CN118067978A - Preparation method of dehydroepiandrosterone sulfate assay kit - Google Patents
Preparation method of dehydroepiandrosterone sulfate assay kit Download PDFInfo
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- CZWCKYRVOZZJNM-USOAJAOKSA-N dehydroepiandrosterone sulfate Chemical compound C1[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 CZWCKYRVOZZJNM-USOAJAOKSA-N 0.000 title claims abstract description 59
- CZWCKYRVOZZJNM-UHFFFAOYSA-N Prasterone sodium sulfate Natural products C1C(OS(O)(=O)=O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CC=C21 CZWCKYRVOZZJNM-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 229950009829 prasterone sulfate Drugs 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000003149 assay kit Methods 0.000 title claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000006249 magnetic particle Substances 0.000 claims abstract description 11
- -1 acridine ester Chemical class 0.000 claims abstract 3
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims abstract 3
- 239000000427 antigen Substances 0.000 claims description 24
- 102000036639 antigens Human genes 0.000 claims description 23
- 108091007433 antigens Proteins 0.000 claims description 23
- 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 description 16
- 239000000243 solution Substances 0.000 claims description 15
- 108010071390 Serum Albumin Proteins 0.000 claims description 13
- 102000007562 Serum Albumin Human genes 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 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 description 10
- 239000011616 biotin Substances 0.000 claims description 8
- 229960002685 biotin Drugs 0.000 claims description 8
- 235000020958 biotin Nutrition 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 7
- 238000002156 mixing Methods 0.000 claims 5
- 238000007865 diluting Methods 0.000 claims 2
- 239000012528 membrane Substances 0.000 claims 2
- 239000008213 purified water Substances 0.000 claims 2
- 238000003756 stirring Methods 0.000 claims 2
- 238000000108 ultra-filtration Methods 0.000 claims 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 2
- 238000011033 desalting Methods 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- 239000011148 porous material Substances 0.000 claims 1
- 239000003761 preservation solution Substances 0.000 claims 1
- 230000002335 preservative effect Effects 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000004907 flux Effects 0.000 abstract 1
- 238000004806 packaging method and process Methods 0.000 abstract 1
- 239000002245 particle Substances 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 abstract 1
- 239000013024 dilution buffer Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000003098 androgen Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000011859 microparticle Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 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
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 230000001919 adrenal effect Effects 0.000 description 2
- 229940030486 androgens Drugs 0.000 description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- FMGSKLZLMKYGDP-UHFFFAOYSA-N Dehydroepiandrosterone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CC=C21 FMGSKLZLMKYGDP-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 208000024447 adrenal gland neoplasm Diseases 0.000 description 1
- 230000001780 adrenocortical effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001548 androgenic effect Effects 0.000 description 1
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 description 1
- 229960005471 androstenedione Drugs 0.000 description 1
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- FMGSKLZLMKYGDP-USOAJAOKSA-N dehydroepiandrosterone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 FMGSKLZLMKYGDP-USOAJAOKSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229960002847 prasterone Drugs 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—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
- G01N33/543—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
- G01N33/74—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
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The patent discloses the technical field related to biological immunity in-vitro diagnosis of medical instruments, in particular to a preparation method of a dehydroepiandrosterone-sulfate assay kit; the method comprises the steps of independently packaging a reagent 1, a reagent 2, a magnetic particle reagent, a calibrator 1 and a calibrator 2, and combining an acridine ester chemiluminescence technology with immunomagnetic particles, so that the method has the advantages of high detection sensitivity, high precision, wide detection range, short reaction time, economy and effectiveness; and can be on full-automatic chemiluminescence appearance simultaneous determination a plurality of samples, realize the high flux of DHEA-S and change the survey fast, dependable performance, sensitivity are high, the linearity scope is wide, can cooperate semi-automatic, full-automatic instrument use.
Description
Technical Field
The invention relates to the technical field related to biological immunity in-vitro diagnosis of medical instruments, in particular to a preparation method of a dehydroepiandrosterone-sulfate assay kit.
Background
Dehydroepiandrosterone sulfate (DHEA-S) is the most abundant adrenal androgen produced by the adrenal cortex and also has the action of a neurosteroid. Dehydroepiandrosterone sulfate is a good indicator reflecting the synthesis of adrenal androgens. Although dehydroepiandrosterone sulfate exhibits only weak androgenic activity, androgens such as testosterone and androstenedione, which are more active, can be metabolically produced. The concentration of dehydroepiandrosterone sulfate in serum decreases with increasing annual growth, and thus can be used as a predictor of progression of severe disease and breast cancer. Elevated levels of dehydroepiandrosterone sulfate can be detected in the plasma of patients with adrenal tumor or congenital adrenocortical hyperplasia. Dehydroepiandrosterone sulfate is also elevated in polycystic ovarian patients. In men, hCG secreting tumors also lead to elevated levels of dehydroepiandrosterone sulfate in the testes, and the currently known methods for measuring dehydroepiandrosterone sulfate antibodies are relatively few, mainly chemiluminescence. But are currently predominantly imported kits. Therefore, the detection kit for dehydroepiandrosterone sulfate has high accuracy, high sensitivity and wide linear range, and can reduce detection cost.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to: the preparation method of the dehydroepiandrosterone-sulfate assay kit has the advantages of high accuracy, strong sensitivity and wide linear range.
In order to achieve the above purpose, the invention adopts the following technical scheme: the direct chemiluminescence technology of magnetic particles is applied, and an indirect method is adopted. Preparing a dehydroepiandrosterone sulfate assay kit, which comprises a reagent 1, a reagent 2, a magnetic particle reagent, a calibrator 1 and a calibrator 2 which are packaged independently; the reagent 1 is diluted to 0.75mg/mL by a dilution buffer solution for DHEA-S biotin antigen, the reagent 2 is diluted to 0.75mg/mL by a dilution buffer solution for DHEA-S acridinium ester labeled antibody, the magnetic particle reagent is diluted to 1mg/mL by a dilution buffer solution for 10mg/mL of chain and a magnetic bead of philic mycin, and the calibrator 1 and the calibrator 2 are prepared by preparing a calibrator from DHEA-S antigen and a 1% -5% animal serum albumin solution and assigning values.
The invention has the following advantages:
1. The indirect sulfuric acid dehydroepiandrosterone assay kit (magnetic particle direct chemiluminescence method) has the advantages that each reagent component comprises a reagent 1, a reagent 2, a magnetic particle reagent, a calibrator 1, a calibrator 2 and each component reagent, the stability is good, the effective period can reach more than 18 months, and the cost is effectively reduced;
2. the detection sensitivity is high, the specificity performance is good, and the linear range is wide;
3. In the invention, a perfect and unified process is obtained through process optimization of a large number of experiments, and production is carried out strictly according to standard production operation rules and quality control rules;
4. The user can obtain reliable results only by carrying out standard operation according to the operation instruction, and the operation is simple and convenient.
Drawings
FIG. 1 is a main graph of example 1;
FIG. 2 is a main graph of example 2;
Fig. 3 is a main graph of example 3.
Detailed Description
Example 1:
a method for preparing a dehydroepiandrosterone-sulfate antibody assay kit comprises the following steps:
1.1, preparation of a calibrator 1:
The DHEA-S antigen is prepared into a calibrator by using 1 to 5 percent bovine serum albumin solution, and the calibrator is assigned to prepare the DHEA-S antigen with the concentration of 50 mug/dL.
1.2, Preparation of a calibrator 2:
the DHEA-S antigen is prepared into a calibrator by using 1 to 5 percent bovine serum albumin solution, and the calibrator is assigned to prepare the DHEA-S antigen with the concentration of 500 mug/dL.
1.3 Preparation of reagent 1
The DHEA-S biotin antigen was diluted to 0.75mg/mL with dilution buffer.
1.4 Preparation of reagent 2
DHEA-S acridinium ester-labeled antibody was diluted to 0.75mg/mL with dilution buffer.
1.5 Preparation of magnetic microparticle reagents
10Mg/mL of the chain and the magnetic beads of the streptavidin were diluted to 1mg/mL with a dilution buffer.
1.6, Assembling:
assembling the reagents into a box, and storing at 2-8 ℃;
1.7, major reagents and materials:
The dehydroepiandrosterone sulfate antigen and the dehydroepiandrosterone sulfate antibody are preserved by the inventor, bovine serum albumin, PB buffer, biotin, tris, sodium chloride, acridinium ester, tween20 and Proclin300 are all Sigma company products, sodium chloride is a national pharmaceutical group chemical reagent company product, HCl is a Guangzhou chemical reagent plant product, and all the used chemical reagents are analytically pure.
Example 2
A method for preparing a dehydroepiandrosterone-sulfate antibody assay kit comprises the following steps:
2.1, preparation of a calibrator 1:
The DHEA-S antigen was formulated as a calibrator with 1% to 5% horse serum albumin solution at 50 μg/dL, respectively.
2.2, Preparation of a calibrator 2:
the DHEA-S antigen was formulated as a calibrator with 1% to 5% horse serum albumin solution at 500 μg/dL, respectively.
2.3 Preparation of reagent 1
The DHEA-S biotin antigen was diluted to 0.75mg/mL with dilution buffer.
2.4 Preparation of reagent 2
DHEA-S acridinium ester-labeled antibody was diluted to 0.75mg/mL with dilution buffer.
2.5 Preparation of magnetic microparticle reagents
10Mg/mL of the chain and the magnetic beads of the streptavidin were diluted to 1mg/mL with a dilution buffer.
2.6, Assembling: assembling the reagents into a box, and storing at 2-8 ℃;
2.7, major reagents and materials:
The dehydroepiandrosterone sulfate antigen and the dehydroepiandrosterone sulfate antibody are preserved by the inventor, horse serum albumin, biotin, tris, acridinium ester, triton-X100 and sodium azide are all Sigma company products, sodium chloride is a national pharmaceutical group chemical reagent company product, HCl is a Guangzhou chemical reagent factory product, and all chemical reagents are analytically pure.
Example 3
A method for preparing a dehydroepiandrosterone-sulfate antibody assay kit comprises the following steps:
3.1, preparation of a calibrator 1:
the DHEA-S antigen is prepared into a calibrator by using a goat serum albumin solution with the concentration of 1% -5%, and assigned to 50 mug/dL.
3.2, Preparing a calibrator 2:
The DHEA-S antigen is prepared into a calibrator by using a 1% -5% goat serum albumin solution, and assigned to prepare the calibrator with the concentration of 500 mug/dL.
3.3 Preparation of reagent 1
The DHEA-S biotin antigen was diluted to 0.75mg/mL with dilution buffer.
3.4 Preparation of reagent 2
DHEA-S acridinium ester-labeled antibody was diluted to 0.75mg/mL with dilution buffer.
3.5 Preparation of magnetic microparticle reagents
10Mg/mL of the chain and the magnetic beads of the streptavidin were diluted to 1mg/mL with a dilution buffer.
3.6, Assembling:
assembling the reagents into a box, and storing at 2-8 ℃;
3.7, major reagents and materials:
the dehydroepiandrosterone sulfate antigen and the dehydroepiandrosterone sulfate antibody are preserved by the inventor, goat serum albumin, biotin, HEPES buffer, acridinium ester, CTAC and Krovin300N are all Sigma company products, sodium chloride is a national pharmaceutical group chemical reagent company product, HCl is a Guangzhou chemical reagent factory product, and all the used chemical reagents are domestic analytical pure.
To further illustrate examples 1-3, further details are as follows: the bovine serum albumin, the horse serum albumin and the goat serum albumin can be summarized as animal serum albumin; the Proclin300, the sodium azide, the Krovin N can be summarized as preservatives; the PB buffer, tris buffer and HEPES buffer can be summarized as a buffer; the Tween20, the Triton-X100, and the CTAC can be summarized as surfactants.
Example 4
The test method of the dehydroepiandrosterone-sulfate antibody assay kit prepared in further specific examples 1 to 3 comprises the following steps:
4.1 reagent treatment
4.1.1, Loading the dehydroepiandrosterone-sulfate antibody assay kit reagent into a full-automatic chemiluminescence reagent instrument manufactured by Shenzhen huge east biomedical engineering Co., ltd.
4.1.2 Determining the name of the reagent of the dehydroepiandrosterone-sulfate-loaded antibody assay kit on a software operating system, loading a test sample on a sample bin, determining a sample test item on the software operating system, and filling the reaction cup.
4.1.3, The reagents should be gently mixed before loading, but the opened reagents are forbidden to be turned upside down; each time the sample size needs to be 10 mu L, the dead volume factor of the detection system is considered, the sample size should be ensured to be more than 200 mu L, and the dead cavity factor of the sample container should be considered.
4.2, Scaling
4.2.1, The kit of each batch of reagent is provided with a bar code for recording the specific calibration information of each batch of reagent and a predetermined main curve.
4.2.2, Scaling interval: kits of different lot numbers must be rescaled. In addition, the following cases must be rescaled: scaling is performed once every kit is replaced; the same kit is used on the tester for more than 28 days; if necessary, the quality control data exceeds the rated limit value.
4.3 Calculation of
The instrument automatically calculates the measured concentration in μg/dL for each sample.
4.4, Detection result:
4.4.1, detection curve:
And drawing a curve by using a four-parameter equation, namely a detection curve. When the linear range of the kit is 3-1500 mug/dL, the drawing is shown.
4.4.2 Sensitivity test: repeating the test for 20 times on the zero calibration point, calculating the average value (M) and Standard Deviation (SD) of 20 times of measured values, and taking M+2SD into a curve, wherein the obtained concentration value is the sensitivity of the kit. The sensitivity of the detection kit of the three embodiments is less than or equal to 1.8 mug/dL.
4.4.3, Accuracy test: accuracy references of (50.+ -.10) μg/dL and (500.+ -.100) μg/dL were measured, with the relative deviations in the three batch examples being within.+ -. 10% of each other. See tables 1-3 for details:
Table 1, accuracy test table for example 1
Table 2, example 2 accuracy test table
Table 3, example 3 accuracy test table
4.5 Principle of inspection:
The product adopts a magnetic particle direct chemiluminescence technology and adopts a competition method. In a first step, the sample, biotinylated dehydroepiandrosterone sulfate antigen and acridinium ester-labeled dehydroepiandrosterone sulfate antibody are reacted to form an antibody-antigen complex. In the second step, streptavidin coated magnetic particles are added to form a solid phase. After washing under the action of a magnetic field, adding a pre-excitation solution and an excitation solution, and measuring the luminescence value of a chemiluminescent reaction, wherein the luminescence intensity of the luminescent value is inversely related to the concentration of dehydroepiandrosterone sulfate in a sample.
From the analysis, the dehydroepiandrosterone-sulfate antibody assay kit prepared by the invention can ensure the storage life under the storage condition of 2-8 ℃, and improves the actual utilization rate of the product; in addition, the linear range of the kit can reach 3-1500 mug/dL, and experimental results show that the sensitivity of the detection kit is less than or equal to 1.8 mug/dL, and the relative deviation is within a range of +/-10%, so the dehydroepiandrosterone-sulfate antibody detection kit has the characteristics of high accuracy, strong sensitivity and wide linear range.
Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. Therefore, the invention is not to be limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (5)
1.A preparation method of a dehydroepiandrosterone-sulfate assay kit is characterized by comprising the following steps of: the magnetic particle calibrator comprises a reagent 1, a reagent 2, a magnetic particle reagent, a calibrator 1 and a calibrator 2 which are packaged independently, wherein the concentration of the reagent 1 is 0.05-1 mug/mL, the concentration of the reagent 2 is 0.05-1 mug/mL, the concentration of the calibrator 1 is 50 mug/dL, and the concentration of the calibrator 2 is 500 mug/dL.
2. The method for preparing the dehydroepiandrosterone-sulfate assay kit according to claim 1, which is characterized in that: the preparation method of the reagent 1 comprises the following steps:
s1, taking 1mg of DHEA-S antigen;
S2, preparing buffer solution (pH 7.0-8.5);
S3, preparing solutions of the antigens in the step 1 and the antigens in the step 2 according to the volume ratio of 1:1, mixing uniformly, and ultrafiltering by an ultrafiltration membrane at 4 ℃ or room temperature;
S4, adding 0.05mg of biotin into the antigen subjected to ultrafiltration in the step 3, adding the buffer solution to enable the final concentration of the antigen to be 1mg/mL, and uniformly mixing for 5min. Placing the mixed solution in a constant temperature cabinet at 24-26 ℃, standing for reaction for 110-130 min, and dialyzing the biotinylated antibody for 16-24 h at 4 ℃ or room temperature by using a buffer solution.
3. The method for preparing the dehydroepiandrosterone-sulfate assay kit according to claim 1, which is characterized in that: the preparation method of the reagent 2 comprises the following steps:
A1, weighing 6.06gTris g and 9.0g of sodium chloride at room temperature, adding purified water and 3.8mL of concentrated HCl, and stirring until the materials are completely dissolved;
a2, adding 1.0mL of surfactant and 5mL of preservative into the completely dissolved liquid in the step 6, finally slowly adding 10.0g of animal serum albumin, stirring until the animal serum albumin is completely dissolved, adjusting the pH value to 7.1-7.3, and fixing the volume of purified water to 1L.
4. The method for preparing the dehydroepiandrosterone-sulfate assay kit according to claim 1, which is characterized in that: the preparation method of the magnetic particle reagent comprises the following steps:
(1) Taking 1mLDHEA-S antibody;
(2) Preparing a buffer solution (pH 7.0-8.5), and mixing the buffer solution (pH 7.0-8.5) prepared in the step 1 according to a volume ratio of 1: ultrafiltering with ultrafilter membrane of pore size smaller than 50um at room temperature or 2-8deg.C;
(3) Diluting the ultrafiltered antibody to 1mg/mL by using a buffer solution, taking 0.1mg of acridine ester, rapidly mixing the antibody and the acridine ester, adding the buffer solution to ensure that the final concentration of the antibody is 0.5mg/mL, and uniformly mixing for 5min;
(4) Placing the solution in a constant temperature box at 24-26 ℃, standing for reaction for 110-130 min, and performing light-shielding operation in the whole reaction process;
(5) Purifying by using a desalting column, diluting the purified acridinium ester labeled antibody to 350ng/mL by using DHEA-S acridinium ester labeled preservation solution, and preserving at 2-8 ℃ for later use.
5. The method for preparing the dehydroepiandrosterone-sulfate assay kit according to claim 1, which is characterized in that: the components of the calibrator 1 and the calibrator 2 have the same concentration and different concentrations, and are prepared by preparing a calibrator from DHEA-S antigen and 1% -5% of animal serum albumin solution and assigning values.
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