# US 2018/0024124 A1 · An Extraction Reagent of Immunosuppressant Drug for Immunoassays > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0024124 A1 - Document ID: US20180024124A1 - Status: Published application - Kind: A1 - Application number: 15/549,952 - Filed: 2015-05-29 - Published: 2018-01-25 - Earliest claimed: 2015-03-30 - Assignees: Shanghai Inzex Biotechnology Co., Ltd. - Applicants: Shanghai Inzex Biotechnology Co.,Ltd. - Inventors: Fengbo Wu; Yongyan Nie; Xiaojuan Shi - Web page: https://patentgrep.com/patent/US20180024124A1 ## CPC - G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding} - G01N 1/4022: {by thermal techniques; Phase changes} - G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition} - G01N 1/44: Sample treatment involving radiation, e.g. heat - G01N 33/487: of liquid biological material - G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q) - G01N 33/54333: {Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction} - G01N 33/577: involving monoclonal antibodies {binding reaction mechanisms characterised by the use of monoclonal antibodies (G01N33/5302 - G01N33/576 take precedence)} - G01N 33/9493: {Immunosupressants} - G01N 33/533: with fluorescent label - G01N 33/5375: {by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning} - G01N 2001/388: {mixing the sample with a tracer} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 An extraction reagent of the immunosuppressant drug in blood sample for immunoassay, comprising a protein denaturant, proteolytic enzyme, surfactant and pH buffer. ### Claim 2 (depends on 1) The extraction reagent according to claim 1, wherein the protein denaturant is selected from urea, guanidine hydrochloride or other non-organic solvent based denaturants. ### Claim 3 (depends on 2) 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. ### Claim 4 (depends on 3) 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. ### Claim 5 (depends on 1) The extraction reagent according to claim 1, wherein the proteolytic enzyme is selected from one or more of subtilisin, protease K and dispase. ### Claim 6 (depends on 5) The extraction reagent according to claim 5, wherein the proteolytic enzyme is subtilisin. ### Claim 7 (depends on 6) 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. ### Claim 8 (depends on 1) The extraction reagent according to claim 1, wherein the surfactant is selected from one or more of Tween-20, saponin and Triton X-100. ### Claim 9 (depends on 8) The extraction reagent according to claim 8, wherein the surfactant is Tween-20. ### Claim 10 (depends on 9) 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). ### Claim 11 (depends on 10) 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). ### Claim 12 (depends on 1) The extraction reagent according to claim 1, wherein the pH value of the buffer is between 6.5 and 8.5. ### Claim 13 (depends on 1) 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. ### Claim 14 (depends on 13) The method according to claim 13, wherein the immunosuppressant comprises tacrolimus, sirolimus, everolimus, zotarolimus, cyclosporin A or other structural analogues. ### Claim 15 (depends on 13) 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. ### Claim 16 (depends on 15) 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. ### Claim 17 (depends on 13) The method according to claim 13, wherein the heating temperature is 50° C.-90° C., and the heating time is 5 min-50 min. ### Claim 18 (depends on 17) The method according to claim 17, wherein the heating temperature is 60° C.-80° C., and the heating time is 10 min-30 min. ### Claim 19 (depends on 1) 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. ### Claim 20 (depends on 19) 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 Eu3+, Sm3+, Tb3+, Dy3+ and chelate ligands thereof.