CN1755365A - A kind of method that detects antibody of HCV - Google Patents
A kind of method that detects antibody of HCV Download PDFInfo
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- CN1755365A CN1755365A CN 200510071806 CN200510071806A CN1755365A CN 1755365 A CN1755365 A CN 1755365A CN 200510071806 CN200510071806 CN 200510071806 CN 200510071806 A CN200510071806 A CN 200510071806A CN 1755365 A CN1755365 A CN 1755365A
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Abstract
The invention discloses a kind of method that adopts Enzyme-multiplied immune technique to detect antibody of HCV, also disclose the hepatitis C virus multilist anti-position chimeric antigen and the enzyme labeling linking arm that adopt in this method.Detection method of the present invention comprises the preparation of hepatitis C virus multi-epitope chimeric antigen, the preparation of chimeric antigen and enzyme labeling linking arm, the preparation of chimeric antigen mark horseradish peroxidase complex, inosculating antibody primordial covering elisa plate check-out console adds steps such as antibody to be measured and enzyme-labelled antigen compound.Method high specificity of the present invention, susceptibility height, good reproducibility, early detection are used to detect hepatitis C virus antibody and can obtain satisfied result, can be widely used in blood donor's screening and clinical diagnosis.
Description
Technical field
The present invention relates to a kind of method that detects antibody of HCV, relate in particular to a kind of method that adopts Enzyme-multiplied immune technique to detect antibody of HCV, also relate to the hepatitis C virus multi-epitope chimeric antigen and the enzyme labeling linking arm that are adopted in this method.
Background technology
(Hepatitis C virus HCV) is the main pathogen that causes the viral non-A non-B hepatitis in blood transfusion back to hepatitis C virus, mainly propagates by blood transfusion, IDU, also can be by close contact and mother-to-baby transmission.The whole world has 1.7 hundred million HCV the infecteds approximately at present.Have half can develop into chronic hepatitis among the sufferer approximately, and then part can develop into cirrhosis and hepatocellular carcinoma.Because also really not effective at present methods of treatment and vaccine are therefore very harmful to prevent its further propagation.According to nearest, only the U.S. has the new cases of infection of 50,000 HCV to take place every year approximately, and there are 25,120 ten thousand people's New Development hepatocarcinoma patients in the whole world every year approximately, and except that HBV infected, HCV was one of main paathogenic factor that causes hepatocellular carcinoma.
Usually, HCV the infected of about 15～20% is an autolimiting infection, can very fast rehabilitation.But, also there is the infected of 80～85% to develop into chronic hepatitis C, 20% developed into liver fibrosis is wherein arranged again, 4～5% patients with liver fibrosis generation hepatocellular carcinoma is finally arranged, endanger very serious.
At present, chronic hepatitis C does not still have other effective medicine except interference have definite curative effect.Recently report in the world, the interferon that PEG modifies adds the Ribavirin therapeutic alliance can the patient about 40～50% obtain reaction preferably, but also have grow the course of treatment, costs an arm and a leg, shortcoming such as the big and easy bounce-back of toxic and side effect.In addition, the development of hcv vaccine also highly makes a variation difficultly because of HCV, and difficulty has breakthrough in a short time.Therefore, detect HCV the infected as early as possible, the propagation of blocking-up HCV is the task of top priority.
The present situation of HCV detection technique
HCV generally propagates by the blood and the blood product that are polluted by HCV, and HCV detection technique commonly used now has dual mode: (1) indirect detection, mainly detect HCV antigen/antibody combination and antigen; (2) directly detect, determine viral existence, for example HCVRNA by the constituent of qualitative or detection by quantitative HCV virion.These two kinds of detection modes are being made a definite diagnosis the HCV infection, select HCV the infected's the therapeutic scheme and the curative effect aspect of estimating anti HCV treatment to play key effect.
The early 1990s in last century, utilize recombinant HCV antigen to develop the HCV antigen/antibody combination detection technique, promptly (enzyme immunoassays, EIAs) technology have developed into the third generation to EIA enzyme immunoassay.This technology can detect in the sample mixed antibody at different HCV antigens (as C district, NS3 district, NS4 district and NS5 district etc.), and the specificity that detects is up to more than 99% at present.
First generation reagent: use HCV-C100 (511) to be antigen, use the indirect method principle and detect HCV antibody.Second generation reagent: use gene engineering expression reorganization or chemically synthesized polypeptide (HCV-C, NS3 NS4) HCV antigen, use the indirect elisa method principle and detect HCV antibody, specificity and susceptibility are higher than first generation reagent.Third generation reagent: (NS4 NS5) detects HCV antibody with the indirect elisa method principle for HCV-C, NS3 to use gene engineering expression recombinant HCV antigen.
In March, 1993, health ministry is issued the documents and is required blood donor HCV detection of antibodies, has tens families to detect the ELISA reagent listing of HCV antibody subsequently, and batch calibrating.But be indirect elisa method entirely, but, caused some mistaken diagnosis and omission, thereby caused the certain harm of bad social influence and society because there are the certain quality problem in the defective of indirect method technology self and the product of each manufacturer.For this reason, should fundamentally solve the quality problems of reagent itself.
At present, dual-antigen sandwich method successfully is used to detect anti-HIV and Tp antibody, and the susceptibility of this type of reagent and specificity all are better than the indirect method of the anti-human immunoglobulin(HIg) of mark.And the indirect ELISA technology is still adopted in the detection of antibody of HCV, and mainly due to the direct mark C hepatitis virus antigen of peroxidase, its sensitivity does not reach requirement, and antigen active is reduced.But there is quick gold marked reagent to adopt the dual-antigen sandwich method principle both at home and abroad, detects HCV antigen/antibody combination by immunochromatography.But sensitivity is lower, can not be used for blood donor's screening.
Summary of the invention
For solving present problem, the invention discloses a kind of method that adopts the double antigens sandwich elisa technique to detect HCV antigen/antibody combination to specificity and susceptibility in the HCV antibody test.The present invention is merged last 4 lysines (4K) with different connected modes on the basis of how former epi-position HCV chimeric antigen, be used for the linking arm of mark horseradish peroxidase (HRP); The present invention has improved antigenic mark horseradish peroxidase (HRP) technology, and sets up double antigens sandwich (ELISA) and detect the antibody of HCV method.
Method of the present invention comprises the steps:
One. the preparation of hepatitis C virus multilist anti-position chimeric antigen and enzyme labeling linking arm:
At first prepare the anti-position of hepatitis C virus multilist chimeric antigen: the HCV epitope is analyzed with the Goldkey CASE(Computer Aided Software Engineering).The amino acid position of determining the different section antigens of the strong HCV of antigenicity (is respectively HCV-C:10-53aa; NS3:1192-1457aa; NS4:1916-1947aa; ), synthetic gene makes up the pBVIL-1 carrier respectively, and antigen is connected into the multi-epitope chimeric antigen, and with inclusion body formal representation HCV multi-epitope antigen (HCV-C, NS3, NS4), by the ion-exchange chromatography purifying, SDS-PAGE identifies purity in E.coli.
Then, on the HCV of above-mentioned clonal expression fused antigen basis, 5 ' end, 3 ' end and the middle enzyme labeling linking arm of introducing at antigen is 4 lysines (4K) respectively.
Synthetic primer at first, primer sequence is seen sequence 1-6 in the sequence table.Then with pILla-NS
4CNS
3Plasmid is a template, carries out pcr amplification, obtains genetic fragment, is inserted into respectively in the carrier after enzyme is cut, and obtains expression plasmid.
With the pIL1-HCV/C plasmid is template, carry out pcr amplification with the F3/R3 primer, obtain genetic fragment, again with behind EcoR I, the BamH I double digestion, be inserted in the pIL1 carrier, obtain expression plasmid pIL1-4K+HCV/C, utilize pIL1 a plurality of genes of interest can be made things convenient for connection characteristics, pIL1-HCV/NS4, pIL1-4K+HCV/C, pIL1-HCV/NS3 are connected, be built into pIL1-NS
4-4KC-NS
3Expression plasmid.
Three expression plasmids of above acquisition are transformed in the Escherichia coli recipient bacterium, efficiently express, extract inclusion body, carry out chromatography with crossing Q-Sepharose-FF negative ion handing-over post, S-Sepharose-FF kation handing-over post, solvent resistant column etc., SDS-PAGE identifies, the Lowy method is measured protein content, lyophilization ,-25 ℃ of preservations.
Two, the preparation of the anti-position of hepatitis C virus multilist chimeric antigen mark horseradish peroxidase:
(7) HRP activation: horseradish peroxidase (HRP) adds the SMPB gentle agitation.
(8) antigen activation: the anti-position of dithiothreitol (DTT) (DTT) activation hepatitis C virus multilist chimeric antigen (HCVAg) gentle agitation.
(9) desalination: to the antigen and the HRP desalination of above-mentioned activation.
(10) combination: antigen after the desalination and HRP combination.
(11) stop: add the iodoacetamide gentle agitation.
(12) preserve: packing ,-20 ℃ of preservations.
Three, dual-antigen sandwich method detects hepatitis C virus antibody
1, double antigens sandwich detects the ultimate principle of antibody of HCV:
The enzyme link detection reagent (double antigens sandwich) of antibody of HCV: with hepatitis C virus epi-position inosculating antibody primordial covering elisa plate, the sick epi-position chimeric antigen of type hepatitis that adds testing sample and enzyme labeling, as there being antibody in the sample, form antigen antibody complex, after the washing, chromogenic enzyme substrate is measured optical density, according to the optical density judged result.
2, detect the search procedure of antibody of HCV method: see accompanying drawing 1.
3, detecting the antibody of HCV kit forms: comprise the antigen coated elisa plate of HCV; HCV antibody positive control serum; HCV negative antibody control serum; Enzyme conjugates; Developer A; Developer B; Stop buffer; Cleansing solution etc.
4, running program:
(1) take out dried bag elisa plate, every hole adds testing sample; Add the enzyme-labelled antigen bond, the yin and yang attribute contrast adds diplopore, and blank well does not add any reagent, water-bath behind the mixing.
(2) wash plate with cleansing solution, pat dry for the last time.
(3) wash plate with 2.
(4) add developer A liquid earlier, add developer B liquid again, the lucifuge colour developing.
(5) every hole adds stop buffer.
5, the result judges:
(1) Instrument measuring: measure each hole OD value (deducting blank calculates) positive control OD＞0.8 with enzyme connection instrument 450nm, negative control OD＜0.10 kit is effective, stops back 10 minutes with interior survey OD value.
(2) critical value: the average OD value of negative control+0.05.If negative control OD value＜0.05 o'clock is calculated by 0.05; If＞0.05, calculate by actual OD value.
Be divided into four groups by connected mode, wherein
A：5’-KKKK-NS3-C-NS4；B：NS3-C-NS4-KKKK-3’；C：5’-KKKK-NS3-C-NS4-KKKK-3’；D：NS3-KKKK-C-NS4
The determination of activity result of four kinds of different connected mode multi-epitope chimeric antigen mark horseradish peroxidase double antigens sandwiches detection antibody of HCV shows four groups of different connected modes and HCV serum reactivity significant difference, and A group connected mode and HCV serum reactivity are better than B, C, D group.With A group echo HPR, be used for double antigens sandwich and detect antibody of HCV.
The HRP mark changes structure antigen dual-antigen sandwich method national third generation standard serum testing result is shown: the double antigens sandwich elisa technique detects specificity and the susceptibility that HCV antigen/antibody combination can improve detectable greatly.
Development double antigens sandwich ELISA detects HCV antigen/antibody combination has following advantage than indirect method:
(1) antigen sandwich method can detect all models other IgG hypotype and IgM of HCV the infected simultaneously, but early diagnosis.Indirect method only can be caught IgG, can not catch IgM.And two anti-use anti-human IgG enzymic-labelled antibody, and an antigen and antibody specific reaction only takes place, and hypotype or the IgM of some IgG often caused omission; (2) the double antigens sandwich antagonist carries out the secondary specific reaction, the false positive that the only enzyme labelled antibody of a specific reaction of reduction indirect method causes.
Method high specificity of the present invention, susceptibility height, can detect IgG hypotype and IgM hypotype at good reproducibility, early detection, are used to detect hepatitis C virus antibody and can obtain satisfied result, can be widely used in blood donor's screening and clinical diagnosis.
Description of drawings
Fig. 1 is for detecting the search procedure synoptic diagram of antibody of HCV method.
Embodiment
The preparation of embodiment one hepatitis C virus multilist anti-position chimeric antigen and enzyme labeling linking arm
One. material:
PIL1 carrier and pIL1a-NS
4CNS
3Plasmid: applied for Chinese patent and obtained the authorization the patent No.: ZL00100695.9, patent name: " expression vector PBVIL1 and construction method thereof and purposes ", and carried out preservation, preserving number: CGMCC No:0437.
The pIL1-HCV/C plasmid:
E.coli HB101：
The Q-Sepharose-FF anion-exchange column:
The S-Sepharose-FF cation exchange column:
Horseradish peroxidase (HRP):
SMPB：
Dithiothreitol (DTT) (DTT):
HCV-Ag：
Iodoacetamide:
Enzyme connection instrument:
EcoR I, BamH I enzyme:
Two, methods and results:
1. the development of the anti-position of hepatitis C virus multilist chimeric antigen:
With the Goldkey CASE(Computer Aided Software Engineering) HCV epitope is analyzed.The amino acid position of having determined the different section antigens of the strong HCV of antigenicity (is respectively HCV-C:10-53aa; NS3:1192-1457aa; NS4:1916-1947aa; ), synthetic gene makes up the pBVIL-1 carrier respectively, and antigen is connected into the multi-epitope chimeric antigen, and with inclusion body formal representation HCV multi-epitope antigen (HCV-C, NS3, NS4), by the ion-exchange chromatography purifying, SDS-PAGE identifies purity in E.coli.Specific operation process is referring to document (1). Song Xiaoguo, Ling Shigan, Zhang Heqiu, Chen Kun. and the research of reorganization C hepatitis virus antigen. institute of Military Medical Science Institute periodical; 2001; 25 (2): 91-95. (2). Song Xiaoguo, Ling Shigan, Zhang Heqiu, Chen Kun. the structure of efficient prokaryotic fusion expression vector (pBVIL1) and the application in HCV expresses. cell and molecular immunology magazine; 2001; 17 (3): 231-233.).
On the HCV of above-mentioned clonal expression fused antigen basis, 5 ' end, 3 ' end and the middle enzyme labeling linking arm of introducing at antigen is 4 lysines (4K) respectively, at first synthetic following primer:
1、F
1 GC
GAATTC ATG
AAAAAAAAAAAA GCACCTGTACGATCACTG
EcoR I 4K IL1 5 ' end
2、R
1 GC
GGATCC TTA
ACACGTATTGCAGTCTAT
BamH I ends NS3 3 ' end
3、F
2 5’GC
GAA TTC ATG
GCA CCT GTA CGA TC
EcoR I IL1 5 ' end
4、R
2 GC
GGATCC TTA
CTTCTTCTTCTT ACACGTATTGCAGTCTAT
BamH I ends 4K NS3
5、F
3 5’GC ACT AGT
AAA AAA AAA AAA GAG GGT GGA TCT
The general linking arm of Spe I KKKK
6、R3：5’CGC
GGA TCC TTA
GGA AGA CAC AAA
BamH I IL1
With pIL1a-NS
4CNS
3Plasmid is template (made up and preserved by Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, vaccine engineering research chamber), carry out pcr amplification with F1/R1, F2/R2, F3/R3 primer respectively, obtain three genetic fragments, again with behind EcoR I, the BamH I double digestion, be inserted into respectively in the pIL1 carrier, obtain three expression plasmid pIL1a/4K-NS
4-CNS
3, pIL1a/NS
4-C-NS
3-4K, pIL1a/4K-NS
4-C-NS
3-4K.
With the pIL1-HCV/C plasmid is template, carry out pcr amplification with the F3/R3 primer, obtain genetic fragment, again with behind EcoR I, the BamH I double digestion, be inserted in the pIL1 carrier, obtain expression plasmid pIL1-4K+HCV/C, utilize pIL1 a plurality of genes of interest can be made things convenient for connection characteristics, pIL1-HCV/NS4, pIL1-4K+HCV/C, pIL1-HCV/NS3 are connected, be built into pIL1-NS
4-4KC-NS
3Expression plasmid,
Three expression plasmids of above acquisition are transformed in the E.coli HB101 recipient bacterium, efficiently express, extract inclusion body, with Q-Sepharose-FF anion-exchange column, S-Sepharose-FF cation exchange column, solvent resistant column carry out chromatography excessively, SDS-PAGE identifies, the Lowy method is measured protein content, lyophilization ,-25 ℃ of preservations.
The preparation of the anti-position of embodiment two hepatitis C virus multilists chimeric antigen mark horseradish peroxidase
One, material: with embodiment one.
Two, methods and results:
(1) HRP activation: 1ml (10mg HRP+1mlPBS PH7.6) horseradish peroxidase (HRP) adds 18-20 ℃ of gentle agitation of 100 μ l SMPB 20 minutes.
(2) antigen activation: DTT activation hepatitis C virus multilist anti-position chimeric antigen (HCVAg): 5mgHCVAg adds 18-20 ℃ of gentle agitation of 115 μ l (30.9mg/ml) dithiothreitol (DTT)s (DTT) 20 minutes.
(3) desalination: lean on respectively antigen and HRP desalination to above-mentioned activation, each 1.5ml of the volume of collection with the PD-10 desalination.
(4) combination: antigen after the desalination and HRP were in conjunction with 18-20 ℃ of gentle agitation 45 minutes.
(5) stop: add 37 ℃ of gentle agitation of 40 μ l 0.1M iodoacetamides 30 minutes.
(6) preserve: packing 100 μ l/ prop up ,-20 ℃ of preservations.
Embodiment three dual-antigen sandwich methods detect hepatitis C virus antibody
One, material: with embodiment one.
Two, methods and results:
1, with the hepatitis C virus MEFA7.1 of purifying, be diluted to 0.33 μ g/ml with the carbonate buffer solution of 0.05M pH9.6,100 μ l/ holes bag is washed plate 2 times by enzyme connection assay plate with damping fluid, 3%BSA sealing 2 hours, room temperature is dried standby.The hepatitis C virus MEFA7.1 of 4 kinds of different linking arms of enzyme labeling, with the enzyme-labelled antigen dilution of special special use with labelled antigen be diluted to respectively 1: 2000 times 4 ℃ standby.4 parts of antibody of HCV positives and 4 parts of cloudy serum are measured respectively.
2, detect the search procedure of antibody of HCV method: see accompanying drawing 1.
3, kit is formed:
(1) the antigen coated elisa plate of HCV is 1
(2) HCV antibody positive control serum 2ml is 1 bottle
(3) HCV negative antibody control serum 2ml is 1 bottle
(4) enzyme conjugates 12ml is 1 bottle
(5) developer A 6ml is 1 bottle
(6) developer B 6ml is 1 bottle
(7) stop buffer 6ml is 1 bottle
(8) 1 bottle of cleansing solution 50ml (20 * concentrate)
4, running program:
(1) take out dried bag elisa plate, every hole adds 50 μ l testing samples; Add 100 μ l enzyme-labelled antigen bonds, yin and yang attribute contrast adds diplopore, respectively adds 100 μ l, and blank well does not add any reagent, and 37 ℃ of water-baths are 60 minutes behind the mixing.
(2) wash plate 5 times with the cleansing solution after the dilution in 1: 20, pat dry for the last time.
(3) wash plate with 2.
(4) add developer A liquid 50 μ l earlier, add developer B liquid 50 μ l again, 37 ℃ of water-bath lucifuges developed the color 20+2 minute.
(5) every hole adds 50 μ l stop buffers.
5, the result judges:
(1) Instrument measuring: measure each hole OD value (deducting blank calculates) positive control OD＞0.8 with enzyme connection instrument 450nm, negative control OD＜0.10 kit is effective, stops back 10 minutes with interior survey OD value.
(2) critical value: the average OD value of negative control+0.05.If negative control OD value＜0.05 o'clock is calculated by 0.05; If＞0.05, calculate by actual OD value.
Four kinds of different connected mode multi-epitope chimeric antigen mark horseradish peroxidases of table 1
Double antigens sandwich detects the determination of activity result of antibody of HCV
| The labelled antigen kind | Measurement result | |||||||
| P1 | P2 | P3 | P4 | N1 | N2 | N3 | N4 | |
| A B C D | 1.238 0.618 1.459 0.402 | 1.096 0.656 1.234 0.294 | 2.259 1.507 2.330 0.583 | 0.275 0.089 0.19l 0.066 | 0.047 0.042 0.052 0.042 | 0.051 0.058 0.052 0.054 | 0.079 0.066 0.081 0.065 | 0.046 0.052 0.054 0.047 |
A：5’-KKKK-NS3-C-NS4；
B：NS3-C-NS4-KKKK-3’；
C：5’-KKKK-NS3-C-NS4-KKKK-3’；
D：NS3-KKKK-C-NS4
The result confirms: four groups of different connected modes and HCV serum reactivity significant difference, A group connected mode and HCV serum reactivity are better than B, C, D organizes.With A group echo HPR, be used for double antigens sandwich and detect antibody of HCV.
Table 2 HRP mark changes structure antigen double antigens sandwich to national third generation standard serum testing result
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
| A B C D E F G H | 0.005 0.005 0.054 0.053 0.005 0.005 2.008 2.015 | 0.076 0.076 0.091 0.080 0.072 0.068 0.077 0.082 | 0.071 0.077 0.080 0.089 0.083 0.078 0.073 0.076 | 0.083 0.080 0.077 0.088 0.088 0.077 0.078 0.076 | 0.093 0.074 0.080 0.075 0.070 0.094 0.071 0.105 | 0.081 0.096 0.098 0.101 0.076 0.072 0.077 0.076 | 0.274 3.414 0.323 0.736 0.177 0.119 0.764 0.274 | 1.201 0.217 0.333 0.116 0.391 0.106 0.274 0.183 | 0.129 0.130 0.159 0.481 1.711 0.155 0.191 0.186 | 0.149 0.209 0.162 0.181 0.447 3.317 0.135 0.176 | 0.970 0.164 0.140 0.144 0.115 0.506 0.313 0.101 |
● A1, B1, E1, F1 are blank well, the negative contrast of C1, D1, the positive contrast of G1, H1;
● A2, B2-H6 are 1～No. 40 negative standard serum; A7, B7-H11 are 1～No. 40 positive criteria serum.Cutoff＝0.102
Sequence table
＜110〉Institute of Basic Medical Sciences, Academy of Military Medical Sciences, PLA
＜120〉a kind of method that detects antibody of HCV
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Claims (7)
1. a method that detects antibody of HCV comprises the steps:
(1) preparation of hepatitis C virus multi-epitope chimeric antigen;
(2) preparation of hepatitis C virus multi-epitope chimeric antigen and enzyme labeling linking arm;
(3) the anti-position of hepatitis C virus multilist chimeric antigen mark horseradish peroxidase;
(4) with hepatitis C virus multi-epitope inosculating antibody primordial covering enzyme linked immunosorbent detection plate;
(5) add antibody to be measured and (3) prepared enzyme-labelled antigen;
(6) add developer, chromogenic enzyme substrate;
(7) color development stopping reaction is measured optical density, according to the optical density judged result.
2. according to the described method of claim 1, it is characterized in that the preparation of described hepatitis C virus multi-epitope chimeric antigen comprises the steps:
(1) determines the amino acid position of the different section antigens of the strong HCV of antigenicity;
(2) the difference synthetic gene makes up the pBVIL-1 carrier, and antigen is connected into the multi-epitope chimeric antigen;
(3) in E.coli with inclusion body formal representation HCV multi-epitope antigen;
(4) by the ion-exchange chromatography purifying, SDS-PAGE identifies purity.
3. method according to claim 2, the amino acid position of the different section antigens of the HCV that wherein antigenicity is strong is HCV-C:10-53aa; NS3:1192-1457aa; NS4:1916-1947aa.
4. according to the described method of claim 1, it is characterized in that the preparation of hepatitis C virus multi-epitope chimeric antigen and enzyme labeling linking arm comprises the steps:
(1) synthetic primer carries out pcr amplification, obtains genetic fragment;
(2) enzyme is cut genetic fragment, is inserted in the carrier preparation expression plasmid;
(3) transformed into escherichia coli is expressed, and extracts inclusion body, carries out purifying.
5. according to the described method of claim 4, wherein the nucleotide sequence of the primer that is synthesized is shown in sequence 1-6 in the sequence table.
6. according to the described method of claim 4, carrier wherein is pIL1.
7. according to the described method of claim 1, wherein the anti-position of hepatitis C virus multilist chimeric antigen mark horseradish peroxidase comprises the steps:
(1) horseradish peroxidase (HRP) activation;
(2) hepatitis C virus multilist anti-position chimeric antigen (HCVAg) activation;
(3) desalination;
(4) combination;
(5) stop;
(6) preserve.
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| CN101410797B (en) * | 2006-03-30 | 2013-04-24 | 英特尔公司 | Method, device and system for transactional memory in out-of-order processors |
| CN103472233A (en) * | 2013-06-21 | 2013-12-25 | 温州大学 | Detection kit for hepatitis C virus and applications thereof |
| CN107247144A (en) * | 2017-05-16 | 2017-10-13 | 上海科华生物工程股份有限公司 | A kind of method and detection kit for pre-processing hepatitis C antigen |
| CN107247144B (en) * | 2017-05-16 | 2019-12-24 | 上海科华生物工程股份有限公司 | Method for pretreating hepatitis C antigen and detection kit |
| EP4047369A4 (en) * | 2019-10-14 | 2022-11-23 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd | Kit and method for detecting hcv antibody |
| CN112014573A (en) * | 2020-08-25 | 2020-12-01 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity determination kit for troponin I in human whole blood sample and kit |
| CN112014573B (en) * | 2020-08-25 | 2023-08-08 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit |
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