CN114002442A - Testosterone detection kit and preparation method thereof - Google Patents
Testosterone detection kit and preparation method thereof Download PDFInfo
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- CN114002442A CN114002442A CN202111270160.4A CN202111270160A CN114002442A CN 114002442 A CN114002442 A CN 114002442A CN 202111270160 A CN202111270160 A CN 202111270160A CN 114002442 A CN114002442 A CN 114002442A
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- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 title claims abstract description 110
- 229960003604 testosterone Drugs 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 131
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 64
- 229960002685 biotin Drugs 0.000 claims abstract description 32
- 235000020958 biotin Nutrition 0.000 claims abstract description 32
- 239000011616 biotin Substances 0.000 claims abstract description 32
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims abstract description 26
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims abstract description 15
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 239000006249 magnetic particle Substances 0.000 claims abstract description 12
- 238000003908 quality control method Methods 0.000 claims abstract description 12
- 239000007983 Tris buffer Substances 0.000 claims abstract description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 9
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 8
- 230000005593 dissociations Effects 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 4
- 239000003085 diluting agent Substances 0.000 claims description 27
- 238000002372 labelling Methods 0.000 claims description 19
- 239000007987 MES buffer Substances 0.000 claims description 16
- 238000011033 desalting Methods 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 12
- 239000003755 preservative agent Substances 0.000 claims description 12
- 230000002335 preservative effect Effects 0.000 claims description 12
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 239000011324 bead Substances 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 239000002981 blocking agent Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000011859 microparticle Substances 0.000 claims description 4
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000006172 buffering agent Substances 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 239000008176 lyophilized powder Substances 0.000 claims 1
- 239000000047 product Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 3
- 206010058359 Hypogonadism Diseases 0.000 description 2
- 102000014034 Transcortin Human genes 0.000 description 2
- 108010011095 Transcortin Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000008448 Congenital adrenal hyperplasia Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000017701 Endocrine disease Diseases 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 206010020112 Hirsutism Diseases 0.000 description 1
- 101600111816 Homo sapiens Sex hormone-binding globulin (isoform 1) Proteins 0.000 description 1
- 108010089417 Sex Hormone-Binding Globulin Proteins 0.000 description 1
- 102100030758 Sex hormone-binding globulin Human genes 0.000 description 1
- 102300044179 Sex hormone-binding globulin isoform 1 Human genes 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 201000010066 hyperandrogenism Diseases 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 1
- 208000006155 precocious puberty Diseases 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- 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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a testosterone detection kit and a preparation method thereof, wherein the kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic particles and Tris buffer, the reagent R2 is a dissociation agent, the reagent R3 has acridinium ester labeled mouse anti-human T antibody and phosphate buffer, and the reagent R4 has biotin labeled T derivative and morpholine ethanesulfonic acid MES; also comprises a calibration material and a quality control material. The detection principle is that a competition method is adopted for detection, the reagent R2 is a dissociating agent, bound testosterone in a sample is released, buffer solutions are arranged in the reagent R1, the reagent R3 and the reagent R4 (in the reagent R4, morpholine ethanesulfonic acid MES is a buffer solution), and in addition, the testosterone detection kit comprises a calibrator and a quality control product, so that the detection result is high in accuracy and better in consistency.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a testosterone detection kit and a preparation method thereof.
Background
Testosterone is a steroid hormone secreted by the male testes or ovaries of women, the adrenal glands are secreted in small amounts, and over 97% of testosterone is present in the blood in a protein-bound state and circulates: most of them have strong affinity to bind with serum proteins such as Sex Hormone Binding Globulin (SHBG) or testosterone-binding globulin (TeBG), testosterone can also bind weakly to Corticosteroid Binding Globulin (CBG) and albumin, and the amount of free testosterone depends on the change of SHBG.
Serum testosterone determination is of great clinical significance in the analysis of some endocrine disorders, and in males testosterone determination is an important reference indicator of hypogonadism or hypogonadism, and is also used to assess precocious puberty or delay in immature males. Normal women have lower serum testosterone levels than men, but women with hyperandrogenism, such as: congenital hirsutism, adrenal hyperplasia, polycystic ovary syndrome and the like, and serum testosterone is also used as an important evaluation index. The detection methods commonly used in clinic and laboratory mainly include enzyme-linked immunosorbent assay, immunofluorescence assay, chemiluminescence assay and the like.
In chinese patent document CN112129955A, a testosterone detection kit is disclosed, which comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 comprises streptavidin magnetic particles and a basic buffer solution; the reagent R2 comprises an anti-testosterone antibody labeled with a detectable label and a basal buffer; the reagent R3 comprises biotinylated testosterone and basal buffer; at least one of the reagent R2 and the reagent R3 further comprises an anti-interference agent, and the anti-interference agent is selected from one or more of biotin and biotin derivatives.
The testosterone detection kit adopting the technical scheme has a not ideal detection effect, and has a close relation with the selection and content of the reagent types.
Disclosure of Invention
The invention aims to provide a testosterone detection kit which can improve the accuracy of testosterone detection, and has good anti-interference performance and better stability.
In order to solve the technical problems, the invention adopts the technical scheme that the testosterone detection kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic particles and Tris buffer, the reagent R2 is a dissociation agent, the reagent R3 has acridinium ester labeled mouse anti-human T antibody and phosphate buffer, and the reagent R4 has biotin labeled T derivative and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
The detection principle is to adopt a competition method for detection, a sample, a reagent R2 and a biotin-labeled mouse anti-human testosterone antibody react under an incubation condition, a reagent R2 (a displacing agent) releases testosterone combined in the sample, the antibody captures antigen in the sample to form an antigen-antibody complex, streptavidin-coated magnetic particles and acridinium ester-labeled testosterone bovine serum albumin conjugate are added to react under the incubation condition, the latter competes with the released testosterone to combine with the biotin-labeled mouse anti-human testosterone antibody to form a solid phase complex, and detection reading is carried out: after the incubation is finished, the magnetic field is added for precipitation, the supernatant is removed, the precipitation compound is washed by a cleaning solution, the waste liquid is sucked dry, the substances which are not combined with the magnetic particles are removed, and the reaction cup is sent into a measuring chamber. Pumping exciting liquid to make the compound produce chemiluminescence signal, measuring luminescence intensity by a photomultiplier, and calculating to obtain detection result by working curve.
In the invention, the reagent R2 is a dissociation agent, releases testosterone combined in a sample, and buffers are arranged in the reagent R1, the reagent R3 and the reagent R4 (in the reagent R4, morpholine ethanesulfonic acid MES is a buffer), and in addition, the testosterone detection kit comprises a calibrator and a quality control product, so that the detection result has high accuracy, good anti-interference performance and better stability.
Preferably, the reagent R2 has 17 alpha-methylandrost-17 beta-ol-3-one and morpholine ethanesulfonic acid MES buffer.
17 alpha-methyl androstane-17 beta-alcohol-3-ketone is used as a displacer.
Preferably, the reagent R1, the reagent R2, the reagent R3 and the reagent R4 all contain a preservative Proclin 300; in the reagent R2, the 17 alpha-methylandrostane-17 beta-alcohol-3-ketone is 0.20mg/L, and the morpholine ethanesulfonic acid MES buffer solution is 50 mmol/L.
Preferably, the calibrator and the quality control product are testosterone antigens in a freeze-dried powder state; the preservative Proclin300 is 1 g/L.
Preferably, in the reagent R1, the streptavidin-coated magnetic particles are 2.5g/L, and the Tris buffer is 50 mmol/L; in the reagent R3, the acridinium ester labeled mouse anti-human T antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled T derivative is 0.78mg/L, and the morpholine ethanesulfonic acid MES is 100 mmol/L.
Another problem to be solved by the present invention is to provide a method for preparing a testosterone detection kit, wherein the testosterone detection kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4, and comprises the following steps:
preparation of reagent R1 described in S1: taking magnetic particles coated with streptavidin with the diameter of 1uM, diluting the concentration by using a magnetic bead diluent, and uniformly stirring;
preparation of reagent R3 described in S2: mixing a mouse anti-human T antibody aiming at a T antigen epitope with acridinium ester, and then labeling the acridinium ester; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a mouse anti-human T antibody labeled by acridinium ester aiming at the T antigen epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope by using a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the T derivative by using a buffer solution, mixing the diluted concentration with biotin, and labeling the biotin; after the labeling is finished, carrying out centrifugal desalting purification to obtain a biotin-labeled T derivative;
diluting the obtained biotin-labeled T derivative with a diluent;
s4 reagent packaging: subpackaging the prepared reagent R1, the prepared reagent R3 and the prepared reagent R4, and storing at the temperature of 2-8 ℃ in a dark place;
the testosterone detection kit also comprises a calibrator and a quality control product, and the reagent R2 is a dissociation agent.
Preferably, in the step S1, the magnetic bead diluent contains Tris as a buffer, BSA as a blocking agent and Proclin300 as a preservative; in the step S2, the diluent comprises sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate as buffering agents, BSA and casein as blocking agents, Proclin300 as a preservative and Tween-20 as a surfactant; in step S3, MES is a main component of the diluent.
Preferably, in step S2 and step S3, centrifugal desalting purification is performed by centrifugal desalting purification using a Zeba desalting centrifugal column; in both of the step S2 and the step S3, the labeling is performed by reacting at 37 ℃ for 90 minutes.
Wherein, the Zeba desalination centrifugal column is 7K MWCO, 0.5mL of the product number: 89883 specification.
Preferably, the labeling method further comprises the step of labeling S5: sticking the radio frequency card on the kit, and then covering the label on the radio frequency card; the radio frequency card contains reagent batch number, box number, service life, project code, project name and reagent specification information.
Preferably, in step S1, the streptavidin-coated magnetic microparticles are diluted with a magnetic bead diluent to a concentration of 2.5 g/L; in the step S2, the molar ratio of each mouse anti-human T antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope is prepared into the concentration of 0.15mg/L by using a diluent; in step S3, the T derivative is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the T derivative to biotin is 1:10, and the obtained biotin-labeled T derivative is diluted with a diluent solution to a concentration of 0.78 mg/L.
Detailed Description
The following is a more detailed description of embodiments of the invention:
the testosterone detection kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic particles and Tris buffer, the reagent R2 is a dissociation agent, the reagent R3 has acridinium ester labeled mouse anti-human T antibody and phosphate buffer, and the reagent R4 has biotin labeled T derivative and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
The reagent R2 has 17 alpha-methylandrost-17 beta-ol-3-one and morpholine ethanesulfonic acid MES buffer.
The reagent R1, the reagent R2, the reagent R3 and the reagent R4 all contain a preservative Proclin 300; in the reagent R2, the 17 alpha-methylandrostane-17 beta-alcohol-3-ketone is 0.20mg/L, and the morpholine ethanesulfonic acid MES buffer solution is 50 mmol/L.
The calibrator and the quality control product are testosterone antigens in a freeze-dried powder state; the preservative Proclin300 is 1 g/L.
In the reagent R1, the streptavidin-coated magnetic particles are 2.5g/L, and the Tris buffer is 50 mmol/L; in the reagent R3, the acridinium ester labeled mouse anti-human T antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled T derivative is 0.78mg/L, and the morpholine ethanesulfonic acid MES is 100 mmol/L.
The preparation method of the testosterone detection kit in the embodiment comprises the following steps:
preparation of reagent R1 described in S1: taking magnetic particles coated with streptavidin with the diameter of 1uM, diluting the concentration by using a magnetic bead diluent, and uniformly stirring;
preparation of reagent R3 described in S2: mixing a mouse anti-human T antibody aiming at a T antigen epitope with acridinium ester, and then labeling the acridinium ester; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a mouse anti-human T antibody labeled by acridinium ester aiming at the T antigen epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope by using a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the T derivative by using a buffer solution, mixing the diluted concentration with biotin, and labeling the biotin; after the labeling is finished, carrying out centrifugal desalting purification to obtain a biotin-labeled T derivative;
diluting the obtained biotin-labeled T derivative with a diluent;
s4 reagent packaging: subpackaging the prepared reagent R1, the prepared reagent R3 and the prepared reagent R4, and storing at the temperature of 2-8 ℃ in a dark place;
the testosterone detection kit also comprises a calibrator and a quality control product, and the reagent R2 is a dissociation agent.
The prepared testosterone detection kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4.
In step S1, the magnetic bead diluent contains Tris as a buffer, BSA as a blocking agent and Proclin300 as a preservative; in the step S2, the diluent comprises sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate as buffering agents, BSA and casein as blocking agents, Proclin300 as a preservative and Tween-20 as a surfactant; in step S3, MES is a main component of the diluent.
In step S2 and step S3, centrifugal desalting purification is performed by using a Zeba desalting centrifugal column; in both of the step S2 and the step S3, the labeling is performed by reacting at 37 ℃ for 90 minutes.
Also comprises a step S5 of labeling: sticking the radio frequency card on the kit, and then covering the label on the radio frequency card; the radio frequency card contains reagent batch number, box number, service life, project code, project name and reagent specification information.
In step S1, the streptavidin-coated magnetic microparticles are diluted to a concentration of 2.5g/L with a magnetic bead diluent; in the step S2, the molar ratio of each mouse anti-human T antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope is prepared into the concentration of 0.15mg/L by using a diluent; in step S3, the T derivative is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the T derivative to biotin is 1:10, and the obtained biotin-labeled T derivative is diluted with a diluent solution to a concentration of 0.78 mg/L.
In this example, the specific components and contents of the reagent R1, the reagent R2, the reagent R3, and the reagent R4 of the kit 1 are shown in table 1 below:
table 1:
the biotinylated testosterone in table 1 is the biotin-labeled T derivative;
in this embodiment, the components and contents of the reagent R1, the reagent R2, the reagent R3 and the reagent R4 of the kit 2 are shown in table 2 below:
table 2:
the biotinylated testosterone in table 2 is the biotin-labeled T derivative;
the anti-interference tests of the kit 1 and the kit 2 are shown in the following table 3:
from the above test results, the anti-interference performance of the kit 1 is good, and the deviation of the kit 1 is small relative to that of the kit 2.
The stability test was performed on the above kit 1 and kit 2, and the results are shown in table 4 below:
table 4:
from the test results, the stability of the kit 1 is better, and the signal retention rate of the kit 1 is higher than that of the kit 2; a, B, C, D, E in the table indicates the number of the calibrator at different concentrations, i.e. the concentration is the number following the number in ng/mL.
The inventor finds that the biotin concentration of the reagent R4 in the kit 1 has an influence on the anti-interference performance of the kit, and the specific data table is shown in the following table 5:
table 5:
the results show that biotin concentrations of 30ng/mL are the best interference-resistant.
When the testosterone detection kit of the embodiment is used for detecting testosterone, after a detection result is obtained, the positive judgment value or the reference interval is shown in the following tables 6 and 7:
serum table 6:
| reference human sample | 95% reference interval (ng/mL) |
| Male sex | 1.75-7.81 |
| Female with a view to preventing the formation of wrinkles | ＜0.100-0.750 |
Plasma, whole blood, table 7:
| reference population | 95% reference interval (ng/mL) |
| Male sex | 1.68-7.58 |
| Female with a view to preventing the formation of wrinkles | ≤0.100-0.900 |
The reference range of the user is suggested to be established due to the differences of geography, race, gender, age and the like; the default units for the results for the testosterone items were ng/mL or nmol/L (conversion factor: ng/mL × 3.47 ═ nmol/L).
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The testosterone detection kit is characterized by comprising a reagent R1, a reagent R2, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic particles and Tris buffer, the reagent R2 is a dissociation agent, the reagent R3 has acridinium ester labeled mouse anti-human T antibody and phosphate buffer, and the reagent R4 has biotin labeled T derivative and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
2. The testosterone detection kit of claim 1, wherein said reagent R2 has 17 α -methylandrost-17 β -ol-3-one and morpholinoethanesulfonic acid MES buffer.
3. The testosterone detection kit according to claim 2, wherein each of said reagent R1, said reagent R2, said reagent R3, and said reagent R4 comprises a preservative Proclin 300; in the reagent R2, the 17 alpha-methylandrostane-17 beta-alcohol-3-ketone is 0.20mg/L, and the morpholine ethanesulfonic acid MES buffer solution is 50 mmol/L.
4. The testosterone detection kit according to claim 3, wherein said calibrator and quality control materials are testosterone antigens in a lyophilized powder state; the preservative Proclin300 is 1 g/L.
5. The testosterone detection kit according to any one of claims 1 to 4, wherein in said reagent R1, said streptavidin-coated magnetic microparticles is 2.5g/L, said Tris buffer is 50 mmol/L; in the reagent R3, the acridinium ester labeled mouse anti-human T antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled T derivative is 0.78mg/L, and the morpholine ethanesulfonic acid MES is 100 mmol/L.
6. The preparation method of the testosterone detection kit is characterized in that the testosterone detection kit comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4, and comprises the following steps:
preparation of reagent R1 described in S1: taking magnetic particles coated with streptavidin with the diameter of 1uM, diluting the concentration by using a magnetic bead diluent, and uniformly stirring;
preparation of reagent R3 described in S2: mixing a mouse anti-human T antibody aiming at a T antigen epitope with acridinium ester, and then labeling the acridinium ester; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a mouse anti-human T antibody labeled by acridinium ester aiming at the T antigen epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope by using a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the T derivative by using a buffer solution, mixing the diluted concentration with biotin, and labeling the biotin; after the labeling is finished, carrying out centrifugal desalting purification to obtain a biotin-labeled T derivative;
diluting the obtained biotin-labeled T derivative with a diluent;
s4 reagent packaging: subpackaging the prepared reagent R1, the prepared reagent R3 and the prepared reagent R4, and storing at the temperature of 2-8 ℃ in a dark place;
the testosterone detection kit also comprises a calibrator and a quality control product, and the reagent R2 is a dissociation agent.
7. The method of preparing a testosterone detection kit according to claim 6, wherein in said step S1, the magnetic bead diluent comprises Tris as a buffer, BSA as a blocking agent and Proclin300 as a preservative; in the step S2, the diluent comprises sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate as buffering agents, BSA and casein as blocking agents, Proclin300 as a preservative and Tween-20 as a surfactant; in step S3, MES is a main component of the diluent.
8. The method for preparing a testosterone detection kit according to claim 6, wherein in step S2 and step S3, centrifugal desalting purification is performed by using a Zeba desalting centrifugal column; in both of the step S2 and the step S3, the labeling is performed by reacting at 37 ℃ for 90 minutes.
9. The method for preparing a testosterone detection kit according to any one of claims 6-8, further comprising step S5 of labeling: sticking the radio frequency card on the kit, and then covering the label on the radio frequency card; the radio frequency card contains reagent batch number, box number, service life, project code, project name and reagent specification information.
10. The method for preparing a testosterone detection kit according to any one of claims 6 to 8, wherein in said step S1, streptavidin-coated magnetic microparticles are diluted with a magnetic bead diluent to a concentration of 2.5 g/L; in the step S2, the molar ratio of each mouse anti-human T antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human T antibody aiming at the T epitope is prepared into the concentration of 0.15mg/L by using a diluent; in step S3, the T derivative is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the T derivative to biotin is 1:10, and the obtained biotin-labeled T derivative is diluted with a diluent solution to a concentration of 0.78 mg/L.
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| US20140072963A1 (en) * | 2011-03-16 | 2014-03-13 | Beijing Unidiag Technology Inc | Electrochemiluminescence immunoassay method |
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
| US20170307605A1 (en) * | 2014-07-24 | 2017-10-26 | Intelligent Fingerprinting Limited | Sample analysing device |
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