CN106324239A - preparation method and application of C-reactive protein immuno latex microsphere - Google Patents
preparation method and application of C-reactive protein immuno latex microsphere Download PDFInfo
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- CN106324239A CN106324239A CN201610604720.8A CN201610604720A CN106324239A CN 106324239 A CN106324239 A CN 106324239A CN 201610604720 A CN201610604720 A CN 201610604720A CN 106324239 A CN106324239 A CN 106324239A
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- reactive protein
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- concussion
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- 102100032752 C-reactive protein Human genes 0.000 title claims abstract description 146
- 239000004005 microsphere Substances 0.000 title claims abstract description 135
- 108010074051 C-Reactive Protein Proteins 0.000 title claims abstract description 117
- 239000004816 latex Substances 0.000 title claims abstract description 92
- 229920000126 latex Polymers 0.000 title claims abstract description 92
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000004793 Polystyrene Substances 0.000 claims abstract description 55
- 229920002223 polystyrene Polymers 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 72
- 230000009514 concussion Effects 0.000 claims description 62
- 230000036039 immunity Effects 0.000 claims description 44
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 36
- 239000000872 buffer Substances 0.000 claims description 35
- 238000012360 testing method Methods 0.000 claims description 31
- 239000007853 buffer solution Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 23
- 150000001718 carbodiimides Chemical class 0.000 claims description 22
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- 239000004471 Glycine Substances 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000007987 MES buffer Substances 0.000 claims description 16
- 239000003755 preservative agent Substances 0.000 claims description 16
- 230000002335 preservative effect Effects 0.000 claims description 16
- 230000002708 enhancing effect Effects 0.000 claims description 15
- 238000004879 turbidimetry Methods 0.000 claims description 15
- 230000004913 activation Effects 0.000 claims description 14
- 239000006193 liquid solution Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 13
- 239000003381 stabilizer Substances 0.000 claims description 13
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 10
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 9
- 229940033663 thimerosal Drugs 0.000 claims description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- IOPFAHJCNYBXMR-UHFFFAOYSA-N O.[Na].C(C)S(=O)(=O)O.OCCN1CCNCC1 Chemical compound O.[Na].C(C)S(=O)(=O)O.OCCN1CCNCC1 IOPFAHJCNYBXMR-UHFFFAOYSA-N 0.000 claims description 6
- QAUPLEKJQJHECR-UHFFFAOYSA-N O.[Na].N1(CCOCC1)CC(CS(=O)(=O)O)O Chemical compound O.[Na].N1(CCOCC1)CC(CS(=O)(=O)O)O QAUPLEKJQJHECR-UHFFFAOYSA-N 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 6
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 239000012190 activator Substances 0.000 claims description 4
- 239000000701 coagulant Substances 0.000 claims description 4
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 4
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 3
- 229960000633 dextran sulfate Drugs 0.000 claims description 3
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 2
- 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 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 4
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 2
- 229910052796 boron Inorganic materials 0.000 claims 2
- 235000009508 confectionery Nutrition 0.000 claims 2
- 230000001934 delay Effects 0.000 claims 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 1
- 230000003053 immunization Effects 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 238000010168 coupling process Methods 0.000 abstract description 17
- 230000008878 coupling Effects 0.000 abstract description 16
- 238000005859 coupling reaction Methods 0.000 abstract description 16
- 238000001514 detection method Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 239000000203 mixture Substances 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 3
- 230000003213 activating effect Effects 0.000 abstract description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 2
- 238000010382 chemical cross-linking Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 125000003277 amino group Chemical group 0.000 abstract 1
- 230000015271 coagulation Effects 0.000 abstract 1
- 238000005345 coagulation Methods 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 abstract 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 32
- 238000002835 absorbance Methods 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000010836 blood and blood product Substances 0.000 description 7
- 229940125691 blood product Drugs 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- XINQFOMFQFGGCQ-UHFFFAOYSA-L (2-dodecoxy-2-oxoethyl)-[6-[(2-dodecoxy-2-oxoethyl)-dimethylazaniumyl]hexyl]-dimethylazanium;dichloride Chemical compound [Cl-].[Cl-].CCCCCCCCCCCCOC(=O)C[N+](C)(C)CCCCCC[N+](C)(C)CC(=O)OCCCCCCCCCCCC XINQFOMFQFGGCQ-UHFFFAOYSA-L 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Substances [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- LLJZKKVYXXDWTB-UHFFFAOYSA-N acetic acid;sodium Chemical compound [Na].[Na].CC(O)=O LLJZKKVYXXDWTB-UHFFFAOYSA-N 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- MCHZKGNHFPNZDP-UHFFFAOYSA-N 2-aminoethane-1,1,1-triol;hydrochloride Chemical compound Cl.NCC(O)(O)O MCHZKGNHFPNZDP-UHFFFAOYSA-N 0.000 description 1
- WFCSWCVEJLETKA-UHFFFAOYSA-N 2-piperazin-1-ylethanol Chemical compound OCCN1CCNCC1 WFCSWCVEJLETKA-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 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
- 239000008118 PEG 6000 Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009692 acute damage Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- -1 trihydroxy methyl amino Chemical group 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (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)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a preparation method and application of C-reactive protein immuno latex microsphere. The preparation method of C-reactive protein immuno latex microsphere optimizes a chemical crosslinking method. The carboxyl of polystyrene microspheres is activated at a low pH, and is coupled with the amino group of a dissolved antibody at a high pH, the mixture of polystyrene microspheres and the dissolved antibody is neutral, after coupling for 12-24h at low temperature, the coupling efficiency of the antibody is ensured, and the damage of an activating agent on antibody activity is greatly reduced. A latex enhanced immunoturbidimetry C-reactive protein determination kit prepared by the immuno latex microsphere which is produced by the C-reactive protein immuno latex microsphere preparation method has the advantagess of high sensitivity, high accuracy, good repeatability and stable property, the quantitative linear range is 0.1-20 mg/L, and the latex enhanced immunoturbidimetry C-reactive protein determination kit can be used in a full-automatic biochemical analyzer or a coagulation analyzer. The operation is fast and simple, it only takes 5 to 10 minutes from detection to result collection, and the prospect for clinical application is good.
Description
Technical field
The present invention relates to medical immunology diagnostic field, in particular to a kind of C reactive protein immunity latex microsphere preparation method
And application.
Background technology
C reactive protein (C-reactive protein, CRP) is a kind of mainly to be produced by liver under the mediation of interleukin-6
Raw acute phase reactive protein, acute stage concentration can raise thousands of times.
No matter CRP is all one of albumen important in Acute reaction protein in human or animal's body, is a kind of sensitive
Marker of inflammation, acute injury and infect time its blood concentration drastically raise.Normal animal serum CRP content is the lowest, but
When infection, inflammatory, operation and tumor, within several hours, raise rapidly, in Canis familiaris L. body 24～48 hours up to peak.Pathological changes
After disappearing, CRP can be dropped rapidly to normally, so the first-selected index that can infect as animal bacteria and virus, prevents from resisting
The health problem that raw element abuse and minimizing food bring.
Within 1977, Sternberg creates rate nephelometry, is to sentence scattering of light degree with the solution measured
The content of antigen in disconnected sample.The light of certain wavelength irradiates along trunnion axis, encounters short grained immune complex and may result in light and dissipate
Penetrating, scattering strength is directly proportional to the content of antigenantibody complex.But immunoturbidimetry does not reaches due to detection sensitivity
Clinical requirement, then occurs in that latex enhancing immune turbidimetry.Its ultimate principle is first to adsorb antibody at a kind of latex
On granule, when running into corresponding antigen, antigen-antibody combines and latex agglutination occurs.The size of single latex particle is in incidence
Within optical wavelength, light-transmissive.When two or more latex particle agglutination can hinder light to pass through, making transmission light reduce, it subtracts
Few degree is directly proportional to the degree of latex agglutination, is also inversely proportional to antigen.
Frequently with polystyrene microsphere coupled antibody in prior art, obtain the emulsion reagent for detection, but coupling is imitated
Rate is the most on the low side, only 60～80%, and during coupling, Carbodiimides activator antagonist activity is destroyed relatively big, causes product
Yield rate is low.
Summary of the invention
Present invention aim to overcome existing C reactive protein immunity latex microsphere preparation method finished product rate low
Defect, it is provided that the C reactive protein immunity latex microsphere preparation side that a kind of coupling efficiency is high, antibody activity impact is little after coupling
Method and the latex enhancing immune turbidimetry C reactive protein by this C reactive protein immunity latex microsphere of employing measure test kit.
For achieving the above object, C reactive protein immunity latex microsphere preparation method provided by the present invention, step is as follows:
1) preparing the polystyrene microsphere of particle size range 50～200nm, first polystyrene microsphere is 50mmol/ by concentration
The MES buffer of L is diluted to final concentration 0.8～1.2%w/v, obtains the microspheres solution that pH is 5.5～6.5；
2) with MES buffer solution carbodiimides, the carbodiimides that concentration is 10mg/mL is obtained molten
Liquid；
3) with the weight of polystyrene microsphere contained in microspheres solution for counting, by every 10mg polystyrene microsphere add 50～
Carbodiimides solution is dropped in microspheres solution by the ratio of 60 μ L, 2～8 DEG C concussion activation 1h, concussion speed be 30～
50r/min, obtains activated polystyrene microspheres solution；
4) being dissolved under the conditions of pH8.5～9.5 by the CRP polyclonal antibody of concentration >=10mg/mL, solvent is 50mmol/L
Borate buffer solution, make antibody final concentration between 0.5～2mg/mL, obtain CRP Anti-TNF-α liquid solution, be stored in 2～8
DEG C standby；
5) CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody and microspheres quality
Ratio is 0.05～0.2:1, and whole dropping process continues 4～7min, makes activated polystyrene microsphere couple with the amino on antibody,
Both mixed liquors are neutrality, afterwards 0～4 DEG C of concussion 12～24h, and concussion speed is 30～50r/min, obtains C reactive protein
Immunity latex microsphere.
Preferably, described step 3) in concussion activation condition be: 4 DEG C of concussion activation 1h, concussion speed is 50r/min.
Preferably, described step 5) in when making activated polystyrene microsphere couple with the amino on antibody, 4 DEG C of concussion 16h,
Concussion speed is 50r/min.
Present invention also offers a kind of latex enhancing immune turbidimetry C reactive protein and measure test kit, react egg including C-
White detectable R1 and C reactive protein detectable R2；
In described C reactive protein detectable R1, each component and content are: 0.01～13% stabilizer 1,10～200mM
The preservative 1 of coagulant 1,0.02～0.1% of buffer 1,1～6%；
In described C reactive protein detectable R2, each component and content are: 0.01～23% stabilizer 2,1～5mg/ml
The preservative 2 of buffer 2,0.02～0.1% of C reactive protein immunity latex microsphere, 10～200mM；
The preparation method of described C reactive protein detectable R2 is:
1) preparing the polystyrene microsphere of particle size range 50～200nm, first polystyrene microsphere is 50mmol/ by concentration
The MES buffer of L is diluted to final concentration 0.8～1.2%w/v, obtains the microspheres solution that pH is 5.5～6.5；
2) with MES buffer solution carbodiimides, the carbodiimides that concentration is 10mg/mL is obtained molten
Liquid,
3) with the weight of polystyrene microsphere contained in microspheres solution for counting, by every 10mg polystyrene microsphere add 50～
Carbodiimides solution is dropped in microspheres solution by the ratio of 60 μ L, 2～8 DEG C concussion activation 1h, concussion speed be 30～
50r/min, obtains activated polystyrene microspheres solution；
4) being dissolved under the conditions of pH8.5～9.5 by the CRP polyclonal antibody of concentration >=10mg/mL, solvent is 50mmol/L
Borate buffer solution, make antibody final concentration between 0.5～2mg/mL, obtain CRP Anti-TNF-α liquid solution, be stored in 2～8
DEG C standby；
5) CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody and microspheres quality
Ratio is 0.05～0.2:1, and whole dropping process continues 4～7min, makes activated polystyrene microsphere couple with the amino on antibody,
Both mixed liquors are neutrality, afterwards 0～4 DEG C of concussion 12～24h, and concussion speed is 30～50r/min, obtains C reactive protein
Immunity latex microsphere；
6) adding containing stabilizer 2 and the confining liquid of buffer 2 in C reactive protein immunity latex microsphere, concussion obtains
Latex solution；
7) latex solution is centrifuged, and removes supernatant, the activator wherein remained with removing, is subsequently adding preservative 2 to storage
Concentration, ultrasonic disperse, to obtain final product.
Preferably, described stabilizer 1 is selected from 0.5～the second two of the BSA of the sodium chloride of 1.8%, 0.1～1%, 1～20mM
One or more in the Triton X-100 of amine tetraacethyl disodium, 0.01～1%；
Described buffer 1 is selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution (Hepes), trihydroxy methyl amino
Methane-hydrochloride buffer (Tris), 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution (Mopso), phosphate
One or more in buffer (PBS), borate buffer solution (BBS), glycine buffer (Glycine)；
The described coagulant 1 one in polyethylene glycol 6000, PEG 8000 or dextran sulfate；
The described preservative 1 one in sodium azide, Proclin300 or thimerosal；
Described stabilizer 2 is selected from 0.5～the BSA of the sodium chloride of 1.8%, 0.2～3%, 1～20mM ethylenediaminetetraacetic acid two
One or more in the glycerol of Triton X-100,1～10% of sodium, 0.01～1%；
Described buffer 2 is selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-salt
Acid buffer, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, phosphate buffer, borate buffer solution,
One or more in glycine buffer；
The described preservative 2 one in sodium azide, Proclin300 or thimerosal.
It is further preferred that each component and content are in described C reactive protein detectable R1: 0.4～0.8%
100～200mM Tris, 1～the PEG of 3% of NaCl, pH7.4 of BSA, 0.5～0.9%, the NaN of 0.05%3；
In described C reactive protein detectable R2, each component and content are: 0.4～the BSA of 0.8%, 1～5mg/ml
C reactive protein immunity latex microsphere, the 25-50mmol/L glycine buffer of pH7.4, the NaN of 0.05%3；
Or the NaN of 0.05% in described C reactive protein detectable R1 and described C reactive protein detectable R23Replace
It is changed to the Proclin 300 of 0.1%.
Further, mentioned reagent box also includes C reactive protein calibration object, and its each component and content is: 0.01～13%
The calibration object of calibration object buffer, 0.02～0.1% of calibration object stabilizer, the C reactive protein of 20mg/L, 10～200mM
Preservative.
Preferably, described calibration object stabilizer is selected from 0.5～the BSA of the sodium chloride of 1.8%, 0.1～1%, 1～20mM
One or more in the Triton X-100 of disodiumedetate, 0.01～1%；
Described calibration object buffer is selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution, trihydroxy methyl amino first
Alkane-hydrochloride buffer, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, phosphate buffer, borate are slow
Rush in liquid, glycine buffer one or more；
Described calibration object preservative one in sodium azide, Proclin300 or thimerosal.
It is further preferred that each component and content are in described C reactive protein calibration object: 0.4～the BSA of 0.8%, 0.5
～the 25-50mmol/L glycine buffer of the C reactive protein of NaCl, 20mg/L of 0.9%, pH7.4, the NaN of 0.05%3；
Or each component and content are in described C reactive protein calibration object: 0.4～the BSA of 0.8%, 0.5～0.9%
The C reactive protein of NaCl, 20mg/L, the 25-50mmol/L glycine buffer of pH7.4, the Proclin 300 of 0.1%.
Preferably, in the technical scheme of mentioned reagent box, described step 3) in concussion activation condition be: 4 DEG C of concussion activation
1h, concussion speed is 50r/min.
Preferably, in the technical scheme of mentioned reagent box, described step 5) in make activated polystyrene microsphere with on antibody
Amino coupling time, 4 DEG C concussion 16h, concussion speed is 50r/min.
Preferably, in the technical scheme of mentioned reagent box, described step 6) in concussion condition be: 25 DEG C of concussion 2h, then
Proceeding to 4 DEG C of concussion 24h, concussion speed is 50r/min.
Beneficial effects of the present invention:
(1) present invention optimizes Chemical Crosslinking Methods, by polystyrene when the C reactive protein immunity latex microsphere of preparation
The carboxyl of microsphere activates at low ph conditions, with high pH condition dissolve antibody on amino couple, both mixed liquors in
Property, coupling 12～24h at low temperatures, both ensure that the coupling efficiency of antibody, the activator that reduces of high degree resists simultaneously
The destruction of body activity.
(2) the latex enhancing immune turbidimetry C reactive protein mensuration test kit of the present invention has highly sensitive, quantitatively accurate
Really, reproducible, the feature of stable in properties, its quantitative range of linearity is 0.1～20mg/L.
(3) can be applicable to automatic clinical chemistry analyzer or blood coagulation analyzer, operation is quick, simple, from detection to gathering knot
Fruit only needs 5～10 minutes, has preferable potential applicability in clinical practice.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
C reactive protein immunity latex microsphere preparation method provided by the present invention is as follows:
1, preparing the polystyrene microsphere of particle diameter 120nm, concentration is 10%, takes 5ml；Polystyrene microsphere is first with dense
Degree is diluted to final concentration 1%w/v for the MES buffer of 50mmol/L, obtains the microsphere that pH is 5.5～6.5 molten
Liquid, the volume of solution is 50mL；
2, with MES buffer solution carbodiimides, the carbodiimides that concentration is 10mg/mL is obtained molten
Liquid；
3, with the weight of polystyrene microsphere contained in microspheres solution for counting, by every 10mg polystyrene microsphere add 50～
Carbodiimides solution is dropped in microspheres solution by the ratio of 60 μ L, drips 250 μ L, 4 DEG C of concussion activation 1h altogether, shakes speed
For 50r/min, obtain activated polystyrene microspheres solution；
4, being dissolved under the conditions of pH8.5～9.5 by CRP polyclonal antibody (commercially available, concentration >=10mg/mL), solvent is
The borate buffer solution of 50mmol/L, makes antibody final concentration at 1.2mg/mL, obtains CRP Anti-TNF-α liquid solution, the body of solution
Long-pending is 50mL, be stored in 4 DEG C standby；
5, CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody and microspheres quality
Ratio is 0.05～0.2:1, and whole dropping process continues 4min, and both mixed liquors are neutrality, afterwards 4 DEG C of concussion 16h, concussion speed
Degree is 50r/min, obtains C reactive protein immunity latex microsphere 1.
Embodiment 2
C reactive protein immunity latex microsphere preparation method provided by the present invention is as follows:
1, preparing the polystyrene microsphere of particle diameter 50nm, concentration is 10%, takes 5ml；Polystyrene microsphere is first with dense
Degree is diluted to final concentration 0.8%w/v for the MES buffer of 50mmol/L, obtains the microsphere that pH is 5.5～6.5 molten
Liquid, the volume of solution is 50mL；
2, with MES buffer solution carbodiimides, the carbodiimides that concentration is 10mg/mL is obtained molten
Liquid；
3, with the weight of polystyrene microsphere contained in microspheres solution for counting, by every 10mg polystyrene microsphere add 50～
Carbodiimides solution is dropped in microspheres solution by the ratio of 60 μ L, drips 240 μ L, 8 DEG C of concussion activation 1h altogether, shakes speed
For 30r/min, obtain activated polystyrene microspheres solution；
4, being dissolved under the conditions of pH8.5～9.5 by CRP polyclonal antibody (commercially available, concentration >=10mg/mL), solvent is
The borate buffer solution of 50mmol/L, makes antibody final concentration at 0.5mg/mL, obtains CRP Anti-TNF-α liquid solution, the body of solution
Long-pending is 50mL, be stored in 8 DEG C standby；
5, CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody and microspheres quality
Ratio is 0.05～0.2:1, and whole dropping process continues 5min, and both mixed liquors are neutrality, afterwards 2 DEG C of concussion 24h, concussion speed
Degree is 50r/min, obtains C reactive protein immunity latex microsphere 2.
Embodiment 3
C reactive protein immunity latex microsphere preparation method provided by the present invention is as follows:
1, preparing the polystyrene microsphere of particle diameter 200nm, concentration is 10%, takes 5ml；Polystyrene microsphere is first with dense
Degree is diluted to final concentration 1.2%w/v for the MES buffer of 50mmol/L, obtains the microsphere that pH is 5.5～6.5 molten
Liquid, the volume of solution is 50mL；
2, with MES buffer solution carbodiimides, the carbodiimides that concentration is 10mg/mL is obtained molten
Liquid；
3, with the weight of polystyrene microsphere contained in microspheres solution for counting, by every 10mg polystyrene microsphere add 50～
Carbodiimides solution is dropped in microspheres solution by the ratio of 60 μ L, drips 300 μ L, 2 DEG C of concussion activation 1h altogether, shakes speed
For 50r/min, obtain activated polystyrene microspheres solution；
4, being dissolved under the conditions of pH8.5～9.5 by CRP polyclonal antibody (commercially available, concentration >=10mg/mL), solvent is
The borate buffer solution of 50mmol/L, makes antibody final concentration at 2mg/mL, obtains CRP Anti-TNF-α liquid solution, the volume of solution
50mL, be stored in 2 DEG C standby；
5, CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody and microspheres quality
Ratio is 0.05～0.2:1, and whole dropping process continues 7min, and both mixed liquors are neutrality, afterwards 0 DEG C of concussion 12h, concussion speed
Degree is 30r/min, obtains C reactive protein immunity latex microsphere 3.
Embodiment 4
With embodiment 1 preparation C reactive protein immunity latex microsphere 1 as raw material, prepare latex enhancing immune turbidimetry survey
Determine the test kit of C reactive protein.Its each component is as follows:
In C reactive protein detectable R1, each component and content are: 0.8%BSA, 0.5%NaCl, 200mM Tris (pH
=7.4), 3%PEG6000,0.05%NaN3；
In C reactive protein detectable R2 each component and content be: the C reactive protein of 0.4%BSA, 2mg/ml is exempted from
Epidemic disease latex microsphere, the glycine buffer (pH=7.4) of 25mmol/L, 0.05%NaN3；
In C reactive protein calibration object, each component and content are: the C-of 0.4%BSA, 0.5%NaCl, 20mg/L reacts egg
In vain, the glycine buffer of 25mmol/L, 0.05%NaN3。
Wherein, the preparation process of C reactive protein detectable R2 is as follows:
1, in the solution of the C reactive protein immunity latex microsphere 1 of 100mL, 50mL confining liquid is added, 25 DEG C of concussion 2h,
Then proceeding to 4 DEG C of concussion 24h, concussion speed is 50r/min, obtains latex solution；Confining liquid component is: the BSA of 3%w/v,
The glycine buffer (pH=8.5 does not contains preservative) of 100mmol/L.
2, latex solution is centrifuged 30～45min with 15000r/min, removes supernatant, is subsequently adding containing NaN3Composition
Stock solution to stock concentration, ultrasonic disperse and get final product.
Embodiment 5
With embodiment 1 preparation C reactive protein immunity latex microsphere 1 as raw material, prepare latex enhancing immune turbidimetry survey
Determine the test kit of C reactive protein.Its each component is as follows:
In C reactive protein detectable R1, each component and content are: 0.8%BSA, 0.9%NaCl, 100mM Tris (pH
=7.4), 1%PEG8000,0.1%Proclin 300；
In C reactive protein detectable R2 each component and content be: the C reactive protein of 0.8%BSA, 2mg/ml is exempted from
Epidemic disease latex microsphere, the glycine buffer (pH=7.4) of 50mmol/L, 0.1%Proclin 300；
In C reactive protein calibration object, each component and content are: the C-of 0.8%BSA, 0.9%NaCl, 20mg/L reacts egg
In vain, the glycine buffer of 50mmol/L, 0.1%Proclin300.
Wherein, the preparation process of C reactive protein detectable R2 is as follows:
1, in the solution of the C reactive protein immunity latex microsphere 1 of 100mL, 50mL confining liquid is added, 25 DEG C of concussion 2h,
Then proceeding to 4 DEG C of concussion 24h, concussion speed is 50r/min, obtains latex solution；Confining liquid component is: the BSA of 3%w/v,
The glycine buffer (pH=8.5 does not contains preservative) of 100mmol/L.
2, latex solution is centrifuged 30～45min with 15000r/min, removes supernatant, is subsequently adding containing Proclin
The stock solution of 300 compositions to stock concentration, ultrasonic disperse and get final product.
Embodiment 6
With embodiment 1 preparation C reactive protein immunity latex microsphere 1 as raw material, prepare latex enhancing immune turbidimetry survey
Determine the test kit of C reactive protein.Its each component is as follows:
In C reactive protein detectable R1, each component and content are: 1%BSA, 20mM disodiumedetate,
The NaCl of 1.8%, the phosphate buffer (PBS) of Triton X-100,10mM of 1%, the 4-hydroxyethyl piperazine second sulphur of 100mM
Acid-sodium hydrate buffer solution (Hepes), the PEG6000 of 1%, the thimerosal of 0.02%；
In C reactive protein detectable R2, the content of each component is: the glycerol of 5%, the C reactive protein of 1mg/ml are exempted from
Epidemic disease latex microsphere, 3-(N-the morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution (Mopso) of 10mM, the sulfur willow of 0.04%
Hydrargyrum；
In C reactive protein calibration object, the content of each component is: the C-of Triton X-100,20mg/L of 1% reacts egg
In vain, the borate buffer solution (BBS) of 200mM, the thimerosal of 0.04%；
Wherein, the preparation process of C reactive protein detectable R2 is as follows:
1, in the solution of the C reactive protein immunity latex microsphere 1 of 100mL, 50mL confining liquid is added, 25 DEG C of concussion 2h,
Then proceeding to 4 DEG C of concussion 24h, concussion speed is 50r/min, obtains latex solution；Containing glycerol and 3-(N-morpholine in confining liquid
Base)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution (Mopso).
2, latex solution is centrifuged 30～45min with 15000r/min, removes supernatant, is subsequently adding containing thimerosal composition
Stock solution to stock concentration 1mg/mL, ultrasonic disperse and get final product.
Embodiment 7
With embodiment 1 preparation C reactive protein immunity latex microsphere 1 as raw material, prepare latex enhancing immune turbidimetry survey
Determine the test kit of C reactive protein.Its each component is as follows:
In C reactive protein detectable R1, the content of each component is: 10mM disodiumedetate, 50mmol/L
Glycine buffer, the dextran sulfate of 6%, the NaN of 0.08%3；
In C reactive protein detectable R2, the content of each component is: NaCl, 3%BSA, 20mM ethylenediamine tetraacetic of 1.8%
Acetic acid disodium, the Triton X-100 of 1%, the glycerol of 10%, the C reactive protein immunity latex microsphere of 5mg/ml, 110mM
Tris, the Proclin 300 of 0.05%；
In C reactive protein calibration object, the content of each component is: the C reactive protein of NaCl, 20mg/L of 1%, 50mM
Phosphate buffer (PBS), the NaN of 0.02%3；
Wherein, the preparation process of C reactive protein detectable R2 is as follows:
1, in the solution of the C reactive protein immunity latex microsphere 1 of 100mL, 50mL confining liquid is added, 25 DEG C of concussion 2h,
Then proceeding to 4 DEG C of concussion 24h, concussion speed is 50r/min, obtains latex solution；Containing NaCl, BSA, ethylenediamine tetraacetic in confining liquid
Acetic acid disodium, Triton X-100, glycerol and Tris.
2, latex solution is centrifuged 30～45min with 15000r/min, removes supernatant, is subsequently adding containing Proclin
The stock solution of 300 compositions to stock concentration 5mg/mL, ultrasonic disperse and get final product.
Test example
One, C reactive protein immunity latex microsphere coupling efficiency and Activity determination
(1) prepare CRP polyclonal antibody and the polystyrene latex particles of 4 parts of particle diameter 120nm of 4 parts of 50mg, adopt for three parts
The preparation method provided by embodiment 1～3, prepares C reactive protein immunity latex microsphere 1～3；
(2) another part uses the preparation method of existing C reactive protein immunity latex microsphere, prepares comparison C reactive protein
Immunity latex microsphere；
The preparation method step of comparison C reactive protein immunity latex microsphere is as follows:
1) surface pH with polystyrene latex particles (particle diameter 120nm) 50mM of carboxylic group of 5ml is taken
The MES buffer of=6.0 is diluted to 50ml, final concentration of 1%w/v.
2) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride of 0.5%w/v and 0.5%w/v are added
Sulfur carries out activating latex, 37 DEG C of water-baths 1 hour for N hydroxysuccinimide.
3) centrifugal, remove supernatant (containing residual activatable crosslinking agent), by MES buffer solution for cleaning one time, add
The CRP polyclonal antibody of 50mg carries out cross-linking reaction, 37 DEG C of water-baths 4 hours, to obtain final product.
(3) to C reactive protein immunity latex microsphere 1～3 and comparison C reactive protein immunity latex microsphere centrifugal point respectively
From, to collect the antibody of non-coupling in supernatant, more above-mentioned microsphere is separately added into confining liquid, closes latex surface residual and live
Change site.
Detection test for above-mentioned immunity latex beads is as follows:
1, by the antibody of the non-coupling of centrifugation before closing, use BCA determination of protein concentration method to obtain non-coupling and resist
The quality of body, can obtain coupling efficiency, obtain the preparation method coupling efficiency of embodiment 1～3 be respectively 93%, 88%,
90%, and the coupling efficiency of existing preparation method is 78.4%.
2, detected the antibody titer of the above-mentioned immune latex microsphere closed by enzyme linked immunosorbent assay, calculate C-
The activity of reactive protein immunity latex microsphere 1～3 is respectively 1:3200,1:1600 and 1:3200, comparison C reactive protein immunity
The activity of latex microsphere is 1:400.
Above-mentioned test proves that the inventive method both ensure that the coupling efficiency of antibody, simultaneously high degree reduce activation
The destruction of agent antagonist activity.
Two, test kit application on protein analyzer
1, the C reactive protein calibration object in embodiment 4 is configured to 5 Concentraton gradient and a blank, uses thoroughly
Penetrating and detect than turbid protein analyzer, detecting step is: each calibration object of 10ul is added separately to the C-equipped with 400ul anti-
Answering in the cuvette of protein assay reagent R1 reagent and stirrer, cuvette is put into detection mouth, instrument automatically detects colorimetric
Cup, adds 80ul C reactive protein detectable R2 reagent and joins in cuvette, instrument automatic stirring mixing 10s, measures
Absorbance OD1, after reaction 1min, measures absorbance OD2, and instrument calculates the difference of absorbance automatically: △ OD=OD2-OD1.Often
Individual Concentration Testing 5 times, the meansigma methods of the △ OD of each concentration calibration product recorded makees vertical coordinate, and corresponding concentration makees abscissa, system
Making concentration-absorbance difference calibration curve, being calculated curvilinear equation by excel is Y=0.0331X+0.0032.
2, be then placed in the blood plasma of product to be tested 1 people, measure and obtain its absorbance difference △ OD, then according to above-mentioned concentration-
Absorbance difference calibration curve, is calculated the concentration of C reactive protein in product to be tested 1.
3, diluting 100 times with a human blood product to be tested 1, then use the test kit of embodiment 4 to detect, mensuration obtains
Its absorbance difference, then according to above-mentioned concentration-absorbance difference calibration curve, is calculated human blood product to be tested 1 and dilutes 100 times
After, the wherein concentration of C reactive protein.
Repeating above-mentioned human blood product to be tested 1 and detect 5 times, the result of gained see table 1
In table 1 human blood product to be tested, CRP Concentration Testing repeats test
4, use the test kit of embodiment 5～7 to detect again with a human blood product to be tested 1, measure and obtain its absorbance
Difference, then according to the concentration-absorbance difference calibration curve of each test kit, is calculated C reactive protein in product to be tested 1
Concentration, be repeated 5 times and average, and calculate Cv%, the result of gained see table 2.
The different test kit of table 1 is for the detection test of same human blood product to be tested
| Test kit embodiment is numbered | Embodiment 4 | Embodiment 5 | Embodiment 6 | Embodiment 7 |
| CRP concentration in human blood product to be tested | 17.27mg/L | 17.25mg/L | 17.40mg/L | 17.18mg/L |
| Cv% | 0.57% | 0.61% | 3.04% | 3.56% |
Above-mentioned test shows, the test kit range of linearity of the present invention is 0.1～20mg/L, highly sensitive, the most accurate, heavy
Renaturation is good, stable in properties.
Claims (10)
1. a C reactive protein immunity latex microsphere preparation method, step is as follows:
1) preparing the polystyrene microsphere of particle size range 50～200nm, first polystyrene microsphere is 50mmol/L's by concentration
MES buffer is diluted to final concentration 0.8～1.2%w/v, obtains the microspheres solution that pH is 5.5～6.5；
2) with MES buffer solution carbodiimides, the carbodiimides solution that concentration is 10mg/mL is obtained；
3) with the weight of polystyrene microsphere contained in microspheres solution for counting, 50～60 μ L are added by every 10mg polystyrene microsphere
Ratio carbodiimides solution is dropped in microspheres solution, 2～8 DEG C concussion activation 1h, concussion speed be 30～50r/
Min, obtains activated polystyrene microspheres solution；
4) being dissolved under the conditions of pH8.5～9.5 by the CRP polyclonal antibody of concentration >=10mg/mL, solvent is the boron of 50mmol/L
Phthalate buffer, makes antibody final concentration between 0.5～2mg/mL, obtains CRP Anti-TNF-α liquid solution, be stored in 2～8 DEG C standby
With；
5) CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody with microspheres quality ratio is
0.05～0.2:1, whole dropping process continues 4～7min, makes activated polystyrene microsphere couple with the amino on antibody, both
Mixed liquor be neutrality, afterwards 0～4 DEG C concussion 12～24h, concussion speed be 30～50r/min, obtain C reactive protein immunity
Latex microsphere.
C reactive protein immunity latex microsphere preparation method the most according to claim 1, it is characterised in that: described step 3)
Middle concussion activation condition is: 4 DEG C of concussion activation 1h, concussion speed is 50r/min.
C reactive protein immunity latex microsphere preparation method the most according to claim 1 and 2, it is characterised in that: described step
Rapid 5), when making activated polystyrene microsphere couple with the amino on antibody in, 4 DEG C of concussion 16h, concussion speed is 50r/min.
4. a latex enhancing immune turbidimetry C reactive protein measures test kit, it is characterised in that: include that C reactive protein is examined
Test agent R1 and C reactive protein detectable R2；
In described C reactive protein detectable R1, each component and content are: 0.01～13% stabilizer 1,10～200mM slow
Rush the preservative 1 of the coagulant 1,0.02～0.1% of liquid 1,1～6%；
In described C reactive protein detectable R2, each component and content are: 0.01～23% stabilizer 2,1～the C-of 5mg/ml
The preservative 2 of the buffer 2,0.02～0.1% of reactive protein immunity latex microsphere, 10～200mM；
The preparation method of described C reactive protein detectable R2 is:
1) preparing the polystyrene microsphere of particle size range 50～200nm, first polystyrene microsphere is 50mmol/L's by concentration
MES buffer is diluted to final concentration 0.8～1.2%w/v, obtains the microspheres solution that pH is 5.5～6.5；
2) with MES buffer solution carbodiimides, the carbodiimides solution that concentration is 10mg/mL is obtained,
3) with the weight of polystyrene microsphere contained in microspheres solution for counting, 50～60 μ L are added by every 10mg polystyrene microsphere
Ratio carbodiimides solution is dropped in microspheres solution, 2～8 DEG C concussion activation 1h, concussion speed be 30～50r/
Min, obtains activated polystyrene microspheres solution；
4) being dissolved under the conditions of pH8.5～9.5 by the CRP polyclonal antibody of concentration >=10mg/mL, solvent is the boron of 50mmol/L
Phthalate buffer, makes antibody final concentration between 0.5～2mg/mL, obtains CRP Anti-TNF-α liquid solution, be stored in 2～8 DEG C standby
With；
5) CRP Anti-TNF-α liquid solution is dropped in activated polystyrene microspheres solution, until antibody with microspheres quality ratio is
0.05～0.2:1, whole dropping process continues 4～7min, makes activated polystyrene microsphere couple with the amino on antibody, both
Mixed liquor be neutrality, afterwards 0～4 DEG C concussion 12～24h, concussion speed be 30～50r/min, obtain C reactive protein immunity
Latex microsphere；
6) adding in C reactive protein immunity latex microsphere containing stabilizer 2 and the confining liquid of buffer 2, concussion obtains latex
Solution；
7) latex solution is centrifuged, and removes supernatant, the activator wherein remained with removing, is subsequently adding preservative 2 to stock concentration,
Ultrasonic disperse, to obtain final product.
Latex enhancing immune turbidimetry C reactive protein the most according to claim 4 measures test kit, it is characterised in that:
Described stabilizer 1 is selected from 0.5～the ethylenediaminetetraacetic acid two of the BSA of the sodium chloride of 1.8%, 0.1～1%, 1～20mM
One or more in the Triton X-100 of sodium, 0.01～1%；
Described buffer 1 delays selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-hydrochloric acid
Rush liquid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, phosphate buffer, borate buffer solution, sweet ammonia
One or more in acid buffer；
The described coagulant 1 one in polyethylene glycol 6000, PEG 8000 or dextran sulfate；
The described preservative 1 one in sodium azide, Proclin300 or thimerosal；
Described stabilizer 2 selected from 0.5～the BSA of the sodium chloride of 1.8%, 0.2～3%, 1～20mM disodiumedetate,
0.01～1% Triton X-100,1～10% glycerol in one or more；
Described buffer 2 delays selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-hydrochloric acid
Rush liquid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, phosphate buffer, borate buffer solution, sweet ammonia
One or more in acid buffer；
The described preservative 2 one in sodium azide, Proclin300 or thimerosal.
Latex enhancing immune turbidimetry C reactive protein the most according to claim 4 measures test kit, it is characterised in that:
In described C reactive protein detectable R1, each component and content are: 0.4～the BSA of 0.8%, 0.5～0.9%
100～200mM Tris, 1～the PEG of 3% of NaCl, pH7.4, the NaN of 0.05%3；
In described C reactive protein detectable R2, each component and content are: 0.4～the C-of the BSA of 0.8%, 1～5mg/ml is anti-
Answer protein immunization latex microsphere, the 25-50mmol/L glycine buffer of pH7.4, the NaN of 0.05%3；
Or the NaN of 0.05% in described C reactive protein detectable R1 and described C reactive protein detectable R23Replace with
The Proclin 300 of 0.1%.
7. measuring test kit according to the latex enhancing immune turbidimetry C reactive protein described in any one of claim 4～6, it is special
Levying and be: described test kit also includes C reactive protein calibration object, its each component and content is: 0.01～13% calibration object steady
Determine the calibration object preservative of calibration object buffer, 0.02～0.1% of agent, the C reactive protein of 20mg/L, 10～200mM.
Latex enhancing immune turbidimetry C reactive protein the most according to claim 7 measures test kit, it is characterised in that:
Described calibration object stabilizer is selected from 0.5～the ethylenediamine tetrem of the BSA of the sodium chloride of 1.8%, 0.1～1%, 1～20mM
One or more in the Triton X-100 of acid disodium, 0.01～1%；
Described calibration object buffer is selected from 4-hydroxyethyl piperazine ethanesulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-salt
Acid buffer, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, phosphate buffer, borate buffer solution,
One or more in glycine buffer；
Described calibration object preservative one in sodium azide, Proclin300 or thimerosal.
Latex enhancing immune turbidimetry C reactive protein the most according to claim 7 measures test kit, it is characterised in that:
In described C reactive protein calibration object, each component and content are: 0.4～the NaCl of the BSA of 0.8%, 0.5～0.9%,
The C reactive protein of 20mg/L, the 25-50mmol/L glycine buffer of pH7.4, the NaN of 0.05%3；
Or each component and content are in described C reactive protein calibration object: 0.4～the NaCl of the BSA of 0.8%, 0.5～0.9%,
The C reactive protein of 20mg/L, the 25-50mmol/L glycine buffer of pH7.4, the Proclin 300 of 0.1%.
Latex enhancing immune turbidimetry C reactive protein the most according to claim 4 measures test kit, it is characterised in that:
Described step 6) in concussion condition be: 25 DEG C concussion 2h, then proceed to 4 DEG C concussion 24h, concussion speed is 50r/min.
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| CN108709988A (en) * | 2018-04-10 | 2018-10-26 | 波音特生物科技（南京）有限公司 | A kind of novel antigens antibody response sensitizer |
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| CN109580936A (en) * | 2018-12-04 | 2019-04-05 | 蓝怡科技集团股份有限公司 | A kind of microballoon reagent and its preparation method and application |
| CN109580936B (en) * | 2018-12-04 | 2022-03-04 | 蓝怡科技集团股份有限公司 | Microsphere reagent and preparation method and application thereof |
| CN109521200A (en) * | 2018-12-29 | 2019-03-26 | 南京新耀医疗技术有限公司 | It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma |
| CN109856067A (en) * | 2019-01-11 | 2019-06-07 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | A kind of c reactive protein assay kit |
| CN109975555A (en) * | 2019-03-11 | 2019-07-05 | 杭州利安生物科技有限公司 | A kind of reagent, method and high thermal stability c reactive protein kit improving c reactive protein kit thermal stability |
| CN109975555B (en) * | 2019-03-11 | 2022-03-08 | 杭州利安生物科技有限公司 | Reagent and method for improving thermal stability of C-reactive protein kit and high-thermal stability C-reactive protein kit |
| CN111751544A (en) * | 2019-03-28 | 2020-10-09 | 北京九强生物技术股份有限公司 | Kit for detecting soluble growth hormone expression gene 2 protein |
| CN111751544B (en) * | 2019-03-28 | 2023-06-09 | 北京九强生物技术股份有限公司 | Kit for detecting soluble growth hormone expressed gene 2 protein |
| CN110031636A (en) * | 2019-05-21 | 2019-07-19 | 贵州盛世康生物科技有限公司 | A kind of c reactive protein measurement reagent and preparation method thereof |
| CN110646618A (en) * | 2019-09-17 | 2020-01-03 | 广州市伊川生物科技有限公司 | C-reactive protein assay kit and preparation method and application thereof |
| CN110646618B (en) * | 2019-09-17 | 2022-11-01 | 广州市伊川生物科技有限公司 | C-reactive protein assay kit and preparation method and application thereof |
| CN111879942A (en) * | 2020-07-06 | 2020-11-03 | 武汉生之源生物科技股份有限公司 | Full-range C-reactive protein latex enhanced immunoturbidimetric assay kit |
| CN112798790A (en) * | 2020-12-24 | 2021-05-14 | 深圳市科曼医疗设备有限公司 | Kit for determining concentration of C-reactive protein and preparation method thereof |
| CN112763724A (en) * | 2020-12-26 | 2021-05-07 | 北京安图生物工程有限公司 | Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine |
| CN113671192A (en) * | 2021-07-29 | 2021-11-19 | 广西康柏莱科技有限公司 | CRP latex enhanced kit and preparation method and application thereof |
| CN114047338A (en) * | 2021-11-10 | 2022-02-15 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
| CN114047338B (en) * | 2021-11-10 | 2024-03-12 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
| CN114137223A (en) * | 2021-11-25 | 2022-03-04 | 安徽大千生物工程有限公司 | Latex enhanced immunoturbidimetry assay kit for rapidly assaying IGF-1 and preparation and use methods thereof |
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