CN116519946A - C-reactive protein assay kit with stable bottle opening performance and detection method - Google Patents
C-reactive protein assay kit with stable bottle opening performance and detection method Download PDFInfo
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- CN116519946A CN116519946A CN202310418466.2A CN202310418466A CN116519946A CN 116519946 A CN116519946 A CN 116519946A CN 202310418466 A CN202310418466 A CN 202310418466A CN 116519946 A CN116519946 A CN 116519946A
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- 102100032752 C-reactive protein Human genes 0.000 title claims abstract description 88
- 108010074051 C-Reactive Protein Proteins 0.000 title claims abstract description 26
- 238000002731 protein assay Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 68
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 60
- 230000000295 complement effect Effects 0.000 claims description 47
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 42
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 28
- 239000000427 antigen Substances 0.000 claims description 27
- 102000036639 antigens Human genes 0.000 claims description 27
- 108091007433 antigens Proteins 0.000 claims description 27
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 27
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 27
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 27
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 27
- 238000002835 absorbance Methods 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 21
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 17
- 229910052708 sodium Inorganic materials 0.000 claims description 17
- 239000011734 sodium Substances 0.000 claims description 17
- 239000003085 diluting agent Substances 0.000 claims description 16
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 239000008213 purified water Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000002202 Polyethylene glycol Substances 0.000 claims description 14
- 229920001223 polyethylene glycol Polymers 0.000 claims description 14
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 claims description 12
- 229960002303 citric acid monohydrate Drugs 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 10
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 10
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 9
- 241001494479 Pecora Species 0.000 claims description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 9
- 239000008139 complexing agent Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 6
- 229960004106 citric acid Drugs 0.000 claims description 5
- 108010052164 Sodium Channels Proteins 0.000 claims description 4
- 102000018674 Sodium Channels Human genes 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 235000015424 sodium Nutrition 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000012895 dilution Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000003814 drug Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000004816 latex Substances 0.000 description 4
- 229920000126 latex Polymers 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000004879 turbidimetry Methods 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 208000035868 Vascular inflammations Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 description 1
- 229960005294 triamcinolone Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of immunonephelometry in-vitro diagnosis, in particular to a C-reactive protein assay kit with stable bottle opening performance and a detection method.
Description
Technical Field
The invention relates to the technical field of immunonephelometry in-vitro diagnosis, in particular to a C-reactive protein assay kit with stable bottle opening performance and a detection method.
Background
In clinic, an index in blood detection is C-reactive protein, CRP for short, and cardiovascular related conditions are hypertension patients and diabetes patients in general, and whether inflammation occurs in vivo or not, the degree of elevation and the severity of the inflammation can be predicted by a C-reactive protein test. If the C-reactive protein is obviously increased, vascular inflammation may exist, atherosclerosis is easy to occur, coronary heart disease and cerebrovascular disease are easy to occur, most of early immunoassay techniques analyze the presence and the content of specific proteins in a sample to be detected through the formation of precipitates, the occurrence of agglutination and hemolysis phenomena and determination of light scattering caused by aggregates, but the methods have complicated operation and poor accuracy.
The immunoturbidimetry is an antigen-antibody binding dynamic assay method which is based on the principle that: when the antigen and the antibody react in a special dilution system and the proportion is proper, the formed soluble immune complex is separated out from a liquid phase under the action of a polymerization promoter in the dilution system to form particles, and the reaction liquid is turbid. When the concentration of the antibody is fixed, the amount of the immunocomplex formed increases with an increase in the amount of the antigen in the sample, and the turbidity of the reaction solution increases.
Most of the kits on the market at present, such as a latex-enhanced turbidimetric immunoassay reagent, a kit and application thereof, have the patent number of CN 201310079651.X; the second one is a kit for measuring latex immune turbidimetry and its application, the patent number is CN 202010528691.8, the third one is a kit for enhancing latex sensitivity CRP, the kit is CN 202110864091.3, the kit base is sensitization latex immune turbidimetry, and the antibodies are easy to precipitate in the preservation process, so that the antibody performance is poor, the repeatability is poor, the variation coefficient is large, and the reagent detection result is inaccurate.
Disclosure of Invention
The invention aims to provide a C-reactive protein assay kit with stable bottle opening performance and a detection method, and aims to solve the problem that the existing immunonephelometry assay kit is poor in detection precision.
To achieve the above object, in a first aspect, the present invention provides a C-reactive protein assay kit with stable performance in opening bottles, comprising a first reagent, a second reagent and a standard diluent for complement CRP antigen.
Wherein the first reagent, the second reagent and the standard substance diluent for complement CRP antigen are all preserved at the temperature of 2-8 ℃.
In a second aspect, the invention also provides a method for detecting the C-reactive protein assay kit with stable bottle opening performance, which comprises the following steps:
adding the first reagent into a sample to be detected, uniformly mixing, incubating at 37 ℃ to obtain a first mixed solution, and reading absorbance A1 of the first mixed solution at a wavelength of 340 nm;
adding the second reagent into the first mixed solution, uniformly mixing, incubating at 37 ℃ to obtain a second mixed solution, and reading absorbance A2 of the second mixed solution at a wavelength of 340 nm;
obtaining the difference value between the absorbance A2 and the absorbance A1 to obtain an absorbance difference;
fitting a standard curve on a full-automatic biochemical analyzer by using the standard substance diluent for the complement CRP antigen, and calculating the content of complement CRP in the sample to be detected based on the absorbance difference.
Wherein, the volume ratio of the sample to be tested to the first reagent is 17:300, the volume ratio of the second reagent to the first reagent is 1:4.
in a third aspect, the invention also provides a preparation method of the C-reactive protein assay kit with stable bottle opening performance, which comprises the following steps:
obtaining polyethylene glycol, sodium azide, tween, purified water, disodium hydrogen phosphate and sodium dihydrogen phosphate, uniformly mixing at 25+/-3 ℃ and filtering to obtain a first reagent;
obtaining water-soluble sodium channel, triton, trehalose, glycerol, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium azide, citric acid, CRP sheep anti-human antibody and purified water, uniformly mixing at 25+/-3 ℃ and filtering to obtain a second reagent;
obtaining citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, disodium hydrogen phosphate, sodium dihydrogen phosphate, polyvinylpyrrolidone, sodium complexing agent and purified water, and diluting the citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, sodium complexing agent and purified water into complement CRP calibrator with different concentrations by using a calibrator to obtain a standard diluent for complement CRP antigen;
and packaging the first reagent, the second reagent and the complement CRP antigen with a standard substance diluent to obtain a kit.
According to the C-reactive protein determination kit and the detection method, the sample to be detected is mixed with the first reagent to obtain absorbance, the mixed solution is mixed with the second reagent again to obtain absorbance, the difference of the absorbance is obtained again, and finally the standard curve is fitted on a full-automatic biochemical instrument through a built-in curve fitting mode by utilizing the standard substance diluent for the complement CRP antigen, so that the content of the complement CRP in the sample to be detected is calculated according to the absorbance difference.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a C-reactive protein assay kit with stable bottle opening performance.
FIG. 2 is a flow chart of a method for detecting a C-reactive protein assay kit with stable bottle opening performance.
FIG. 3 is a flow chart of a method for preparing a C-reactive protein assay kit with stable opening performance.
In the figure: 1-first reagent, 2-second reagent, 3-complement CRP antigen with standard dilution.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Referring to fig. 1, in a first aspect, the present invention provides a C-reactive protein assay kit with stable performance in opening bottles, which comprises a first reagent 1, a second reagent 2 and a standard diluent 3 for complement CRP antigen.
In this embodiment, the first reagent 1, the second reagent 2 and the standard substance dilution 3 for complement CRP antigen are all stored at 2-8 ℃, the standard substance dilution 3 for complement CRP antigen (s1=0 mg/L; s1=9.8 mg/L; s2=28.1 mg/L; s3=56.2 mg/L; s1=115.2 mg/L) is obtained by mixing the sample to be tested with the first reagent 1, the mixed solution is mixed again with the second reagent 2 to obtain absorbance again, and the difference of the absorbance is obtained for two times, finally, the standard curve is fitted on a fully automatic biochemical analyzer by using the standard substance dilution 3 for complement CRP antigen, and the content of complement CRP in the sample to be tested is calculated according to the difference of absorbance.
Referring to fig. 2, in a second aspect, the present invention further provides a method for detecting a C-reactive protein assay kit with stable bottle opening performance, comprising the following steps:
s01, adding the first reagent 1 into a sample to be detected, uniformly mixing, incubating at 37 ℃ to obtain a first mixed solution, and reading absorbance A1 of the first mixed solution at 340nm wavelength;
specifically, the volume ratio of the sample to be tested to the first reagent 1 is 17:300.
s02, adding the second reagent 2 into the first mixed solution, uniformly mixing, incubating at 37 ℃ to obtain a second mixed solution, and reading absorbance A2 of the second mixed solution at a wavelength of 340 nm;
specifically, the volume ratio of the second reagent 2 to the first reagent 1 is 1:4.
s03, obtaining a difference value between the absorbance A2 and the absorbance A1 to obtain an absorbance difference;
specifically, the difference Δa in absorbance, Δa=a2-A1, is obtained.
S04, fitting a standard curve on a full-automatic biochemical analyzer by using the standard substance diluent (3) for the complement CRP antigen, and calculating the content of the complement CRP in the sample to be detected based on the absorbance difference.
Referring to fig. 3, in a third aspect, the present invention further provides a preparation method of a C-reactive protein assay kit with stable bottle opening performance, comprising the following steps:
s11, obtaining polyethylene glycol, sodium azide, tween, purified water, disodium hydrogen phosphate and sodium dihydrogen phosphate, uniformly mixing at 25+/-3 ℃ and filtering to obtain a first reagent 1;
specifically, 5-6 parts of polyethylene glycol, 3-12 parts of sodium azide, 2-15 parts of tween, 1000-1650 parts of purified water, 0.45-0.55 parts of disodium hydrogen phosphate and 15-23 parts of sodium dihydrogen phosphate are respectively weighed, uniformly mixed at 25+/-3 ℃ and filtered to obtain a first reagent 1.
S12, obtaining water-soluble sodium channel, triton, trehalose, glycerol, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium azide, citric acid, CRP sheep anti-human antibody and purified water, uniformly mixing at 25+/-3 ℃ and filtering to obtain a second reagent 2;
specifically, 3-8 parts of water-soluble sodium channel, 1-4 parts of triton, 2-5 parts of trehalose, 2-6 parts of glycerol, 1-4 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 1-2 parts of sodium dihydrogen phosphate, 1-2 parts of sodium azide, 1-3 parts of citric acid, 3-20 parts of CRP sheep anti-human antibody and 40-55 parts of purified water are respectively weighed, uniformly mixed at 25+/-3 ℃ and filtered to obtain a second reagent 2;
s13, obtaining citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, disodium hydrogen phosphate, sodium dihydrogen phosphate, polyvinylpyrrolidone, sodium complexing agent and purified water, and diluting the citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, sodium complexing agent and purified water into complement CRP calibrator with different concentrations by using a calibrator to obtain a standard product diluent 3 for complement CRP antigen;
specifically, 3 to 6 parts of citric acid monohydrate, 5 to 18 parts of sodium chloride, 1 to 6 parts of sodium azide, 2.3 to 6 parts of sodium hydroxide, 3.7 to 9 parts of triatomic acid, 2 to 15 parts of disodium hydrogen phosphate, 3 to 16 parts of sodium dihydrogen phosphate, 3 to 7 parts of polyvinylpyrrolidone, 3 to 8 parts of sodium complexing agent and 40 to 55 parts of purified water are respectively weighed, and the mixture is diluted into complement CRP calibrator with different concentrations by using a calibrator to obtain a standard diluent 3 for complement CRP antigen;
s14, packaging the first reagent 1, the second reagent 2 and the complement CRP antigen with a standard substance diluent 3 to obtain a kit.
The sources of the raw materials involved in the complement CRP detection kit provided by the invention are as follows:
raw materials | Source |
Polyethylene glycol | Guangdong Guanghua Sci-Tech Co.,Ltd. |
Sodium azide | SHANGHAI SEEBIO BIOTECH Inc. |
Tween-type oil | SHANGHAI SEEBIO BIOTECH Inc. |
Disodium hydrogen phosphate | SHANGHAI SEEBIO BIOTECH Inc. |
Sodium dihydrogen phosphate | SHANGHAI SEEBIO BIOTECH Inc. |
Sodium chloride | SHANGHAI SEEBIO BIOTECH Inc. |
Citric acid | SHANGHAI SEEBIO BIOTECH Inc. |
CRP sheep anti-human antibody | Hall (Beijing) diagnostic technology Co., ltd |
Sodium hydroxide | SHANGHAI SEEBIO BIOTECH Inc. |
Polyvinyl pyrrolidone | SHANGHAI SEEBIO BIOTECH Inc. |
Cursor-pull medicine | SHANGHAI SEEBIO BIOTECH Inc. |
Complex protein sodium | Hall (Beijing) diagnostic technology Co., ltd |
Cursor-pull medicine | SHANGHAI SEEBIO BIOTECH Inc. |
Trehalose | SHANGHAI SEEBIO BIOTECH Inc. |
Glycerol | SHANGHAI SEEBIO BIOTECH Inc. |
Specific examples are as follows:
example 1
Complement CRP detection kit
First reagent
Reagent name | Dosage of |
Polyethylene glycol | 3 parts of |
Sodium azide | 4 parts of |
Tween-type oil | 1 part of |
Disodium hydrogen phosphate | 2.3 parts of |
Sodium dihydrogen phosphate | 2.3 parts of |
Second reagent
Reagent name | Dosage of |
Polyethylene glycol | 5 parts of |
Sodium azide | 7 parts of |
CRP sheep anti-human antibody | 2 parts of |
Disodium hydrogen phosphate | 3 parts of |
Sodium dihydrogen phosphate | 3 parts of |
Sodium chloride | 9 parts of |
Complex protein sodium | 6 parts of |
Cursor-pull medicine | 2 parts of |
Trehalose | 3 parts of |
Glycerol | 2 parts of |
Calibration material:
standard dilutions of complement CRP antigen (3 parts citric acid monohydrate, 7 parts sodium chloride, 1 part sodium azide, 2.3 parts sodium hydroxide, 3.7 parts triton, 2 parts disodium hydrogen phosphate, 3 parts sodium dihydrogen phosphate, 5 parts polyvinylpyrrolidone, 3 parts sodium complexing agent,).
Example 2
Complement CRP detection kit
First reagent
Reagent name | Dosage of |
Polyethylene glycol | 4 parts of |
Sodium azide | 5 parts of |
Tween-type oil | 3 parts of |
Disodium hydrogen phosphate | 5 parts of |
Sodium dihydrogen phosphate | 6 parts of |
Second reagent
Reagent name | Dosage of |
Polyethylene glycol | 4 parts of |
Sodium azide | 5 parts of |
CRP sheep anti-human antibody | 7 parts of |
Disodium hydrogen phosphate | 6 parts of |
Sodium dihydrogen phosphate | 8 parts of |
Sodium chloride | 7 parts of |
Complex protein sodium | 5 parts of |
Trehalose | 1 part of |
Cursor-pull medicine | 3 parts of |
Glycerol | 4 parts of |
Calibration material:
standard dilutions of complement CRP antigen (3 parts citric acid monohydrate, 9 parts sodium chloride, 2 parts sodium azide, 2.3 parts sodium hydroxide, 3.7 parts triton, 3 parts disodium hydrogen phosphate, 4 parts sodium dihydrogen phosphate, 6 parts polyvinylpyrrolidone, 3 parts sodium complexing).
Example 3
Complement CRP detection kit
First reagent
Reagent name | Dosage of |
Polyethylene glycol | 5 parts of |
Sodium azide | 5 parts of |
Tween-type oil | 4 parts of |
Disodium hydrogen phosphate | 8 parts of |
Sodium dihydrogen phosphate | 7 parts of |
Second reagent
Reagent name | Dosage of |
Polyethylene glycol | 6 parts of |
Sodium azide | 11 parts of |
CRP sheep anti-human antibody | 8 parts of |
Disodium hydrogen phosphate | 13 parts of |
Sodium dihydrogen phosphate | 11 parts of |
Sodium chloride | 7 parts of |
Complex protein sodium | 9 parts of |
Trehalose | 5 parts of |
Cursor-pull medicine | 2 parts of |
Glycerol | 3 parts of |
Calibration material:
standard dilution for complement CRP antigen (4 parts citric acid monohydrate, 7 parts sodium chloride, 1 part sodium azide, 2.9 parts sodium hydroxide, 3.9 parts triton, 2.4 parts disodium hydrogen phosphate, 3.8 parts sodium dihydrogen phosphate, 5.9 parts polyvinylpyrrolidone, 5 parts sodium collaterals protein)
Example 4
Complement CRP detection kit
First reagent
Reagent name | Dosage of |
Polyethylene glycol | 8 parts of |
Sodium azide | 7 parts of |
Tween-type oil | 4 parts of |
Disodium hydrogen phosphate | 6 parts of |
Sodium dihydrogen phosphate | 7 parts of |
Second reagent
Calibration material:
standard dilution for complement CRP antigen (3.8 parts citric acid monohydrate, 8 parts sodium chloride, 1.8 parts sodium azide, 2.7 parts sodium hydroxide, 3.7 parts triamcinolone, 4 parts disodium hydrogen phosphate, 5 parts sodium dihydrogen phosphate, 6 parts polyvinylpyrrolidone, 7 parts sodium collaterals protein)
Example 5
Complement CRP detection kit
First reagent
Reagent name | Dosage of |
Polyethylene glycol | 15 parts of |
Sodium azide | 12 parts of |
Tween-type oil | 17 parts of |
Disodium hydrogen phosphate | 13 parts of |
Sodium dihydrogen phosphate | 12 parts of |
Second reagent
Reagent name | Dosage of |
Polyethylene glycol | 15 parts of |
Sodium azide | 18 parts of |
CRP sheep anti-human antibody | 2 parts of |
Disodium hydrogen phosphate | 3 parts of |
Sodium dihydrogen phosphate | 3 parts of |
Sodium chloride | 9 parts of |
Complex protein sodium | 7 parts of |
Trehalose | 4 parts of |
Cursor-pull medicine | 2 parts of |
Glycerol | 6 parts of |
Calibration material:
standard dilution for complement CRP antigen (6 parts citric acid monohydrate, 9 parts sodium chloride, 1 part sodium azide, 3 parts sodium hydroxide, 7 parts triton, 8 parts disodium hydrogen phosphate, 8 parts sodium dihydrogen phosphate, 4 parts polyvinylpyrrolidone, 2 parts sodium collaterals protein)
Comparison of test results in different embodiments
1. One sample with 35mg/L complement CRP concentration was prepared, and the sample was repeatedly tested 7 times with the kit of example 1, and the test 9 results are shown in the table:
it can be seen that the concentration of complement CRP measured in example 1 at 4 months, 6 months and 12 months is close to the true value, and the relative deviation is less than 5%, which indicates that the kit of the invention has good repeatability and accurate measurement.
Example 1 is the optimum formulation ratio.
2. One sample with a CRP concentration of 67.63mg/L was prepared, and the sample was repeatedly tested 7 times with the kit of example 2, and the test 9 results are shown in the table:
/ | for 0 month | For 4 months | 8 months of | For 12 months | 13 months of | For 14 months |
1 | 67.75 | 67.94 | 67.66 | 67.63 | 57.71 | 47.73 |
2 | 67.79 | 67.65 | 67.98 | 67.58 | 57.78 | 47.57 |
3 | 67.79 | 67.77 | 67.82 | 67.90 | 57.79 | 47.97 |
Theoretical concentration | 67.63 | 67.63 | 67.63 | 67.63 | 67.63 | 67.63 |
Relative deviation 1 | 0.18% | 0.46% | 0.04% | 0.00% | -14.67% | -29.42% |
Relative deviation 2 | 0.24% | 0.03% | 0.52% | -0.07% | -14.56% | -29.66% |
Relative deviation 3 | 0.24% | 0.21% | 0.28% | 0.40% | -14.55% | -29.07% |
It can be seen from the table that the concentration of complement CRP measured in example 2 at 4 months, 6 months and 12 months is close to the true value, and the relative deviation is less than 5%, which indicates that the kit of the invention has good repeatability and accurate measurement.
3. One sample with a CRP concentration of 10.00mg/L was prepared, and the sample was repeatedly tested 7 times with the kit of example 3, and the test 9 results are shown in the table:
/ | for 0 month | For 4 months | 8 months of | For 12 months | 13 months of | For 14 months |
1 | 10.47 | 10.75 | 9.93 | 10.47 | 8.17 | 7.14 |
2 | 9.98 | 9.85 | 10.48 | 9.82 | 8.43 | 7.76 |
3 | 10.78 | 10.41 | 10.65 | 9.59 | 8.85 | 7.51 |
Theoretical concentration | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
Relative deviation 1 | 4.70% | 7.50% | -0.70% | 4.70% | -18.30% | -28.60% |
Relative deviation 2 | -0.20% | -1.50% | 4.80% | -1.80% | -15.70% | -22.40% |
Relative deviation 3 | 7.80% | 4.10% | 6.50% | -4.10% | -11.50% | -24.90% |
As can be seen from the table, the concentration of complement CRP measured in example 3 at 1 month, 6 months and 12 months is close to the true value, and the relative deviation is less than 5%, which indicates that the kit of the invention has good repeatability and accurate measurement.
4. One sample with a complement CRP concentration of 9.26mg/L was prepared separately, and the sample was repeatedly tested 7 times with the kit of example 4, with the test 9 results shown in the table:
/ | for 0 month | For 4 months | 8 months of | For 12 months | 13 months of | For 14 months |
1 | 9.34 | 8.68 | 7.90 | 7.91 | 6.03 | 4.61 |
2 | 9.22 | 8.93 | 7.18 | 7.59 | 7.08 | 5.66 |
3 | 9.34 | 7.00 | 7.95 | 8.40 | 6.25 | 7.26 |
Theoretical concentration | 9.26 | 9.26 | 9.26 | 9.26 | 9.26 | 9.26 |
Relative deviation 1 | 0.86% | -6.26% | -14.69% | -14.58% | -34.88% | -50.22% |
Relative deviation 2 | -0.43% | -3.56% | -22.46% | -18.03% | -23.54% | -38.88% |
Relative deviation 3 | 0.86% | -24.41% | -14.15% | -9.29% | -32.51% | -21.60% |
As can be seen from Table 15, the concentration of complement CRP measured at the beginning of 4 months in example 4 all deviate from the true value, and the relative deviation is greater than 15% when left for 4 months, indicating that the kits of the invention have poor reproducibility and inaccurate measurements.
5. One sample with a CRP concentration of 70.2mg/L was prepared, and the sample was repeatedly tested 7 times with the kit of example 5, and the test 9 results are shown in the table:
/ | for 0 month | For 4 months | 8 months of | For 12 months | 13Month of moon | For 14 months |
1 | 70.41 | 72.35 | 63.54 | 58.29 | 58.08 | 55.99 |
2 | 70.02 | 70.89 | 61.88 | 57.9 | 61.93 | 54.53 |
3 | 71.56 | 70.91 | 60.88 | 56.35 | 59.14 | 55.11 |
Theoretical concentration | 70.02 | 70.02 | 70.02 | 70.02 | 70.02 | 70.02 |
Relative deviation 1 | 0.56% | 3.33% | -9.25% | -16.75% | -17.05% | -20.04% |
Relative deviation 2 | 0.00% | 1.24% | -11.63% | -17.31% | -11.55% | -22.12% |
Relative deviation 3 | 2.20% | 1.27% | -13.05% | -19.52% | -15.54% | -21.29% |
As can be seen from the table, the concentration of complement CRP measured at 1 month, 6 months and 12 months in example 5 deviates from the true value, and the relative deviation is greater than 15% when left for 8 months, indicating that the kit of the invention has poor reproducibility and inaccurate measurement.
In summary, the bottle opening performance verification of example 2 with the best performance was selected:
it can be seen from the table that the concentration of complement CRP measured in example 1 at 0 to 40 days is close to the true value, and the relative deviation is less than 5%, which indicates that the kit of the invention has good repeatability and accurate measurement.
The above disclosure is only a preferred embodiment of a C-reactive protein assay kit and a detection method with stable performance, but the scope of the invention is not limited thereto, and those skilled in the art will understand that all or part of the procedures for implementing the above embodiments are equivalent and still fall within the scope of the invention.
Claims (5)
1. A C-reactive protein assay kit with stable bottle opening performance is characterized in that,
comprises a first reagent, a second reagent and a standard diluent for complement CRP antigen.
2. A stable C-reactive protein assay kit according to claim 1, wherein,
the first reagent, the second reagent and the standard substance diluent for complement CRP antigen are all preserved at the temperature of 2-8 ℃.
3. A method for detecting a C-reactive protein assay kit with stable bottle opening performance, which is applied to the C-reactive protein assay kit with stable bottle opening performance as claimed in claim 1, and is characterized by comprising the following steps:
adding the first reagent into a sample to be detected, uniformly mixing, incubating at 37 ℃ to obtain a first mixed solution, and reading absorbance A1 of the first mixed solution at a wavelength of 340 nm;
adding the second reagent into the first mixed solution, uniformly mixing, incubating at 37 ℃ to obtain a second mixed solution, and reading absorbance A2 of the second mixed solution at a wavelength of 340 nm;
obtaining the difference value between the absorbance A2 and the absorbance A1 to obtain an absorbance difference;
fitting a standard curve on a full-automatic biochemical analyzer by using the standard substance diluent for the complement CRP antigen, and calculating the content of complement CRP in the sample to be detected based on the absorbance difference.
4. The C-reactive protein assay kit and the assay method for stable performance in opening bottles of claim 3, wherein,
the volume ratio of the sample to be tested to the first reagent is 17:300, the volume ratio of the second reagent to the first reagent is 1:4.
5. a method for preparing a C-reactive protein assay kit with stable opening performance, for preparing a C-reactive protein assay kit with stable opening performance according to claim 1, comprising the steps of:
obtaining polyethylene glycol, sodium azide, tween, purified water, disodium hydrogen phosphate and sodium dihydrogen phosphate, uniformly mixing at 25+/-3 ℃ and filtering to obtain a first reagent;
obtaining water-soluble sodium channel, triton, trehalose, glycerol, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium azide, citric acid, CRP sheep anti-human antibody and purified water, uniformly mixing at 25+/-3 ℃ and filtering to obtain a second reagent;
obtaining citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, disodium hydrogen phosphate, sodium dihydrogen phosphate, polyvinylpyrrolidone, sodium complexing agent and purified water, and diluting the citric acid monohydrate, sodium chloride, sodium azide, sodium hydroxide, triton, sodium complexing agent and purified water into complement CRP calibrator with different concentrations by using a calibrator to obtain a standard diluent for complement CRP antigen;
and packaging the first reagent, the second reagent and the complement CRP antigen with a standard substance diluent to obtain a kit.
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