CN109324184A - A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit and preparation method thereof - Google Patents
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit and preparation method thereof Download PDFInfo
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- CN109324184A CN109324184A CN201811483460.9A CN201811483460A CN109324184A CN 109324184 A CN109324184 A CN 109324184A CN 201811483460 A CN201811483460 A CN 201811483460A CN 109324184 A CN109324184 A CN 109324184A
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- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 61
- 239000000427 antigen Substances 0.000 title claims abstract description 54
- 102000036639 antigens Human genes 0.000 title claims abstract description 54
- 108091007433 antigens Proteins 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 45
- 208000006454 hepatitis Diseases 0.000 title claims abstract description 41
- 231100000283 hepatitis Toxicity 0.000 title claims abstract description 40
- 241000700605 Viruses Species 0.000 title claims abstract description 38
- 239000004005 microsphere Substances 0.000 claims abstract description 73
- 239000007788 liquid Substances 0.000 claims abstract description 51
- 238000007865 diluting Methods 0.000 claims abstract description 50
- 241001494479 Pecora Species 0.000 claims abstract description 45
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 19
- 241000711549 Hepacivirus C Species 0.000 claims description 105
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 23
- 239000000872 buffer Substances 0.000 claims description 23
- 238000012360 testing method Methods 0.000 claims description 21
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 16
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 9
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 7
- 229910017604 nitric acid Inorganic materials 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000007689 inspection Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 2
- -1 aminopropyl Chemical group 0.000 claims 1
- 239000012467 final product Substances 0.000 claims 1
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 208000005176 Hepatitis C Diseases 0.000 description 10
- 230000003612 virological effect Effects 0.000 description 8
- 229920003043 Cellulose fiber Polymers 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108700039791 Hepatitis C virus nucleocapsid Proteins 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention discloses a kind of C hepatitis virus antigen fluorescence immune chromatography detection kits and preparation method thereof, which includes following components: antibody of HCV 1, antibody of HCV 2, sheep anti mouse capture antibody, phosphate buffer and the sample diluting liquid of fluorescent microsphere label；The preparation method of the detection kit is the following steps are included: prepare sample diluting liquid；Prepare the antibody of HCV 1 of fluorescent microsphere label；Preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label；Preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody；Standard items assemble to get fluorescence immune chromatography detection kit；The detection kit is, it can be achieved that the quantitative detection for carrying out C hepatitis virus antigen of scene quickly, easy；Detection kit of the invention has preferable detection sensitivity, preci-sion and accuracy.
Description
Technical field
The present invention relates to technical field of immune assay, specifically a kind of C hepatitis virus antigen fluorescence immune chromatography
Detection kit and preparation method thereof.
Background technique
Hepatitis C is caused by Hepatitis C Virus (HCV), and hepatitis C is through blood born.China has 40,000,000 at present
Above hepatitis C virus infection, wherein 70% hepatitis patient evolution is chronic hepatitis, 20% development is cirrhosis,
12% development is liver cancer, and harm is serious.So early finding, early diagnosis, early treatment have great importance for hepatitis patient.
Hepatitis C Virus early detection method common at present includes detection of nucleic acids and antigen detection.Detection of nucleic acids is being set
It is standby and technically more demanding, it is easy false sun, so being difficult to promote in routine and laboratories.Antigen detecting agent mostly with
Based on enzyme linked immunological, chemiluminescence or colloidal gold, as the ELISA kit of Ortho company of the U.S., Hunan scape are limited up to pharmacy
ELISA detection kit, the chemiluminescence Hepatitis C virus core antigen of Abbott (HCV Core Ag) detection kit of company
Deng, but enzyme linked immunological and chemiluminescence are required in Spot detection labs, and colloid gold reagent can not be quantified.
Summary of the invention
The purpose of the present invention is to provide a kind of C hepatitis virus antigen fluorescence immune chromatography detection kit and its systems
Preparation Method, to solve the problems of the prior art.
To achieve the above object, the invention provides the following technical scheme:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
As optimization, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder
15g/L, Triton X-1003ml/L and SDS 150g/L.
As optimization, the concentration of Tris-HCl buffer is 1mol/L, pH 8.0.
As optimization, the concentration of phosphate buffer is 0.01mol/L, pH 7.2-7.4.
A kind of preparation method of C hepatitis virus antigen fluorescence immune chromatography detection kit, fluorescence immune chromatography inspection
The preparation method of test agent box the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, CHAPS dry powder, Triton X-100 and SDS to be added and hold
It is uniformly mixed in device, constant volume is to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: fluorescent microsphere is taken, with 1- ethyl-(3- dimethyl
Aminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activation, Hepatitis C Virus is then added thereto
Antibody 1, on label after sealed again with sealing fluid, be centrifuged, save the antibody of HCV 1 marked to get fluorescent microsphere, it is standby
With；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then coated in load
On body 1, it is lyophilized to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody: by Hepatitis C Virus
Antibody 2 and sheep anti mouse capture antibody are diluted with phosphate buffer respectively, then draw it onto carrier 2 with film instrument is drawn,
Drying is to get the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
As optimization, a kind of preparation method of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence
The preparation method of immunochromatographytest test kit the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Triton X-100 and 150g SDS are added to the container uniformly mixed, then are settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
On the carrier 1, with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.0-1.5 μ l/cm onto carrier 2, is dried overnight to get antibody of HCV 2 is coated with
With the carrier 2 of sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
As optimization, the volume ratio of fluorescent microsphere marks in step (3) antibody of HCV 1 and sample diluting liquid
It is 1: 50.
As optimization, carrier 1 is glass in step (3), and carrier 2 is nitric acid cellulose fiber film (NC film) in the step (4).
The application method of C hepatitis virus antigen fluorescence immune chromatography detection kit of the invention detects program
Are as follows:
(A) sample process: 200ul sample diluting liquid and 100ul serum sample is added in 1.5ml Ep is managed, is uniformly mixed
Afterwards, 30min is handled at 56 DEG C；
(B) it is loaded: taking out a fluorescence immune chromatography test paper card, draw 80ul step (A) resulting sample, be added drop-wise to glimmering
In the well of light immune chromatography test card, 15min is reacted at room temperature；
(C) readings: according to T/C's as a result, dry type fluorescence immunoassay readings instrument directly displays out sample results.
Compared with prior art, the beneficial effects of the present invention are: a kind of C hepatitis virus antigen fluorescence immunoassay of the present invention
Detection kit is chromatographed, it can be achieved that the quantitative detection for carrying out C hepatitis virus antigen of scene quickly, easy；Examination of the invention
Whether the quantitative detection of C hepatitis virus antigen of the agent box suitable for human serum, blood plasma, auxiliary diagnosis infect hepatitis C
Virus is suitable for clinical and on-site test；The experimental results showed that C hepatitis virus antigen fluorescence immune chromatography of the invention is examined
Test agent box has preferable detection sensitivity, and sensitivity can achieve 20pg/ml；With preferable detection precision, cV (becomes
Different coefficient) value be 8.8%；With preferable accuracy in detection, testing result accuracy is 100%.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
Wherein, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder 15g/
L, Triton X-1003ml/L and SDS 150g/L；The concentration of Tris-HCl buffer is 1mol/L, pH 8.0；Phosphate is slow
The concentration of fliud flushing is 0.01mol/L, pH 7.2.
The preparation method of the above-mentioned fluorescence immune chromatography detection kit the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Tri ton X-100 and 150g SDS be added to the container it is uniformly mixed, then with deionized water be settled to 1L to get sample dilution
Liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
It is glimmering with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label on glass
The antibody of HCV 1 of light microballoon label and the volume ratio of sample diluting liquid are 1: 50；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.0 μ l/cm onto nitric acid cellulose fiber film, is dried overnight anti-to get Hepatitis C Virus is coated with
The carrier 2 of body 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
Embodiment 2:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
Wherein, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100m1/L, CHAPS dry powder 15g/
L, Triton X-1003ml/L and SDS 150g/L；The concentration of Tris-HCl buffer is 1mol/L, pH 8.0；Phosphate is slow
The concentration of fliud flushing is 0.01mol/L, pH 7.3.
The preparation method of above-mentioned C hepatitis virus antigen fluorescence immune chromatography detection kit, fluorescence immune chromatography inspection
The preparation method of test agent box the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Triton X-100 and 150g SDS are added to the container uniformly mixed, then are settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
It is glimmering with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label on glass
The antibody of HCV 1 of light microballoon label and the volume ratio of sample diluting liquid are 1: 50；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.1 μ l/cm onto nitric acid cellulose fiber film, is dried overnight anti-to get Hepatitis C Virus is coated with
The carrier 2 of body 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
Embodiment 3:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
Wherein, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder 15g/
L, Triton X-1003ml/L and SDS 150g/L；The concentration of Tris-HCl buffer is 1mol/L, pH 8.0；Phosphate is slow
The concentration of fliud flushing is 0.01mol/L, pH 7.4.
The preparation method of above-mentioned C hepatitis virus antigen fluorescence immune chromatography detection kit, fluorescence immune chromatography inspection
The preparation method of test agent box the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Triton X-100 and 150g SDS are added to the container uniformly mixed, then are settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
It is glimmering with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label on glass
The antibody of HCV 1 of light microballoon label and the volume ratio of sample diluting liquid are 1: 50；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.2 μ l/cm onto nitric acid cellulose fiber film, is dried overnight anti-to get Hepatitis C Virus is coated with
The carrier 2 of body 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
Embodiment 4:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
Wherein, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder 15g/
L, Triton X-1003ml/L and SDS 150g/L；The concentration of Tris-HCl buffer is 1mol/L, pH 8.0；Phosphate is slow
The concentration of fliud flushing is 0.01mol/L, pH 7.2.
The preparation method of above-mentioned C hepatitis virus antigen fluorescence immune chromatography detection kit, fluorescence immune chromatography inspection
The preparation method of test agent box the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Triton X-100 and 150g SDS are added to the container uniformly mixed, then are settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
It is glimmering with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label on glass
The antibody of HCV 1 of light microballoon label and the volume ratio of sample diluting liquid are 1: 50；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.4 μ l/cm onto nitric acid cellulose fiber film, is dried overnight anti-to get Hepatitis C Virus is coated with
The carrier 2 of body 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
Embodiment 5:
A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, the fluorescence immune chromatography detection kit packet
Include following components: antibody of HCV 1, antibody of HCV 2, the sheep anti mouse capture antibody of fluorescent microsphere label
(nature controlling line antibody), phosphate buffer and sample diluting liquid.
Wherein, sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder 15g/
L, Triton X-1003ml/L and SDS 150g/L；The concentration of Tris-HCl buffer is 1mol/L, pH 8.0；Phosphate is slow
The concentration of fliud flushing is 0.01mol/L, pH 7.4.
The preparation method of above-mentioned C hepatitis virus antigen fluorescence immune chromatography detection kit, fluorescence immune chromatography inspection
The preparation method of test agent box the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml of 100ml 1mol/L
Triton X-100 and 150g SDS are added to the container uniformly mixed, then are settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3-
Dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, 160 are then added thereto
The antibody of HCV 1 of μ g reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4
DEG C the antibody of HCV 1 that marks to get fluorescent microsphere is saved, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: will be obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of fluorescent microsphere label is diluted, and is then uniformly applied
It is glimmering with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label on glass
The antibody of HCV 1 of light microballoon label and the volume ratio of sample diluting liquid are 1: 50；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody (nature controlling line antibody): will
Antibody of HCV 2 and sheep anti mouse capture antibody are diluted to 1mg/ml with phosphate buffer respectively, then use it and draw
Film instrument is successively drawn with the discharge rate of 1.5 μ l/cm onto nitric acid cellulose fiber film, is dried overnight anti-to get Hepatitis C Virus is coated with
The carrier 2 of body 2 and sheep anti mouse capture antibody；
(5) it assembles: the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Hepatitis C is assembled into the carrier 2 for being coated with antibody of HCV 2 and sheep anti mouse capture antibody obtained by step (4)
Viral antigen fluorescence immune chromatography detection kit.
Effect example 1:
The detection sensitivity of C hepatitis virus antigen fluorescence immune chromatography detection kit of the invention is tested,
Obtaining concentration with 20% NBS (N-bromosuccinimide) dilution HCV (Hepatitis C Virus) recombinant antigen is respectively 5ng/
The sample of ml, 1ng/ml, 500pg/ml, 100pg/ml and 20pg/ml utilize the obtained hepatitis C of the embodiment of the present invention 1
Viral antigen fluorescence immune chromatography detection kit detects above-mentioned sample, and the results are shown in Table 1 for test experience.
The sensitivity test result of 1 kit of table
| Concentration of specimens | Fluorescent value |
| 5ng/ml | 351500 |
| 1ng/ml | 110098 |
| 500pg/ml | 59609 |
| 100pg/m1 | 12738 |
| 20pg/ml | 2610 |
From table 1 it follows that the obtained C hepatitis virus antigen fluorescence immune chromatography of the embodiment of the present invention 1 detects
Kit has preferable detection sensitivity, and sensitivity can achieve 20pg/ml.
Effect example 2:
The detection precision of C hepatitis virus antigen fluorescence immune chromatography detection kit of the invention is tested,
HCV recombinant antigen sample based on 100pg/ml concentration, with the obtained C hepatitis virus antigen fluorescence of the embodiment of the present invention 2
Immunochromatographytest test kit be repeatedly detected 10 times to above-mentioned sample, and the results are shown in Table 2 for test experience.
The precision test result of 2 kit of table
| Number | Concentration of specimens | Fluorescent value |
| 1 | 100pg/ml | 12738 |
| 2 | 100pg/ml | 11002 |
| 3 | 100pg/ml | 13201 |
| 4 | 100pg/ml | 10981 |
| 5 | 100pg/ml | 12126 |
| 6 | 100pg/ml | 11765 |
| 7 | 100pg/ml | 9865 |
| 8 | 100pg/ml | 11463 |
| 9 | 100pg/ml | 12721 |
| 10 | 100pg/ml | 10985 |
| CV | 8.8% |
From Table 2, it can be seen that the obtained C hepatitis virus antigen fluorescence immune chromatography of the embodiment of the present invention 2 detects
Kit has preferable detection precision, and CV (coefficient of variation) value is 8.8%.
Effect example 3:
The accuracy in detection of C hepatitis virus antigen fluorescence immune chromatography detection kit of the invention is tested,
Based on 5 parts of positive samples (P1-P5) and 5 parts of negative samples (N1-N5), with the obtained hepatitis C virus of the embodiment of the present invention 3
Malicious antigen fluorescence immune chromatography detection kit detects above-mentioned sample, and the results are shown in Table 3 for test experience.
The accuracy test result of 3 kit of table
| Sample | Fluorescent value |
| P1 | 3896 |
| P2 | 12654 |
| P3 | 5873 |
| P4 | 25985 |
| P5 | 16901 |
| N1 | 984 |
| N2 | 760 |
| N3 | 532 |
| N4 | 609 |
| N5 | 674 |
From table 3 it is observed that the obtained C hepatitis virus antigen fluorescence immune chromatography of the embodiment of the present invention 3 detects
Kit has preferable accuracy in detection, and testing result accuracy is 100%.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any label in claim should not be construed as limiting the claims involved.
Claims (8)
1. a kind of C hepatitis virus antigen fluorescence immune chromatography detection kit, which is characterized in that fluorescence immune chromatography inspection
Test agent box includes following components: antibody of HCV 1, the antibody of HCV 2, sheep anti mouse of fluorescent microsphere label
Capture antibody, phosphate buffer and sample diluting liquid.
2. a kind of C hepatitis virus antigen fluorescence immune chromatography detection kit according to claim 1, feature exist
In the sample diluting liquid includes the component of following concentration: Tris-HCl buffer 100ml/L, CHAPS dry powder 15g/L,
Triton X-1003ml/L and SDS 150g/L.
3. a kind of C hepatitis virus antigen fluorescence immune chromatography detection kit according to claim 2, feature exist
In: the concentration of the Tris-HCl buffer is 1mol/L, pH 8.0.
4. according to claim 1 or a kind of C hepatitis virus antigen fluorescence immune chromatography detection reagent described in any one of 3
Box, it is characterised in that: the concentration of the phosphate buffer is 0.01mol/L, pH 7.2-7.4.
5. a kind of preparation method of C hepatitis virus antigen fluorescence immune chromatography detection kit, which is characterized in that the fluorescence
The preparation method of immunochromatographytest test kit the following steps are included:
(1) it prepares sample diluting liquid: Tris-HCl buffer, CHAPS dry powder, Triton X-100 and SDS being taken to be added to the container
It is uniformly mixed, constant volume is to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: fluorescent microsphere is taken, with 1- ethyl-(3- dimethylamino
Propyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activation, antibody of HCV is then added thereto
1, on label after sealed again with sealing fluid, be centrifuged, save the antibody of HCV 1 marked to get fluorescent microsphere, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: by fluorescence obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of microballoon label is diluted, and is then coated in carrier 1
On, it is lyophilized to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody: by antibody of HCV 2
It is diluted with phosphate buffer with sheep anti mouse capture antibody, then draws it onto carrier 2 with film instrument is drawn respectively, it is dry,
It is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody to obtain the final product；
(5) it assembles: the carrier 1 and step of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Suddenly the carrier 2 that antibody of HCV 2 and sheep anti mouse capture antibody are coated with obtained by (4) is assembled into Hepatitis C Virus
Antigen fluorescence immune chromatography detection kit.
6. a kind of preparation side of C hepatitis virus antigen fluorescence immune chromatography detection kit according to claim 5
Method, which is characterized in that the preparation method of the fluorescence immune chromatography detection kit the following steps are included:
(1) it prepares sample diluting liquid: taking Tris-HCl buffer, the 15g CHAPS dry powder, 3ml Triton of 100ml 1mol/L
X-100 and 150g SDS is added to the container uniformly mixed, then is settled to 1L with deionized water to get sample diluting liquid；
(2) it prepares the antibody of HCV 1 of fluorescent microsphere label: the fluorescent microsphere of 100 μ l is taken, with 1- ethyl-(3- diformazan
Base aminopropyl) carbodiimide hydrochloride and N- hydroxy thiosuccinimide activated, then it is added 160g's thereto
Antibody of HCV 1 reacts 5h at room temperature, then seals 1h with sealing fluid, and centrifugation is placed in suspension, 4 DEG C of guarantors
It deposits to get the antibody of HCV 1 of fluorescent microsphere label, it is spare；
(3) preparation is coated with the carrier 1 of the antibody of HCV 1 of fluorescent microsphere label: by fluorescence obtained by step (2)
The sample diluting liquid obtained by step (1) of antibody of HCV 1 of microballoon label is diluted, and is then evenly coated in load
On body 1, with freeze dryer freeze-drying overnight to get the carrier 1 for the antibody of HCV 1 for being coated with fluorescent microsphere label；
(4) preparation is coated with the carrier 2 of antibody of HCV 2 and sheep anti mouse capture antibody: by antibody of HCV 2
It is diluted to 1mg/ml with phosphate buffer respectively with sheep anti mouse capture antibody, then it is used and draws film instrument successively with 1.0-1.5 μ
The discharge rate of l/cm is drawn to being dried overnight on carrier 2 to be coated with antibody of HCV 2 and sheep anti mouse capture antibody
Carrier 2；
(5) it assembles: the carrier 1 and step of the antibody of HCV 1 of fluorescent microsphere label will be coated with obtained by step (3)
Suddenly the carrier 2 that antibody of HCV 2 and sheep anti mouse capture antibody are coated with obtained by (4) is assembled into Hepatitis C Virus
Antigen fluorescence immune chromatography detection kit.
7. a kind of preparation side of C hepatitis virus antigen fluorescence immune chromatography detection kit according to claim 6
Method, it is characterised in that: the volume of fluorescent microsphere marks in the step (3) antibody of HCV 1 and sample diluting liquid
Than being 1: 50.
8. a kind of C hepatitis virus antigen fluorescence immune chromatography detection reagent according to any one of claims 5 to 7
The preparation method of box, it is characterised in that: carrier 1 is glass in the step (3), and carrier 2 is that nitric acid element is fine in the step (4)
Tie up film.
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Application publication date: 20190212 |