CN106093402A - Hepatitis C virus antigen-antibody combined detection kit - Google Patents
Hepatitis C virus antigen-antibody combined detection kit Download PDFInfo
- Publication number
- CN106093402A CN106093402A CN201610378119.1A CN201610378119A CN106093402A CN 106093402 A CN106093402 A CN 106093402A CN 201610378119 A CN201610378119 A CN 201610378119A CN 106093402 A CN106093402 A CN 106093402A
- Authority
- CN
- China
- Prior art keywords
- hcv
- diluting liquid
- sample diluting
- hepatitis
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 55
- 241000711549 Hepacivirus C Species 0.000 title claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 112
- 238000007865 diluting Methods 0.000 claims abstract description 98
- 239000000427 antigen Substances 0.000 claims abstract description 52
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- 239000003398 denaturant Substances 0.000 claims abstract description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 30
- 230000003213 activating effect Effects 0.000 claims abstract description 27
- 238000002372 labelling Methods 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 239000003446 ligand Substances 0.000 claims abstract description 21
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 22
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 20
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 16
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 13
- 229960002685 biotin Drugs 0.000 claims description 11
- 235000020958 biotin Nutrition 0.000 claims description 11
- 239000011616 biotin Substances 0.000 claims description 11
- 239000004202 carbamide Substances 0.000 claims description 10
- 235000013877 carbamide Nutrition 0.000 claims description 10
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 9
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 9
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 9
- 108090001008 Avidin Proteins 0.000 claims description 8
- -1 dodecyl alanine Chemical compound 0.000 claims description 8
- 150000002596 lactones Chemical class 0.000 claims description 8
- YWFWDNVOPHGWMX-UHFFFAOYSA-N n,n-dimethyldodecan-1-amine Chemical group CCCCCCCCCCCCN(C)C YWFWDNVOPHGWMX-UHFFFAOYSA-N 0.000 claims description 8
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 7
- 239000002585 base Substances 0.000 claims description 7
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 7
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 7
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 6
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 6
- 235000004279 alanine Nutrition 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 229960000800 cetrimonium bromide Drugs 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- 239000013504 Triton X-100 Substances 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 4
- QEDXSHCYPROEOK-UHFFFAOYSA-N 3-phosphanylpropanoic acid Chemical compound OC(=O)CCP QEDXSHCYPROEOK-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 3
- 229960003180 glutathione Drugs 0.000 claims description 3
- 235000003969 glutathione Nutrition 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims 2
- 239000004094 surface-active agent Substances 0.000 claims 2
- 239000002574 poison Substances 0.000 claims 1
- 231100000614 poison Toxicity 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000000523 sample Substances 0.000 description 99
- 238000000034 method Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 229940005654 nitrite ion Drugs 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 208000005176 Hepatitis C Diseases 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 238000007689 inspection Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 5
- 230000012447 hatching Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 230000009871 nonspecific binding Effects 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 206010067268 Post procedural infection Diseases 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- SNGZVXPRGLTKNU-RGMNGODLSA-N [Na].C(CC)N[C@@H](CCO)C(=O)O Chemical compound [Na].C(CC)N[C@@H](CCO)C(=O)O SNGZVXPRGLTKNU-RGMNGODLSA-N 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 229940127121 immunoconjugate Drugs 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- RZYKUPXRYIOEME-UHFFFAOYSA-N CCCCCCCCCCCC[S] Chemical compound CCCCCCCCCCCC[S] RZYKUPXRYIOEME-UHFFFAOYSA-N 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 241000872931 Myoporum sandwicense Species 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 101800001020 Non-structural protein 4A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 238000007037 hydroformylation reaction Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091069025 single-strand RNA Proteins 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940048098 sodium sarcosinate Drugs 0.000 description 1
- ZUFONQSOSYEWCN-UHFFFAOYSA-M sodium;2-(methylamino)acetate Chemical compound [Na+].CNCC([O-])=O ZUFONQSOSYEWCN-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Communicable Diseases (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of hepatitis C virus antigen-antibody combined detection kit, including: the first HCV-Ab IgG cAg monoclonal antibody, a HCV restructuring chimeric antigen, the first sample diluting liquid, the second sample diluting liquid, the second HCV-Ab IgG cAg monoclonal antibody of the first enzyme labelling, the 2nd HCV restructuring chimeric antigen, the Ligands of second enzyme labelling, developer and the stop buffer of the first ligand-labeled；First sample diluting liquid includes reducing agent, first surface activating agent and the first denaturant；Second sample diluting liquid includes second surface activating agent and the second denaturant.Former dose of this hepatitis C virus antigen-antibody combined detection kit is by opening in envelope antigen and sample the disulfide bond of mispairing in antigen antibody complex in the first sample diluting liquid, second sample diluting liquid destroys HCV-cAg and HCV antibody complex, discharge more antigen, thus improve the sensitivity of detection.
Description
Technical field
The present invention relates to field of biological detection, especially relate to a kind of hepatitis C virus antigen-antibody combined detectable
Box.
Background technology
Hepatitis C is that a kind of liver acute and chronic caused by hepatitis C virus (Hepatitis C virus, HCV) is scorching
Disease, shows according to WHO statistics, there are about 200,000,000 people the most in the world and infect hepatitis C virus, account for the 3% of population in the world, China is about
Have 40,000,000～60,000,000.Hepatitis C relatively hepatitis B is more easy to liver cirrhosis, fibrosis occur, causes hepatocarcinoma, has predictive display,
Replacement hepatitis B is become the another big virus threatening human health after HIV (human immunodeficiency virus) by hepatitis C in the near future
Killer.Although hepatitis C virus is very big to human health risk, currently also there is no vaccine can prevent, HCV early diagnosis and
Source of infection examination has directive function to clinical treatment and Disease epizootic.Therefore, find simple to operate, sensitive quick, cheap,
Method accurately of testing realizes the detection to HCV, for the whole world, especially developing country, has great meaning.
HCV is the positive single strand RNA virus having peplos spherical in shape, belongs to flaviviridae family, total length about 9500 base.HCV base
Because group both sides are respectively 5 ' and 3 ' noncoding regions, centre is opening code-reading frame (ORF), is divided into structural area and non-structural district.Structure
District includes core protein district (C) and Liang Ge envelope protein district (E1, E2), is separately encoded core protein and envelope protein.Non-structural
Protein region includes NS2, NS3, NS4 and NS5 district, and encoding function albumen, such as protease (NS2, NS3 and NS4A district), helicase
And the RNA polymerase (NS5B district) of dependenc RNA (NS3).Tetracycline regulated system, containing about 190aa, rises in virus replication
To very important effect.Generally use gene engineering expression said structure and non-structural protein as envelope antigen, set up
The ELISA method of detection of anti-HCV.HCV genome has significant heterogeneity, and same gene group difference field-mutation degree has
Marked difference.5 ' noncoding regions are the most conservative, it has also become the focus of HCV molecular diagnosis research.
Traditional hepatitis C virus detection technique mainly has: (1) PCR method detection HCV RNA；(2) immunity
Method detection C hepatitis virus antigen or antibody.The detection of PCR method is mainly used in the selection of antiviral therapy and supervises with curative effect
Surveying, but it need to operate in strict accordance with PCR rule of operation, and testing staff need to be through professional training and obtain corresponding qualification, and sample
Quality control requirement high, it is necessary to being cryogenic conditions censorship in latter 2 hours of blood sampling, aseptic extracting RNA, easily because operation, setting
The factors such as standby and environment cause error, thus produce false positive or false negative.The detection of hepatitis C immunological method divides again antibody test
And Detection of antigen.HCV antibody test is the most the most frequently used detection method, but its fatal defects is to there is " window phase ", i.e. HCV
Producing the middle time having 40-70 days to HCV antibody after infection, blood donor is the most infected and has infectiousness, but uses mesh
Before antibody test reagent cannot detect, this stage be referred to as infecting after window phase (Perseroconversion before seropositive conversion
Window Phase,PWP).The existence of window phase is the main cause of blood transfusion infection.The blood transfusion postoperative infection of hepatitis C accounts at present
The 70% of hepatitis cases, and more have 80-90% to be belonging to postoperative infection of transfusing blood in hepatitis C virus infection；And hepatitis C antigen
Detection can make the window phase of detection about 50 days averagely in advance, shorten the risk of window phase HCV infection.Occur at HCV nucleic acid
After 1-2 days in, arise that HCV-cAg in the infected's body, and have to the level of HCV nucleic acid certain relevant
Property, can be as the mark of HCV detection.
Hepatitis C virus antigen-antibody combined detectable is to detect HCV-cAg and HCV antibody simultaneously,
Not only can shorten the window phase of detection, it is also possible to differentiation is HCV Current Infection or previous infection, can be used for the early stage of HCV
Detection and treatment are evaluated, and provide important reference for guidance clinic；Meanwhile, this reagent can be used for the examination of blood source, can effectively carry
Height blood and transfusion safety, antigen-antibody detects simultaneously and also improves work efficiency, saved cost.
But, owing to patient (average 49 days) after infecting hepatitis C virus about a period of time can produce antibody, formed
Antigenantibody complex, after producing antigenantibody complex, because free antigen reduces, makes the inspection of HCV antigen
Go out rate to be substantially reduced.General detection HCV antigen all can add detergent and denaturant in sample diluting liquid, destroys complex, releases
Put antigen, but this detergent and denaturant the most also destroy the HCV antibody in sample, cause cannot detecting in joint inspection reagent
HCV antibody, causes the missing inspection of antagonist.
Hepatitis C virus antigen-antibody combined detection kit the most on the market is all to try to use gentle de-sludging
Agent and denaturant, reduce the impact of HCV antibody in testing sample as far as possible, if but detergent and denaturant intensity are strong or
Not, it is the most incomplete that the immune complex in specimen to be measured destroys concentration, and HCV-cAg can not discharge completely
Coming, the HCV-cAg content in testing sample is the fewest, and the detection sensitivity for HCV-cAg and HCV antibody is relatively low,
Easily cause missing inspection.
Summary of the invention
Based on this, it is necessary to providing a kind of can improve for HCV-cAg and the associating of the detection sensitivity of HCV antibody
Detection kit.
A kind of hepatitis C virus antigen-antibody combined detection kit, is used for detecting in sample whether contain hepatitis C
Virus or antibody of HCV, including: the first HCV-Ab IgG cAg monoclonal antibody, HCV restructuring chimeric antigen, first sample
Product diluent, the second sample diluting liquid, the second HCV-Ab IgG cAg monoclonal antibody of the first enzyme labelling, the second of the first ligand-labeled
HCV restructuring chimeric antigen, the Ligands of second enzyme labelling, developer and stop buffer；
Described first HCV-Ab IgG cAg monoclonal antibody, the second HCV-Ab IgG cAg monoclonal antibody of described first enzyme labelling, described
2nd HCV restructuring chimeric antigen of the oneth HCV restructuring chimeric antigen and described first ligand-labeled does not the most occur to intersect instead
Should, described first part can be combined with described Ligands；
Described first sample diluting liquid according to mass percentage concentration include the reducing agent of 0.05%～5%, 0.005%～
The first surface activating agent of 0.1% and first denaturant of 0.005%～0.5%；
Described second sample diluting liquid includes second surface activating agent and the second denaturant；
In described second sample diluting liquid, the mass percentage concentration of described second surface activating agent is 0.5%～10%, institute
The concentration stating the second denaturant is 0.5mol/L～10mol/L.
In one embodiment, described reducing agent is selected from dithiothreitol, DTT, three (2-carboxyethyl) phosphine, cysteine, β-mercapto
At least one in base ethanol and reduced glutathion；
In described first sample diluting liquid, the mass percentage concentration of described reducing agent is 0.3%～1%.
In one embodiment, described first surface activating agent is selected from dodecyl-dimethyl amine second lactone, dodecyl
At least one in alanine, sarcosyl, dodecyl sodium sulfate and cetrimonium bromide；
In described first sample diluting liquid, the mass percentage concentration of described first surface activating agent be 0.01%～
0.08%.
In one embodiment, described first denaturant is selected from guanidine hydrochloride, carbamide, Tween-20 and Polyethylene Glycol octyl group benzene
At least one in base ether；
In described first sample diluting liquid, the mass percentage concentration of described first denaturant is 0.01%～0.1%.
In one embodiment, described second surface activating agent is selected from dodecyl-dimethyl amine second lactone, dodecyl
At least one in alanine, sarcosyl, dodecyl sodium sulfate and cetrimonium bromide；
In described second sample diluting liquid, the mass percentage concentration of described second surface activating agent is 1%～5%.
In one embodiment, described second denaturant is selected from guanidine hydrochloride, carbamide, Tween-20 and Polyethylene Glycol octyl group benzene
At least one in base ether；
In described second sample diluting liquid, the concentration of described second denaturant is 1mol/L～5mol/L.
In one embodiment, the solvent of described first sample diluting liquid is phosphate buffer or carbonate buffer solution,
The solvent of described second sample diluting liquid is phosphate buffer or carbonate buffer solution.
In one embodiment, described sample, described first sample diluting liquid and the use of described second sample diluting liquid
Ratio is 1～10:1～18:1～18.
In one embodiment, described first enzyme is horseradish peroxidase or alkali phosphatase, and described second enzyme is Radix Cochleariae officinalis
Peroxidase or alkali phosphatase, described first part is biotin or Avidin, and described Ligands is biotin or affine
Element.
This hepatitis C virus antigen-antibody combined detection kit is beaten by the reducing agent in the first sample diluting liquid
Open that envelope antigen is internal or the disulfide bond of mispairing between antigen, open in sample mispairing two sulfur in antigen antibody complex simultaneously
Key, so that outside epi-position is preferably exposed to, it is simple to immunity association reaction, the first denaturant in the first sample diluting liquid is permissible
Renaturation antigen, improves the specific recognition capability of antigen, can also reduce the non-specific binding of antibody, the second diluted sample simultaneously
Second surface activating agent and the second denaturant in liquid can destroy HCV-cAg and HCV antibody complex, discharge more multi-resistance
Former, thus improve the sensitivity of Detection of antigen.Relative to current business-like hepatitis C virus antigen-antibody combined detection examination
Agent box, this hepatitis C virus antigen-antibody combined detection kit is sensitive for the detection of HCV-cAg and HCV antibody
Spend higher.
Accompanying drawing explanation
Fig. 1 is to add the principle schematic after the first sample diluting liquid processes during sample detection, wherein,Be first resist
HCV-cAg monoclonal antibody,It is HCV restructuring chimeric antigen (mispairing disulfide-bonded),It is that a HCV restructuring is embedding
Close antigen,For the HCV-cAg in sample and HCV antibody conjugates (mispairing disulfide-bonded),For in sample
Free HCV-cAg,For the free HCV antibody in sample；
Fig. 2 is to add the principle schematic after the second sample diluting liquid processes during sample detection, wherein,In sample
HCV-cAg and HCV antibody conjugates (non-specific binding).
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
Whether the hepatitis C virus antigen-antibody combined detection kit of one embodiment, be used for detecting in sample and contain
Hepatitis C virus and antibody of HCV, including: the first HCV-Ab IgG cAg monoclonal antibody, a HCV restructuring inosculating antibody
Former, the second HCV-Ab IgG cAg monoclonal antibody of the first sample diluting liquid, the second sample diluting liquid, the first enzyme labelling, the first part mark
2nd HCV restructuring chimeric antigen, the Ligands of second enzyme labelling, developer and the stop buffer of note.
First HCV-Ab IgG cAg monoclonal antibody, the second HCV-Ab IgG cAg monoclonal antibody of the first enzyme labelling, a HCV recombinate embedding
The most there is not cross reaction in the 2nd HCV restructuring chimeric antigen closing antigen and the first ligand-labeled, and the first part can
To be combined with Ligands.
Second HCV-Ab IgG cAg monoclonal antibody of the first enzyme labelling, the first ligand-labeled the 2nd HCV restructuring chimeric antigen and
The Ligands of second enzyme labelling can be prepared voluntarily, it is also possible to directly buys.
In the application, the first HCV-Ab IgG cAg monoclonal antibody, HCV restructuring chimeric antigen, a second HCV-Ab IgG cAg
Monoclonal antibody and the 2nd HCV restructuring chimeric antigen are purchased from Meridian Life Science company；The Ligands of second enzyme labelling
Purchased from Nanjing Pan Gu's gene biological Engineering stock Co., Ltd；Second HCV-Ab IgG cAg monoclonal antibody of the first enzyme labelling and first
2nd HCV restructuring chimeric antigen of ligand-labeled is all to prepare voluntarily.
Preferably, the first enzyme is horseradish peroxidase (HRP) or alkali phosphatase, and second enzyme is horseradish peroxidase
(HRP) or alkali phosphatase, the first part is biotin or Avidin, and Ligands is biotin or Avidin.
First sample diluting liquid includes the reducing agent of 0.05%～5%, 0.005%～0.1% according to mass percentage concentration
First surface activating agent and first denaturant of 0.005%～0.5%.
The solvent of the first sample diluting liquid can be phosphate buffer or carbonate buffer solution.
In conjunction with Fig. 1, the first sample diluting liquid contains reducing agent and the first denaturant, and reducing agent is primarily to open and be coated
Antigen internal or between the disulfide bond of mispairing, open the mispairing disulfide bond of antigen antibody complex in specimen to be measured simultaneously, from
And outside making epi-position preferably be exposed to, it is simple to immunoreation；First denaturant major function is renaturation antigen, improves the spy of antigen
Opposite sex identification ability, can also reduce the non-specific binding of antibody simultaneously.
Reducing agent selected from dithiothreitol, DTT (DTT), three (2-carboxyethyl) phosphine (TCEP), cysteine, beta-mercaptoethanol and
At least one in reduced glutathion.
Preferably, in the first sample diluting liquid, the mass percentage concentration of reducing agent is 0.3%～1%.
Particularly preferred, in the first sample diluting liquid, reducing agent is beta-mercaptoethanol, and the percent mass of beta-mercaptoethanol is dense
Degree is 0.5%.
First surface activating agent is selected from dodecyl-dimethyl amine second lactone, dodecyl alanine, dodecyl flesh
At least one in propylhomoserin sodium (NLS), dodecyl sodium sulfate and cetrimonium bromide (CTAB).
Preferably, in the first sample diluting liquid, the mass percentage concentration of first surface activating agent is 0.01%～0.08%.
Particularly preferred, in the first sample diluting liquid, first surface activating agent is dodecyl sodium sulfate, dodecyl sulphur
The mass percentage concentration of acid sodium is 0.05%.
At least one in guanidine hydrochloride, carbamide, Tween-20 and Triton X-100 of first denaturant.
Preferably, in the first sample diluting liquid, the mass percentage concentration of the first denaturant is 0.01%～0.1%.
Particularly preferred, in the first sample diluting liquid, the first denaturant is that the percent mass of Tween-20, Tween-20 is dense
Degree is 0.03%.
Second sample diluting liquid includes second surface activating agent and the second denaturant.In second sample diluting liquid, the second table
The mass percentage concentration of face activating agent is 0.5%～10%, and the concentration of the second denaturant is 0.5mol/L～10mol/L.
The solvent of the second sample diluting liquid can be phosphate buffer or carbonate buffer solution.
In conjunction with Fig. 2, containing second surface activating agent and the second denaturant in the second sample diluting liquid, second surface activating agent
It is to destroy HCV-cAg and HCV antibody complex with the second denaturant, discharges more antigen, thus improve antigen inspection
The sensitivity surveyed.
Second surface activating agent is selected from dodecyl-dimethyl amine second lactone, dodecyl alanine, dodecyl flesh
At least one in propylhomoserin sodium, dodecyl sodium sulfate and cetrimonium bromide.
Preferably, in the second sample diluting liquid, the mass percentage concentration of second surface activating agent is 1%～5%.
Particularly preferred, in the second sample diluting liquid, second surface activating agent is sarcosyl, dodecyl
The mass percentage concentration of sodium sarcosinate is 3%.
At least one in guanidine hydrochloride, carbamide, Tween-20 and Triton X-100 of second denaturant.
Preferably, in the second sample diluting liquid, the concentration of the second denaturant is 1mol/L～5mol/L.
Particularly preferred, in the second sample diluting liquid, the second denaturant is carbamide, and the concentration of carbamide is 3mol/L.
First sample diluting liquid, the second sample diluting liquid are used to the dilution of sample, sample, the first sample diluting liquid and
The use ratio of two sample diluting liquids is 1～10:1～18:1～18.
Preferably, the use ratio of sample, the first sample diluting liquid and the second sample diluting liquid be 7～10:3～10:3～
10。
Particularly preferred, the use ratio of sample, the first sample diluting liquid and the second sample diluting liquid is 10:5:5.
Developer and stop buffer select conventional reagent.
This hepatitis C virus antigen-antibody combined detection kit is beaten by the reducing agent in the first sample diluting liquid
Open envelope antigen internal or between the disulfide bond of mispairing, open the mispairing disulfide bond of antigen antibody complex in sample simultaneously,
So that outside epi-position is preferably exposed to, it is simple to immunoreation, the first denaturant in the first sample diluting liquid can resist with renaturation
Former, improve the specific recognition capability of antigen, the non-specific binding of antibody can also be reduced, in the second sample diluting liquid simultaneously
Second surface activating agent and the second denaturant can destroy HCV-cAg and HCV antibody complex, discharge more antigen, from
And improve the sensitivity of Detection of antigen.Relative to current business-like hepatitis C virus antigen-antibody combined detection kit,
The detection sensitivity of this hepatitis C virus antigen-antibody combined detection kit is higher.
This hepatitis C virus antigen-antibody combined detection kit, it is possible to ensure that the antigen-antibody in testing sample is multiple
Compound is opened, and discharges HCV-cAg, can not destroy again the HCV antibody in testing sample, greatly improve antigen and antibody
The sensitivity of detection.
Below the using method of above-mentioned hepatitis C virus antigen-antibody combined detection kit is briefly described.
The using method of above-mentioned hepatitis C virus antigen-antibody combined detection kit and the most business-like third type liver
Scorching virus antigen-antibody combined detection kit is similar, hatch after differing only in addition the first sample diluting liquid 30min～
50min, does not wash plate and adds the second sample diluting liquid and hatch.
Detailed process is as follows: the mixing preparing the first HCV-Ab IgG cAg monoclonal antibody and HCV restructuring chimeric antigen is molten
Liquid, joins on microwell plate, through adsorbing, wash, close, completing after drying to be coated, then adds sample and the first sample diluting liquid,
Do not wash plate after hatching 30min～50min and add the second sample diluting liquid, after hatching 30min, wash plate, be subsequently added into the first enzyme mark
Second HCV-Ab IgG cAg monoclonal antibody, the 2nd HCV chimeric antigen of the first ligand-labeled and the Ligands of second enzyme labelling of note
Mixed solution, wash plate after hatching 30min, add after nitrite ion fully reacts and add stop buffer, finally measure OD value.
Being below specific embodiment, the various instruments occurred in embodiment and reagent if not otherwise specified, all use
This area conventional instrument or reagent.
The the first HCV-Ab IgG cAg monoclonal antibody used in embodiment is designated as AB1, AB1 purchased from U.S. meridian life
Science company (article No.: C8A216M)；Second HCV-Ab IgG cAg monoclonal antibody is designated as AB2, AB2 purchased from U.S. meridian
Life science company (article No.: C8A017M)；Oneth HCV restructuring chimeric antigen is designated as AG1, AG1 purchased from U.S. meridian
Life science company (article No.: R01599)；2nd HCV restructuring chimeric antigen is designated as AG2, AG2 purchased from U.S. meridian
Life science company (article No.: R01600)；The Avidin of HRP labelling is limited purchased from Nanjing Pan Gu's gene biological engineering share
Company, article No.: TRP-10005.
The most there is not cross reaction in AB1, AB2, AG1, AG2.
AB2 uses HRP labelling, and the AB2 of HRP labelling uses sodium periodate labelling method to prepare, and detailed process is as follows: (1) claims
Take 5mgHRP to be dissolved in 1mL distilled water；(2) in upper liquid, the 0.1M NaIO that 0.2mL newly joins is added4Solution, lucifuge under room temperature
Stir 20 minutes.(3) loading in bag filter by above-mentioned solution, dialyse the sodium-acetate buffer of 1mM PH4.4,4 DEG C overnight.
(4) add 20 μ L 0.2M PH9.5 carbonate buffer solutions, make the PH of above hydroformylation HRP be increased to 9.0～9.5, add immediately after
In 10mg AB2 to 1mL0.01M carbonate buffer solution, room temperature lucifuge is gently mixed 2 hours.(5) 4mg/mL that 0.1mL newly joins is added
NaBH4Liquid, mixing, then put 4 DEG C 2 hours.(6) above-mentioned liquid is loaded in bag filter, 0.15M PH7.4PBS is dialysed, 4 DEG C of mistakes
Night.(7) 3000rpm is centrifuged half an hour, abandons supernatant.Precipitate semi-saturation ammonium sulfate washes secondary, and last precipitate is dissolved on a small quantity
In the PBS of 0.15M PH7.4.(8) above-mentioned solution is loaded in bag filter, the PB buffer saline of 0.15M PH7.4 is dialysed, goes
Except (detecting with Nai Shi reagent) after ammonium ion, 10,000rpm are centrifuged 30 minutes removal precipitations, and supernatant is enzyme conjugates；(9)
Add equivalent glycerol, after subpackage, stored frozen.
AG2 uses biotin labeling, and the preparation process of biotin labeled AG2 is as follows:
(1) PH 7.4 20mmPB buffer 4 degree dialysis antigen 2 hours.(2) mix according to AG2:BIO (1:5) mass ratio
Place 1.5 hours at 4 degree after conjunction.(3) PH 7.4 20mmPB buffer 4 degree dialysis AG2 and BIO mixture 3 hours.After subpackage,
Stored frozen.
Choose 10 parts of positive samples and 10 parts of ' negative ' specimens carry out antigen-antibody joint inspection test, positive sample and ' negative ' specimens
Being all that the test kit assert through Bureau of Drugs Supervision confirms, specifying information is as follows:
Embodiment 1
Using phosphate buffered saline the first sample diluting liquid and the second sample diluting liquid, concrete composition is as follows.
The beta-mercaptoethanol of the first sample diluting liquid: 0.5wt%, the dodecyl sodium sulfate of 0.05wt%, 0.03wt%
Tween-20.
The sarcosyl of the second sample diluting liquid: 3wt% and the carbamide of 3mol/L.
Diluting AB1 with carbonate buffer solution is 3 μ g/mL to concentration, and diluting AG1 with carbonate buffer solution is 6 μ g/ to concentration
ML, by the two according to volume ratio be 1:1 mixing, obtain being coated liquid.
Then will be coated liquid and add in polystyrene micropore plate, through adsorbing, wash, close, completing after drying to be coated.
Every hole adds 100 μ L sample/positive control/negative control and 50 μ L the first sample diluting liquids, after 37 DEG C hatch 30min
Do not wash the every hole of plate and add 50 μ L the second sample diluting liquids, 37 DEG C hatch 30min after wash plate 6 times.
Add the AB2 of 200 μ L HRP labellings, the Avidin mixed solution of biotin labeled AG2 and HRP labelling, 37 DEG C
Plate is washed 6 times after hatching 30min.
Every hole 100 μ L nitrite ion A liquid and 100 μ L nitrite ion B liquid, after 37 DEG C of lucifuge reaction 30min, every hole adds 50 μ L terminations
Liquid, after 10min, surveys OD value at 450nm and 630nm dual wavelength.
Embodiment 2
Using phosphate buffered saline the first sample diluting liquid and the second sample diluting liquid, concrete composition is as follows.
The dithiothreitol, DTT of the first sample diluting liquid: 0.3wt%, the dodecyl sodium sulfate of 0.01wt%, 0.01wt%
Guanidine hydrochloride.
The dodecyl-dimethyl amine second lactone of the second sample diluting liquid: 1wt% and the carbamide of 5mol/L.
Diluting AB1 with carbonate buffer solution is 3 μ g/mL to concentration, and diluting AG1 with carbonate buffer solution is 6 μ g/ to concentration
ML, by the two according to volume ratio be 1:1 mixing, obtain being coated liquid.
Then will be coated liquid and add in polystyrene micropore plate, through adsorbing, wash, close, completing after drying to be coated.
Every hole adds 100 μ L sample/positive control/negative control and 50 μ L the first sample diluting liquids, after 37 DEG C hatch 30min
Do not wash the every hole of plate and add 50 μ L the second sample diluting liquids, 37 DEG C hatch 30min after wash plate 6 times.
Add AB2, the mixed solution of biotin labeled AG2 and the Avidin of HRP labelling of 200 μ L HRP labellings, 37
Plate is washed 6 times after DEG C hatching 30min.
Every hole 100 μ L nitrite ion A liquid and 100 μ L nitrite ion B liquid, after 37 DEG C of lucifuge reaction 30min, every hole adds 50 μ L terminations
Liquid, after 10min, surveys OD value at 450nm and 630nm dual wavelength.
Embodiment 3
Using phosphate buffered saline the first sample diluting liquid and the second sample diluting liquid, concrete composition is as follows.
The cysteine of the first sample diluting liquid: 1wt%, the dodecyl sodium sulfate of 0.08wt%, the hydrochloric acid of 0.1wt%
Guanidine.
The dodecyl-dimethyl amine second lactone of the second sample diluting liquid: 5wt% and the Polyethylene Glycol octyl group benzene of 1mol/L
Base ether.
Diluting AB1 with carbonate buffer solution is 3 μ g/mL to concentration, and diluting AG1 with carbonate buffer solution is 6 μ g/ to concentration
ML, by the two according to volume ratio be 1:1 mixing, obtain being coated liquid.
Then will be coated liquid and add in polystyrene micropore plate, through adsorbing, wash, close, completing after drying to be coated.
Every hole adds 100 μ L sample/positive control/negative control and 50 μ L the first sample diluting liquids, after 37 DEG C hatch 30min
Do not wash the every hole of plate and add 50 μ L the second sample diluting liquids, 37 DEG C hatch 30min after wash plate 6 times.
Add AB2 and the mixed solution of biotin labeled AG2 of 200 μ L HRP labellings, 37 DEG C hatch 30min after wash plate
6 times.
Every hole 100 μ L nitrite ion A liquid and 100 μ L nitrite ion B liquid, after 37 DEG C of lucifuge reaction 30min, every hole adds 50 μ L terminations
Liquid, after 10min, surveys OD value at 450nm and 630nm dual wavelength.
According to embodiment 1～3, obtain table 1 below.
Table 1: the OD value under 450nm and 630nm dual wavelength in embodiment 1～3.
As can be seen from Table 1, embodiment 1～3 can realize the detection of HCV antigen-antibody, and in embodiment 1 first
No matter the component ratio of sample diluting liquid and the second sample diluting liquid is detection HCV-cAg or HCV antibody effects is all
Optimal.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a hepatitis C virus antigen-antibody combined detection kit, is used for detecting in sample whether contain hepatitis C virus
Poison or antibody of HCV, it is characterised in that including: the first HCV-Ab IgG cAg monoclonal antibody, a HCV restructuring inosculating antibody
Former, the second HCV-Ab IgG cAg monoclonal antibody of the first sample diluting liquid, the second sample diluting liquid, the first enzyme labelling, the first part mark
2nd HCV restructuring chimeric antigen, the Ligands of second enzyme labelling, developer and the stop buffer of note；
Described first HCV-Ab IgG cAg monoclonal antibody, the second HCV-Ab IgG cAg monoclonal antibody of described first enzyme labelling, described first
The most there is not cross reaction in the 2nd HCV restructuring chimeric antigen of HCV restructuring chimeric antigen and described first ligand-labeled, institute
State the first part to be combined with described Ligands；
Described first sample diluting liquid includes reducing agent, 0.005%～0.1% of 0.05%～5% according to mass percentage concentration
First surface activating agent and first denaturant of 0.005%～0.5%；
Described second sample diluting liquid includes second surface activating agent and the second denaturant；
In described second sample diluting liquid, the mass percentage concentration of described second surface activating agent is 0.5%～10%, described
The concentration of two denaturants is 0.5mol/L～10mol/L.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described reduction
Agent is selected from least in dithiothreitol, DTT, three (2-carboxyethyl) phosphine, cysteine, beta-mercaptoethanol and reduced glutathion
Kind；
In described first sample diluting liquid, the mass percentage concentration of described reducing agent is 0.3%～1%.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described first
Surfactant is selected from dodecyl-dimethyl amine second lactone, dodecyl alanine, sarcosyl, dodecane
At least one in base sodium sulfonate and cetrimonium bromide；
In described first sample diluting liquid, the mass percentage concentration of described first surface activating agent is 0.01%～0.08%.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described first
At least one in guanidine hydrochloride, carbamide, Tween-20 and Triton X-100 of denaturant；
In described first sample diluting liquid, the mass percentage concentration of described first denaturant is 0.01%～0.1%.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described second
Surfactant is selected from dodecyl-dimethyl amine second lactone, dodecyl alanine, sarcosyl, dodecane
At least one in base sodium sulfonate and cetrimonium bromide；
In described second sample diluting liquid, the mass percentage concentration of described second surface activating agent is 1%～5%.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described second
At least one in guanidine hydrochloride, carbamide, Tween-20 and Triton X-100 of denaturant；
In described second sample diluting liquid, the concentration of described second denaturant is 1mol/L～5mol/L.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described first
The solvent of sample diluting liquid is phosphate buffer or carbonate buffer solution, and the solvent of described second sample diluting liquid is phosphate
Buffer or carbonate buffer solution.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described sample
The use ratio of product, described first sample diluting liquid and described second sample diluting liquid is 1～10:1～18:1～18.
Hepatitis C virus antigen-antibody combined detection kit the most as claimed in claim 1, it is characterised in that described first
Enzyme is horseradish peroxidase or alkali phosphatase, and described second enzyme is horseradish peroxidase or alkali phosphatase, and described first joins
Body is biotin or Avidin, and described Ligands is biotin or Avidin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610378119.1A CN106093402A (en) | 2016-05-31 | 2016-05-31 | Hepatitis C virus antigen-antibody combined detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610378119.1A CN106093402A (en) | 2016-05-31 | 2016-05-31 | Hepatitis C virus antigen-antibody combined detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106093402A true CN106093402A (en) | 2016-11-09 |
Family
ID=57230692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610378119.1A Pending CN106093402A (en) | 2016-05-31 | 2016-05-31 | Hepatitis C virus antigen-antibody combined detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106093402A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109738648A (en) * | 2018-12-29 | 2019-05-10 | 山东莱博生物科技有限公司 | Stability and high efficiency expresses engineering cell strain and its application of core antigen of C type hepatitis virus antibody |
| CN109917124A (en) * | 2018-10-30 | 2019-06-21 | 郑州安图生物工程股份有限公司 | A kind of hepatitis C virus antigen-antibody combined detection kit |
| CN110045116A (en) * | 2018-10-30 | 2019-07-23 | 郑州安图生物工程股份有限公司 | A kind of viral antigen coating promotor |
| CN110261616A (en) * | 2019-04-30 | 2019-09-20 | 广东菲鹏生物有限公司 | A kind of hepatitis c virus detection reagent box |
| CN110927391A (en) * | 2019-12-19 | 2020-03-27 | 新疆维吾尔自治区人民医院 | A detection kit for diagnosing vascular inflammatory hypertension and its application |
| CN111521804A (en) * | 2019-02-01 | 2020-08-11 | 科美诊断技术股份有限公司 | Treating agent and application thereof |
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
| CN111830259A (en) * | 2020-07-07 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | Alpha 1 microglobulin determination kit |
| CN111965356A (en) * | 2019-11-28 | 2020-11-20 | 上海荣盛生物药业有限公司 | Sulfhydryl reducing composition, fluorescent microsphere chromatography test paper and application |
| CN117310164A (en) * | 2023-11-27 | 2023-12-29 | 山东康华生物医疗科技股份有限公司 | Hepatitis C virus core antigen detection test strip and kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1274424A (en) * | 1998-07-30 | 2000-11-22 | 株式会社先端生命科学研究所 | Determination method of hepatitis C virus |
| CN101975857A (en) * | 2010-09-29 | 2011-02-16 | 中国医学科学院医学生物学研究所 | Virus disaggregating agent and method for disaggregating virus antigen-antibody complex and detecting HCV (Hepatitis C Virus) antigen |
| CN102955032A (en) * | 2012-10-16 | 2013-03-06 | 武汉康苑生物医药科技有限公司 | Enzyme-linked immunosorbent assay direct labeled antigen detection hepatitis C virus antigen-antibody and detection kit |
| CN103018455A (en) * | 2012-10-08 | 2013-04-03 | 张年 | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit |
| CN103630690A (en) * | 2013-12-17 | 2014-03-12 | 山东莱博生物科技有限公司 | Hepatitis C virus antigen-antibody combined detection kit and detection method |
| CN103674657A (en) * | 2013-12-24 | 2014-03-26 | 山东莱博生物科技有限公司 | Kit for processing antigen-antibody immune complex in serum or plasma sample and application of kit |
| US20160109447A1 (en) * | 2009-05-20 | 2016-04-21 | Ortho-Clinical Diagnostics, Inc. | Reagents For HCV Antigen-Antibody Combination Assays |
-
2016
- 2016-05-31 CN CN201610378119.1A patent/CN106093402A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1274424A (en) * | 1998-07-30 | 2000-11-22 | 株式会社先端生命科学研究所 | Determination method of hepatitis C virus |
| US20160109447A1 (en) * | 2009-05-20 | 2016-04-21 | Ortho-Clinical Diagnostics, Inc. | Reagents For HCV Antigen-Antibody Combination Assays |
| CN101975857A (en) * | 2010-09-29 | 2011-02-16 | 中国医学科学院医学生物学研究所 | Virus disaggregating agent and method for disaggregating virus antigen-antibody complex and detecting HCV (Hepatitis C Virus) antigen |
| CN103018455A (en) * | 2012-10-08 | 2013-04-03 | 张年 | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit |
| CN102955032A (en) * | 2012-10-16 | 2013-03-06 | 武汉康苑生物医药科技有限公司 | Enzyme-linked immunosorbent assay direct labeled antigen detection hepatitis C virus antigen-antibody and detection kit |
| CN103630690A (en) * | 2013-12-17 | 2014-03-12 | 山东莱博生物科技有限公司 | Hepatitis C virus antigen-antibody combined detection kit and detection method |
| CN103674657A (en) * | 2013-12-24 | 2014-03-26 | 山东莱博生物科技有限公司 | Kit for processing antigen-antibody immune complex in serum or plasma sample and application of kit |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109917124A (en) * | 2018-10-30 | 2019-06-21 | 郑州安图生物工程股份有限公司 | A kind of hepatitis C virus antigen-antibody combined detection kit |
| CN110045116A (en) * | 2018-10-30 | 2019-07-23 | 郑州安图生物工程股份有限公司 | A kind of viral antigen coating promotor |
| CN109738648B (en) * | 2018-12-29 | 2021-09-17 | 山东莱博生物科技有限公司 | Engineering cell strain for stably and efficiently expressing hepatitis C virus core antigen antibody and application thereof |
| CN109738648A (en) * | 2018-12-29 | 2019-05-10 | 山东莱博生物科技有限公司 | Stability and high efficiency expresses engineering cell strain and its application of core antigen of C type hepatitis virus antibody |
| CN111521804B (en) * | 2019-02-01 | 2024-01-19 | 科美诊断技术股份有限公司 | Treating agent and application thereof |
| CN111521804A (en) * | 2019-02-01 | 2020-08-11 | 科美诊断技术股份有限公司 | Treating agent and application thereof |
| CN111521779A (en) * | 2019-02-01 | 2020-08-11 | 科美诊断技术股份有限公司 | Hepatitis C virus antigen-antibody joint inspection method and kit |
| CN111521779B (en) * | 2019-02-01 | 2024-02-23 | 科美诊断技术股份有限公司 | Combined detection method and kit for hepatitis C virus antigen and antibody |
| CN110261616A (en) * | 2019-04-30 | 2019-09-20 | 广东菲鹏生物有限公司 | A kind of hepatitis c virus detection reagent box |
| WO2020221098A1 (en) * | 2019-04-30 | 2020-11-05 | 广东菲鹏生物有限公司 | Hepatitis c virus detection kit |
| CN110261616B (en) * | 2019-04-30 | 2021-07-20 | 广东菲鹏生物有限公司 | A kind of hepatitis C virus detection kit |
| CN111965356A (en) * | 2019-11-28 | 2020-11-20 | 上海荣盛生物药业有限公司 | Sulfhydryl reducing composition, fluorescent microsphere chromatography test paper and application |
| CN111965356B (en) * | 2019-11-28 | 2023-09-08 | 上海荣盛生物药业股份有限公司 | Sulfhydryl reduction composition, fluorescent microsphere chromatography test paper and application |
| CN110927391A (en) * | 2019-12-19 | 2020-03-27 | 新疆维吾尔自治区人民医院 | A detection kit for diagnosing vascular inflammatory hypertension and its application |
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
| CN111830259A (en) * | 2020-07-07 | 2020-10-27 | 武汉生之源生物科技股份有限公司 | Alpha 1 microglobulin determination kit |
| CN117310164A (en) * | 2023-11-27 | 2023-12-29 | 山东康华生物医疗科技股份有限公司 | Hepatitis C virus core antigen detection test strip and kit |
| CN117310164B (en) * | 2023-11-27 | 2024-02-27 | 山东康华生物医疗科技股份有限公司 | Hepatitis C virus core antigen detection test strip and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106093402A (en) | Hepatitis C virus antigen-antibody combined detection kit | |
| CN103630690B (en) | Hepatitis C virus antigen-antibody combined detection kit and detection method | |
| US8865398B2 (en) | Combination hepatitis C virus antigen and antibody detection method | |
| CN104237520B (en) | A kind of hepatitis C virus antigen-antibody combined detection kit and preparation method thereof | |
| CN101294147B (en) | Monoclone antibody of swine fever virus resistant wild strain E2 protein, preparation method and application thereof | |
| CN101419238A (en) | Hepatitis C virus core antigen chemiluminescence ELISA detection kit | |
| CN101144818A (en) | Method for detecting classical swine fever virus specific antibody and enzyme-linked immunosorbent kit | |
| Favorov et al. | Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides | |
| CN101196518B (en) | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method | |
| CN103819565B (en) | HCV restructuring fused antigen and expressing gene and preparation method | |
| CN109374887A (en) | Bovine viral diarrhea virus antigen colloidal gold detection kit and its application | |
| CN101407813B (en) | Protein gene of hepatitis C virus F and use thereof | |
| WO2004070387A1 (en) | Improved method of detection of hcv antibodies in combination assay or sole antibody assay | |
| Lemos et al. | Hepatitis E virus in Cuba | |
| CN102181438A (en) | Nucleotide detection sequence and detection method thereof | |
| CN107247144A (en) | A kind of method and detection kit for pre-processing hepatitis C antigen | |
| ES2303496B1 (en) | PERFECTED ANALYTICAL METHOD FOR THE DETECTION OF HIDDEN HEPATITIS, APPLICATIONS OF THE SAME AND ITS CORRESPONDING DIAGNOSTIC KIT. | |
| CN109212220A (en) | Antibody of HCV Rapid detection test strip | |
| Sukhdeo | Improvement in the antibody detection sensitivity of the Murex Ab/Ag combination HCV assay | |
| Wang et al. | Establishment and methodological evaluation of a chemiluminescence assay for detection of anti‐envelope protein (E1, E2) antibodies in the serum of hepatitis C virus‐infected patients | |
| Mohamedin et al. | Isolation of Hepatitis C Virus Antigen El and Establishment of a Comparison of its Level in Different Fibrosis Stages in Patients Infected with Chronic Hepatitis C | |
| CN102043048A (en) | HCV antigen detection board and method for detecting HCV antigen by using same | |
| Adah | Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C Downloaded from | |
| BLOOD | RAPID HBSAG/HIV I&II/HCV/SYPHILIS PANEL TEST | |
| Mikhailov | The ELISA test for hepatitis C is questionable. False positive ifa hepatitis C. Molecular diagnostics of hepatitis C virus in morphological substrates |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |