WO2016155111A1 - 用于免疫分析的免疫抑制剂药物提取试剂 - Google Patents

用于免疫分析的免疫抑制剂药物提取试剂 Download PDF

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WO2016155111A1
WO2016155111A1 PCT/CN2015/080330 CN2015080330W WO2016155111A1 WO 2016155111 A1 WO2016155111 A1 WO 2016155111A1 CN 2015080330 W CN2015080330 W CN 2015080330W WO 2016155111 A1 WO2016155111 A1 WO 2016155111A1
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extraction reagent
extraction
blood sample
concentration
sample
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PCT/CN2015/080330
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English (en)
French (fr)
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吴冯波
乜永艳
史小娟
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上海云泽生物科技有限公司
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Priority to US15/549,952 priority Critical patent/US20180024124A1/en
Priority to KR1020177010977A priority patent/KR101976964B1/ko
Priority to JP2017564786A priority patent/JP6605627B2/ja
Publication of WO2016155111A1 publication Critical patent/WO2016155111A1/zh
Priority to US16/838,889 priority patent/US11573223B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9493—Immunosupressants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G01N2001/386—Other diluting or mixing processes
    • G01N2001/388—Other diluting or mixing processes mixing the sample with a tracer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning

Definitions

  • the present invention relates to the field of in vitro diagnostic reagents, and in particular to an improved immunosuppressant drug extraction reagent, and an extraction method and an immunoassay kit using the same, which can be used to determine the concentration level of an immunosuppressive agent in a blood sample of a patient.
  • Immunosuppressant is a chemical or biological substance that reduces tissue damage by inhibiting cellular and humoral immune responses. It is widely used in anti-rejection after organ transplantation and in the treatment of autoimmune diseases (eg rheumatoid, lupus erythematosus). , ankylosing spondylitis and autoimmune hemolytic anemia, etc.).
  • immunosuppressants are mainly divided into five categories: microbial metabolites, glucocorticoids, antimetabolites, anti-lymphocyte antibodies, alkylating agents, etc.
  • Tacrolimus (FK506) and cyclosporin (Cyclosporin) A, CsA), rapamycin (RPM) and other microbial metabolites are most widely used, and they mainly exert their immunity by inhibiting the activity of cytosolic calcineurin and blocking the transcription of a series of cytokines such as IL2. Inhibition, effectively inhibiting the activation and proliferation of T lymphocytes.
  • the blood concentration of immunosuppressive agents is too low, which may lead to immune rejection. If the blood concentration of immunosuppressive agents is too high, it may cause toxicity to liver, kidney and other organs, and cause a series of infections and tumors. Adverse clinical events, therefore, the rational use of immunosuppressive agents in transplant patients requires accurate detection of blood levels.
  • immunosuppressive agents such as tacrolimus, sirolimus, cyclosporin A, although they have different structures, they are mainly present in the blood cells in the form of protein conjugates in the blood, and these immunitys should be accurately detected.
  • the blood concentration of the inhibitor must dissociate the immunosuppressant from its binding protein, which is a common requirement for the detection of blood levels of such immunosuppressants.
  • methods for determining the blood concentration of immunosuppressants include receptor binding method, HPLC-MS, MEIA, chemiluminescence microparticle immunoassay (CMIA), and enzyme-linked immunosorbent assay. (ELISA) and the like.
  • the receptor binding method is mainly used for drug research; the HPLC-MS method is accurate and sensitive, but its operation is cumbersome, the detection time is long, the detection cost is high, and expensive equipment is needed. It is mainly used in scientific research or as a reference. method.
  • MEIA and CMIA are currently commonly used detection methods, and their detection is highly automated and the detection results are accurate.
  • an organic solvent such as methanol, acetonitrile or diethyl ether is used as a sample extraction reagent in the detection process to dissolve the cells and extract the drug
  • the extraction step not only brings inconvenience to the experimental operation and the post-experimental treatment, but also has different degrees.
  • Reduce the sensitivity and accuracy of immunoassays In order to enter the immune reaction system, the organic solvent will inevitably inhibit the immuno-binding reaction between the antibody and the drug, thereby reducing the binding ability of the antibody, thereby reducing the detection sensitivity; in addition, the volatilization of the organic solvent in the extraction reagent is also likely to cause the drug concentration. Fluctuations affect the accuracy of test results.
  • Robert W. Siegel et al. (Clinical Chemistry, 2008, 54: 6, 1008-1017; Pat. No.: US 8022188B2) complemented antibodies by gene mutagenesis.
  • the area is modified to improve the detection sensitivity; however, the method requires complex steps such as nucleic acid extraction, amplification, sequencing, gene mutagenesis, and cloning screening, and the utility is not strong.
  • the DMSO used in the method still severely inhibits the immunoassay system.
  • the antibody-drug binding reaction reduces the detection sensitivity; in addition, like all the above organic solvent extraction reagents, the lysis of the extraction reagent depends on a high concentration of divalent metal ions (10 mM-100 mM) and the concentration of divalent metal ions The change of the extraction results in a change in the extraction effect. Therefore, the concentration change of the commonly used complex anticoagulant (such as EDTA) in the whole blood sample may cause a change in the concentration of the divalent metal ion in the extraction reagent, thereby causing the drug extraction effect of different samples. Differences, causing measurement errors.
  • the samples treated by the above extraction reagent are all non-homogeneous solutions, and need to be centrifuged to remove solid impurities before use; this step also increases the difficulty of operation and prolongs the detection time.
  • FK506 detection method using RAPA as a replacement reagent for FK506 (US Pat. 6187547B1); since FK506 and RAPA mainly bind to FKBP (FK binding protein) in blood, a small amount binds to serum albumin and lipoprotein. And the highly lipophilic FK506 and RAPA can penetrate the cell membrane quickly.
  • FKBP FK binding protein
  • the high concentration of RAPA in the immunoassay system can quickly and effectively displace FK506 in the blood without lysis and protein denaturation; due to anti-FK506 antibody
  • the high specificity, high concentration of RAPA does not interfere with the immunoassay of FK506; since no drug extraction is required, the method of detecting FK506 is simpler and faster, and can accurately detect most samples without interference factors, but for some strong interference samples, In particular, patient samples using antibody-based immunosuppressive agents often exhibit large errors in detecting FK506 by this method even if corresponding anti-interference measures are used.
  • the present invention provides a novel blood sample immunosuppressant drug extraction reagent and method for immunoassay.
  • the drug extraction reagent consists of a protein denaturant, a proteolytic enzyme, a surfactant, and a pH buffer.
  • the extraction method comprises mixing and incubating the sample and the extraction reagent in a certain ratio, and lysing the cells under the action of the protein denaturant and the protease to release the intracellular drug.
  • the invention eliminates the need for an organic solvent, effectively dissolves blood cells, and releases intracellular drugs.
  • the drug extraction reagents and methods provided by the invention not only avoid the problem of drug concentration change caused by solvent evaporation, but also significantly reduce the inhibitory effect of the solution medium on antibody-drug immunological binding.
  • the whole blood sample treated by the method is a completely transparent homogeneous solution, and the processed sample can be directly used without centrifugation, which simplifies the sample processing step and shortens the detection time.
  • a second object of the present invention is to provide a method for detecting the concentration of an immunosuppressive agent in a blood sample.
  • a third object of the present invention is to provide a kit for detecting the concentration of an immunosuppressive agent in a blood sample.
  • the present invention provides the following technical solutions:
  • an extraction reagent for extracting an immunosuppressive agent from a blood sample comprises a protein denaturant, a proteolytic enzyme, a surfactant, and a pH buffer.
  • the protein denaturant is selected from the group consisting of urea, guanidine hydrochloride, or other non-organic solvent-based protein denaturant.
  • the protein denaturant is urea.
  • the concentration of the urea in the extraction reagent is 4 mol / L - 12 mol / L, and the molar concentration of the guanidine hydrochloride in the extraction reagent is 1 mol / L - 8 mol / L; preferably, the urea
  • the concentration in the extraction reagent is 6 mol/L to 8 mol/L, and the molar concentration of the guanidine hydrochloride in the extraction reagent is 2 mol/L to 6 mol/L.
  • the proteolytic enzyme is selected from the group consisting of proteases such as subtilisin, proteinase K, and dispase, or a mixture thereof; preferably, the proteolytic enzyme is a subtilisin.
  • the amount of the subtilisin in the extraction reagent is from 1 U/ml to 20 U/ml; preferably, from 2.5 U/ml to 10 U/ml.
  • the surfactant is selected from one or more of the group consisting of: Tween-20, saponin, Triton X-100; preferably, Tween-20.
  • the Tween-20 is 0.005% to 1% (v/v) by volume in the extraction reagent; preferably, 0.02% to 0.1% (v/v).
  • the pH of the pH buffer is between 6.5 and 8.5; preferably between 7.0 and 8.0.
  • a method for detecting an immunosuppressant drug concentration in a blood sample comprising treating a blood sample under heating conditions using the extraction reagent, through a protein denaturant, a proteolytic enzyme, a surface
  • the synergistic action of the active agent converts the blood sample into a homogeneous solution while dissolving the blood cells and releasing the drug, and the homogeneous solution can be directly applied to the immunoassay without centrifugation to obtain an accurate concentration of the immunosuppressive drug.
  • the immunosuppressive agent comprises tacrolimus, sirolimus, everolimus, zotarolimus, cyclosporine or other structural analogues.
  • the blood sample is from an organ transplant patient or other patient taking an immunosuppressive agent.
  • the volume ratio of the blood sample/the extraction reagent is 1/1-1/10; preferably, 1/2-1/5.
  • the blood sample is treated by heating at a heating temperature of from 50 ° C to 90 ° C; preferably from 60 ° C to 80 ° C.
  • the blood sample is treated by heating for a heating time of from 5 min to 50 min; preferably from 10 min to 30 min.
  • the extraction reagent and the extraction method function to dissolve the cells, release the drug, and convert the blood sample into a homogeneous solution.
  • the immunoassay is a competitive immunoassay method in which an immunosuppressant in a blood sample competes with a fixed amount of an immunosuppressant to bind a limited amount of an anti-immunosuppressive antibody.
  • the immunoassay adopts a solid phase immunoassay method, which is implemented by: immobilizing an immunological reagent on the surface of the solid phase container, and realizing the test object by washing and separating the free detection reagent and the binding detection reagent after the competitive binding reaction is completed. Concentration detection.
  • the solid phase container refers to a microwell, test tube or other form of container.
  • the immunoassay comprises the following steps:
  • the signal generated by the tracer substance contained in the immune complex captured by the solid phase reagent is detected, and the immunosuppressant concentration is determined by a relationship between the signal intensity and the immunosuppressant concentration curve (calibration curve).
  • the blood sample refers to an anticoagulated whole blood sample, including EDTA-K (Na), sodium citrate (potassium), heparin anticoagulated whole blood sample; preferably, EDTA-K (Na) anticoagulated whole blood.
  • EDTA-K Na
  • sodium citrate potassium
  • heparin anticoagulated whole blood sample preferably, EDTA-K (Na) anticoagulated whole blood.
  • a kit for detecting the concentration of an immunosuppressive agent in a blood sample comprising: a) an anti-immunosuppressive antibody; b) the extraction reagent; c) immunization Inhibitor; d) calibrator; e) buffer solution.
  • an immunosuppressive antibody or an immunosuppressant is labeled with a tracer as a detection reagent.
  • the anti-immunosuppressive antibody refers to an antibody capable of specifically binding to an immunosuppressive drug, and may be a polyclonal antibody or a monoclonal antibody; preferably, a monoclonal antibody.
  • the extraction reagent consists of a protein denaturant, a proteolytic enzyme, a surfactant and a pH buffer.
  • the detection reagent is indicative of a trace-labeled antibody, antigen or immunosuppressive hapten.
  • the tracer substance refers to a substance capable of inducing a detectable signal, including an enzyme, a chemiluminescent substance, a radioactive substance, a fluorescent substance, a rare earth ion such as Eu 3+ , Sm 3+ , Tb 3+ , Dy 3+ , and
  • the chelate ligand may also be a small molecule indirect tracer such as biotin or digoxin.
  • the buffer solution is composed of a pH buffer solution, a protein, a surfactant, and a detection interference canceling agent, and is used for diluting the detection reagent, reducing the detection background signal and eliminating the influence of an interfering substance such as a heterophilic antibody on the detection. .
  • the calibrator refers to a solution or lyophilized product containing a known concentration of an immunosuppressant to establish a calibration curve; to reduce the matrix effect, the present invention prepares a calibrator from human whole blood.
  • the sample extraction reagent provided by the invention has the following advantages that the conventional organic solvent-based extraction reagent does not have:
  • the treated blood sample is a homogeneous solution, which can be directly detected without centrifugation, which simplifies the operation and shortens the detection time;
  • the detection method of the extraction reagent provided by the invention can be more conveniently applied to the fully automatic detection device, or based on the detection method of the extraction reagent provided by the invention, the accessory method can be more easily developed. Fully automatic testing equipment.
  • the effectiveness of the immunosuppressant extraction reagent of the present invention results from its efficient cytolysis, protein denaturation, and corresponding drug release ability. Therefore, the present invention can be applied not only to the extraction of immunosuppressive agents for human blood samples, but also to the extraction of drugs or non-drug substances in a bound state in vivo other than immunosuppressive agents of human blood and various animal blood samples.
  • Figure 1 shows the effect of different concentrations of Tween-20 on the extraction of FK506 in the extraction reagent.
  • Figure 2 shows the effect of different concentrations of protein denaturant on the extraction of FK506 in the extraction reagent.
  • Figure 3 shows the effect of different concentrations of protease in the extraction reagent on the extraction of FK506.
  • Figure 4 shows the effect of different incubation temperatures on the calibration curve during sample extraction.
  • Figure 5 shows the effect of different incubation times on the calibration curve.
  • Figure 6 shows the effect of different three pretreatment solutions on the calibration curve.
  • Figure 7A is a correlation of FK506-TRFIA detection values and HPLC-MS measurements based on the extraction reagent of the present invention.
  • Figure 7B is a correlation of the FK506-TRFIA detection value based on the ABBOTT-i2000 pretreatment solution with the HPLC-MS measurement.
  • Figure 7C is a correlation of FK506-TRFIA detection values based on Sorin's pretreatment solution with HPLC-MS measurements.
  • the immunosuppressive agent in the present invention refers to various drugs which can inhibit the proliferation and related functions of immune cells in the body and reduce the immune response of the body; preferably, the immunosuppressive agent of the present invention refers to tacrolimus, sirolimus, Everolimus, his left Moss or cyclosporin.
  • the extraction reagent of the invention is a mixture of a protein denaturant, a proteolytic enzyme, a surfactant and a pH buffer; the synergistic action of the protein denaturant, the proteolytic enzyme and the surfactant can rapidly dissolve the cells and release the protein binding.
  • Immunosuppressant is a mixture of a protein denaturant, a proteolytic enzyme, a surfactant and a pH buffer; the synergistic action of the protein denaturant, the proteolytic enzyme and the surfactant can rapidly dissolve the cells and release the protein binding. Immunosuppressant.
  • the immunosuppressant extraction according to the present invention refers to a process of extracting a drug in a bound state in a sample and using it as a detectable component by using the extraction reagent.
  • the incubation temperature selected is the optimum temperature at which the proteolytic enzyme acts; the incubation temperature and incubation time are selected to ensure that the immunosuppressant is sufficiently dissociated and the proteolytic enzyme is effectively inactivated.
  • the incubation temperature is from 50 ° C to 90 ° C
  • the incubation time is from 5 min to 50 min; preferably, incubation is carried out at 60 ° C to 80 ° C for 10 min to 30 min.
  • the mixture of the blood sample and the extraction reagent becomes a homogeneous solution and can be directly applied to the immunoassay.
  • Immunoassay refers to an analytical method for detecting various substances by using an antibody-antigen (or hapten) specific binding reaction.
  • the immunoassay of the present invention is used for quantitative determination of an immunosuppressive agent;
  • the immunoassay kit mainly comprises an extraction reagent, an antibody, a fixed amount of a test substance for competing for binding to an antibody with a test substance, and an analysis buffer. And calibrators; depending on the mode of detection, the labeled antibody or analyte can be selected.
  • the effectiveness of the extraction reagent of the present invention is derived from its efficient cytolysis, protein denaturation and corresponding drug release ability. Therefore, the present invention can be applied not only to the extraction of immunosuppressants of human blood samples, but also to humans. Extraction of a drug or non-drug substance in a bound state other than an immunosuppressive agent of blood and various animal blood samples; preferably, the test sample is an anticoagulated whole blood sample of a patient taking an immunosuppressive agent.
  • the immunoassay kit of the present invention also includes instructions for explaining the operation of the kit.
  • the instructions may be attached to the outer packaging material or stored in a separate leaflet in the kit.
  • the instructions may be printed or handwritten text materials, or any medium that may store the instructions and communicate the information to an end user, including but not limited to electronic storage media such as optical disks, magnetic disks, and the like.
  • Urea is a major component of the extraction reagent; as a commonly used protein denaturant, urea can form a double hydrogen bond with the carbonyl oxygen atom of two adjacent peptide bonds on the protein backbone at a high concentration, destroying the protein.
  • the second and third-order structure makes the protein chain fully stretch and loses the original physical and chemical properties and biological activity of the protein.
  • urea is low, the denaturing effect of urea on the protein is significantly reduced; based on this characteristic of urea, 6 mol/ can be used.
  • L-10mol/L high concentration of urea dissolves cells, denatures blood proteins and releases immunosuppressive agents.
  • Subtilisin is another major component of the extraction reagents of the present invention.
  • Subtilisin can exert maximum proteolytic activity at 60 ° C - 80 ° C, and the enzyme activity also decreases rapidly at this temperature ( ⁇ , Han Baoqin. Purification and enzymatic properties of subtilisin, World of Biotechnology, 2014, 3, 11-12), therefore, in this temperature range, subtilisin can concentrate on enzymatic hydrolysis in a short period of time, promoting lysis and protein denaturation, At the same time, the enzyme is partially inactivated during this process; in addition, when the extraction reagent containing the enzyme is cooled to room temperature, the activity of the enzyme is also lowered by the temperature drop (Niu Shuzhen, Han Baoqin.
  • Surfactants are another important component of the extraction reagents of the present invention.
  • the main function of the surfactant in the present invention is to promote cell rupture by synergistic action with urea and protease, and to make the blood sample after cell lysis in a homogeneous state; strong surfactant (such as SDS) has high cell rupture. Function, but its strong protein denaturation can seriously affect the activity of the antibody; the nonionic surfactants used in the present invention, such as Tween-20, exhibit good synergistic lysis and solubilization at optimized working concentrations. At the same time, it helps to reduce the background signal of the immunoassay.
  • the % involved is the mass to volume ratio (w/v) unless otherwise specified.
  • the FK506-TRFIA is a competitive immunoassay method based on a solid phase second antibody, that is, a treated calibrator/sample, anti-FK506 is added to the micro-pore coated with the goat anti-mouse second antibody.
  • Monoclonal antibody and biotinylated FK506, biotinylated FK506 competes with FK506 in the calibrator or sample to bind a limited amount of anti-FK506 monoclonal antibody, and the resulting immune complex is captured by the goat anti-mouse secondary antibody to the microporous surface.
  • the extraction reagent was 50 mM Tris-HCl buffer, pH 8.0, containing urea 8 mol/L, subtilisin 5 U/ml and 0.05% Tween-20.
  • TBST-BSA buffer containing 2 ⁇ g/ml SA-Eu 3+ (Suzhou Xinbo Biotechnology Co., Ltd.) was added, and the plate was incubated for 20 min to connect with the biotin on the surface of the microwell.
  • the microplate was washed 6 times with TBST buffer; the enhancement solution (Suzhou Xinbo Biotechnology Co., Ltd.) was added, the plate was incubated for 5 min, and the fluorescence intensity was measured by a time-resolved fluorescence analyzer (Victor 1420, Perkin-Elmer) according to the fluorescence intensity. And the calibration curve calculates the concentration of the drug contained in the sample to be tested.
  • the FK506 calibrator concentration is taken as the X-axis, and the detected fluorescence intensity signal value is the Y-axis, and a four-parameter fit is performed to obtain a regression equation and a fitting curve.
  • the sample concentration can be obtained by substituting the signal value of the sample to be tested into the regression equation.
  • the analysis of the detection results in the present invention can also utilize professional calculation analysis software, such as ELISA Calc software, to facilitate rapid analysis of a large number of samples.
  • Example 2 Effect of different concentrations of Tween-20 on the extraction of FK506 in extracting reagents
  • the extraction reagent is 50 mM Tris-HCl buffer, pH 8.0, urea containing 8 mol/L, subtilisin 5 U/ml and different concentrations of Tween-20; the sample to be tested is prepared for healthy human whole blood medium.
  • FIG. 1 shows that with the increase of Tween concentration in the extraction reagent, the fluorescence value shows a significant downward trend; when the Tween concentration is greater than 0.10% (v/v), the fluorescence value drops especially; since it contains 0.10%-0.20% (v /v)
  • the Tween-20 extraction reagent introduced into the reaction system has a Tween-20 concentration of only about 0.01%-0.02% (v/v). This low concentration of Tween-20 does not occur in most immune reaction systems. Causes solid phase antibody dissociation, and does not cause significant damage to antibody activity. Therefore, this phenomenon may be related to the urea contained in the system. Under the synergistic action of urea and Tween-20, the dissociation of the antibody on the surface of the solid phase is caused, resulting in a decrease in the detection signal.
  • the surfactant Tween-20 concentration in the extraction reagent of the present invention is preferably 0.02% to 0.1% (v/v).
  • Example 3 Effect of different concentrations of protein denaturant on the extraction of FK506 in extraction reagent
  • the extraction reagent is 50 mM Tris-HCl buffer solution of pH 8.0, containing Tween-20 0.05% (v/v), subtilisin 5 U/ml and different concentrations of urea or guanidine hydrochloride;
  • the sample is a calibrator containing different concentrations of FK506 prepared from a healthy human whole blood medium; the procedure is the same as in Example 1. The results are shown in Figure 2.
  • Figure 2 shows that as the concentration of protein denaturant (urea or guanidine hydrochloride) increases, the fluorescence intensity of each calibrator decreases.
  • the inhibition of the protein-FK506 immunoreactivity by protein denaturant and the release of protein by the protein denaturant The increase in the concentration of FK506 is the main cause of the decrease in the fluorescence intensity of each calibrator.
  • each calibrator For extracting reagents containing no protein denaturant (protease only), each calibrator has higher fluorescence intensity and lower inhibition rate, indicating that the protease contained in the extraction reagent containing no protein denaturant under the experimental conditions is insufficient to fully release.
  • the FK506 bound to the protein in the whole blood sample in addition, the whole blood sample treated with the extraction reagent containing no protein denaturant is a turbid solution containing a bulky impurity, which is inconvenient to sample, and the detection precision is poor.
  • the extraction reagent of the present invention is preferably 6 mol/L to 8 mol. /L urea is a protein denaturant.
  • the concentration of protease in the extraction reagent should be sufficient to lyse the cells in a short time, to promote protein denaturation, release protein-binding drugs; at the same time, the protease residual activity after sample treatment should not respond to immune binding reaction. Significant adverse effects.
  • the extraction reagent is 50 mM Tris-HCl buffer solution of pH 8.0, containing Tween-20 0.05% (v/v), urea 8 mol/L and different concentrations of subtilisin; the sample to be tested is a healthy person.
  • Figure 3 shows that when the extraction reagent does not contain subtilisin, the inhibition rate of different FK506 concentration calibrators is low, and the change is not regular, indicating that the FK506 in the sample is mostly bound; The homogeneity of the sample after treatment with the protease extracting agent was also unsatisfactory.
  • concentration of subtilisin in the extraction reagent is from 2.5 U/ml to 10.0 U/ml, the inhibition rate of each point of the calibrator tends to be uniform, and each calibrator is an amber, fully transparent homogeneous liquid. It can be seen from Fig.
  • the amount of subtilisin in the extraction reagent of the present invention is preferably from 2.5 U/ml to 10 U/ml.
  • the extraction reagent is 50 mM Tris-HCl buffer, pH 8.0, containing Tween-20 0.05% (v/v), urea 8 mol/L, subtilisin 5 U/ml, and the extraction reagent is mixed with the sample. After that, incubate for 20 min at different temperatures.
  • the sample to be tested is a calibrated product containing different concentrations of FK506 prepared from healthy human whole blood medium and 12 samples of EDTA anticoagulated whole blood measured by ABBOTT ARCHITECT i2000 system special reagent (FK506 chemiluminescent microparticle immunoassay reagent).
  • ABBOTT ARCHITECT i2000 system special reagent FK506 chemiluminescent microparticle immunoassay reagent.
  • the invention adopts the heating and culturing method to treat the blood sample, in order to fully exert the enzymatic hydrolysis of the protease in a short time, and at the same time partially inactivate the protease during the heating and incubation to reduce the influence of the protease on the antibody activity in the subsequent immune reaction.
  • Table 1 and Figure 4 show that when the incubation temperature is lower than 60 °C, the fluorescence intensity decreases significantly as the incubation temperature decreases, and the measured value of the sample is lower than the reference method (HPLC-MS method) and the change is irregular; It indicates that too low incubation temperature during sample processing is insufficient to fully dissolve cells, denatured proteins and release drugs; meanwhile, when the incubation temperature is low, residual protease activity after sample extraction against antibodies (solid phase secondary antibody and anti-FK506 antibody) The immunoreactive activity has a large effect, resulting in a decrease in fluorescence intensity. When the incubation temperature is in the range of 70-80 °C, the calibration curve has higher fluorescence intensity and the inhibition rate is relatively stable.
  • the heating incubation temperature of the sample treated with the extraction reagent of the present invention is selected from 60 ° C to 80 ° C.
  • the extraction reagent is 50 mM Tris-HCl buffer, pH 8.0, containing Tween-20 0.05% (v/v), urea 8 mol/L, subtilisin 5 U/ml, and the extraction reagent is mixed with the sample.
  • test sample was a calibrator containing different concentrations of FK506 prepared for healthy human whole blood medium, and 12 ABBOTT ARCHITECT i2000 system special reagents (FK506 chemiluminescent microparticles described in Example 5) Immunodetection reagent)
  • FK506 chemiluminescent microparticles described in Example 5 Immunodetection reagent
  • Figure 5 shows that the fluorescence intensity of each point of the calibrator increases with the incubation time of the sample from 5 min to 40 min during sample processing.
  • the thermal incubation time is extended from 5 min to 10 min, the fluorescence intensity increases more significantly, indicating that When the incubation time is less than 10 min, the residual protease activity of the system may reduce the detection signal by enzymatically decomposing the antibody.
  • the sample measurements showed good agreement with the HPLC-MS and ABBOTT-i2000 measurements for different incubation times from 5 min to 40 min (Table 2); the present invention was less active due to the longer protease activity at longer incubation times.
  • the sample treatment heat incubation time is selected from 10 min to 30 min.
  • This Comparative Example Sorin Corporation (PRO-Trac TM II Tacrolimus ELISA , DiaSorin), Abbott (ABBOTTARCHITECT i2000 System) and the pretreatment liquid of the present invention, calibration curve samples and measurements in the present invention, the FK506-TRFIA The impact of the value.
  • ABBOTT pretreatment liquid treatment sample 200 ⁇ l whole blood sample and 200 ⁇ l whole blood precipitant (purchased from Abbott Trading (Shanghai) Co., Ltd., 309221) were added to the centrifuge tube, shake and mix for 10 seconds, centrifuge at 10000 ⁇ g for 5-6 min; Take the supernatant as a sample.
  • Example 1 The sample of the extraction reagent of the present invention was treated as in Example 1.
  • the ABBOTT-i2000 pretreatment liquid containing organic solvent as a main component has a great influence on the detection, and the calibration curve has the lowest fluorescence intensity, and the parallel 4 holes are used to measure the fluorescence intensity of each calibrator.
  • FIG. 7A, 7B, and 7C show the FK506-TRFIA detection value and HPLC-MS determination of the extraction reagent, ABBOTT-i2000 pretreatment solution, and Sorin pretreatment solution according to the present invention in 55 samples processed by the three methods.
  • the correlation coefficients of the values were 0.981, 0.957, and 0.951, respectively, indicating that the FK506-TRFIA detection value based on the extraction reagent of the invention is in agreement with the HPLC-MS method.

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Abstract

一种免疫分析用的血液样品免疫抑制剂药物提取试剂，提取试剂包括蛋白变性剂、蛋白水解酶、表面活性剂和pH缓冲液；还提供了包括使用所述提取试剂检测全血样品中免疫抑制剂浓度的方法以及包含所述提取试剂的免疫检测试剂盒。该提取试剂无需使用传统提取方法中的有机溶剂，从而避免了有机溶剂对检测体系中抗体活性的影响；该药物提取过程无需离心，处理后的样品可直接用于免疫检测，操作简单，检测结果准确可靠。

Description

用于免疫分析的免疫抑制剂药物提取试剂 技术领域
本发明涉及体外诊断试剂领域，特别地，涉及改进的免疫抑制剂药物提取试剂，以及使用该提取试剂的提取方法和免疫检测试剂盒，可用于测定患者血液样品中免疫抑制剂的浓度水平。
背景技术
免疫抑制剂(Immunosuppressant)是一类通过抑制细胞及体液免疫反应而减轻组织损伤的化学或生物物质，广泛应用于器官移植后的抗排斥反应和治疗自身免疫性疾病(如：类风湿、红斑狼疮、强直性脊柱炎和自身免疫性溶血贫血等)。
目前常用的免疫抑制剂主要分为微生物代谢产物、糖皮质激素、抗代谢物、抗淋巴细胞抗体、烷化剂等五类，其中他克莫司(Tacrolimus，FK506)、环孢菌素(Cyclosporin A，CsA)、雷帕霉素(Rapamycin，RPM)等微生物代谢产物应用最广，它们主要通过抑制胞浆内神经钙蛋白的活性,阻断IL2等一系列细胞因子的转录，从而发挥其免疫抑制作用，有效抑制T淋巴细胞的活化与增殖。
对于移植患者的临床用药，免疫抑制剂的血液浓度过低则容易导致免疫排斥，而免疫抑制剂的血液浓度过高，则可能对肝、肾等器官产生毒性，并引发感染、肿瘤等一系列不良临床事件，因此，移植患者免疫抑制剂的合理用药要求精确检测其血液浓度。对于他克莫司、西罗莫司、环孢菌素A等常用免疫抑制剂,虽然它们结构各不相同，但在血液中均主要以蛋白结合物方式存在于红细胞中，要准确检测这些免疫抑制剂的血药浓度，必须使免疫抑制剂从其结合蛋白上解离下来，这是这类免疫抑制剂血药浓度检测的共同要求。
目前测定免疫抑制剂血药浓度的方法主要有受体结合法、HPLC-质谱联用(HPLC-MS)、微粒子酶免疫测定(MEIA)、化学发光微粒子免疫测定(CMIA)以及酶联免疫吸附法(ELISA)等。其中受体结合法主要用于药物研究；HPLC-MS法测定血药浓度准确、灵敏，但其操作繁琐，检测时间长，检测成本高，需要昂贵设备，主要用于科研领域或作为一种参考方法。MEIA和CMIA是目前常用的检测方法，其检测自动化程度高，检测结果准确。然而，上述方法在检测过程中均需使用甲醇、乙腈、乙醚等有机溶剂作为样品提取试剂，以溶解细胞和提取药物，该提取步骤不仅给实验操作和实验后处理带来不便，而且会不同程度地降低免疫检测的灵敏度和准确性，因 为进入免疫反应体系的有机溶剂会不可避免地抑制抗体-药物之间的免疫结合反应，降低抗体的结合力，进而降低检测敏感度；另外，提取试剂中有机溶剂的挥发也容易引起药物浓度的波动，影响检测结果的准确性。
为降低有机溶剂对抗体结合FK506的抑制作用，Robert W.Siegel等人(Clinical Chemistry，2008,54:6,1008-1017；Pat.No.:US 8022188B2)通过基因诱变方式对抗体的互补决定区加以改造，使检测灵敏度得到改善；但该方法需要核酸提取、扩增、测序、基因诱变、克隆筛选等复杂步骤，实用性不强。为了降低有机溶剂挥发引起的药物浓度波动，Frank C.Grenier等人采用低挥发性的DMSO配制血液药物提取试剂(US2008/0020401A1)，但该方法中所用的DMSO仍会严重抑制免疫检测体系中的抗体-药物间的结合反应，从而降低检测灵敏度；另外，同上述所有有机溶剂提取试剂一样，提取试剂的溶胞作用依赖高浓度的二价金属离子(10mM-100mM),且二价金属离子浓度的变化会导致提取效果的改变，因此全血样品中常用络合物抗凝剂(如：EDTA)的浓度变化可能引起提取试剂中二价金属离子的浓度变化，进而导致不同样品药物提取效果的差异，造成测量误差。
上述提取试剂处理后的样品均为非均质溶液，使用前需要离心去除固体杂质；该步骤也增加了操作的难度，延长了检测时间。
为避免有机溶剂对免疫检测的影响，
Figure PCTCN2015080330-appb-000001
Legay等人提出了以RAPA作为FK506的置换试剂的FK506检测方法(US Pat.6187547B1)；由于FK506和RAPA在血液中均主要与FKBP(FK结合蛋白)结合，少量与血清白蛋白和脂蛋白结合，且高度脂溶性的FK506和RAPA均可以快速穿透细胞膜，因此，免疫分析体系中高浓度的RAPA可以快速、有效地将血液中的FK506置换出来，且无需溶胞和蛋白变性；由于抗FK506抗体的高度特异性，高浓度的RAPA不干扰FK506的免疫检测；由于无需药物提取，该方法检测FK506更简单、快捷，可以准确检测大部分不含干扰因子的样品，但对于某些强干扰样品，尤其是使用了抗体类免疫抑制剂的患者样品，即使使用了相应的抗干扰措施，以该方法检测FK506仍常出现很大的误差。
因此，免疫抑制剂血药浓度的检测迫切需要一种可以避免使用有机溶剂，又能够准确、快速、简便地检测药物浓度的药物提取方法。目前，国内外未见具备上述特点的提取试剂和提取方法的报道。
发明内容
为了克服以有机溶剂提取药物的上述弊端，本发明提供了一种新的用于免疫分析的血液样品免疫抑制剂药物提取试剂和方法。所述药物提取试剂由蛋白变性剂、蛋白水解酶、表面活性剂和pH缓冲液组成。所述提取方法包括将样品与提取试剂按一定比例混合、孵育，在蛋白变性剂和蛋白酶作用下溶解细胞，释放胞内药物。本发明无需使用有机溶剂，能有效溶解血液细胞，释放胞内药物。同现有的提取试剂相比，本发明提供的药物提取试剂和方法，不仅避免了溶剂挥发导致的药物浓度改变问题，同时显著降低了溶液介质对抗体-药物间免疫结合的抑制作用。另外，本方法处理后的全血样品为全透明的均质溶液，处理后的样品可直接使用，无需离心，有效简化了样品处理步骤，缩短了检测时间。
因此，本发明的第一个目的在于提供一种从血液样品中提取免疫抑制剂的提取试剂。
本发明的第二个目的在于提供一种用于检测血液样品中免疫抑制剂浓度的方法。
本发明的第三个目的在于提供一种用于检测血液样品中免疫抑制剂浓度的试剂盒。
为了达到上述目的，本发明提供了如下技术方案：
根据本发明的第一个方面，一种从血液样品中提取免疫抑制剂的提取试剂，包括蛋白变性剂、蛋白水解酶、表面活性剂和pH缓冲液。
根据本发明，所述蛋白变性剂选自：尿素、盐酸胍、或者其他非有机溶剂类蛋白变性剂。
根据本发明的优选实施例，所述蛋白变性剂为尿素。
根据本发明，所述尿素在提取试剂中的浓度为4mol/L-12mol/L，所述盐酸胍在所述提取试剂中的摩尔浓度为1mol/L-8mol/L；优选的，所述尿素在所述提取试剂中的浓度为6mol/L-8mol/L，所述盐酸胍在所述提取试剂中的摩尔浓度为2mol/L-6mol/L。
根据本发明，所述蛋白水解酶选自：枯草杆菌蛋白酶、蛋白酶K、分散酶等蛋白酶类或者其混合物；优选的，所述蛋白水解酶为枯草杆菌蛋白酶。
根据本发明，所述枯草杆菌蛋白酶在所述提取试剂中的量为1U/ml-20U/ml；优选的，为2.5U/ml-10U/ml。
根据本发明，所述表面活性剂选自：吐温-20、皂素、Triton X-100中的一种或多种；优选的，为吐温-20。
根据本发明，所述吐温-20在所述提取试剂中按体积占比为0.005％-1％(v/v)；优选的，为0.02％-0.1％(v/v)。
根据本发明，所述pH缓冲液的pH在6.5-8.5之间；优选的，为7.0-8.0。
根据本发明的第二个方面，一种用于血液样品中免疫抑制剂药物浓度检测的方法，包括使用所述的提取试剂在加热条件下处理血液样品，通过蛋白变性剂、蛋白水解酶、表面活性剂的协同作用，在溶解血液细胞、释放药物的同时，将血液样品转变为均质溶液，该均质溶液可以无需离心，直接应用于免疫分析，获得准确的免疫抑制剂药物浓度。
根据本发明，所述免疫抑制剂包括他克莫司、西罗莫司、依维莫司、佐他莫司、环孢霉素或其他结构类似物。
根据本发明，所述血液样品来自器官移植患者或者其他服用免疫抑制剂的患者。
根据本发明，所述提取试剂和血液样品混合时，所述血液样品/所述提取试剂的体积比为1/1-1/10；优选地，为1/2-1/5。
根据本发明，所述通过加热方式处理血液样品，其加热温度为50℃-90℃；优选的，为60℃-80℃。
根据本发明，所述通过加热方式处理血液样品，其加热时间为5min-50min；优选的，为10min-30min。
根据本发明，所述提取试剂和提取方法，其作用在于溶解细胞、释放药物，并将血液样品转变为均质溶液。
根据本发明，所述免疫分析为竞争免疫分析方法，其实施方式为：血液样品中的免疫抑制剂与固定量的免疫抑制剂共同竞争结合限量的抗免疫抑制剂抗体。
根据本发明，所述免疫分析采用固相免疫分析方法，其实施方式为：在固相容器表面固定一种免疫试剂，竞争结合反应完成后通过洗涤分离游离检测试剂和结合检测试剂实现待测物浓度检测。
根据本发明，所述固相容器指微孔、试管或其它形式的容器。
根据本发明，所述免疫分析包括以下步骤：
1)血液样品处理。将样品与提取试剂混合，加热后恢复到室温；
2)免疫反应。在固定有免疫试剂的固相容器中加入经处理的样品、标记的抗免疫抑制剂抗体和固定量的免疫抑制剂；或，加入经处理的样品、标记的固定量的免疫抑制剂和抗免疫抑制剂抗体；固定量的免疫抑制剂与样品中的免疫抑制剂竞争结合限量的抗免疫抑制剂抗体，免疫复合物同时被固相试剂捕获。
3)分离。洗涤分离的游离检测试剂和结合的检测试剂。
4)检测。检测固相试剂捕获的免疫复合物所含的示踪物质产生的信号，通过信号强度与免疫抑制剂浓度关系曲线(校准曲线)测定免疫抑制剂浓度。
所述的血液样品指抗凝全血样品，包括EDTA-K(Na)、柠檬酸钠(钾)、肝素抗凝的全血样品；优选的，为EDTA-K(Na)抗凝全血。
根据本发明的第三个方面，一种用于检测血液样品中免疫抑制剂浓度的试剂盒，所述试剂盒包括：a)抗免疫抑制剂抗体；b)所述的提取试剂；c)免疫抑制剂；d)校准品；e)缓冲溶液。其中，免疫抑制剂抗体或免疫抑制剂以示踪剂标记，作为检测试剂。
根据本发明的优选实施例，所述的抗免疫抑制剂抗体指能特异性结合免疫抑制剂药物的抗体，可为多克隆抗体或单克隆抗体；优选的，为单克隆抗体。
根据本发明，所述的提取试剂由蛋白变性剂、蛋白水解酶、表面活性剂和pH缓冲液组成。
根据本发明，所述的检测试剂指示踪物标记的抗体、抗原或免疫抑制剂半抗原。
根据本发明，所述示踪物质指能引发可检测信号的物质，包括酶、化学发光物质、放射性物质、荧光物质、稀土离子如Eu3+、Sm3+、Tb3+、Dy3+及其螯合物配基，也可以是生物素、地高辛等小分子间接示踪物。
根据本发明，所述的缓冲溶液由pH缓冲溶液、蛋白质、表面活性剂、检测干扰消除剂组成，用于稀释检测试剂，降低检测本底信号和消除异嗜性抗体等干扰物质对检测的影响。
根据本发明，所述的校准品指含有已知浓度免疫抑制剂的溶液或冻干品，用以建立校准曲线；为降低基质效应，本发明以人全血为基质配制校准品。
有益效果：
本发明提供的血液样品提取试剂保留了传统的基于有机溶剂的提取试剂的以下优点：
具有一致的快速溶解细胞、释放药物的效果；
具有一致的灭活样品干扰物质的效果；在提取试剂的作用下，样品中的
异嗜性抗体、类风湿因子等干扰物质均被有效灭活；
同时，本发明提供的样品提取试剂又具备了传统的基于有机溶剂的提取试剂所不具备的以下优点：
1)不含有机溶剂，避免了有机试剂挥发对药物浓度的影响；
2)不含有机溶剂，有效降低了提取介质对抗体结合药物的抑制作用，使样品提取和实验后的样品处理更加简单；
3)不含二价金属离子，消除了不同样品间具有络合作用的抗凝剂浓度的差异对药物提取效果的影响；
4)处理后的血液样品为均质溶液，无需离心便可直接检测，简化了操作，缩短了检测时间；
5)可获得更可靠的检测结果；
6)基于上述特性，使用本发明提供的提取试剂的检测方法，可更方便地应用于全自动的检测设备，或基于本发明提供的提取试剂的检测方法，可更容易地研制与之配套的全自动的检测设备。
本发明所述的免疫抑制剂提取试剂的有效性，源自于其高效的细胞溶解、蛋白变性和与之相应的药物释放能力。因此，本发明不仅可用于人血液样品免疫抑制剂的提取，也可用于人血液和各种动物血液样品的免疫抑制剂以外的其它在体内呈结合状态的药物或非药物物质的提取。
附图说明
图1为提取试剂中不同浓度吐温-20对FK506提取效果的影响。
图2为提取试剂中不同浓度蛋白变性剂对FK506提取效果的影响。
图3为提取试剂中不同浓度蛋白酶对FK506提取效果的影响。
图4为样品提取过程中不同孵育温度对校准曲线的影响。
图5为不同孵育时间对校准曲线的影响。
图6为不同三种前处理液对校准曲线的影响。
图7A为基于本发明所述提取试剂的FK506-TRFIA检测值与HPLC-MS测定值的相关性。
图7B为基于ABBOTT-i2000前处理液的FK506-TRFIA检测值与HPLC-MS测定值的相关性。
图7C为基于索林前处理液的FK506-TRFIA检测值与HPLC-MS测定值的相关性。
具体实施方式
除非另作说明，在权利要求和说明书中按如下阐述使用的定义。
Ⅰ、免疫抑制剂
本发明中所述免疫抑制剂指能抑制机体免疫细胞的增殖和相关功能，降低机体免疫反应的各种药物；优选的，本发明所述免疫抑制剂指他克莫司、西罗莫司、依维莫司、他左莫司或者环孢菌素。
Ⅱ、提取试剂
本发明所述提取试剂为蛋白变性剂、蛋白水解酶、表面活性剂、pH缓冲液组成的混合物；通过蛋白变性剂、蛋白水解酶和表面活性剂的协同作用，可快速溶解细胞，释放蛋白结合的免疫抑制剂。
Ⅲ、免疫抑制剂提取
本发明所述免疫抑制剂提取，指应用所述的提取试剂将样品中结合状态的药物提取出来并成为可检测成分的过程。
将待测血样和提取试剂按适当比例混合；优选地，将10μl-50μl血样与50μl-200μl提取试剂混合；更为优选地，将25μl血样与170μl提取试剂混合；涡旋混匀后，加热孵育。选用的孵育温度为蛋白水解酶发挥作用的最适温度；选用的孵育温度和孵育时间要既保证免疫抑制剂被充分解离，又能使蛋白水解酶被有效灭活。一般地，孵育温度为50℃-90℃，孵育时间为5min-50min；优选地，选用60℃-80℃孵育10min-30min。孵育结束后，血样与提取试剂的混合物成为均质溶液，可直接应用于免疫分析。
Ⅳ、免疫检测
免疫检测指利用抗体-抗原(或半抗原)间特异性结合反应检测各种物质的分析方法。本发明所述的免疫检测用于免疫抑制剂的定量测定；所述的免疫检测试剂盒主要包括提取试剂、抗体、固定量的用以与待测物竞争结合抗体的待测物、分析缓冲液和校准品；根据检测模式的不同，可选择标记抗体或待测物。
本发明所述的提取试剂的有效性源自于其高效的细胞溶解、蛋白变性和与之相应的药物释放能力，因此，本发明不仅可用于人血液样品免疫抑制剂的提取，也可用于人血液和各种动物血液样品的免疫抑制剂以外的其它呈结合状态的药物或非药物物质的提取；优选的，测试样品为服用免疫抑制剂的患者的抗凝全血样品。
本发明的免疫检测试剂盒也包括对本试剂盒操作进行说明的说明书。所述的说明书可以固定在外包装材料上，或以单独的活页形式存放在试剂盒内。所述的说明书可以是印刷或者手写的文字材料，或任何可以存储本说明书并且将信息传递给终端用户的媒介，包括但不限于电子存储媒介，如光盘、磁盘等。
尿素是所述提取试剂的一种主要组份；作为一种常用的蛋白变性剂，尿素在高浓度下可以与蛋白质骨架上两个相邻肽键的羰基氧原子形成双氢键，破坏蛋白质的二、三级结构，使蛋白质链充分伸展，失去蛋白质原有的理化性状和生物活性；而当尿素为低浓度时，尿素对蛋白质的变性作用显著降低；基于尿素的该特性，可以使用6mol/L-10mol/L的高浓度尿素溶解细胞，使血液蛋白变性，释放免疫抑制剂；同时，少量含尿素的提取试剂样品加入反应容器后被缓冲液所稀释，其尿素浓度降低为原浓度的1/10-1/5，尿素对反应容器内的抗体活性的影响得以显著降低。
枯草杆菌蛋白酶是本发明所述提取试剂的另一种主要组份。枯草杆菌蛋白酶在60℃-80℃可以发挥最大的蛋白水解活性，且在该温度下酶活性也快速降低(牛树壹， 韩宝芹。枯草杆菌蛋白酶的纯化及酶学性质，生物技术世界，2014,3,11-12)，因此，在该温度区间枯草杆菌蛋白酶可以在短时间内集中发挥酶解作用，促使溶胞和蛋白变性，同时，在此过程中酶被部分灭活；另外，当含有该酶的提取试剂冷却至室温后，该酶活性也因温度下降而降低(牛树壹，韩宝芹。枯草杆菌蛋白酶的纯化及酶学性质，生物技术世界，2014,3,11-12)；另外，少量提取试剂样品加入反应容器后蛋白酶被缓冲液所稀释；以上三个因素使提取后的样品内残留的酶活性对反应容器内的抗体活性的影响得以显著降低。
表面活性剂是本发明所述提取试剂的另一种重要组份。表面活性剂在本发明中的主要作用是通过与尿素、蛋白酶的协同作用促使细胞破裂，并使细胞裂解后的血液样品处于均质状态；强烈的表面活性剂(如SDS)具有高效的细胞破裂作用，但其强烈的蛋白变性作用会严重影响抗体活性；本发明使用的非离子型表面活性剂，如吐温-20，在经优化的工作浓度下表现出良好的协同溶胞和增溶作用的同时，并有助于降低免疫分析的本底信号。
以下结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非限制本发明的范围。
以下实施例中，除特别注明外，首先使用本发明所述的提取试剂对血液样品进行处理，然后进行免疫抑制剂浓度检测。
以下实施例中，除特别注明外，所涉及的％为质量体积比(w/v)。
实施例1、FK506-TRFIA(Time Resolved Fluorescence Immunoassay)
本实施例中，所述的FK506-TRFIA是基于固相第二抗体的竞争免疫分析方法，即：在羊抗鼠第二抗体包被的微孔中加入处理后的校准品/样品、抗FK506单克隆抗体和生物素标记的FK506，生物素标记的FK506与校准品或样品中的FK506竞争结合限量的抗FK506单克隆抗体，形成的免疫复合物被羊抗鼠第二抗体捕获到微孔表面。洗涤去除未结合的生物素标记的FK506，加入铕离子(Eu3+)标记的链霉亲合素(SA-Eu3+)，使其与微孔内表面免疫复合物中的生物素结合；洗涤去除未结合的SA-Eu3+，免疫复合物上的Eu3+经解离增强形成稳定的荧光配合物，以荧光强度对FK506浓度建立标准曲线，通过校准曲线确定样品中FK506浓度。
提取试剂为50mM Tris-HCl缓冲液，pH8.0，含尿素8mol/L，枯草杆菌蛋白酶5U/ml和0.05％吐温-20。
将170μl提取试剂和25μl血液样品或校准品混合，涡旋使之充分混匀，70℃水浴孵育20min，取出，恢复至室温。
在包被有羊抗鼠二抗(Abcam公司)的微孔内加入25μl处理好的血样/校准品和100μl 0.2μg/ml TBST-BSA(50mM Tris-HCl，pH7.5，含0.9％NaCl，0.05％吐温-20，0.05％NaN3，0.5％BSA)稀释的FK506单克隆抗体(上海强耀公司)，混匀，室温(20℃-25℃)振板孵育30min；
在各微孔中加入50μl含0.05μg/ml生物素标记FK506(浙江海正药业有限公司)的TBST-BSA溶液，继续反应30min；
用TBST缓冲液洗涤微孔2次；
加入150μl含2μg/ml SA-Eu3+(苏州新波生物技术有限公司)的TBST-BSA缓冲液，振板孵育20min，使其与微孔表面的生物素相连接；
用TBST缓冲液洗涤微孔板6次；加入增强液(苏州新波生物技术有限公司)，振板孵育5min，以时间分辨荧光分析仪(Victor 1420,Perkin-Elmer)检测荧光强度，根据荧光强度及校准曲线计算被测样品所含的药物浓度。
检测结果分析
以FK506校准品浓度为X轴，以检测的荧光强度信号值为Y轴，进行四参数拟合，得到回归方程及拟合曲线。将待测样品信号值代入回归方程，即可求得样品浓度。本发明中的检测结果的分析还可以利用专业计算分析软件，如ELISACalc软件，便于大量样品的快速分析。
实施例2、提取试剂中不同浓度吐温-20对FK506提取效果的影响
在本实施例中，提取试剂为50mM Tris-HCl缓冲液，PH 8.0，含尿素8mol/L，枯草杆菌蛋白酶5U/ml和不同浓度的吐温-20；被测样品为健康人全血介质配制的含不同FK506浓度的校准品；操作步骤同实施例1。结果示于图1。
图1显示，随着提取试剂中吐温浓度的增加，荧光值呈显著下降趋势；吐温浓度大于0.10％(v/v)时，荧光值下降尤为明显；由于含0.10％-0.20％(v/v)吐温-20的提取试剂引入反应体系的吐温-20浓度仅约0.01％-0.02％(v/v)，这种低浓度的吐温-20在大多数免疫反应体系中不会造成固相抗体解离，也不会对抗体活性产生明显损害， 因此该现象可能与体系所含的尿素有关，在尿素和吐温-20的协同作用下，造成了固相表面抗体的解离，从而导致检测信号降低。
当提取试剂中不含吐温-20时，校准曲线各校准品点的荧光强度和抑制率良好，但此时部分样品(约20％)在检测过程出现血块粘连微孔孔壁的现象，导致检测精密性降低和测量值偏离，而提取试剂中少量的吐温-20可避免这一现象。根据本实施例，本发明所述提取试剂中表面活性剂吐温-20浓度优选为0.02％-0.1％(v/v)。
实施例3、提取试剂中不同浓度蛋白变性剂对FK506提取效果的影响
在本实施例中，提取试剂为50mM pH8.0的Tris-HCl缓冲液，含吐温-20 0.05％(v/v)，枯草杆菌蛋白酶5U/ml和不同浓度的尿素或盐酸胍；被测样品为健康人全血介质配制的含有不同浓度FK506的校准品；操作步骤同实施例1。结果示于图2。
图2显示，随着蛋白变性剂(尿素或盐酸胍)浓度的增加，各校准品荧光强度均呈下降趋势；蛋白变性剂对抗体-FK506间免疫反应的抑制以及蛋白变性剂促使药物释放所导致的FK506浓度的升高是各校准品荧光强度下降的主因。对于不含蛋白变性剂(仅含蛋白酶)的提取试剂，各校准品荧光强度较高，抑制率较低，显示本实验条件下不含蛋白变性剂的提取试剂中所含的蛋白酶不足以充分释放全血样品中蛋白质所结合的FK506；另外，以不含蛋白变性剂的提取试剂处理后的全血样品为含有块状杂质的混浊溶液，不便于取样，且检测精密性较差。
从图2可知，当提取试剂中尿素浓度从2mol/L增加到8mol/L，不含FK506的校准品A以及低浓度FK506校准品(如：3ng/mL)的荧光强度下降幅度很小，说明尿素对抗体-FK506间的免疫反应的抑制作用很低，但对于高浓度的FK506校准品(20ng/mL-30ng/mL)，随着尿素浓度的增加其荧光强度明显降低，说明只有高浓度的尿素才能充分促使蛋白质变性和FK506的释放；当提取试剂中尿素浓度从6mol/L增加到8mol/L，各FK506校准品的荧光强度和抑制率均变化不大，表明提取试剂中6mol/L-8mol/L的尿素对标本的变性作用已近饱和。同尿素相比，盐酸胍显示了更强的蛋白变性作用；当提取试剂中盐酸胍浓度从1mol/L增加到6mol/L时，不含FK506的校准品A的荧光强度下降明显，表明盐酸胍对抗体-FK506间的免疫反应的抑制作用很强；同时，随着盐酸胍浓度的升高，高浓度FK506校准品的荧光强度的降低幅度大于低浓度FK506校准品的荧光强度降低幅度，也体现了药物的充分释放对盐酸胍浓度有一定依赖性。
由于基于尿素的提取试剂对抗体-FK506免疫结合反应的抑制微弱，同时又能高效释放FK506，且处理后的样品呈全透明的均质溶液，故本发明所述提取试剂优选6mol/L-8mol/L尿素为蛋白变性剂。
实施例4、提取试剂中不同浓度蛋白酶对FK506提取效果的影响
在尿素、盐酸胍等变性剂的存在下，提取试剂中的蛋白酶浓度应足以在短时间内裂解细胞，促使蛋白变性，释放蛋白结合药物；同时，样品处理后的蛋白酶残留活性不应对免疫结合反应产生明显不良影响。
在本实施例中，提取试剂为50mM pH 8.0的Tris-HCl缓冲液，含吐温-20 0.05％(v/v)，尿素8mol/L和不同浓度的枯草杆菌蛋白酶；被测样品为健康人全血介质配制的含有不同浓度FK506的校准品；操作步骤同实施例1。检测结果示于图3。
图3显示，当提取试剂中不含有枯草杆菌蛋白酶时，不同FK506浓度校准品的抑制率均呈低水平，且变化欠规律，表明此时样品中的FK506多为结合状态；另外，不含枯草杆菌蛋白酶的提取试剂处理后的样品均质性也不理想。当提取试剂中枯草杆菌蛋白酶浓度为2.5U/ml-10.0U/ml时，校准品各点的抑制率趋于一致，各校准品均为琥珀色、全透明的均质液体。从图3可以看出，提取试剂中枯草杆菌蛋白酶用量为5U/ml-10U/ml时，校准品各点的荧光强度和抑制率均趋于一致，而当枯草杆菌蛋白酶用量>10U/ml时，校准品各点的荧光强度呈快速下降趋势，提示高浓度的枯草杆菌蛋白酶将损害抗体-抗原间的结合反应。基于以上现象，本发明所述提取试剂中枯草杆菌蛋白酶用量优选为2.5U/ml-10U/ml。
实施例5、不同孵育温度对校准曲线和血液样品测量值的影响
在本实施例中，提取试剂为50mM Tris-HCl缓冲液，pH 8.0，含吐温-20 0.05％(v/v)，尿素8mol/L，枯草杆菌蛋白酶5U/ml，提取试剂与样品混匀后，在不同温度下孵育20min。
被测样品为健康人全血介质配制的含有不同浓度FK506的校准品以及12份ABBOTT公司ARCHITECT i2000系统专用试剂(FK506化学发光微粒子免疫检测试剂)测定过的EDTA抗凝全血临床样本(来源于中国人民解放军第二军医大学第三附属医院)(测定值示于表1)。
参考李鹏飞等人方法(李鹏飞，刘丽宏,马萍，等；高效液相色谱-串联质谱法在他克莫司临床血药浓度监测中的应用，质谱学报，2008,29,137-143)，以HPLC-MS/MS法复测上述样品，测定值示于表1。
表1 样品提取过程中不同孵育温度对血液样品测定值(ng/mL)的影响
Figure PCTCN2015080330-appb-000002
本发明采用加热孵育方式处理血液样品，是为了在较短时间内充分发挥蛋白酶的酶解作用，同时使蛋白酶在加热孵育过程中部分失活，以降低蛋白酶对后续免疫反应中抗体活性的影响。表1和图4显示，当孵育温度低于60℃时，随着孵育温度的降低，荧光强度降低明显，样品测量值低于参考方法(HPLC-MS法)测量值且变化无规律；该现象表明，样品处理过程中过低的孵育温度不足以充分溶解细胞、变性蛋白和释放药物；同时，孵育温度较低时，样品提取后残留的蛋白酶活性对抗体(固相第二抗体和抗FK506抗体)的免疫反应活性产生了较大影响，导致荧光强度降低。当孵育温度处于70-80℃区间时，校准曲线荧光强度较高，抑制率相对稳定；在孵育温度为70℃时，样品测量值与HPLC-MS和ABBOTT i2000测量值均具有较好的一致性；当孵育 温度为90℃时，样品测量值有降低趋势。综上，以本发明所述的提取试剂处理样品的加热孵育温度选用60℃-80℃。
实施例6、不同孵育时间对校准曲线和血液样品测量值的影响
在本实施例中，提取试剂为50mM Tris-HCl缓冲液，pH 8.0，含吐温-20 0.05％(v/v)，尿素8mol/L，枯草杆菌蛋白酶5U/ml，提取试剂与样品混匀后，在70℃下孵育不同时间；被测样品为健康人全血介质配制的含有不同浓度FK506的校准品，以及实施例5中所述12份ABBOTT公司ARCHITECT i2000系统专用试剂(FK506化学发光微粒子免疫检测试剂)测定过的EDTA抗凝全血临床样本(来源于中国人民解放军第二军医大学第三附属医院)。
图5显示，样品处理过程中随着加热孵育时间从5min延长至40min，校准品各点的荧光强度呈现上升趋势；当热孵育时间从5min延长至10min时，荧光强度增加更为明显，表明当加热孵育时间不足10min时，体系残留的蛋白酶活性可能通过酶解抗体而降低检测信号。对于5min-40min内的不同孵育时间，样品测量值均与HPLC-MS和ABBOTT-i2000测量值呈现良好的一致性(表2)；由于较长的孵育时间下残留的蛋白酶活性更小，本发明的样品处理加热孵育时间选用10min-30min。
表2 样品提取过程中不同孵育时间对血液样品测量值(ng/mL)的影响
Figure PCTCN2015080330-appb-000003
实施例7、不同前处理液的实验对比
本实施例对比了索林公司(PRO-TracTM II Tacrolimus ELISA，DiaSorin)、雅培公司(ABBOTTARCHITECT i2000 System)和本发明所述前处理液在本发明所述FK506-TRFIA中对校准曲线和样品测量值的影响。
索林前处理液处理样品：在离心试管中加入50μl全血样品和300μl酶解液(购自索林医疗(上海)有限公司，批号305102)，震荡混匀20秒，室温静置15min，转至75℃水浴锅，加热15min，取出试管，震荡混匀20秒，1800×g离心10min；取上清液为样品。
ABBOTT前处理液处理样品：在离心试管中加入200μl全血样品和200μl全血沉淀剂(购自雅培贸易(上海)有限公司，309221)，震荡混匀10秒，10000×g离心5-6min；取上清液为样品。
本发明所述提取试剂处理样品：同实施例1。
采用上述三种不同方法处理55份经HPLC-MS法测定过的EDTA抗凝全血临床样本(来源于中国人民解放军第二军医大学第三附属医院)，按实施例1所述步骤检测处理后的各样品；以HPLC-MS作为参照方法，考察不同前处理液在FK506-TRFIA中对校准曲线(各校准品平行4孔测定)(图6)和测量值的影响(图7A，7B，7C)。
从图6可知，在所述FK506检测方法中，以有机溶剂为主要成分的ABBOTT-i2000前处理液对检测影响较大，校准曲线荧光强度最低，平行4孔测定各校准品的荧光强度的平行性较差，提示有机溶剂对反应体系存在不良影响；基于索林前处理液与本发明所述提取试剂的FK506检测有接近平行的校准曲线，校准品各点精密性良好，但以本发明所述提取试剂获得的荧光强度为最高，说明本发明所述提取试剂既能有效释放药物，其介质对反应体系的影响更为温和。
图7A，7B，7C显示，在三种方法处理的55份样品中，基于本发明所述提取试剂、ABBOTT-i2000前处理液、索林前处理液的FK506-TRFIA检测值与HPLC-MS测定值的相关系数分别为0.981、0.957、0.951，说明基于发明所述的提取试剂的FK506-TRFIA检测值与HPLC-MS法测定值一致性最好。

Claims (20)

  1. 一种免疫分析用的血液样品免疫抑制剂药物提取试剂，包括蛋白变性剂、蛋白水解酶、表面活性剂和pH缓冲液。
  2. 根据权利要求1所述的提取试剂，其特征在于，所述蛋白变性剂选自：尿素、盐酸胍或其它非有机溶剂类蛋白变性剂。
  3. 根据权利要求2所述的提取试剂，其特征在于，所述尿素在所述提取试剂中的摩尔浓度为4mol/L-12mol/L，所述盐酸胍在所述提取试剂中的摩尔浓度为1mol/L-8mol/L。
  4. 根据权利要求3所述的提取试剂，其特征在于，所述尿素在所述提取试剂中的摩尔浓度为6mol/L-8mol/L，所述盐酸胍在所述提取试剂中的摩尔浓度为2mol/L-6mol/L。
  5. 根据权利要求1所述的提取试剂，其特征在于，所述蛋白水解酶选自：枯草杆菌蛋白酶、蛋白酶K、分散酶中的一种或多种。
  6. 根据权利要求5所述的提取试剂，其特征在于，所述蛋白水解酶为枯草杆菌蛋白酶。
  7. 根据权利要求6所述的提取试剂，其特征在于，所述枯草杆菌蛋白酶在所述提取试剂中的量为2.5U/ml-10U/ml。
  8. 根据权利要求1所述的提取试剂，其特征在于，所述表面活性剂选自吐温-20、皂素、Triton X-100中的一种或多种。
  9. 根据权利要求8所述的提取试剂，其特征在于，所述表面活性剂为吐温-20。
  10. 根据权利要求9所述的提取试剂，其特征在于，所述吐温-20在所述提取试剂中按体积占比为0.005％-1％(v/v)。
  11. 根据权利要求10所述的提取试剂，其特征在于，所述吐温-20在所述提取试剂中按体积占比为0.02％-0.1％(v/v)。
  12. 根据权利要求1所述的提取试剂，其特征在于，所述pH缓冲液的pH在6.5-8.5之间。
  13. 一种用于血液样品中免疫抑制剂药物浓度检测的方法，其特征在于，使用如权利要求1-12中任一项所述的提取试剂通过加热方式处理血液样品，然后通过免疫分析测定其中所含的药物浓度。
  14. 根据权利要求13所述的方法，其特征在于，所述免疫抑制剂包括他克莫司、西罗莫司、依维莫司、佐他莫司、环孢霉素或其他结构类似物。
  15. 根据权利要求13所述的方法，其特征在于，所述提取试剂和所述血液样品混合时，所述血液样品与所述提取试剂的体积比为1/1-1/10。
  16. 根据权利要求15所述的方法，其特征在于，所述提取试剂和所述血液样品混合时，所述血液样品与所述提取试剂的体积比为1/2-1/5。
  17. 根据权利要求13所述的方法，其特征在于，所述加热的温度为50℃-90℃，所述加热的时间为5min-50min。
  18. 根据权利要求17所述的方法，其特征在于，所述加热的温度为60℃-80℃，所述加热的时间为10min-30min。
  19. 一种用于检测血液样品中免疫抑制剂浓度的试剂盒，其特征在于，所述试剂盒包括：a)特异性结合免疫抑制剂药物的抗体，b)含有所述免疫抑制剂的校准品；c)如权利要求1-12中任一项所述的提取试剂；d)检测试剂，所述检测试剂为示踪物标记的抗体、抗原或半抗原。
  20. 根据权利要求19所述的试剂盒，其特征在于，所述示踪物包括酶、化学发光物质、放射性物质、荧光物质、稀土离子、生物素、地高辛，所述稀土离子包括Eu3+、Sm3+、Tb3+、Dy3+及其螯合物配基。
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