CN114002444A - Beta-human chorionic gonadotropin detection kit and preparation method thereof - Google Patents
Beta-human chorionic gonadotropin detection kit and preparation method thereof Download PDFInfo
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- 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
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- 238000011534 incubation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
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- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 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 description 2
- 108010061174 Thyrotropin Proteins 0.000 description 2
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- 239000003270 steroid hormone Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
Classifications
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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
- G01N33/74—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
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- 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
- G01N33/53—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
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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
- G01N33/58—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a beta-human chorionic gonadotropin detection kit, which comprises a reagent R1, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic microparticles and Tris buffer, the reagent R3 has acridinium ester-labeled mouse anti-human HCG antibody and phosphate buffer, and the reagent R4 has biotin-labeled mouse anti-human HCG antibody and morpholine ethanesulfonic acid MES; also comprises a calibration material and a quality control material. The reagent R1, the reagent R3 and the reagent R4 are all provided with buffer solution (in the reagent R4, morpholine ethanesulfonic acid MES is the buffer solution), and in addition, the beta-human chorionic gonadotrophin detection kit comprises a calibrator and a quality control product, so that the detection result has high accuracy, good anti-interference performance and good stability.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a beta-human chorionic gonadotropin detection kit and a preparation method thereof.
Background
Human chorionic gonadotropin (hCG) is a glycoprotein with two non-covalently bound subunits. Its alpha subunit is similar to that of Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), and Thyroid Stimulating Hormone (TSH). The beta subunit of human chorionic gonadotropin is distinct from other pituitary glycoprotein hormones and exhibits unique biochemical and immunological properties. The human chorionic gonadotropin is synthesized by cells of placenta and can maintain the function of corpus luteum in pregnancy, is a good pregnancy detection marker and can be detected in one week after pregnancy.
During pregnancy, human chorionic gonadotropin (hCG) levels rise exponentially and continue until approximately 8 to 10 weeks after the last menstruation. In the late phase of pregnancy, at about 12 weeks after conception, the hCG concentration begins to decrease as the placenta begins to produce steroid hormones. The decrease of human chorionic gonadotropin is indicative of abortion, ectopic pregnancy, etc.
At present, the detection of human chorionic gonadotropin in clinical laboratories mainly adopts a chemiluminescence method and an enzyme-linked immunofluorescence method. 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 CN107677837A, a method for preparing a detection kit for β human chorionic gonadotropin is disclosed, which comprises preparing a reagent R1 and a reagent R2; the preparation of the reagent R1 comprises the following steps: adding sodium azide, sucrose, Tween-80 and PEG-8000 into MES buffer solution to prepare a reagent R1; the preparation of the reagent R2 comprises the following steps: (1) adding NaCl, sodium azide, bovine serum albumin and glycerol into the MES buffer solution to prepare a latex microsphere dissolving reagent; (2) coating the beta-human chorionic gonadotropin antibody into latex microspheres to prepare antibody latex; (3) and centrifuging the antibody latex to remove supernatant, dissolving the precipitate by using a latex microsphere dissolving reagent, and performing ultrasonic dispersion to prepare a reagent R2.
The detection effect of the beta human chorionic gonadotropin detection kit in the technical scheme is not ideal, and the detection effect is closely related to the reason, the selection of the types of reagents and the content of the reagents.
Disclosure of Invention
The invention aims to provide a beta-human chorionic gonadotropin detection kit which can improve the accuracy of testosterone detection and has good anti-interference performance and good stability.
In order to solve the technical problems, the invention adopts the technical scheme that the beta-human chorionic gonadotropin detection kit comprises a reagent R1, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic microparticles and Tris buffer, the reagent R3 has acridinium ester-labeled mouse anti-human HCG antibody and phosphate buffer, and the reagent R4 has biotin-labeled mouse anti-human HCG antibody and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
The detection principle is to adopt a double-antibody sandwich method for detection, a sample, a biotin-labeled HCG + beta monoclonal antibody and streptavidin-coated magnetic particles react under an incubation condition, the antibody captures an antigen in the sample to form an antigen-antibody complex, the antigen-antibody complex is bound to the magnetic particles, after the incubation is finished, a magnetic field is added for precipitation, supernatant is removed, a washing solution is used for washing the precipitated complex, waste liquid is sucked dry, substances which are not bound with the magnetic particles are removed, another HCG + beta monoclonal antibody which is labeled by acridinium ester is added to react under the incubation condition to form the antigen-antibody sandwich 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. The instrument automatically pumps excitation liquid to enable the compound to generate a chemiluminescent signal, the photomultiplier is used for measuring the luminous intensity, and the instrument automatically calculates a detection result through a working curve.
In the invention, the reagent R1, the reagent R3 and the reagent R4 are all provided with buffer solution (in the reagent R4, morpholine ethanesulfonic acid MES is the buffer solution), and in addition, the beta-human chorionic gonadotropin detection kit comprises a calibrator and a quality control product, so that the detection result has high accuracy, good interference resistance and good stability.
Preferably, the reagent R1, the reagent R3 and the reagent R4 each contain a preservative Proclin 300.
Preferably, the calibrator and the quality control product are human chorionic gonadotropin recombinant antigens obtained by in-vitro recombination 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 HCG antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled mouse anti-human HCG antibody was 0.78mg/L and the morpholine ethanesulfonic acid MES was 100 mmol/L.
Preferably, the reagent R1, the reagent R3, and the reagent R4 each contain bovine serum albumin BSA at 10 g/L.
Another problem to be solved by the present invention is to provide a method for preparing a β -hcg assay kit, wherein the β -hcg assay kit comprises a reagent R1, 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 HCG antibody aiming at HCG 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 HCG antibody labeled by acridinium ester aiming at HCG epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope with a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the mouse anti-human HCG antibody with a buffer solution, mixing the diluted concentration with biotin, and labeling with biotin; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a biotin-labeled mouse anti-human HCG antibody;
diluting the obtained biotin-labeled mouse anti-human HCG antibody 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 beta-human chorionic gonadotropin detection kit also comprises a calibrator and a quality control product.
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 using a Zeba desalting centrifugal column; in the step S2, when labeling is performed, the labeling is performed by reacting at 37 ℃ for 90 minutes; in the step S3, the labeling is carried out by reacting at 37 ℃ for 60 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 HCG antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope is prepared into the concentration of 0.15mg/L by using diluent; in step S3, the mouse anti-human HCG antibody is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the mouse anti-human HCG antibody to biotin is 1:10, and the obtained biotin-labeled mouse anti-human HCG antibody is diluted with a diluent to a concentration of 0.78 mg/L.
Detailed Description
The following is a more detailed description of embodiments of the invention:
the kit for detecting the beta-human chorionic gonadotropin comprises a reagent R1, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic microparticles and Tris buffer, the reagent R3 has acridinium ester-labeled mouse anti-human HCG antibody and phosphate buffer, and the reagent R4 has biotin-labeled mouse anti-human HCG antibody and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
The reagent R1, the reagent R3 and the reagent R4 all contain a preservative Proclin 300.
The calibrator and the quality control product are human chorionic gonadotropin recombinant antigens obtained by in-vitro recombination 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 HCG antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled mouse anti-human HCG antibody was 0.78mg/L and the morpholine ethanesulfonic acid MES was 100 mmol/L.
The reagent R1, the reagent R3, and the reagent R4 each contain bovine serum albumin BSA at 10 g/L.
The preparation method of the above-mentioned β -human chorionic gonadotropin detection kit of this embodiment, said β -human chorionic gonadotropin detection kit comprising reagent R1, reagent R3 and reagent R4, 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 HCG antibody aiming at HCG 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 HCG antibody labeled by acridinium ester aiming at HCG epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope with a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the mouse anti-human HCG antibody with a buffer solution, mixing the diluted concentration with biotin, and labeling with biotin; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a biotin-labeled mouse anti-human HCG antibody;
diluting the obtained biotin-labeled mouse anti-human HCG antibody 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 beta-human chorionic gonadotropin detection kit also comprises a calibrator and a quality control product.
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 the step S2, when labeling is performed, the labeling is performed by reacting at 37 ℃ for 90 minutes; in the step S3, the labeling is carried out by reacting at 37 ℃ for 60 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 HCG antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope is prepared into the concentration of 0.15mg/L by using diluent; in step S3, the mouse anti-human HCG antibody is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the mouse anti-human HCG antibody to biotin is 1:10, and the obtained biotin-labeled mouse anti-human HCG antibody is diluted with a diluent to a concentration of 0.78 mg/L.
In this example, the specific components and contents of the reagent R1, the reagent R3, and the reagent R4 of the kit 1 are shown in table 1 below:
TABLE 1
The biotin-labeled mouse anti-monoclonal antibody in table 1 is a biotin-labeled mouse anti-human HCG antibody; the acridinium ester marked mouse anti-monoclonal antibody is a mouse anti-human HCG antibody marked by acridinium ester of HCG epitope;
in this embodiment, the components and contents of the reagent R1, the reagent R3 and the reagent R4 of the kit 2 are shown in table 2 below:
TABLE 2
The biotin-labeled mouse anti-monoclonal antibody in table 2 is a biotin-labeled mouse anti-human HCG antibody; the acridinium ester marked mouse anti-monoclonal antibody is a mouse anti-human HCG antibody marked by acridinium ester of HCG epitope;
the anti-interference tests of the kit 1 and the kit 2 are shown in the following table 3:
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 30mIU/mL are the best interference-resistant.
When the beta-hcg detection kit of the embodiment is used for detecting hcg, the positive judgment value or the reference interval after the detection result is obtained is shown in the following table 6:
TABLE 6
Due to differences in geography, race, gender, age, etc., it is recommended that each laboratory establish its own reference range.
1. The default result unit for the human chorionic gonadotropin project is used mIU/mL (or IU/L);
2. due to different methodologies or antibody specificity, there may be deviations between the test results of reagents from different manufacturers, and therefore they should not be directly compared with each other to avoid erroneous medical interpretation;
3. when the concentration of HCG + beta in the sample exceeds 8000mIU/mL, the sample can be diluted by using a sample diluent (the recommended dilution multiple is 100 times) and then detected;
4. human chorionic gonadotropin concentrations below the lower limit of detection are reported to be < 0.600mIU/mL, while concentrations above the upper limit of detection are reported to be > 8000 mIU/mL.
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 kit for detecting the beta-human chorionic gonadotropin is characterized by comprising a reagent R1, a reagent R3 and a reagent R4; wherein the reagent R1 has streptavidin-coated magnetic microparticles and Tris buffer, the reagent R3 has acridinium ester-labeled mouse anti-human HCG antibody and phosphate buffer, and the reagent R4 has biotin-labeled mouse anti-human HCG antibody and morpholine ethanesulfonic acid MES;
also comprises a calibration material and a quality control material.
2. The β -human chorionic gonadotropin detection kit according to claim 1, wherein preservative Proclin300 is contained in each of said reagent R1, said reagent R3 and said reagent R4.
3. The beta-hcg assay kit according to claim 2, wherein the calibrator and quality control are hcg recombinant antigens obtained by in vitro recombination in a lyophilized powder state; the preservative Proclin300 is 1 g/L.
4. The detection kit for detecting beta-human chorionic gonadotropin according to any one of claims 1 to 3, wherein in the reagent R1, the streptavidin-coated magnetic particle is 2.5g/L, and the Tris buffer solution is 50 mmol/L; in the reagent R3, the acridinium ester labeled mouse anti-human HCG antibody is 0.15mg/L, and the phosphate buffer solution is 100 mmol/L; in the reagent R4, the biotin-labeled mouse anti-human HCG antibody was 0.78mg/L and the morpholine ethanesulfonic acid MES was 100 mmol/L.
5. The β -human chorionic gonadotropin detection kit according to claim 4, wherein each of said reagent R1, said reagent R3, and said reagent R4 comprises bovine serum albumin BSA, wherein said bovine serum albumin BSA is 10 g/L.
6. The preparation method of the beta-human chorionic gonadotropin detection kit is characterized in that the beta-human chorionic gonadotropin detection kit comprises a reagent R1, 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 HCG antibody aiming at HCG 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 HCG antibody labeled by acridinium ester aiming at HCG epitope;
diluting the concentration of the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope with a diluent;
preparation of reagent R4 described in S3: diluting the concentration of the mouse anti-human HCG antibody with a buffer solution, mixing the diluted concentration with biotin, and labeling with biotin; after the labeling is finished, centrifugal desalting and purifying are carried out to obtain a biotin-labeled mouse anti-human HCG antibody;
diluting the obtained biotin-labeled mouse anti-human HCG antibody 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 beta-human chorionic gonadotropin detection kit also comprises a calibrator and a quality control product.
7. The method of claim 6, wherein in 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 β -human chorionic gonadotropin detection kit according to claim 6, wherein in both of said step S2 and said step S3, centrifugal desalting purification is performed using a Zeba desalting centrifugal column; in the step S2, when labeling is performed, the labeling is performed by reacting at 37 ℃ for 90 minutes; in the step S3, the labeling is carried out by reacting at 37 ℃ for 60 minutes.
9. The method for preparing a kit for detecting beta-hcg according to any one of claims 6 to 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 of preparing a β -hcg assay kit according to any one of claims 6-8, wherein 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 HCG antibody to acridinium ester is 1:10, and the obtained acridinium ester labeled mouse anti-human HCG antibody aiming at HCG epitope is prepared into the concentration of 0.15mg/L by using diluent; in step S3, the mouse anti-human HCG antibody is diluted with a buffer solution to a concentration of 0.78mg/L, the molar ratio of the mouse anti-human HCG antibody to biotin is 1:10, and the obtained biotin-labeled mouse anti-human HCG antibody is diluted with a diluent to a concentration of 0.78 mg/L.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048475A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit |
| CN107796803A (en) * | 2017-11-03 | 2018-03-13 | 太原瑞盛生物科技有限公司 | A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of free β human chorionic gonadotrophins |
| CN109001471A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method |
| CN109061203A (en) * | 2018-08-07 | 2018-12-21 | 泰州泽成生物技术有限公司 | A kind of human chorionic gonadotrophin detection kit and preparation method thereof and application method |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| WO2019184247A1 (en) * | 2018-03-27 | 2019-10-03 | 苏州长光华医生物医学工程有限公司 | Electrochemiluminescence kit for detecting mechano-growth factor and its e peptide and preparation method therefor |
| CN112710858A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | Kit and preparation method and application thereof |
-
2021
- 2021-11-09 CN CN202111319749.9A patent/CN114002444A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048475A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit |
| CN107796803A (en) * | 2017-11-03 | 2018-03-13 | 太原瑞盛生物科技有限公司 | A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of free β human chorionic gonadotrophins |
| WO2019184247A1 (en) * | 2018-03-27 | 2019-10-03 | 苏州长光华医生物医学工程有限公司 | Electrochemiluminescence kit for detecting mechano-growth factor and its e peptide and preparation method therefor |
| CN109001471A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method |
| CN109061203A (en) * | 2018-08-07 | 2018-12-21 | 泰州泽成生物技术有限公司 | A kind of human chorionic gonadotrophin detection kit and preparation method thereof and application method |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| CN112710858A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | Kit and preparation method and application thereof |
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Address after: 210000 No. 2, Qiande Road, Jiangning District, Nanjing, Jiangsu Province Applicant after: LANSION BIOTECHNOLOGY Co.,Ltd. Applicant after: Guangxi Aimin Biotechnology Co.,Ltd. Address before: 210000 No. 2, Qiande Road, Jiangning District, Nanjing, Jiangsu Province Applicant before: LANSION BIOTECHNOLOGY Co.,Ltd. Applicant before: Guangxi Lanyu Biotechnology Co.,Ltd. |
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Application publication date: 20220201 |