CN118937682B - Kit for heparin binding protein detection and preparation method thereof - Google Patents
Kit for heparin binding protein detection and preparation method thereof Download PDFInfo
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- CN118937682B CN118937682B CN202410934689.9A CN202410934689A CN118937682B CN 118937682 B CN118937682 B CN 118937682B CN 202410934689 A CN202410934689 A CN 202410934689A CN 118937682 B CN118937682 B CN 118937682B
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- binding protein
- heparin binding
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- sodium azide
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- 102100030009 Azurocidin Human genes 0.000 title claims abstract description 60
- 101710154607 Azurocidin Proteins 0.000 title claims abstract description 59
- 238000002331 protein detection Methods 0.000 title claims abstract description 14
- 238000003257 protein preparation method Methods 0.000 title description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 108
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000004816 latex Substances 0.000 claims abstract description 27
- 229920000126 latex Polymers 0.000 claims abstract description 27
- 239000004094 surface-active agent Substances 0.000 claims abstract description 25
- 239000002245 particle Substances 0.000 claims abstract description 18
- 239000003381 stabilizer Substances 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims abstract description 9
- 239000007983 Tris buffer Substances 0.000 claims description 27
- 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 26
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 26
- 229930195725 Mannitol Natural products 0.000 claims description 26
- 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 26
- 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 26
- 239000000594 mannitol Substances 0.000 claims description 26
- 235000010355 mannitol Nutrition 0.000 claims description 26
- WOXWUZCRWJWTRT-UHFFFAOYSA-N 1-amino-1-cyclohexanecarboxylic acid Chemical compound OC(=O)C1(N)CCCCC1 WOXWUZCRWJWTRT-UHFFFAOYSA-N 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 24
- 229940045944 sodium lauroyl glutamate Drugs 0.000 claims description 23
- IWIUXJGIDSGWDN-UQKRIMTDSA-M sodium;(2s)-2-(dodecanoylamino)pentanedioate;hydron Chemical compound [Na+].CCCCCCCCCCCC(=O)N[C@H](C([O-])=O)CCC(O)=O IWIUXJGIDSGWDN-UQKRIMTDSA-M 0.000 claims description 23
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 22
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 22
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000013504 Triton X-100 Substances 0.000 claims description 9
- 229920004890 Triton X-100 Polymers 0.000 claims description 9
- 238000001514 detection method Methods 0.000 abstract description 23
- 239000000872 buffer Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 24
- 230000000052 comparative effect Effects 0.000 description 23
- 238000005259 measurement Methods 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 230000007547 defect Effects 0.000 description 4
- 230000006916 protein interaction Effects 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 102000022382 heparin binding proteins Human genes 0.000 description 3
- 108091012216 heparin binding proteins Proteins 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000003399 chemotactic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000793686 Homo sapiens Azurocidin Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010062255 Soft tissue infection Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- -1 polyoxyethylene lauryl ether Polymers 0.000 description 1
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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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)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供了一种用于肝素结合蛋白检测试剂盒及其制备方法，属于生物检测技术领域。所述试剂盒包括彼此独立的试剂R1和试剂R2，其特征在于：包括的成分及相应含量为：试剂R1：三羟甲基氨基甲烷缓冲液30‑60mmol/L；聚乙二醇6000 1‑3g/L；表面活性剂A15‑25g/L；叠氮钠0.05‑0.15g/L；试剂R2：三羟甲基氨基甲烷缓冲液30‑60mmol/L；包被有鼠抗人肝素结合蛋白抗体的胶乳颗粒1‑3g/L；表面活性剂B 15‑25g/L；稳定剂5‑10g/L；叠氮钠0.05‑0.15g/L；该试剂盒通过对试剂1和试剂2中表面活性剂和稳定剂的选择，使得到的试剂盒具有较高的精密度和稳定性，且抗干扰能力也明显提高。The invention provides a heparin binding protein detection kit and a preparation method thereof, belonging to the field of biological detection technology. The kit comprises a reagent R1 and a reagent R2 which are independent of each other, characterized in that the components and corresponding contents thereof are as follows: reagent R1: tris hydroxymethylaminomethane buffer 30-60mmol/L; polyethylene glycol 6000 1-3g/L; surfactant A 15-25g/L; sodium azide 0.05-0.15g/L; reagent R2: tris hydroxymethylaminomethane buffer 30-60mmol/L; latex particles coated with mouse anti-human heparin binding protein antibody 1-3g/L; surfactant B 15-25g/L; stabilizer 5-10g/L; sodium azide 0.05-0.15g/L; the kit has higher precision and stability through the selection of surfactants and stabilizers in reagents 1 and 2, and the anti-interference ability is also significantly improved.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting heparin binding protein based on a latex immunoturbidimetry and a preparation method thereof.
Background
Heparin-binding protein (HBP), also known as azurocidin or CAP37, is a neutrophil-derived granule protein, and the chemotactic activity, bactericidal capacity and heparin-binding capacity of HBP are all related to the strong positive charge of HBP, and HBP is used as a chemotactic substance, can activate monocytes and macrophages, induce vascular leakage and tissue edema, and can induce Ca 2+ -dependent cytoskeletal rearrangement and formation of single-layer endothelial cell gaps in vitro. HBP increases vascular endothelial cell permeability because HBP has a strong positive charge and contact with endothelial cells causes rapid activation of intracellular Ca 2+ of the endothelial cells to form actin tensioning fibers, which leads to leakage of the bystander cell. In addition to the severe vascular leakage caused by the excessive concentration of HBP, HBP is related to various infections such as soft tissue infection, which indicates that HBP has a close relationship with serious bacterial infection and is very likely to become a clinical diagnosis index and a drug treatment target.
Currently, the common methods for clinically detecting the content of heparin binding protein are an enzyme-linked immunosorbent assay, an immunoturbidimetry and an immunofluorescence method. The ELISA method has the defects of high detection sensitivity, complicated operation process, long detection period and high detection labor cost, the turbidimetric immunoassay method has the defects of low detection sensitivity, narrow linear range and long detection period, so that clinical requirements cannot be effectively met, and the method also has the defects of high antibody consumption, high reagent cost, low sensitivity of most products in a low-value region and poor repeatability near clinical reference values, so that the heparin binding protein is difficult to accurately and quantitatively detect.
Chinese patent CN115980331a discloses a kit for detecting heparin binding proteins. The sample treating agent comprises sodium alginate with the mass concentration of 0.01-0.5%, polyoxyethylene lauryl ether with the mass concentration of 0.1-10g/L and Tris buffer solution with the mass concentration of 30-100mmol/L, wherein the pH value of the sample treating agent is 7.8-8.2. The kit for detecting heparin binding protein can detect heparin binding protein in serum, plasma and whole blood simultaneously, does not need to carry out centrifugal treatment and other treatment modes on blood samples, greatly shortens detection time, can extract heparin binding protein more efficiently and reduce background interference, and has the advantages of high sensitivity, good repeatability, good specificity, wide linear range, low cost and the like.
The Chinese patent CN115656518A discloses a detection kit for detecting heparin binding proteins in human blood plasma and urine, which comprises a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 comprises a blocking agent, salts and a preservative for resisting the interference of homologous proteins in human blood plasma and urine, the reagent R2 comprises a first polystyrene latex microsphere, a second polystyrene latex microsphere, a protective agent and a preservative, the kit can eliminate the interference of homologous samples, can be used for detecting urine samples, and has the characteristics of high detection speed, good linear range and good stability.
However, the precision, stability and anti-interference capability of the existing kit cannot meet the requirements better, so that a kit for detecting the heparin binding protein based on a latex immunoturbidimetry, which has higher precision and stability and good linear relation, and a preparation method thereof are required to be developed.
Disclosure of Invention
Based on the defects existing in the prior art, the invention aims to provide a kit for detecting heparin binding proteins based on a latex immunoturbidimetry and a preparation method thereof, and the kit has higher precision and stability and good linear relationship through the selection of surfactants in the reagent 1 and the reagent 2.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
In one aspect, the invention provides a kit for detecting heparin binding protein (latex immunoturbidimetry), comprising reagents R1 and R2 independent of each other, comprising the following components in percentage by weight:
Reagent R1 comprises 30-60mmol/L of tris buffer solution, 6000-3 g/L of polyethylene glycol, 15-25g/L of surfactant A and 0.05-0.15g/L of sodium azide;
Reagent R2, 30-60mmol/L of tris buffer solution, 1-3g/L of latex particles coated with mouse anti-human heparin binding protein antibody, 15-25g/L of surfactant B, 5-10g/L of stabilizer and 0.05-0.15g/L of sodium azide.
Preferably, the kit comprises a reagent R1 and a reagent R2 which are independent from each other, and comprises the following components in percentage by weight:
Reagent R1, 50mmol/L of tris buffer solution, 6000 g/L of polyethylene glycol, 20g/L of surfactant A and 0.1g/L of sodium azide;
Reagent R2, 50mmol/L of tris buffer solution, 2g/L of latex particles coated with mouse anti-human heparin binding protein antibody, 20g/L of surfactant B, 6g/L of stabilizer and 0.1g/L of sodium azide.
Wherein the surfactant A is a mixture of Tween 20 and sodium lauroyl glutamate, and the concentration ratio of the Tween 20 to the sodium lauroyl glutamate is 6-8:1, preferably 7:1.
The surfactant A in the reagent 1 adopts a mixture of Tween 20 and sodium lauroyl glutamate, the Tween 20 has the function of renaturation antigen, the specific recognition capability can be improved, the protein structure is not damaged, the damage to the original interaction between proteins can be reduced, the sodium lauroyl glutamate contains hydrophilic and hydrophobic long chains, and steric hindrance exists to influence the interaction between proteins, but the invention surprisingly discovers that the mixture of Tween 20 and sodium lauroyl glutamate is added into the reagent 1 as the surfactant in the implementation process, the concentration ratio of the Tween 20 to the sodium lauroyl glutamate is controlled to be 6-8:1, and the detection precision and the sensitivity of the kit can be obviously improved.
The surfactant B is Triton X-100;
the stabilizer is a mixture of mannitol, trehalose and 1-aminocyclohexane carboxylic acid, and the concentration ratio of the mannitol to the trehalose to the 1-aminocyclohexane carboxylic acid is 1-3:5:1-2, preferably 2:5:1.
The invention takes the mixture of mannitol, trehalose and 1-aminocyclohexane carboxylic acid as a stabilizer, and is matched with sodium azide for use, so that the structure of antibody protein can be protected, protein degradation can be prevented, microorganism breeding can be inhibited, and a good antibacterial effect is achieved, and the concentration ratio of mannitol, trehalose and 1-aminocyclohexane carboxylic acid is controlled to be 1-3:5:1-2, and the stability of the reagent can be better improved through interaction among the mannitol, the trehalose and the 1-aminocyclohexane carboxylic acid, so that the accuracy and the anti-interference performance of the kit are improved.
As some preferred embodiments, the kit comprises a reagent R1 and a reagent R2 which are independent from each other, and comprises the following components in percentage by weight:
Reagent R1 comprises 30-60mmol/L of tris buffer solution, 6000-3 g/L of polyethylene glycol, 14-22g/L of Tween, 1-3g/L of sodium lauroyl glutamate and 0.05-0.15g/L of sodium azide;
Reagent R2, 30-60mmol/L of tris buffer solution, 1-3g/L of latex particles coated with mouse anti-human heparin binding protein antibody, 15-25g/L of Triton X-100, 5-10g/L of stabilizer and 0.05-0.15g/L of sodium azide;
the stabilizer is a mixture of mannitol, trehalose and 1-aminocyclohexane carboxylic acid with the concentration ratio of 1-3:5:1-2.
As a preferred embodiment, the kit comprises a reagent R1 and a reagent R2 which are independent from each other, and comprises the following components in percentage by weight:
reagent R1 comprises 50mmol/L of tris buffer solution, 6000 g/L of polyethylene glycol, 17.5g/L of Tween 20, 2.5g/L of sodium lauroyl glutamate and 0.1g/L of sodium azide;
Reagent R2, 50mmol/L of tris buffer solution, 2g/L of latex particles coated with a mouse anti-human heparin binding protein antibody, 100 g/L of Triton X-20 g/L, 1.5g/L of mannitol, 3.75g/L of trehalose, 0.75g/L of 1-aminocyclohexane carboxylic acid and 0.1g/L of sodium azide.
The volume ratio of the sample, the reagent R1 and the reagent R2 in the kit is 1:18-22:4-6, preferably 1:20:5.
Further, the heparin binding protein detection kit also comprises a calibrator and a quality control product, wherein the calibrator and the quality control product comprise phosphate buffer, sodium azide and HBP antigen.
On the other hand, the invention also provides a preparation method of the kit, which comprises the following steps:
(1) Preparing a buffer solution, regulating the pH value of the buffer solution to 6.0-8.0, respectively adding polyethylene glycol 6000, a surfactant A and sodium azide into the buffer solution, and dissolving to obtain the reagent 1;
(2) Preparing a reagent 2, namely preparing a buffer solution, regulating the pH value of the buffer solution to 6.0-8.0, respectively adding latex particles coated with a mouse anti-human heparin binding protein antibody, a surfactant B, a stabilizer and sodium azide into the buffer solution, and dissolving to obtain the reagent 2.
In yet another aspect, the invention further provides a method for using the above kit:
The kit is suitable for:
hitachi series 7080/7100/7180/7600/3100/3110/3500/LST006/LST008AS/LST008a;
Beckman series:
AU400/AU480/AU640/AU680/AU2700/AU5400/AU5421/AU5800、DXC 700AU、DXC800;
Toshiba series TBA-40FR/120FR/2000FR;
the Roche series: MODULAR, cobas c311, cobas c501, cobas c502, cobas c701, cobas c702;
The Michael series BS320/380/400/420/2800 (M)/2000 (M);
Mei Lian series ：MS-480、MS-480B、MS-880、MS-880B、MS-300、MS-200、MS-1280、MS-2080、MS-1880、MS-1680、MS-680、MS-600、MS-520、MS-450、MS-L8080、MS-L7280、MS-L8060、MS-L8000、MS-380、MS-400、MS-420、MS-380P、MS-400P、MS-420P;
Runkang series, RC-480, RC-460, RC-400, RC-680, RC-600, RC-520, RC-450, RC-880, RC-860, RC-800, RC-2080, RC-1880, RC1680;
Siemens of dimensions RXL, dimension EXL, dimension X-PAND, ADVIA, XPT, ADVIA 1800, ADVIA 2400, atellica;
The Hizimel series is BX-3010 and BX-4000;
The yaban series is c8000/c4000/c16000/ci4100/ci8200/ci16200/Alinity;
canon series TBA-FX8 and TBA-1500FR.
Test conditions:
The operation steps are as follows:
The principle of the method is that heparin binding protein HBP in a sample is combined with corresponding antibodies bound on latex particles in a reagent to generate agglutination reaction to form antigen-antibody complex, and a certain turbidity is generated in a linear range, the turbidity is directly proportional to the concentration of HBP in the sample when a sufficient amount of antibody exists, and the concentration of HBP in blood can be calculated by measuring a specific wavelength absorbance value and referring to a calibration curve.
Compared with the prior art, the invention has the technical effects that:
(1) The invention adds the surfactant in the reagent 1 and the reagent 2, improves the uniformity of the reagent, adopts the mixture of Tween 20 and sodium lauroyl glutamate in the reagent 1, has the function of renaturation antigen, can improve the specific recognition capability, can not destroy the protein structure, can reduce the damage to the original interaction between proteins, and has the long chain of hydrophilicity and hydrophobicity, thereby influencing the interaction between proteins, but the invention surprisingly discovers that the mixture of Tween 20 and sodium lauroyl glutamate is added in the reagent 1 as the surfactant in the implementation process, and controls the concentration ratio of the Tween 20 and the sodium lauroyl glutamate to be 6-8:1, thereby obviously improving the detection precision and the sensitivity of the kit.
(2) According to the invention, the stabilizer is added into the reagent 2, the mixture of mannitol, trehalose and 1-aminocyclohexane carboxylic acid is used as the stabilizer, and sodium azide is matched to be added into the reagent 2, so that the structure of antibody protein can be protected, protein degradation can be prevented, microorganism breeding can be inhibited, and a good antibacterial effect is achieved, and the concentration ratio of mannitol, trehalose and 1-aminocyclohexane carboxylic acid is controlled to be 1-3:5:1-2, so that the stability of the reagent can be better improved through interaction among the mannitol, the trehalose and the 1-aminocyclohexane carboxylic acid, and the accuracy and anti-interference performance of the kit are further improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Latex particles coated with murine anti-human heparin-binding protein antibody, used in the examples below, were purchased from Shaoxing, inc. of the Biotechnology of the wound-rotor, inc., at a specification of 50g/L or more
Example 1A kit for heparin-binding protein detection (latex immunoturbidimetry)
The reagent R1 and the reagent R2 which are independent of each other are included, and the components and the corresponding contents are as follows:
reagent R1 comprises 30mmol/L of tris buffer solution, 6000 g/L of polyethylene glycol, 13.2g/L of Tween 20, 2.2g/L of sodium lauroyl glutamate and 0.05g/L of sodium azide;
reagent R2, 30mmol/L of tris buffer solution, 1g/L of latex particles coated with a mouse anti-human heparin binding protein antibody, 15g/L of Triton X-100, 0.8g/L of mannitol, 4g/L of trehalose, 0.8g/L of 1-aminocyclohexane carboxylic acid and 0.05g/L of sodium azide.
The volume ratio of sample, reagent R1 and reagent R2 was 1:18:4, i.e., 12. Mu.L of sample, 216. Mu.L of reagent R1 and 48. Mu.L of reagent R2.
The preparation method comprises the following steps:
(1) Preparing a reagent 1, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding polyethylene glycol 6000, tween 20, sodium lauroyl glutamate and sodium azide into the buffer solution, and dissolving to obtain the reagent 1;
(2) Preparing a reagent 2, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding latex particles coated with a mouse anti-human heparin binding protein antibody, triton X-100, mannitol, trehalose, 1-aminocyclohexane carboxylic acid and sodium azide into the buffer solution, and dissolving to obtain the reagent 2.
Example 2A kit for heparin-binding protein detection (latex immunoturbidimetry)
The reagent R1 and the reagent R2 which are independent of each other are included, and the components and the corresponding contents are as follows:
Reagent R1 comprises 60mmol/L of tris buffer solution, 6000 g/L of polyethylene glycol, 21.6g/L of Tween, 2.7g/L of sodium lauroyl glutamate and 0.15g/L of sodium azide;
Reagent R2, 60mmol/L of tris buffer solution, 3g/L of latex particles coated with mouse anti-human heparin binding protein antibody, 25g/L of Triton X-100, 3g/L of mannitol, 5g/L of trehalose, 2g/L of 1-aminocyclohexane carboxylic acid and 0.15g/L of sodium azide.
The volume ratio of sample, reagent R1 and reagent R2 was 1:22:6, i.e., 12. Mu.L of sample, 264. Mu.L of reagent R1 and 72. Mu.L of reagent R2.
The preparation method comprises the following steps:
(1) Preparing a reagent 1, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding polyethylene glycol 6000, tween 20, sodium lauroyl glutamate and sodium azide into the buffer solution, and dissolving to obtain the reagent 1;
(2) Preparing a reagent 2, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding latex particles coated with a mouse anti-human heparin binding protein antibody, triton X-100, mannitol, trehalose, 1-aminocyclohexane carboxylic acid and sodium azide into the buffer solution, and dissolving to obtain the reagent 2.
Example 3A kit for heparin-binding protein detection (latex immunoturbidimetry)
The reagent R1 and the reagent R2 which are independent of each other are included, and the components and the corresponding contents are as follows:
reagent R1 comprises 50mmol/L of tris buffer solution, 6000 g/L of polyethylene glycol, 17.5g/L of Tween 20, 2.5g/L of sodium lauroyl glutamate and 0.1g/L of sodium azide;
Reagent R2, 50mmol/L of tris buffer solution, 2g/L of latex particles coated with a mouse anti-human heparin binding protein antibody, 100 g/L of Triton X-20 g/L, 1.5g/L of mannitol, 3.75g/L of trehalose, 0.75g/L of 1-aminocyclohexane carboxylic acid and 0.1g/L of sodium azide.
The volume ratio of sample, reagent R1 and reagent R2 was 1:20:5, i.e., 12. Mu.L of sample, 240. Mu.L of reagent R1 and 60. Mu.L of reagent R2.
The preparation method comprises the following steps:
(1) Preparing a reagent 1, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding polyethylene glycol 6000, tween 20, sodium lauroyl glutamate and sodium azide into the buffer solution, and dissolving to obtain the reagent 1;
(2) Preparing a reagent 2, namely preparing a tris buffer solution, regulating the pH value of the buffer solution to 7.0, respectively adding latex particles coated with a mouse anti-human heparin binding protein antibody, triton X-100, mannitol, trehalose, 1-aminocyclohexane carboxylic acid and sodium azide into the buffer solution, and dissolving to obtain the reagent 2.
Comparative example 1
The difference from example 3 is that the surfactant in the reagent R1 is only Tween 20, namely Tween 20g/L, and the content of other components is the same as that in example 3.
Comparative example 2
The difference with example 3 is that the mass ratio of Tween 20 and sodium lauroyl glutamate in the reagent R1 is 1:7, namely 17.5g/L of sodium lauroyl glutamate, 2.5g/L of Tween 20, and the content of other components is the same as example 3.
Comparative example 3
The difference with example 3 is that the stabilizer in reagent R2 is mannitol and trehalose, namely mannitol 1g/L and trehalose 5g/L, and the content of other components is the same as that of example 3.
Comparative example 4
The difference from example 3 is that the mass ratio of mannitol, trehalose and 1-aminocyclohexane carboxylic acid is 2:1:3, namely mannitol 2g/L, trehalose 1g/L and 1-aminocyclohexane carboxylic acid 3g/L, and the other components are contained in the same amounts as in example 3.
Comparative example 5
The difference from example 3 is that the mass ratio of mannitol, trehalose and 1-aminocyclohexane carboxylic acid is 4:1:1, namely mannitol 4g/L, trehalose 1g/L and 1-aminocyclohexane carboxylic acid 1g/L, and the other components are contained in the same amounts as in example 3.
Comparative example 6
The difference from example 3 is that the volume ratio of sample, reagent R1 and reagent R2 is 3:40:10, i.e. 15. Mu.L of sample, 200. Mu.L of reagent R1 and 50. Mu.L of reagent R2, the other components are contained in the same amounts as in example 3.
And (3) effect test:
1. linearity of
The experiment requires that the correlation coefficient r should be more than or equal to 0.990 in the linear range of 6-340 ng/mL.
Experimental method
High value samples (340 ng/mL) near the upper limit of the linear range were mixed with zero concentration samples to 8 diluted concentrations, 340ng/mL, 250ng/mL, 200ng/mL, 150ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 6ng/mL, respectively. The test was repeated 3 times for each sample of each concentration to obtain a concentration value, the measurement results of each sample were recorded, and the average value (yi) of the measurement values of 3 times for each sample was calculated. The linear regression equation was solved using the dilution concentration (x i) as an independent variable and the measurement result mean (y i) as a dependent variable. And calculating a correlation coefficient (r) of the linear regression according to the formula (1), wherein the correlation coefficient (r) meets the experimental requirement, and the detection data is shown in the following table 1.
Wherein:
r-correlation coefficient;
x i -dilution ratio;
y i -the average value of the measurement results of each sample;
-means of dilution ratio;
-total mean of sample measurement results.
TABLE 1
| Correlation coefficient r | |
| Example 1 | 0.9985 |
| Example 2 | 0.9988 |
| Example 3 | 0.9995 |
| Comparative example 1 | 0.9856 |
| Comparative example 2 | 0.9972 |
| Comparative example 3 | 0.9978 |
| Comparative example 4 | 0.9980 |
| Comparative example 5 | 0.9981 |
| Comparative example 6 | 0.9863 |
As can be seen from the detection results in Table 1, the test results of the linear correlation coefficients (r) of the test kits prepared in examples 1-3 of the present invention are not lower than 0.990, and the linear relationship is good, especially the test results of the linear correlation coefficients (r) of the test kits prepared in example 3 are 0.9995, and the linear relationship is good. Therefore, the linear range of the kits prepared in examples 1-3 can be defined as 6-340ng/mL, while the test using the kits prepared in comparative examples 1-6 shows that the experimental result of the linear correlation coefficient (r) is lower than 0.9900, and the linear relationship is not satisfactory although it is better.
2. Accuracy (stability)
The heparin-binding protein detection kits described in examples 1-3 and comparative examples 1-6 were prepared, respectively, stored in a light-protected environment at 2-8 ℃ without corrosive gas, and heparin-binding protein antigen sample reagents were detected at 1 month, 3 months, 6 months, 12 months, and 24 months of storage, respectively, with the monthly No. 1 detection accuracy, and the detection data are shown in table 2.
Experimental method
The heparin binding protein antigen standard solution A with the concentration of 300ng/mL (the allowable deviation is +/-10%) is added into the sample B with the concentration of 180ng/mL, the volume ratio between the added heparin binding protein antigen standard solution and the sample B is 1:9, the recovery rate R is calculated according to the formula (2), the recovery rate is in the range of 85% -115%, and the specific detection results are shown in the table 2 below.
Wherein:
R-recovery;
V-volume of standard a fluid;
V 0 -volume of sample B;
the average value of 3 times of measurement after the C-sample B liquid is added into the standard A liquid;
Average of 3 measurements of C 0 -sample B;
C S -concentration of standard A solution.
TABLE 2
| 1 Month | For 3 months | 6 Months of | For 12 months | 24 Months of | |
| Example 1 | 96.12% | 97.54% | 97.85% | 98.27% | 99.35% |
| Example 2 | 96.46% | 97.85% | 97.47% | 98.14% | 98.72% |
| Example 3 | 98.14% | 96.28% | 97.16% | 98.76% | 100.24% |
| Comparative example 1 | 98.63% | 97.56% | 95.42% | 108.36% | 120.53% |
| Comparative example 2 | 97.27% | 98.12% | 97.34% | 104.76% | 118.42% |
| Comparative example 3 | 88.48% | 92.15% | 94.28% | 88.46% | 125.34% |
| Comparative example 4 | 92.45% | 95.26% | 97.42% | 85.63% | 84.12% |
| Comparative example 5 | 96.41% | 97.24% | 98.38% | 105.72% | 120.45% |
| Comparative example 6 | 95.64% | 97.58% | 80.12% | 89.47% | 78.26% |
According to the detection results of the table 2, it can be seen that the detection kit prepared in the embodiment 1-3 of the invention has the recovery rate in the range of 85% -115% by adding the heparin binding protein antigen standard with known concentration into a low-value sample, and the detection kit still can meet the requirements after being placed for 24 months, which means that the stability of the kit is relatively high, the detection accuracy of the kit prepared in the comparative embodiment 1-6 is reduced to different degrees after being placed for 24 months, the recovery rate obviously fails to meet the requirements, and the change of the type or mass ratio of the reagent in the kit can obviously influence the stability of the kit, and the recovery rate after being placed for 24 months is lower than 85% or higher than 115%, which does not meet the requirements.
3. Precision test
The heparin-binding protein detection kit prepared in examples 1-3 and comparative examples 1-5 was repeatedly assayed for the same sample to be tested in a batch having a content of 250ng/mL, and the results obtained were calculated for SD and CV, and the detection data are shown in Table 3.
TABLE 3 Table 3
As can be seen from the detection results of Table 3, the detection kit prepared in examples 1-3 of the invention repeatedly measures the same sample to be detected with the content of 250ng/mL in batch, the CV is less than or equal to 5%, the requirement is met, the precision of the kit is high, the precision of the repeated measurement of the same sample to be detected by the kit prepared in comparative examples 1-5 is reduced to different degrees, and the change of the type or mass ratio of the reagent in the kit can obviously influence the precision of the kit.
It should be emphasized that the embodiments described herein are illustrative rather than limiting, and that this invention encompasses embodiments not limited to those specifically described, but rather falls within the scope of the present invention as defined by the appended claims.
Claims (6)
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