CN109682975A - A kind of hepatitis B detection reagent and its application in hepatitis B detection - Google Patents
A kind of hepatitis B detection reagent and its application in hepatitis B detection Download PDFInfo
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- CN109682975A CN109682975A CN201910038787.3A CN201910038787A CN109682975A CN 109682975 A CN109682975 A CN 109682975A CN 201910038787 A CN201910038787 A CN 201910038787A CN 109682975 A CN109682975 A CN 109682975A
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Abstract
The present invention provides a kind of hepatitis B detection reagents, including H1: being not higher than 5.0%SDS, pH 7.5-8.0；H2:A is mixed with B according to 1:1, and wherein the glycosidase of A:2 ~ 5U/ μ L and the NP-40 1:20 mixed preparing by volume not higher than 5%, B: mix the NaCNBH3 of the APTS and 1M of the 20mM of same volume；NH4AC, 1 ~ 2mU/ μ L sialidase and the hydrogen peroxide of H3:80-100mM is mixed according to volume 5:1:14；H4: buffer.The present invention also provides a kind of application of composition in hepatitis B detection reagent, the composition is made of NGA2FB and NG1A2F, and the composition detects hepatitis B by the ratio of NGA2FB/NG1A2F.Detection method of the invention has the characteristics that high sensitivity, easy to operate, reproducible, stability is good and high-throughput, compared with prior art, has higher accuracy, reaches 80.4% to the accuracy in detection of hepatitis B.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of hepatitis B detection reagent and its in hepatitis B
Application in detection.
Background technique
Hepatitis B (Chronic Hepatitis B, CHB) be due to a variety of pathogenic factors, such as virus, chemical toxicant,
Drug, alcohol, role of autoimmune factors etc. destroy liver cell, and the function of liver is caused to be damaged, and cause body one
The exception of serial malaise symptoms and liver function index.Chronic hepatitis B (CHB) is by hepatitis B (hepatitis B
Virus, HBV) caused by prolonged infection.The people that the whole world has more than 400,000,000 infects hepatitis B, and there are about nearly 200,000,000 people for China
For hepatitis B chronic infection.Most of the infecteds do not have symptom, do not know to be infected, but in every 10 chronic infections, just
There may be 3 people the severe complication of threat to life, such as cirrhosis and chronic hepatitis hepatic injury occur.Hepatitis B is Chinese every
Year leads to 330,000 many cases cancer related mortalities.It is detected before more serious disease not yet occurs in chronic infection and treatment is
When the vital work of previous item.
The clinical detection of hepatitis depends on blood testing and iconography detection.The goldstandard of diagnosis is that hepatic tissue is living
Inspection, but it is traumatic to have the following deficiencies: that pathologic finding has, and the complication such as easily cause bleeding, infect；There are sampling errors, because
Liver impedance rheograph center only accounts for the 1/200000-1/5 ten thousand of liver, cannot reflect entire hepatic disease；Patient's interdependence is poor, single disease
Reason inspection result cannot reflect liver damageization degree.The specific test of virus marker object should be the initial discrimination test of hepatitis.Blood
It is clear to learn diagnosis, by detection serum alt, AST, total bilirubin, prothrombin time, total bilirubin, albumin, globulin,
Cholinesterase, alkaline phosphatase and transpeptidase etc. test liver function, understand hepatic injury degree.Above-mentioned diagnostic method specificity is not
Height can only play booster action to the diagnosis of hepatic injury.Above-mentioned diagnostic method has the limitation of itself, and urgent need, which is explored, to be used for
The method that auxiliary diagnosis hepatitis B starts an inflammation of the liver and the Novel noninvasive index with higher sensitivity and specificity.
Hepatitis diagnosis detection method mainly uses enzyme-linked immunization and hepatitis B virus DNA detection method two currently on the market
Kind.Above two detection method can carry out qualitative and quantitative detection to hepatitis virus respectively, but can not detect hepatitis virus
To hepar damnification degree.Glycosylation refers to that N- oligosaccharide can be by multiple between hundreds of enzyme, transcription factor, ion channel and albumen
Miscellaneous interaction and formed, and participate in different kinds of molecules process, as protein folding, cell adherence, molecule transfer, signal turn
Lead, adjust receptor active etc.；The change of N- oligonucleotide chain on glycoprotein is related to disease.
Summary of the invention
Following N- oligonucleotide chains is selected from for detecting the composition for suffering from chronic hepatitis B or High risk group:
NGA2FB(anagalactosylated, core-a-1,6-fucosylated bisecting biantennary) and
NG1A2F(monogalactosylated, core-a-1,6-fucosylated biantennary).In following embodiment,
Using the ratio of NGA2FB/NG1A2F come diagnosing chronic hepatitis B.The N- oligonucleotide chain map of human serum is probably shown closely
10 N- oligonucleotide chain peaks, different N- oligonucleotide chains show different migrations because of the difference of molecular size, obtain N- oligosaccharides
Different peaks on chain map then represent different N- oligonucleotide chains；The relative concentration content of N- oligonucleotide chain then shows measured
Peak height.
Aiming at the problem that in the presence of the detection of existing hepatitis B, as blood test and iconography detection specificity and
Accuracy is not high, pathologic finding have it is traumatic, sampling have error, patient's interdependence difference etc.；The present invention provides a kind of apply
Hepatitis B detection reagent in hepatitis B detection.
The technical solution adopted by the invention is as follows:
A kind of hepatitis B detection reagent, including H1: 5.0% SDS, pH 7.5-8.0 are not higher than；H2:A and B is mixed according to 1:1
It closes, wherein the glycosidase of A:2 ~ 5U/ μ L and the NP-40 1:20 mixed preparing by volume not higher than 5%, B: mix same volume
The NaCNBH3 of the APTS and 1M of 20mM；NH4AC, 1 ~ 2mU/ μ L sialidase and the hydrogen peroxide of H3:80-100mM is according to volume
5:1:14 mixing；H4: buffer.
It is 4 μ L, H3 volumes be 2 μ L, H4 volumes is 200 μ L that the volume of the H1, which is the volume of 2 μ L, H2,.
Reagent preparation:
H1: it is configured to containing not higher than solution of 5% SDS, the pH 7.5 ~ 8.0.
H2:A is mixed with B according to 1:1.
The glycosidase of A: concentration 2 ~ 5U/ μ L and the NP-40 1:20 mixed preparing by volume not higher than 5%.
B: 20mM APTS and the 1M NaCNBH3 of same volume are mixed.
H3: concentration 80 ~ 100mM NH4AC, 1 ~ 2 mU/ μ L sialidase of concentration and hydrogen peroxide according to
Volume 5:1:14 mixing.
H4: buffer.
A kind of application of composition in hepatitis B detection reagent, the composition are made of NGA2FB and NG1A2F,
The composition detects hepatitis B by the ratio of NGA2FB/NG1A2F.
The detection of N- sugar group map:
1. the denaturation of N- oligonucleotide chain: the 2 μ L of serum inactivated at 90 ~ 95 DEG C is taken, the H1 of 2 μ L is added,
It mixes and places 5min.
2. the label of N- oligonucleotide chain: in the H2 that 4 μ L are 1. added, being not less than at 50 DEG C and carry out fluorescent marker 3h
Afterwards, the H4 that 200 μ L are added terminates reaction.
3. label post-processing: it takes labeled good 2 μ L of sample, is added the H3 of 2 μ L, it is anti-at 40 ~ 45 DEG C
3h is answered, the H4 for finally adding 200 μ L terminates reaction.
4. N- oligosaccharides chain separation is analyzed: taking 10 μ L of fluid sample 3., carried out under ABI3500dx instrument
N- oligosaccharides chain separation, to obtain N- sugar group map.
5. Data Management Analysis: obtained N- sugar group map being carried out peak value quantization, then uses function
NGA2FB/NG1A2F carries out further statistical analysis, to detect hepatitis B.
Beneficial effects of the present invention:
(1) detection method of the invention has high sensitivity, inspection easy to operate, reproducible, stability is good and high-throughput
Survey method passes through what is established to (606, chronic hepatitis B sample, 600, non-chronic hepatitis B sample), 1206 samples
The N- sugar group map of experimenter's serum, obtains the peak value of the NGA2FB and NG1A2F in experimenter's serum, passes through function NGA2FB
The ratio of/NG1A2F makes detection to chronic hepatitis B sample.This detection method compared with prior art, has higher
Accuracy reaches 80.4% to the accuracy in detection of hepatitis B.
(2) detection method of the invention is used, numerous hepatitis B patients can be allowed to receive conventional, Non-invasive detection, thus
It helps doctor and patient to detect the generation of hepatitis B and the progress of the state of an illness in time, is expected to promote the use of in clinic.
Detailed description of the invention
Fig. 1 is the N- oligonucleotide chain map of serum of hepatitis B sample；
Fig. 2 is the N- oligonucleotide chain map of healthy control group serum sample；
Fig. 3 is that detection sample passes through ROC indicatrix made by NGA2FB/NG1A2F；Detecting total sample number is 1206,
Wherein 606, chronic hepatitis B sample, 600, healthy control group sample；AUC=0.804 of area under the curve.
Specific embodiment
This detection method is described in further detail combined with specific embodiments below.It should be noted that the following example is only used
In illustrating rather than limit the scope of the invention.Test method without specific conditions in following embodiment, usually according to
Normal condition test, or the condition suggested according to manufacturer, reagent is all that laboratory is dedicated.
Embodiment one:
(1) experimental facilities:
ABI3500dx analyzer (Applied Biosystems), PCR, centrifuge
(2) prepared by reagent:
H1: it is configured to containing not higher than solution of 5% SDS, the pH 7.5 ~ 8.0.
H2:A is mixed with B according to 1:1.
The glycosidase of A: concentration 2 ~ 5U/ μ L and the NP-40 1:20 mixed preparing by volume not higher than 5%.
B: the NaCNBH3 of the APTS and 1M of the 20mM of same volume are mixed.
H3: NH4AC, concentration 1 ~ 2 mU/ μ L sialidase and hydrogen peroxide of the concentration in 80 ~ 100mM
It is mixed according to volume 5:1:14.
H4: buffer.
(3) N- sugar group map detects:
1. the denaturation of N- oligonucleotide chain: the 2 μ L of serum inactivated at 90 ~ 95 DEG C is taken, the H1 of 2 μ L is added,
It mixes and places 5min.
2. the label of N- oligonucleotide chain: in the H2 that 4 μ L are 1. added, being not less than at 50 DEG C and carry out fluorescent marker 3h
Afterwards, the H4 that 200 μ L are added terminates reaction.
3. label post-processing: it takes labeled good 2 μ L of sample, is added the H3 of 2 μ L, it is anti-at 40 ~ 45 DEG C
3h is answered, the H4 for finally adding 200 μ L terminates reaction.
4. N- oligosaccharides chain separation is analyzed: taking 10 μ L of fluid sample 3., carried out under ABI3500dx instrument
N- oligosaccharides chain separation, to obtain N- sugar group map.
5. Data Management Analysis: obtained N- sugar group map being carried out peak value quantization, then uses function
NGA2FB/NG1A2F carries out further statistical analysis, to detect hepatitis B.
This detection method compared with prior art, has higher accuracy, reaches to the accuracy in detection of hepatitis B
80.4%.Using detection method of the invention, numerous hepatitis B patients can be allowed to receive conventional, Non-invasive detection, to help to cure
Raw and patient detects the generation of hepatitis B and the progress of the state of an illness in time, is expected to promote the use of in clinic.
The specific embodiment in conjunction with described in attached drawing above carries out the purpose of the present invention, technical scheme and beneficial effects
It is further described, it should be understood that the above is only a specific embodiment of the present invention, but not to the present invention
The limitation of protection scope, those skilled in the art should understand that, all within the spirits and principles of the present invention, do not need to pay
Any modification, equivalent substitution, improvement and etc. that creative work can be made, should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of hepatitis B detection reagent, it is characterised in that: including H1: being not higher than 5.0% SDS, pH 7.5-8.0；H2:A
It is mixed with B according to 1:1, wherein the glycosidase of A:2 ~ 5U/ μ L and the NP-40 1:20 mixed preparing by volume not higher than 5%, B:
Mix the NaCNBH3 of the APTS and 1M of the 20mM of same volume；The NH4AC of H3:80-100mM, 1 ~ 2mU/ μ L sialidase and double
Oxygen water is mixed according to volume 5:1:14；H4: buffer.
2. hepatitis B detection reagent according to claim 1, it is characterised in that: the volume of the H1 is the body of 2 μ L, H2
It is 2 μ L, H4 volumes is 200 μ L that product, which is 4 μ L, H3 volumes,.
3. a kind of application of composition in hepatitis B detection reagent, the composition are made of NGA2FB and NG1A2F, institute
Composition is stated by the ratio of NGA2FB/NG1A2F to detect hepatitis B.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023040868A1 (en) * | 2021-09-15 | 2023-03-23 | 江苏先思达生物科技有限公司 | Liver failure detection reagent and use thereof in liver failure detection |
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| CN107505295A (en) * | 2017-07-21 | 2017-12-22 | 江苏先思达生物科技有限公司 | A kind of liver cancer monitoring kit and its application method |
| CN109100507A (en) * | 2017-06-20 | 2018-12-28 | 江苏先思达生物科技有限公司 | The method for building up of the seroglycoid N- sugar group spectrum model of chronic hepatitis hepatic injury |
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2019
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Patent Citations (4)
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| CN102565318A (en) * | 2012-01-11 | 2012-07-11 | 陈翠英 | Reagent for liver cancer monitoring, staging and prognosis risk assessment and method thereof |
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| 房萌: "肝纤维化、肝癌的N-糖组学动态研究。", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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| WO2023040868A1 (en) * | 2021-09-15 | 2023-03-23 | 江苏先思达生物科技有限公司 | Liver failure detection reagent and use thereof in liver failure detection |
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Inventor after: Chen Cuiying Inventor after: Tan Zongnan Inventor after: Zhu Hongzhang Inventor after: Zhang Fanghong Inventor after: Liu Xueen Inventor after: Zhuang Hui Inventor before: Chen Cuiying Inventor before: Tan Zongnan Inventor before: Zhu Hongzhang Inventor before: Zhang Fanghong Inventor before: Liu Xueen Inventor before: Zhuang Hui |
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