CN103018455A - Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit - Google Patents
Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit Download PDFInfo
- Publication number
- CN103018455A CN103018455A CN2012103769651A CN201210376965A CN103018455A CN 103018455 A CN103018455 A CN 103018455A CN 2012103769651 A CN2012103769651 A CN 2012103769651A CN 201210376965 A CN201210376965 A CN 201210376965A CN 103018455 A CN103018455 A CN 103018455A
- Authority
- CN
- China
- Prior art keywords
- antigen
- hepatitis
- hcv
- antibody
- virus
- 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
Abstract
The invention discloses hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and a detection kit. 2 strains of anti-HCV antigen monoclonal antibodies (AbI and AbII) and 2 kinds of HCV recombinant antigens (AgI and AgII) are obtained by analyzing an HCV sequence and do not undergo cross reaction. The anti-HCV antigen monoclonal antibody AbI and the HCV recombinant antigen AgI are used as coating raw materials, the anti-HCV antigen monoclonal antibody AbII and the HCV recombinant antigen AgII are used as labeling raw materials, the HCV antigens are detected by using a double-antibody sandwich method, and the HCV antibodies are detected by using a double-antigen sandwich method.
Description
Technical field:
The present invention relates to the direct labelled antigen of chemoluminescence method and detect hepatitis C virus antigen-antibody and detection kit, belong to chemoluminescence method and in-vitro diagnosis detection field.
Background technology:
(Hepatitis C virus, HCV) is spherical in shape for hepatitis C virus, and diameter (is 36～40nm in liver cell less than 80nm, be 36-62nm in blood), be single strand plus RNA virus, outside nucleocapsid, hold the cyst membrane that contains lipid, furcella is arranged on the cyst membrane.Hepatitis C virus can cause that hepatitis C is mainly through blood transfusion, acupuncture, the propagation such as drug abuse.HCV infects the normally lifelong infection of continuation, and antibody test is a very effective method.But after HCV infection, anti-HCV occurs slower, generally speaking, some months just occured after the Virus mutation of antibody can be deferred to and expose, the false negative (that is to say before antibody can detect or among Virus mutation i.e. so-called " window phase ") of anti-HCV can occur this moment, has any problem so diagnose in early days the third liver to infect.The HCV-cAg detection time is early than antibody, and HCV-cAg detects the additional reagent that can be used as the HCV antibody test.
The detection method of hepatitis C mainly contains three classes at present: 1. antibody to hepatitis C detects: its shortcoming is after infecting hepatitis C virus HCV, the latent period that 2-26 week is arranged, generally (turning sun) occur to HCV antigen/antibody combination long window phase is arranged, average out to 70d, the patient's window phase that has can extend to 6-9 month or longer.Because the existence of window phase, to latent period and stealthy the infected causing easily undetected, behind the organism infection HCV, before HCV antigen/antibody combination occurs about about 2 weeks, virion can appear in the blood circulation, this moment, the RNA of hepatitis C virus HCV can be positive through the PCR method detection, and blood has infectivity, and can't measure with antibody detection method at all.2. hepatitis C virus HCV detection of nucleic acids: infect 1-2 week behind the hepatitis C virus HCV, can detect HCV-RNA in the serum.Therefore, HCV detection of nucleic acids (HCV RT-PCR detection) can be used for the early diagnosis of hepatitis C, and can monitor its clinical efficacy by the quantitative detection to viral copy number.Because it is more that the PCR method detects the HCV-RNA influence factor, in sample collection, storage and context of detection strict requirement is arranged, and the detection operating process of PCR is complicated, time-consuming.3. hepatitis C antigen detects: the hepatitis C virus HCV-cAg is by part coding the most conservative in the HCV gene, hepatitis C virus HCV antigen namely appears in the 1-2d after HCV-RNA occurs, and parallel with the level of HCV-RNA, can be used as the sign that HCV copies.There are some researches show that HCV antigen detects with the HCV antigen/antibody combination detection and compares, the detection of HCV antigen can make the window phase of detection on average shift to an earlier date 49 days, shortens window phase HCV the infected's the risk of donating blood.Compare with the RT-PCR method have easy and simple to handle, the time short, characteristics low to environmental requirement, can be used for clinically early stage acute hepatitis C diagnosis.
Summary of the invention:
The purpose of this invention is to provide the direct labelled antigen of a kind of chemoluminescence method and detect hepatitis C virus antigen-antibody and detection kit, solve mainly that the sensitivity that prior art exists is low, poor stability, operation is loaded down with trivial details and the technical matters such as contaminated environment.The problem of main solution is to shorten window phase, can be used for early stage acute hepatitis C diagnosis.
The present invention adopts chemoluminescence method, directly labelled antigen joint-detection hepatitis C virus antigen-antibody.This invention is set up on the basis of double antibodies sandwich, and here, at first coated anti-type-C hepatitis virus antigen monoclonal antibody AbI, the hepatitis C virus recombinant antigen AgI on microwell plate of the antigen-antibody in the sample catches.Washing the other parts of sample off, add again HRP label (HRP mark anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII), just can form monoclonal antibody AbI-C hepatitis virus antigen-monoclonal antibody AbII HRP marker complex and recombinant antigen AgI-antibody of HCV-recombinant antigen AgII HRP marker complex, wash plate and add A liquid, B liquid, measure luminous value with chemiluminescent analyzer, the antigen-antibody total concentration is proportionate in luminous value and the sample, compare with critical value, thereby judge yin and yang attribute.
Compared with prior art, the present invention has following outstanding advantages:
1. the present invention adopts the antibody combined detection of hepatitis C antigen greatly to shorten window phase.The present stage detection of hepatitis C generally is to detect antibody to hepatitis C: its shortcoming is after infecting hepatitis C virus, the latent period that 2-26 week is arranged, generally (turning sun) occur to HCV antigen/antibody combination long window phase is arranged, average out to 70d, the patient's window phase that has can extend to 6-9 month or longer.Because the existence of window phase is to latent period and stealthy the infected causing easily undetected.The present invention adopts hepatitis C antigen to detect, the hepatitis C virus HCV-cAg is by part coding the most conservative in the HCV gene, hepatitis C virus HCV antigen namely appears in the 1-2d after HCV-RNA occurs, and parallel with the level of HCV-RNA, can be used as the sign that HCV copies.There are some researches show that HCV antigen detects with the HCV antigen/antibody combination detection and compares, the detection of HCV antigen can make the window phase of detection on average shift to an earlier date 49 days, shortens window phase HCV the infected's the risk of donating blood.The present invention uses joint-detection can be with the advantage of antigen and antibody test according to bringing into play, and the present invention have easy and simple to handle, the time short, characteristics low to environmental requirement, can be used for clinically early stage acute hepatitis C diagnosis.
2. the present invention adopts advanced chemoluminescence method.Chemoluminescence method is highly sensitive, the range of linearity is wide, the term of validity of label long, "dead" harm.
Technical scheme of the present invention is: need finish following preliminary work before detecting:
At first preparation is coated with plate, and used coated plate is the coated microwell plate of specific antigen-antibody, and the preparation of hepatitis C virus antigen-antibody combined detection coated slab may further comprise the steps:
(1) anti-type-C hepatitis virus antigen monoclonal antibody AbI, the hepatitis C virus recombinant antigen AgI with purifying dilutes with the 50mmol/L carbonate buffer solution, be diluted to 0.1-10 μ g/ml, then add in each hole of coated slab, after absorption, washing, sealing, drying, obtain hepatitis C virus antigen-antibody combined detection coated slab;
(2) hepatitis C virus antigen-antibody combined detection coated slab is packed into special-purpose aluminum foil sack seals and refrigerates for subsequent use.
Next is the preparation of hepatitis C virus antigen-antibody combined detection HRP label, may further comprise the steps: get 5mg HRP and be dissolved in 1.0ml NaHCO
3Solution fully dissolves HRP.Add an amount of 0.06M NaIO
4The room temperature lucifuge stirred 30 minutes.Move into bag filter, use 0.01M, pH9.6 carbonate buffer solution dialysed overnight.Get purifying mark raw material (anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII are mixing in 1: 1 according to mass ratio) 5mg, with the NaHCO of 0.011M
3, solution fully dissolves, and mixes with enzyme liquid, and the bag filter of packing into is used 0.01M, and 4 ℃ of lucifuge dialysis of pH9.6 carbonate buffer solution 24 hours are changed liquid 4 times.It is rear in conjunction with liquid to get dialysis, adds 0.2ml 0.4%NaBH
4Stir after 30 minutes, placed 4 hours for 4 ℃.With Sephadex G-200 chromatographic column on the dislysate, use 0.01M, the PBS wash-out of PH7.2 is collected first eluting peak, dilute for behind the HRP label working fluid 2-8 ℃ save backup.
Detect operating process
1) preparation of reagent
2) hepatitis C virus antigen-antibody yin and yang attribute reference substance and sample to be checked are sequentially added in the micropore reaction bar aperture and stick on mounting.
3) water-bath
4) wash plate.
5) add the label working fluid
6) water-bath is hatched
7) wash plate
8) add A liquid, B liquid
9) measured value
10) with the relatively rear judged result of critical value.
The implementation example:
The coated plate of 1 preparation, used coated plate is the coated microwell plate of specific antigen-antibody, the preparation of hepatitis C virus antigen-antibody combined detection coated slab may further comprise the steps:
(1) with the anti-type-C hepatitis virus antigen monoclonal antibody AbI of purifying with the 50mmol/L carbonate buffer solution be diluted to 3 μ g/ml, hepatitis C virus recombinant antigen AgI is diluted to 6 μ g/ml with the 50mmol/L carbonate buffer solution, then add in each hole of coated slab after two kinds of coating buffers being mixed according to 1: 1 ratio, after absorption, washing, sealing, drying, obtain hepatitis C virus antigen-antibody combined detection coated slab;
(2) hepatitis C virus antigen-antibody combined detection coated slab is packed into special-purpose aluminum foil sack seals and refrigerates for subsequent use.
The preparation of 2 hepatitis C virus antigen-antibody combined detection enzyme labeling things may further comprise the steps: get 5mg HRP and be dissolved in 1.0mlNaHCO
3Solution fully dissolves HRP.Add an amount of 0.06M NaIO
4The room temperature lucifuge stirred 30 minutes.Move into bag filter, use 0.01M, pH9.6 carbonate buffer solution dialysed overnight.Get purifying mark raw material (anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII are mixing in 1: 1 according to mass ratio) 5mg, with the NaHCO of 0.011M
3, solution fully dissolves, and mixes with enzyme liquid, and the bag filter of packing into is used 0.01M, and 4 ℃ of lucifuge dialysis of pH9.6 carbonate buffer solution 24 hours are changed liquid 4 times.It is rear in conjunction with liquid to get dialysis, adds 0.2ml 0.4%NaBH4 and stirs after 30 minutes, places 4 hours for 4 ℃.With Sephadex G-200 chromatographic column on the dislysate, use 0.01M, the PBS wash-out of PH7.2 is collected first eluting peak, dilute for behind the HRP label working fluid 2-8 ℃ save backup.
4.HCV the preparation of antigen-antibody yin and yang attribute reference substance
5.20X concentrated cleaning solution
6.A the preparation of liquid.
7.B the preparation of liquid.
8. detection operating process
1) preparation of reagent
2) hepatitis C virus antigen-antibody yin and yang attribute reference substance and sample to be checked are sequentially added in the micropore reaction bar aperture and stick on mounting.
3) water-bath
4) wash plate.
5) add the label working fluid
6) water-bath is hatched
7) wash plate
8) add A liquid, B liquid
9) measured value
10) with the relatively rear judged result of critical value.
Claims (6)
1. " the direct labelled antigen of chemoluminescence method detects hepatitis C virus antigen-antibody ", it is characterized in that following detection principle: the present invention adopts chemoluminescence method, the joint-detection hepatitis C virus antigen-antibody.This invention is set up on the basis of double antibodies sandwich, and here, at first coated anti-type-C hepatitis virus antigen monoclonal antibody AbI, the hepatitis C virus recombinant antigen AgI on microwell plate of the antigen-antibody in the sample catches.Washing the other parts of sample off, add again HRP label (HRP mark anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII), just can form monoclonal antibody AbI-C hepatitis virus antigen-monoclonal antibody AbII HRP marker complex and recombinant antigen AgI-antibody of HCV-recombinant antigen AgII HRP marker complex, wash plate and add A liquid, B liquid, measure luminous value with chemiluminescent analyzer, the antigen-antibody total concentration is proportionate in luminous value and the sample, compare with critical value, thereby judge yin and yang attribute.
2. the direct labelled antigen of chemoluminescence method detects the hepatitis C virus antigen-antibody detection kit, it is characterized in that comprising box body, is located at the coated plate in the box body and is located at reagent and adhesive sticker mounting and operation instructions in the box body.
3. described coated plate according to claim 2, it is characterized in that coated on the coated plate is anti-type-C hepatitis virus antigen monoclonal antibody and hepatitis C virus recombinant antigen, preparation technology dilutes coated antibody as coating buffer with coated damping fluid, is coated with reaction plate, and seals with confining liquid.
4. described coated damping fluid according to claim 3, its feature is comprising Tris-HCl damping fluid, carbonate buffer solution, phosphate buffer.
5. describedly according to claim 2 be located at reagent in the box body, it is characterized in that described reagent comprises: HRP label (HRP mark anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII), HCV antigen-antibody negative control product, HCV antigen-antibody positive reference substance, A liquid, B liquid, 20X concentrated cleaning solution.
6. described HRP label according to claim 5 is characterized in that the preparation method is: get 5mg HRP and be dissolved in 1.0ml NaHCO
3Solution fully dissolves HRP.Add an amount of 0.06M NaIO
4The room temperature lucifuge stirred 30 minutes.Move into bag filter, use 0.01M, pH9.6 carbonate buffer solution dialysed overnight.Get purifying mark raw material (anti-type-C hepatitis virus antigen monoclonal antibody AbII, hepatitis C virus recombinant antigen AgII are mixing in 1: 1 according to mass ratio) 5mg, with the NaHCO of 0.011M
3, solution fully dissolves, and mixes with enzyme liquid, and the bag filter of packing into is used 0.01M, and 4 ℃ of lucifuge dialysis of pH9.6 carbonate buffer solution 24 hours are changed liquid 4 times.It is rear in conjunction with liquid to get dialysis, adds 0.2ml 0.4%NaBH
4Stir after 30 minutes, placed 4 hours for 4 ℃.With Sephadex G-200 chromatographic column on the dislysate, use 0.01M, the PBS wash-out of PH7.2 is collected first eluting peak, and-20 ℃ save backup.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103769651A CN103018455A (en) | 2012-10-08 | 2012-10-08 | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103769651A CN103018455A (en) | 2012-10-08 | 2012-10-08 | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103018455A true CN103018455A (en) | 2013-04-03 |
Family
ID=47967294
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012103769651A Pending CN103018455A (en) | 2012-10-08 | 2012-10-08 | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103018455A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104360063A (en) * | 2014-12-08 | 2015-02-18 | 山东博科生物产业有限公司 | HCV core antigen detection kit based on magnetic micro-particle chemiluminescence method |
| CN106093402A (en) * | 2016-05-31 | 2016-11-09 | 湖南康润药业有限公司 | Hepatitis C virus antigen-antibody combined detection kit |
| CN113189348A (en) * | 2021-06-08 | 2021-07-30 | 中山生物工程有限公司 | Hepatitis C virus antibody detection kit and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1877330A (en) * | 2005-06-10 | 2006-12-13 | 湖南景达基因有限公司 | Enzyme-linked immunologic diagnosis kit for core antigen of C type hepatitis virus and method for preparing same |
| CN101196518A (en) * | 2006-12-07 | 2008-06-11 | 北京科美东雅生物技术有限公司 | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method |
| CN101419238A (en) * | 2008-12-04 | 2009-04-29 | 山东莱博生物科技有限公司 | Hepatitis C virus core antigen chemiluminescence ELISA detection kit |
| CN101477126A (en) * | 2008-09-24 | 2009-07-08 | 湖南景达生物工程有限公司 | Hepatitis C virus antigen-antibody combined detection method |
| CN101551397A (en) * | 2009-05-13 | 2009-10-07 | 郑州安图绿科生物工程有限公司 | Reagent box for detecting nature of third type hepatitis virus antibody by using chemiluminescence method |
| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
-
2012
- 2012-10-08 CN CN2012103769651A patent/CN103018455A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1877330A (en) * | 2005-06-10 | 2006-12-13 | 湖南景达基因有限公司 | Enzyme-linked immunologic diagnosis kit for core antigen of C type hepatitis virus and method for preparing same |
| CN101196518A (en) * | 2006-12-07 | 2008-06-11 | 北京科美东雅生物技术有限公司 | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method |
| CN101477126A (en) * | 2008-09-24 | 2009-07-08 | 湖南景达生物工程有限公司 | Hepatitis C virus antigen-antibody combined detection method |
| CN101419238A (en) * | 2008-12-04 | 2009-04-29 | 山东莱博生物科技有限公司 | Hepatitis C virus core antigen chemiluminescence ELISA detection kit |
| CN101551397A (en) * | 2009-05-13 | 2009-10-07 | 郑州安图绿科生物工程有限公司 | Reagent box for detecting nature of third type hepatitis virus antibody by using chemiluminescence method |
| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 曹诚等: "应用双抗原夹心法检测抗- HCV抗体", 《中华医学检验杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104360063A (en) * | 2014-12-08 | 2015-02-18 | 山东博科生物产业有限公司 | HCV core antigen detection kit based on magnetic micro-particle chemiluminescence method |
| CN106093402A (en) * | 2016-05-31 | 2016-11-09 | 湖南康润药业有限公司 | Hepatitis C virus antigen-antibody combined detection kit |
| CN113189348A (en) * | 2021-06-08 | 2021-07-30 | 中山生物工程有限公司 | Hepatitis C virus antibody detection kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nicol et al. | Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: Two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech) | |
| CN101196518B (en) | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method | |
| CN102735833B (en) | Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof | |
| CN101419238B (en) | Hepatitis C virus core antigen chemiluminescence ELISA detection kit | |
| CN105004862B (en) | Reagent for high throughput combined detection of hepatitis c virus antigen-antibody | |
| CN101551397A (en) | Reagent box for detecting nature of third type hepatitis virus antibody by using chemiluminescence method | |
| CN104237520B (en) | A kind of hepatitis C virus antigen-antibody combined detection kit and preparation method thereof | |
| CN111551712A (en) | Novel test strip and kit for quickly detecting coronavirus IgM/IgG two-in-one and preparation method of test strip and kit | |
| CN103575891A (en) | Kit for comprehensively detecting HE4 and CA125 and application of kit | |
| CN103575902B (en) | A kind of time-resolved fluorescence method four comprehensive detection oophoroma kits and application thereof | |
| CN115176162B (en) | Novel coronavirus antigen and detection use thereof | |
| CN101735319A (en) | Monoclonal antibody against GP73 protein, preparation method and application thereof | |
| CN102236020A (en) | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit | |
| CN105929157A (en) | Diagnostic kit for jointly detecting HIV antigen and HIV antibody and preparation method of diagnostic kit | |
| CN1963512A (en) | Chemiluminescence method for qualitative and quantitative detection of hepatitis B virus | |
| CN104407143B (en) | A kind of C hepatitis virus antigen-antibody combined detection kit | |
| CN102955032A (en) | Enzyme-linked immunosorbent assay direct labeled antigen detection hepatitis C virus antigen-antibody and detection kit | |
| CN105938146A (en) | HBV surface antibody time-resolved immunofluorescence assay kit and preparation method thereof | |
| CN101178404A (en) | Human immunodeficiency virus antibody chemiluminescence immune analyzing diagnose reagent box and method of producing the same | |
| CN103018455A (en) | Hepatitis C virus (HCV) antigen antibodies detected by using chemiluminescent directly-labeled antigens and detection kit | |
| CN109633163B (en) | procalcitonin/C reactive protein two-in-one detection kit | |
| US20040152070A1 (en) | Method of detection of HCV antibodies in combination assay or sole antibody assay | |
| CN103033616A (en) | Detection method of hemorrhagic fever with renal syndrome IgM antibodies and reagent kit | |
| CN102181438A (en) | Nucleotide detection sequence and detection method thereof | |
| CN102890154A (en) | Time-resolved immunofluorescence analysis method for hepatitis c virus core antigen and detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Zhang Nian Document name: Notification of Patent Invention Entering into Substantive Examination Stage |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130403 |