CN1869702A - Integral detection reaction plate and protein chip kit of hapetitis and cirrhosis - Google Patents
Integral detection reaction plate and protein chip kit of hapetitis and cirrhosis Download PDFInfo
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- CN1869702A CN1869702A CN 200510102468 CN200510102468A CN1869702A CN 1869702 A CN1869702 A CN 1869702A CN 200510102468 CN200510102468 CN 200510102468 CN 200510102468 A CN200510102468 A CN 200510102468A CN 1869702 A CN1869702 A CN 1869702A
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Abstract
The invention relates to an integrated detection reaction orifice plate and protein chip reagent box for detecting six indexes of diagnosis, forecast and prognosis of hepatitis, hepatocirrhosis and liver cancer, including hepatitis B virus surface antigen (HBsAg), hepatitis C virus antibody (HCVAb), alpha-fetoprotein (AFP), alpha-L-Fucosidase (AFU), mono amine oxidase (MAO), hyaluronic acid (HA). And the orifice plate comprises a substrate and reaction orifices on the substrate, where the reaction orifices comprise 2-384 sample orifices and 2-8 standard product orifices, and at the bottom of each reaction orifice is solid carrier coated with micro lattice of HBsAg, HCVAg, AFP, AFU,MAO,and HA antigens /antibodies or more. And the reaction plate and reagent can simply and conveniently, quickly and accurately implement simultaneous detection of the six indexes of diagnosis, forecast and prognosis of hepatitis, hepatocirrhosis and liver cancer for many persons.
Description
Technical field
The invention belongs to external clinical examination and biochip technology field, exactly, is a kind of six index (HBsAg that are applicable to diagnosis, prediction and the prognosis of hepatitis, cirrhosis and liver cancer, HCVAb, AFU, AFP, MAO, and HA) reaction plate and the kit of integrated detection.
Background technology
Virus hepatitis is legal Category B notifiable disease, has that infectiousness is strong, characteristics such as complicated, popular wide general, the incidence of disease height in route of transmission; Part is B-mode, hepatitis C patients can develop into chronicly, and can develop into cirrhosis and primary hepatoma, and is very big to people ' s health harm.
China is hepatitis B big country, and the hepatitis B patient in the whole world 75% all concentrates on China.People more than 600,000,000 had infected hepatitis B, and about 1.3 hundred million people carry hepatitis B, and wherein 3,000 ten thousand people suffer from chronic hepatitis, 30% the chronic hepatitis patient of having an appointment again develops into cirrhosis, wherein liver cancer can take place in part, and annual nearly 300,000 because of the number of hepatopathy death, wherein half is a liver cancer.That especially serious is the pregnant woman who carries hepatitis B surface antigen, and wherein about 40% can directly make infections in infants by mother and baby's vertical transmission, has every year 800000～1,000,000 neonates to become the hepatitis carrier approximately.
Hepatitis C mainly causes propagation by approach such as blood transfusion and blood product and unclean injections, and infected patient easily develops into chronic hepatitis, cirrhosis and liver cancer.Latest survey shows that there are 4,000 ten thousand third liver the infecteds in the whole nation.Discover that according to clinical observation acute third liver infection back about 50%～70% can transfer to chronic, 20% people finally develops into cirrhosis or liver cancer, and its speed that develops into liver cancer is come fast than the hepatitis B causer.
The clinical diagnosis of hepatitis B and hepatitis C is based on aetology and serodiagnosis, and hepatitis b virus s antigen (HBsAg) is the specificity immunology diagnostic markers of hepatitis B, and anti-HCV is the leading indicator that detects hepatitis C.
The chronic liver that hepatitis B and hepatitis C cause decreases the generation that all can cause cirrhosis.The pathological change majority of human cirrhosis is comparatively slow, from hepatocellular damage, inflammation even necrosis, to the paraplasm and the deposition of extracellular matrix (ECM), needs the time through several years even many decades, average about 3～5 years time.Because liver has very strong compensation, even cirrhosis is in the progress active stage sometimes, patient's clinical manifestation is not true to type yet; Even patient's performance has symptom, also often lack specificity.So far, the pathological diagnosis based on liver biopsy still is the goldstandard of liver cirrhosis diagnosis.But it is traumatic diagnosis, has damage of causing even potential lethal danger, and patient's biddability is poor, and expense is more expensive.In addition, also there are following shortcomings.It at first is the possibility that sample misleads, typical in the hepatic disease of dispersivity, detecting a Fibrotic stage, less as biopsy specimen, sample also may take place to be misled, as when having one or two header and converging high district, or have a hepatocellular leaflet, but the extracellular matrix around not having; In addition, between different hepatic pathology scholars, in the difference that aspect the judge of the classification of liver fibrosis and degree, also can take place up to 25%; The 3rd, liver biopsy once only provides static information, can not the reacting cells epimatrix produce and degraded between the dynamic change of balance, can not disclose the aetology mechanism of potentiality fully.Based on above various reasons, the non-invasive diagnosis of liver fibrosis more and more causes gazing at of doctor and patient.
At present, using more clinically is MAO (monoamine oxidase), HA (hyaluronic acid), the glutinous albumen of layer indexs such as (LN).Measure their serum content, can reflect liver endothelial cell, fat-storing cell and fibroblastic variation, usually point out the patient may have liver fibrosis and cirrhosis if their serum levels raises.
During liver fibrosis, obvious variation has all taken place in the composition of multiple ECM and content, and participating in the ECM metabolic enzymes also has corresponding change.Detection by to related enzymes in the serum has diagnostic significance to liver fibrosis.Monoamine oxidase (MAO) is the oxidase of some monoamine materials, is distributed in the mitochondria of organs such as liver, brain and in the blood plasma.When collagen synthesized, the lysyl-residue on the MAO catalysis tropocollagen molecule promoted in the tropocollagen molecule and intermolecular covalent cross-linking, forms stable collagenous fibres.In the liver fibrosis later stage, MAO can raise; Portal vein-central vein fibrous septum former 80% raises, and the pseudolobuli former is most to raise.
Hyaluronic acid (HA) is by the synthetic a kind of big molecule Portugal amine polysaccharide of interstitial cell, is distributed in the various connective tissues.Endothelial cell in the liver is the main place that HA removes in the blood.The liver fibrosis later stage, the fenestra of sinusoidal endothelial cell was because of capillary minimizing or disappearance during to cirrhosis, and the HA in the serum can significantly increase.So, in case HA obviously increases, often point out fibrosis heavier, or in the cirrhosis stage.
Because serological index occurs Zao than tangible clinical symptoms, regular or the irregular detection of carrying out hepatitis, the relevant serum index of cirrhosis in healthy population, discovery morning, early treatment for liver diseases have significance, help the prevention of hepatopathy, improve the prognosis situation.On the other hand, owing to hepatitis B, third liver are brought out liver cancer easily, it is also significant to detect the liver cancer index of correlation when hepatitis is detected the hepatitis index.
Alpha-fetoprotein (AFP) is widely used in the early diagnosis of primary carcinoma of liver and monitoring, following up a case by regular visits to of postoperative and the following up a case by regular visits to of people at highest risk of operation of liver cancer curative effect.Virus hepatitis, liver cirrhosis patient AFP concentration have rising in various degree, but its level is usually less than 100 μ g/L.Its rising reason mainly is that along with impaired hepatocellular reparation, it is normal that AFP recovers gradually because when the liver cell regeneration of damaged and primitivization, liver cell just has the ability that produces AFP again.
That be worth proposition is alpha-L-fucosidase (AFU), serum AFU measures with it detecting the hypersensitivity of small liver cancer, high specific to the concurrent liver cancer of forecast cirrhosis, with the good complementarity of measuring with AFP, and more and more be acknowledged as diagnosing cancer of liver, follow up a case by regular visits to the indispensable means with the cirrhosis monitoring.Domestic 24 routine hepatocellular carcinoma patients are carried out serum AFU and AFP correlative study, find that AFU diagnosis sensitivity must be 87%, specificity is 78%, and AFP then is respectively 65% and 89%, and (diagnosis threshold is, 20ug/L).The susceptibility of prompting AFU diagnosis is good, and specificity is low than AFP.AFU activity and positive rate and liver cancer diameter do not have obvious correlativity in the serum.Small liver cancer group serum AFU positive rate 70.8% is significantly higher than AFP (37.5%).AFP feminine gender and AFP raise and are not enough to diagnosing primary liver cancer person, and the positive rate of its serum AFU reaches 80.8%.The hepatic tissue biopsy turns out to be primary carcinoma of liver person, and the positive rate of serum AFU is more than 3 times of AFP positive rate.Detect AFU in liver cirrhosis patient, as its active increasing, have more value to finding some less tumours, this moment, AFP can not make diagnosis to less tumour.Therefore, use and promote AFU as the new diagnosis index of primary carcinoma of liver, have great importance, especially AFP diagnostic value negative and cellule liver cancer is bigger.Because of not having obviously relevant can complementary the diagnosis with AFP, joint-detection can improve the recall rate of liver cancer.
Present serology detection method can only detect single index based on the ELISA method at every turn, is carrying out time-consuming, effort, expensive in many indexs detections, and the check market that is difficult to satisfy large contingent is unfavorable for the day-to-day of health check-up, popular popularizing.The protein chip technology diagnosis protein chip technology that produces that combines with immune diagnostic technique is emerging high-tech technology recent years, aspect the parallel fast detecting of many indexs incomparable advantage is being arranged, having characteristics such as integrated, high flux, robotization, microminiaturization, informationization, is the developing direction of immunodiagnosis.The present invention is by realizing the diagnosis to hepatitis, cirrhosis and liver cancer to the fast detecting of six indexs, prediction and prognosis, prevention of disease and treat important directive significance is arranged.
Summary of the invention
The purpose of this invention is to provide a kind of can the prediction hepatitis B and C, cirrhosis and liver cancer simultaneously, the method that diagnosis and prognosis are judged.Our kit passes through to detect the HBsAg in serum or the blood plasma, HCVAb, and AFU, AFP, these six of MAO and HA or six above indexs reach this purpose.
In a first aspect of the present invention, the diagnosis of a kind of hepatitis B and C, cirrhosis and liver cancer is provided and has predicted six index integral investigating reaction plates, the reaction orifice plate comprises substrate and the reacting hole that is positioned on the substrate, described reacting hole comprises 2-384 sample aperture, 1-300 standard items hole, wherein, it is characterized in that: solid phase carrier is arranged at the bottom of each reacting hole, and bag is by anti-HBsAg on solid phase carrier, HCVAg, anti-AFP, anti-AFU, anti-MAO, the microarray of six kinds of anti-HA etc. or above antigen or antibody.In each reacting hole, also be provided with a location reference point.
In a preference, described solid phase carrier is NC film or PDVF film.
In another preference, the quantity of described sample aperture is 1-300.
In another preference, described anti-HBsAg, HCVAg, anti-AFP, anti-AFU, anti-MAO, six kinds of antigens such as anti-HA or antibody respectively have 2-8 point of sample on solid phase carrier.
In another preference, the every hole of described reacting hole is provided with a positioning reference point.
In a second aspect of the present invention, the diagnosis of a kind of hepatitis B and C, cirrhosis and liver cancer is provided and has predicted six integrated detection kit of index, it comprises the diagnosis of the above-mentioned hepatitis B and C of the present invention, cirrhosis and liver cancer and predicts six index integral investigating reaction plates.
In another preference, also comprise: enzyme mark working fluid, detection liquid and standard items.
In another preference, described enzyme mark working fluid is made up of the corresponding antigens or the monoclonal antibody of HRP mark, and described detection liquid is made up of shiner and two kinds of reagent of hydrogen peroxide, mixes before using.
In another preference, described shiner is luminol, different luminol or derivatives thereof.
Description of drawings
Fig. 1 is the point sample synoptic diagram of six indexs of hepatitis B and C on a kind of solid phase carrier of the present invention, cirrhosis and liver cancer detection
Fig. 2 is each antigen that carries out in the reacting hole of the present invention---the synoptic diagram of antibody response
Embodiment
The key of hepatitis B and C, cirrhosis and six integrated detections of index of liver cancer is to make sensitivity that six indexs detect and specificity all reach requirement guaranteeing the result accurately and reliably, and corresponding difficult point is how respectively the detect index of concentration difference up to 10000 times to be incorporated in the hole and to detect.The extensive studies through going deep into, the present invention adopts two kinds of different reactive modes to detect, and the detection reaction plate done improvement, the microarray detection technique that the present invention simultaneously adopts, enzyme immunity sandwich method and chemiluminescence detection technology, improve the sensitivity and the specificity that detect greatly, thereby solved the problems referred to above.
Particularly, for improving hepatitis B and C, cirrhosis and liver cancer index (HBsAg more than six or six kinds, HCVAb, AFU, AFP, MAO and HA) detection efficiency, the present invention uses the microarray detection technique, desmoenzyme immunity sandwich method and chemiluminescence detection technical design a kind of hepatitis B and C, six index integral investigating reaction plates of cirrhosis and liver cancer, the reaction orifice plate comprises substrate and the reacting hole that is positioned on the substrate, described reacting hole comprises 2-384 sample aperture, 1-300 standard items hole wherein, is characterized in that: on the base plane of each reacting hole solid phase carrier is arranged, and bag is by anti-HBsAg on solid phase carrier, HCVAg, anti-AFP, anti-AFU, anti-MAO, the microarray of six kinds of anti-HA etc. or above antigen or antibody.
The quantity of each reacting hole is not particularly limited, and sample aperture is 2-384 usually, preferably 2-50.Standard items hole 1-300, preferably 2-8.
In the present invention, the shape and the size of reacting hole are not particularly limited, and can be circular, square, sexangle, ellipse or other shapes.Size is generally diameter 0.1nm-8cm, and the degree of depth is 0.1nm-4cm.
In the present invention, the material of reaction plate substrate can be selected this area various materials commonly used for use, and preferable baseplate material comprises glass, plastics, pottery etc.
In the present invention, bag can be selected for use this area various solid phase carriers commonly used by the solid phase carrier of hepatitis B and C, cirrhosis and liver cancer antigen or antibody, comprise (but being not limited to): glass sheet, plastics, cellulose nitrate (NC) film, PDVF film etc., preferable solid phase carrier comprises NC film, PDVF film.
On solid phase carrier, anti-HBsAg is respectively arranged, HCVAg, anti-AFP, anti-AFU, anti-MAO, six kinds of antigens such as anti-HA or an antibody 2-8 point of sample, preferably 2-5 point of sample.In addition, preferably on solid phase carrier, also comprise an anchor point (see figure 1).Distance between each point of sample is generally 0.1nm-20mm.
On the other hand, the present invention also provides a kind of hepatitis B and C, cirrhosis and six of liver cancer or the integrated detection protein chip kit of above index, and this kit can be realized the parallelization of many indexs, microminiaturization, high throughput testing.It comprises reaction orifice plate of the present invention, cleansing solution, enzyme mark working fluid, detects liquid (A, B), standard items etc.Wherein, enzyme mark working fluid is made up of the corresponding antigens or the monoclonal antibody of HRP mark, detects liquid and is made up of shiner and two kinds of reagent of hydrogen peroxide, and shiner is luminol, different luminol or derivatives thereof, mixes before using.
In the present invention, the detection of each index adopts enzyme to exempt from sandwich method or competition law, detects principle as shown in Figure 2.Be described in detail as follows:
1) specific antibody is connected with solid phase carrier, forms insolubilized antibody.Unconjugated site is removed in sealing.
2) add and examined serum and enzyme mark solution, enzyme mark solution comprises six corresponding enzyme labelled antibodies (sandwich method) that detect indexs.
The index that adopts sandwich method to detect, special enzyme labelled antibody and the antigen in the serum form compound, and further form the double antibodies sandwich compound with insolubilized antibody.
3) unconjugated antibody, antigen and impurity are removed in washing.
4) add substrate: the substrate for enzymatic activity on the solid phase, chemiluminescence, CCD detects, and the amount of being examined antigen in light intensity and the sample is proportionate.
The present invention has fully utilized protein microarray detection technique dexterously, and enzyme immunity sandwich method and competition law, and chemiluminescence detection technology detect when having realized hepatitis B and C, cirrhosis and six indexs of liver cancer.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The preparation of detection reaction plate
Get 96 orifice plates, 42-84 sample aperture is set, 1-8 standard items hole.Place a solid phase carrier (NC film or PDVF film) in the bottom of each reacting hole, and on solid phase carrier, wrap by anti-HBsAg, HCVAg, anti-AFP, anti-AFU, anti-MAO, the little array of protein (Fig. 1) of six kinds of antigens such as anti-HA or antibody.
Embodiment 2
The kit preparation
Preparation contains the kit of a hepatitis B and C, cirrhosis and six integrated detections of index of liver cancer, and it contains one of the reaction orifice plate (8-200 person-portion) of embodiment 1,1 bottle of concentrated solution for washing, and 1 bottle of enzyme mark working fluid detects one bottle of liquid A, detects one bottle of liquid B, the standard items bottle.
Concentrated solution for washing, enzyme mark working fluid, detection liquid A, detection liquid B
Embodiment 3
Hepatitis B and C, cirrhosis and six integrated detections of index of liver cancer
By the following method each test blood please be detected by sample:
1. add detect blood please or standard items in the respective reaction hole, the 100ul/ hole.
2. enzyme-added mark working fluid, the 75ul/ hole.
3.30 ℃ insulation oscillating reactions 60 minutes.
4. with washing lotion washing 4-6 time, each 5 minutes, vibration.
5. add and detect liquid 40ul/ hole, make and detect the bottom that liquid is uniformly distributed in the hole.
6.CCD detect, look luminous signal intensity exposure 3-20 second.
The result judges:
According to the CutOff value of different indexs, normative reference product gradient curve, the yin and yang attribute of judgement sample serum.
Meanwhile, with conventional method the blood serum sample of testing is carried out conventional sense.The result shows that the test result that draws with kit of the present invention is identical with conventional method.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the appended claims of the application institute restricted portion equally.
Claims (9)
1. the diagnosis of a hepatitis, cirrhosis and liver cancer and predict six index integral investigating reaction plates is characterized in that, the reaction orifice plate comprises substrate and is positioned at reacting hole on the substrate, described reacting hole comprises 2-384 sample aperture, 1-300 standard items hole wherein, is characterized in that: solid phase carrier is arranged at the bottom of each reacting hole, and bag is by anti-HBsAg on solid phase carrier, HCVAg, anti-AFU, anti-AFP, anti-MAO, the microarray of six kinds of anti-HA etc. or above antigen or antibody.In each reacting hole, also be provided with a location reference point.
2. reaction plate as claimed in claim 1 is characterized in that, described solid phase carrier is NC film or PDVF film.
3. reaction plate as claimed in claim 1 is characterized in that, the quantity of described sample aperture is 1-300.
4. reaction plate as claimed in claim 1 is characterized in that, described anti-HBsAg, and HCVAg, anti-AFU, anti-AFP, anti-MAO, antigen such as anti-HA or antibody respectively have 2-8 point of sample on solid phase carrier.
5. reaction plate as claimed in claim 1 is characterized in that, also is provided with a location reference point in each reacting hole.
6. six integrated detection protein chip kits of the diagnosis of a hepatitis, cirrhosis and liver cancer is characterized in that, it comprises the diagnosis of the described hepatitis of claim 1, cirrhosis and liver cancer and predicts six index integral investigating reaction plates.
7. kit as claimed in claim 6 is characterized in that, also comprises: enzyme mark working fluid, detect liquid and standard items.
8. kit as claimed in claim 7 is characterized in that, described enzyme mark working fluid is made up of the corresponding antigens or the monoclonal antibody of HRP mark, and described detection liquid is made up of shiner and two kinds of reagent of hydrogen peroxide, mixes before using.
9. kit as claimed in claim 8 is characterized in that, described shiner is luminol, different luminol or derivatives thereof.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510102468 CN1869702A (en) | 2005-05-24 | 2005-09-14 | Integral detection reaction plate and protein chip kit of hapetitis and cirrhosis |
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| CN200510026126.7 | 2005-05-24 | ||
| CN200510026126 | 2005-05-24 | ||
| CN 200510102468 CN1869702A (en) | 2005-05-24 | 2005-09-14 | Integral detection reaction plate and protein chip kit of hapetitis and cirrhosis |
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| CN1869702A true CN1869702A (en) | 2006-11-29 |
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| CN 200510102468 Pending CN1869702A (en) | 2005-05-24 | 2005-09-14 | Integral detection reaction plate and protein chip kit of hapetitis and cirrhosis |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779128A (en) * | 2007-06-14 | 2010-07-14 | 弗拉芒区生物技术研究所 | Diagnostic test for the detection of early stage liver cancer |
| CN102216776A (en) * | 2008-09-26 | 2011-10-12 | 匹兹堡大学高等教育联邦体系 | Urinary biomarkers for predicting long-term dialysis |
| RU2488113C1 (en) * | 2012-06-18 | 2013-07-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Бурятский государственный университет" | Method for prediction of postresection hepatic failure in patients with focal hepatic lesions |
| CN108982853A (en) * | 2018-09-12 | 2018-12-11 | 南京工业大学 | A kind of paper base device and the preparation method and application thereof detecting monoamine oxidase content |
| WO2021134302A1 (en) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | Immunoassay instrument and method for hcv detection, and kit |
-
2005
- 2005-09-14 CN CN 200510102468 patent/CN1869702A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779128A (en) * | 2007-06-14 | 2010-07-14 | 弗拉芒区生物技术研究所 | Diagnostic test for the detection of early stage liver cancer |
| CN102216776A (en) * | 2008-09-26 | 2011-10-12 | 匹兹堡大学高等教育联邦体系 | Urinary biomarkers for predicting long-term dialysis |
| CN102216776B (en) * | 2008-09-26 | 2015-01-28 | 匹兹堡大学高等教育联邦体系 | Urinary biomarkers for predicting long-term dialysis |
| RU2488113C1 (en) * | 2012-06-18 | 2013-07-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Бурятский государственный университет" | Method for prediction of postresection hepatic failure in patients with focal hepatic lesions |
| CN108982853A (en) * | 2018-09-12 | 2018-12-11 | 南京工业大学 | A kind of paper base device and the preparation method and application thereof detecting monoamine oxidase content |
| WO2021134302A1 (en) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | Immunoassay instrument and method for hcv detection, and kit |
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