CN113671175A - Reagent and kit for detecting thromboxane B2 - Google Patents
Reagent and kit for detecting thromboxane B2 Download PDFInfo
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 61
- XNRNNGPBEPRNAR-JQBLCGNGSA-N thromboxane B2 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1OC(O)C[C@H](O)[C@@H]1C\C=C/CCCC(O)=O XNRNNGPBEPRNAR-JQBLCGNGSA-N 0.000 title claims abstract description 37
- XNRNNGPBEPRNAR-UHFFFAOYSA-N Thromboxane B2 Natural products CCCCCC(O)C=CC1OC(O)CC(O)C1CC=CCCCC(O)=O XNRNNGPBEPRNAR-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 239000000243 solution Substances 0.000 claims abstract description 40
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 230000001681 protective effect Effects 0.000 claims abstract description 22
- 239000011324 bead Substances 0.000 claims abstract description 15
- 102000008186 Collagen Human genes 0.000 claims abstract description 13
- 108010035532 Collagen Proteins 0.000 claims abstract description 13
- 229920001436 collagen Polymers 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 9
- 239000011592 zinc chloride Substances 0.000 claims description 8
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical group Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 4
- VSMDINRNYYEDRN-UHFFFAOYSA-N 4-iodophenol Chemical compound OC1=CC=C(I)C=C1 VSMDINRNYYEDRN-UHFFFAOYSA-N 0.000 claims description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 3
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 2
- 238000003860 storage Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000002035 prolonged effect Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 15
- 238000005406 washing Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- RZWIIPASKMUIAC-VQTJNVASSA-N thromboxane Chemical compound CCCCCCCC[C@H]1OCCC[C@@H]1CCCCCCC RZWIIPASKMUIAC-VQTJNVASSA-N 0.000 description 5
- DSNBHJFQCNUKMA-SCKDECHMSA-N thromboxane A2 Chemical compound OC(=O)CCC\C=C/C[C@@H]1[C@@H](/C=C/[C@@H](O)CCCCC)O[C@@H]2O[C@H]1C2 DSNBHJFQCNUKMA-SCKDECHMSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 101000862089 Clarkia lewisii Glucose-6-phosphate isomerase, cytosolic 1A Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 210000001772 blood platelet Anatomy 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- KAQKFAOMNZTLHT-OZUDYXHBSA-N prostaglandin I2 Chemical compound O1\C(=C/CCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-OZUDYXHBSA-N 0.000 description 3
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 3
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 206010047139 Vasoconstriction Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 208000029078 coronary artery disease Diseases 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000025033 vasoconstriction Effects 0.000 description 2
- -1 1-ethyl- (3-dimethylaminopropyl) Chemical group 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 206010014824 Endotoxic shock Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
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- 238000003149 assay kit Methods 0.000 description 1
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- 201000011510 cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- HNGDOSBFYRVIEY-UHFFFAOYSA-N ethanesulfonic acid;hydrate Chemical compound O.CCS(O)(=O)=O HNGDOSBFYRVIEY-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing 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
- G01N33/543—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
- G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application belongs to the technical field of in-vitro diagnostic reagents, and particularly discloses a reagent and a kit for detecting thromboxane B2. The reagent for detecting the thromboxane B2 comprises a thromboxane B2 antibody coupled with magnetic beads and a protective solution, wherein the protective solution comprises 0.08-0.13% (w/v) of porcine collagen, 0.8-1.3% (v/v) of glycerol and a buffer solution with the pH value of 7.5-8.0. The application has at least one of the following beneficial effects: according to the reagent for detecting the thromboxane B2, the protection solution is added, so that the reagent is prevented from being oxidized, the stability of the reagent is improved, and the storage time of the reagent is prolonged.
Description
Technical Field
The application belongs to the technical field of in-vitro diagnostic reagents, and more particularly relates to a reagent and a kit for detecting thromboxane B2.
Background
Thromboxane B2: thromboxane (TXA 2) is one of prostaglandins, produced by platelets, and has platelet aggregation and vasoconstriction effects, in contrast to prostaglandin, which are dynamically balanced to maintain vasoconstriction and platelet aggregation. The biological half-life of TXA2 is only 30s, and it is rapidly converted into inactive thromboxane B2 (TXB 2). The level change of the thromboxane B2 is seen in the increase of atherosclerosis, angina pectoris, coronary heart disease, diabetes, hyperlipidemia and the like.
An elevated TXA2/PGI2 ratio is prone to platelet aggregation, thrombosis, and contributes to atherosclerosis and coronary heart disease. The plasma of animals with hemorrhage, injury and endotoxic shock has a significant increase in TXB2, which is associated with increased resistance to pulmonary circulation during shock. TXA2 in artery tissue of malignant tumor patient is changed, and PGI2 and TXA2 can prevent tumor cells from invading blood platelets and adhering to the surface of blood vessels when normal. The factors for inhibiting the generation of the platelet TXA2 and increasing the generation of the vascular endothelial cell PGI2 have the function of resisting tumor metastasis. Therefore, the detection of the content of the thromboxane B2 has important significance for clinical judgment.
Disclosure of Invention
At present, when a kit containing magnetic beads is used for detecting thromboxane, detection results are often inaccurate due to turbidity, precipitation and the like of detection reagents in the kit. Therefore, in order to improve the accuracy of the thromboxane B2, the application provides a reagent for detecting the thromboxane B2, the reagent can be stored for more than 6 months, and the detection result is more accurate.
The application is realized by the following scheme:
the application provides a reagent for detecting thromboxane B2, which comprises a thromboxane B2 antibody coupled with magnetic beads and a protective solution, wherein the protective solution comprises 0.08-0.13% (w/v) of porcine collagen, 0.8-1.3% (v/v) of glycerol and a buffer solution with the pH value of 7.5-8.0. The protection solution which is mainly composed of the pig collagen and the glycerol can prevent the thromboxane coupled with the magnetic beads from being oxidized and deteriorated, forming precipitates and the like, and prolong the shelf life of the thromboxane coupled with the magnetic beads under the condition of normal-temperature storage.
In one embodiment of the present application, the protective solution further comprises Mg2+And Zn2+. The addition of metal ions makes the thromboxane coupled with magnetic beads more stable.
In one embodiment of the present application, the buffer is Tris-HCl.
In one embodiment of the present application, the protective solution further comprises 0.08-0.12mol/L MgCl2And 0.08-0.12mol/L ZnCl2。
In one embodiment of the present application, the protective solution comprises 0.1mol/L MgCl2，0.1mol/L ZnCl21% (w/v) porcine collagen and 1% (v/v) glycerol.
In one embodiment of the present application, the concentration of the magnetic bead-coupled thromboxane B2 antibody is 0.3 to 0.5 mg/ml. For example, the concentration of the magnetic bead-coupled thromboxane B2 antibody is 0.3mg/ml, 0.4mg/ml or 0.5 mg/ml.
In one embodiment of the present application, the concentration of the magnetic bead-coupled thromboxane B2 antibody is 0.4 mg/ml.
In one embodiment of the present application, the reagent further comprises a chemiluminescent solution.
In one embodiment of the present application, the chemiluminescent solution comprises luminol, p-iodophenol and hydrogen peroxide.
In one embodiment of the present application, the reagent further comprises horseradish peroxidase-labeled thromboxane B2 antigen.
In one embodiment of the present application, the reagent further comprises a concentrated washing solution for washing the magnetic beads, wherein the concentrated washing solution comprises phosphate buffer and tween 20.
In another aspect, the present application provides a kit prepared by including the above reagent.
The reagent for detecting the thromboxane B2 provided by the application has at least one of the following beneficial effects:
according to the reagent for detecting the thromboxane B2, the protection solution is added, so that the reagent is prevented from being oxidized, the stability of the reagent is improved, and the storage time of the reagent is prolonged.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The technical solutions of the present application will be described clearly and completely in conjunction with the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Porcine collagen from the following reagents was purchased from bio-technologies ltd, beijing bolmey, model Prionex (10%); Tris-HCl (0.01M) pH 7.8; MgCl2The concentration of (A) is 1 mol/L; ZnCl2The concentration of (2) is 1 mol/L.
Example 1 reagent for detecting thromboxane B2
The specific preparation process of the reagent for detecting thromboxane B2 is as follows:
0.1ml of magnetic beads (10 mg/ml, MS160, Beijing Boelmi Biotechnology Co., Ltd.) was added with 0.2ml of MES (2- (N-morpholine) ethanesulfonic acid monohydrate, 50mM, pH 6.0) and washed, and the supernatant was removed; adding NHS (N-hydroxysuccinimide, 20 mg/mL) 15ul, mixing, adding EDC (1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, 20 mg/mL) 15ul, mixing, incubating at room temperature for 30min, removing residual reagent, adding 0.2mL PBST buffer (0.02M PB +1% TW 20) for washing, adding 0.1mg of thromboxane B2 antibody, mixing rapidly, and coupling overnight. Before blocking, the mixture is subjected to ultrasonic oscillation for 2min, then 20% BSA solution is added, and the mixture is incubated at room temperature for 1h for blocking. The supernatant was removed by a magnetic stand, and 250ul of protective solution was added after PBST buffer washing to obtain a reagent for detecting thromboxane B2, which was diluted at a ratio of 1:20 when used in this example.
The following examples were prepared using 100ml of the protective solution, and the specific components and contents of the protective solution in each case are shown in Table 1.
TABLE 1 specific ingredients and contents of protective solutions
In 100ml of the protective solution, the protective solution is dissolved,
note "-" indicates no inclusion.
The stability of the reagents was observed during the time when the reagents of the above cases 1 to 8 and the reagents of the comparative examples 1 to 2 were left at room temperature for 10 months, and the experimental results are shown in the following table 2:
table 2 reagent stability results
As can be seen from Table 1, the agent can also be stored for more than 6 months when the protective solution contains only porcine collagen and glycerol (cases 7 and 8); when the porcine collagen content is less than 0.08% (comparative example 1), a small amount of precipitate is formed after preparation, and when the porcine collagen content is more than 0.15% (comparative example 2), the reagent is precipitated after being left for 1 month, which is not favorable for clinical application.
When the protective solution is 0.08% (w/v) of porcine collagen, 0.8% (v/v) of glycerol, 0.01M Tris-HCl (pH7.8), 0.08mol/L MgCl2And 0.08mol/L ZnCl2In case 1, precipitation occurred at 8 months, while in other cases no precipitation occurred at 9 months, and thus, when the components of the protective solution were 0.08-0.13% (w/v) porcine collagen, 0.8-1.3% (v/v) glycerol, 0.01M Tris-HCl (pH 7.8), 0.08-0.12mol/L MgCl2And 0.08-0.12mol/L ZnCl2When the prepared reagent can be preserved for more than 8 months at room temperature, and when the components of the protective solution are 0.1% (w/v) of porcine collagen, 1% (v/v) of glycerol, 0.01M Tris-HCl and 0.1mol/L MgCl2And 0.1mol/L ZnCl2In addition, the prepared reagent is not precipitated after being stored for 10 months at room temperature, and the storage time is longest.
EXAMPLE 2 kit
The reagent prepared in example 1 can be applied to the detection of thromboxane B2 (TXB 2) by a detection method including a competition method, a sandwich method and the like. In this embodiment, the kit suitable for detection by the competitive method is exemplified, and the kit specifically includes the following components and preparation processes:
(1) horseradish peroxidase (HRP) labeled antigen
HRP activation: weighing 1mg of horseradish peroxidase, adding 250ul of triple distilled water, mixing well, adding 51ul of sodium periodate (0.1M), and reacting at 4 ℃ in a dark place for 30 min. After further addition of 1.5mL of thromboxane B2 antigen (1 mg/mL) and 30ul of DMSO (50 mM), the reaction was carried out at 37 ℃ for 10 min. And (4) desalting by using a G-50 column to obtain the HRP labeled antigen. When in use, the enzyme diluent is used for diluting 1:1000 for standby.
(2) Luminescent substrate A liquid
0.4g of luminol, 0.075g N- (4-aminobutyl) -N-ethyliisoluminol, 0.06g of p-iodophenol and 0.035g of phenolphthalein were weighed out and made up to 100ml with borax-borate (7.8).
(3) Luminescent substrate B liquid
3ml of hydrogen peroxide was taken and made up to 100ml with 0.02M PB, pH7.4.
(4) Preparation of concentrated washing liquid
In use, the concentrated washing solution is diluted 10 times.
(5) Preparation of quality control product thromboxane B2
Preparing a diluent of a calibrator
The quality control product thromboxane B2 solution is prepared by the diluent, and the concentration is 0 (not containing the quality control product thromboxane B2), 11.1, 33.3, 100, 300 or 900pg/ml respectively.
The reagent prepared in the example 1, the horseradish peroxidase labeled antigen, the luminescent substrate A solution, the luminescent substrate B solution, the concentrated washing solution and the quality control product form a kit.
The detection method and detection range of the kit will be described below by taking the reagent preparation kit prepared in case 2 as an example.
The detection method comprises the following steps:
standard curve: sucking 50ul of the quality control product thromboxane B2 with different concentrations, respectively adding 50ul of the diluted reagent in case 2, and incubating at 37 ℃ for 10 min; then adding 50ul of diluted HRP labeled antigen, incubating and reacting for 10min at 37 ℃, and repeatedly washing magnetic beads by using diluted concentrated washing liquor after magnetic separation.
And (3) a light emitting process: 50ul of luminescent substrate A solution and 50ul of luminescent substrate B solution are respectively added into the washed magnetic beads, and a luminescence value is measured by a full-automatic chemiluminescence immunoassay analyzer.
When the sample is detected, the quality control product is replaced by the sample to be detected.
Detection range:
(1) the absolute value of the correlation coefficient (r) of the dose-response curve in the linear range of 0-900pg/ml should not be less than 0.9900;
(2) the repeatability should not be higher than 15%;
(3) accuracy: the recovery rate is 85-110%;
(4) specificity:
(5) cutoff value: greater than 35.0pg/ml
(6) Stability: the result of 7-day acceleration at 37 ℃ meets the above standard.
The test kits prepared from the reagents prepared in cases 2 and 7 were used to test 108 parts of clinically confirmed patient serum and 60 parts of normal serum, and the test results are shown in table 3 using a plate chemiluminescence test kit (beijing taige technologies and biology ltd., kyo mechanical standard 20172401095) as a control.
TABLE 3 test results
As can be seen from table 3, the detection results of the test samples prepared by using the reagents in cases 2 and 7 were all above 90%. The detection result of the kit prepared by the reagent in case 2 has no significant difference with the result detected by the contrast; the detection result of the kit prepared by the reagent in case 7 is obviously higher than that of the control group, and the kit is more in line with the actual result.
In summary, the reagent for detecting thromboxane B2 provided by the application not only can prevent the reagent from being oxidized, improve the stability of the reagent and prolong the storage time of the reagent, but also has more accurate detection result.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A reagent for detecting thromboxane B2 is characterized by comprising a thromboxane B2 antibody coupled with magnetic beads and a protective solution, wherein the protective solution comprises 0.08-0.13% (w/v) of porcine collagen, 0.8-1.3% (v/v) of glycerol and a buffer solution with the pH value of 7.5-8.0.
2. The reagent of claim 1, wherein the protective solution further comprises Mg2+And Zn2+。
3. The reagent of claim 1, wherein the protective solution comprises 0.08-0.12mol/L MgCl2And 0.08-0.12mol/L ZnCl2。
4. The reagent according to claim 1, wherein the buffer is Tris-HCl.
5. The reagent of claim 1, wherein the protective solution comprises 0.1mol/L MgCl2，0.1mol/L ZnCl20.1% (w/v) porcine collagen and 1% (v/v) glycerol.
6. The reagent according to claim 1, wherein the concentration of the thromboxane B2 antibody having magnetic beads coupled thereto is 0.3 to 0.5 mg/ml.
7. The reagent of any one of claims 1 to 6, wherein the reagent further comprises a chemiluminescent solution.
8. The reagent of claim 7, wherein the chemiluminescent solution comprises luminol, p-iodophenol and hydrogen peroxide.
9. The reagent of any one of claims 1 to 6, wherein the reagent further comprises horseradish peroxidase-labeled thromboxane B2 antigen.
10. A kit comprising the reagent of any one of claims 1 to 9.
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Citations (12)
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