blood sample for immunoassay, comprising a protein denaturant, proteolytic enzyme, surfactant and pH buffer. 
     
     
         2 . The extraction reagent according to  claim 1 , wherein the protein denaturant is selected from urea, guanidine hydrochloride or other non-organic solvent based denaturants. 
     
     
         3 . The extraction reagent according to  claim 2 , wherein the molar concentration of the urea in the extraction reagent is 4 mol/L to 12 mol/L; and alternatively the molar concentration of the guanidine hydrochloride in the extraction reagent is about 1 mol/L to 8 mol/L. 
     
     
         4 . The extraction reagent according to  claim 3 , wherein the molar concentration of the urea in the extraction reagent is 6 mol/L to 8 mol/L; and alternatively the molar concentration of the guanidine hydrochloride in the extraction reagent is 2 mol/L to 6 mol/L. 
     
     
         5 . The extraction reagent according to  claim 1 , wherein the proteolytic enzyme is selected from one or more of subtilisin, protease K and dispase. 
     
     
         6 . The extraction reagent according to  claim 5 , wherein the proteolytic enzyme is subtilisin. 
     
     
         7 . The extraction reagent according to  claim 6 , wherein the amount of the subtilisin used in the extraction reagent is 2.5 U/ml to 10 U/ml. 
     
     
         8 . The extraction reagent according to  claim 1 , wherein the surfactant is selected from one or more of Tween-20, saponin and Triton X-100. 
     
     
         9 . The extraction reagent according to  claim 8 , wherein the surfactant is Tween-20. 
     
     
         10 . The extraction reagent according to  claim 9 , wherein the volume ratio of the Tween-20 in the extraction reagent is 0.005% to 1% (v/v). 
     
     
         11 . The extraction reagent according to  claim 10 , wherein the volume ratio of the Tween-20 in the extraction reagent is 0.02% to 0.1% (v/v). 
     
     
         12 . The extraction reagent according to  claim 1 , wherein the pH value of the buffer is between 6.5 and 8.5. 
     
     
         13 . A method for detecting immunosuppressant drug in whole blood sample, wherein the blood sample is processed via heating with the extraction reagent of  claim 1 , and then determining the concentration of a drug contained therein by immunoassay. 
     
     
         14 . The method according to  claim 13 , wherein the immunosuppressant comprises tacrolimus, sirolimus, everolimus, zotarolimus, cyclosporin A or other structural analogues. 
     
     
         15 . The method according to  claim 13 , wherein when mixing the extraction reagent and the blood sample, the volume ratio of the blood sample to the extraction reagent is 1/1 to 1/10. 
     
     
         16 . The method according to  claim 15 , wherein when mixing the extraction reagent and the blood sample, the volume ratio of the blood sample to the extraction reagent is 1/2 to 1/5. 
     
     
         17 . The method according to  claim 13 , wherein the heating temperature is 50° C.-90° C., and the heating time is 5 min-50 min. 
     
     
         18 . The method according to  claim 17 , wherein the heating temperature is 60° C.-80° C., and the heating time is 10 min-30 min. 
     
     
         19 . A kit for measuring the immunosuppressant concentration in a blood sample, wherein the kit comprises: a) an antibody specifically binding to the immunosuppressant drug; b) a set of calibrators containing the immunosuppressant; c) the extraction reagent of  claim 1 ; d) a detection reagent, the detection reagent is an antibody, antigen or hapten coupled to a tracer material. 
     
     
         20 . The kit according to  claim 19 , wherein the tracer material comprises an enzyme, chemiluminescent substance, radioactive substance, fluorescent substance, rare earth ion, biotin and digoxin, the rare earth ion comprises Eu 3+ , Sm 3+ , Tb 3+ , Dy 3+  and chelate ligands thereof.