CN110333351A - It is a kind of for HBV infection or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis - Google Patents
It is a kind of for HBV infection or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis Download PDFInfo
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- CN110333351A CN110333351A CN201910705480.4A CN201910705480A CN110333351A CN 110333351 A CN110333351 A CN 110333351A CN 201910705480 A CN201910705480 A CN 201910705480A CN 110333351 A CN110333351 A CN 110333351A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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Abstract
The present invention relates to the early diagnosis technical fields of liver diseases, it provides a kind of for HBV infection or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis, present invention firstly discovers that by the concentration of detection blood oxidation type apoA-1, result can be used for HBV infection or hepatocarcinoma early diagnosis；This detection kit sensitivity with higher, diagnostic result accurate and effective when distinguishing Healthy People and HBV infection or Healthy People and early liver cancer patient；The present invention provides new idea and method for the detection of HBV infection or the early diagnosis of liver cancer.
Description
Technical field
The present invention relates to the early diagnosis technical fields of liver diseases, in particular it relates to which a kind of be used for HBV infection
Or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis.
Background technique
Cancer is the serious public health problem in the world, and about 8,000,000 people of the annual whole world die of cancer, and wherein liver cancer is dead
The high malignant tumour of rate, epithelium or mesenchymal tissue originating from liver, the cause of disease of primary carcinoma of liver and definite molecular mechanism are still
It imperfectly understands, it is now recognized that its morbidity is multifactor, multi-step complex process, is influenced by environment and diet double factor.
Epidemiology and experimental study data show hepatitis type B virus (HBV) and Hepatitis C Virus (HCV) infection, aflatoxins,
Contaminated drinking water, alcohol, cirrhosis, sex hormone, nitrosamines substance, microelement etc. are all related to onset of liver cancer.
China is the big country of HBV infection and the big country of liver cancer.It is proved according to reconnaissance information, liver cancer annual death rate is only second to
Gastric cancer and lung cancer occupy third position.And liver is the metabolism center of a variety of important substances of human body, while liver there are also removing toxic substances, secretion,
The critical functions such as excretion, once there is cancerous swelling in liver, and it is very big to the health hazard of human body.Medical statistics show China's primary
80% or more liver cancer is all the hepatitis B patient of the HBsAg positive, that is to say, that for liver cancer, possible hepatitis B continues
Infection is most important reason.The country in the research of liver cancer to being also noted that: hepatitis ratio is high in the crowd of High Phc Incidence Area, liver cancer
Hepatitis B surface antibody (HbsAg) positive is significantly higher than HbsAg negative patient in patient.If hepatitis B patient cannot get for a long time
Alleviate, liver function further damages, in fact it could happen that the performance of cirrhosis.And if a kind of this symptom continues to deteriorate, and not only can
There is renal failure, heart disease etc., can also cause the symptom of liver cancer in the damage for forming other internal organs.Certainly
All eventually development is cirrhosis and liver cancer to the not all people for having infected hepatitis B, it is important to hepatitis B is controlled in time, and
Control hepatitis B premise be timely and effectively find it is hepatitis B infected.
The early diagnosis of liver cancer at present mainly has ultrasonic examination or serum alpha-fetoprotein (AFP) measurement, but it is positive
Predicted value is generally less than 50%, and the diagnosis of HBV infection mainly has Hepatitis B virus inspection, but can there is presently no one
To detect the inspection method of above two project simultaneously.
Summary of the invention
In view of the above-mentioned problems in the prior art, the present invention is based in research apoA-1 (aPoA-I) this apolipoprotein
During find, the apoA-1 of oxidized form has high expression, and its table in the blood of HBV infection person and early liver cancer patient
It is enough to distinguish healthy population and HBV infection person or early liver cancer patient up to difference, thus discloses following technical scheme:
Oxidized form Genetyping poA-1 is as HBV infection or the purposes of the molecule marker of hepatocarcinoma early diagnosis.
Further, on this basis, the invention discloses a kind of for HBV infection or the reagent of hepatocarcinoma early diagnosis
Box, including at least a kind of for detecting the reagent of oxidized form Genetyping poA-1.
Further, the above-mentioned kit for being used for HBV infection or hepatocarcinoma early diagnosis, the diagnostic kit composition
It is as follows:
(1) for carrying out the reagent that pretreatment obtains sample to sample to be tested；
(2) for the detection reagent that oxidized form Genetyping poA-1 expression quantity is detected in sample；
(3) label or operation instructions, the label or operation instructions indicate the kit for diagnose HBV or
Early liver cancer.
Further, the above-mentioned kit for HBV infection or hepatocarcinoma early diagnosis, in the step (2) for pair
The detection reagent that oxidized form Genetyping poA-1 expression quantity is detected in sample is that the oxidized form of biotin labeling carries rouge egg
White apoA-1 monoclonal antibody.
Further, the above-mentioned kit for being used for HBV infection or hepatocarcinoma early diagnosis, further includes having standard items, washing
Liquid, substrate solution, developing solution, terminate liquid and oxidized form Genetyping poA-1 polyclonal antibody coated elisa plate.
Further, the above-mentioned kit for being used for HBV infection or hepatocarcinoma early diagnosis, the oxidized form apolipoprotein
ApoA-1 polyclonal antibody coated elisa plate is made of following steps: by coating oxidized form Genetyping poA-1 Anti-TNF-α
Body 0.1M glycine buffer, PH9.0；Each hole of ELISA Plate is added after dilution, every hole 100ul, absorption is stayed overnight, with the sweet ammonia of 0.1M
Acid buffer washes version；Enzyme mark is coated with to get the oxidized form Genetyping poA-1 polyclonal antibody overnight with confining liquid closing
Plate.
Further, the above-mentioned kit for HBV infection or hepatocarcinoma early diagnosis, the sample to be tested be blood,
Serum or plasma sample.
Further, the above-mentioned kit for being used for HBV infection or hepatocarcinoma early diagnosis, the sample to be tested are people periphery
Blood.
Further, a method of using the above-mentioned kit for being used for HBV infection or hepatocarcinoma early diagnosis, including with
Lower step:
(1) venous collection peripheral blood；
(2) collected peripheral blood is pre-processed；
(3) oxidized form Genetyping poA-1 antibody incubation is added；
(4) Incubating Solution is added in ELISA Plate together with comparison liquid and is analysed and compared.
Further, above-mentioned using HBV infection or the method for the kit of hepatocarcinoma early diagnosis, in the step (2)
Carrying out pretreated method to collected peripheral blood is to be centrifuged 15 minutes in 30 minutes in 1000 × g after acquiring, and takes supernatant.
The invention has the advantages that:
1, can be used in present invention firstly provides the content by oxidized form Genetyping poA-1 in detection blood sample
HBV infection or hepatocarcinoma early diagnosis, in blood sample the expression quantity of oxidized form Genetyping poA-1 distinguish Healthy People with
HBV infection person or when early liver cancer patient sensitivity and specificity with higher, diagnostic result is precisely effective, is liver cancer
Early diagnosis provides new method, to realize the detection of HBV infection and early diagnosis, the early treatment of liver cancer, improves diagnosis and treatment
Efficiency extends patient survival, improves prognosis and lays the foundation.
2, peripheral blood has many advantages, such as materials convenience, non-invasive, can continuously detect, and peripheral blood is utilized to detect HBV infection
Perhaps early liver cancer can find that clinical HBV or hepatocarcinoma early diagnosis are increased to by the generation of HBV infection or liver cancer early
One new level.
Detailed description of the invention
Fig. 1 is the concentration standard curve of 1 Plays product of embodiment；
Fig. 2 is in embodiment 2, and 10 Healthy Peoples compare the peripheral blood of 10 HBV infection persons and 10 early liver cancer patients
The comparison of oxidized form Genetyping poA-1 expression quantity.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention；Made in following embodiments
Experimental method is conventional method unless otherwise specified；The materials, reagents and the like used in the following examples, such as without special
Illustrate, is commercially available.
Liver cancer early stage of the present invention, corresponding to the Tis-T4 phase in liver cancer TNM stage, the HBV infection refers to hepatitis B two
Half-and-half check to be HBsAg, HBeAg and HBV DNA positive.
Embodiment 1
Verify example
Oxidized form Genetyping poA-1 kit disclosed by the invention is solid phase sandwich method enzyme-linked immunosorbent assay
(ELISA) standard items of test substance concentration known to, unknown concentration sample be added in micropore ELISA Plate and detected.First will
The antibody of test substance and biotin labeling incubates simultaneously.After washing, the labeled HRP of Avidin is added.Using incubate and
Washing, removes unbonded enzyme conjugates, and substrate A, substrate B (3,3', 5,5'- tetramethyl benzidine) and enzyme knot is then added
Object is closed to act on simultaneously.Generate color.The concentration of test substance is in proportionate relationship in the depth and sample of color.The following are specific realities
Test step:
1. acquiring peripheral blood, 1000 × g is centrifuged 15 minutes, and taking supernatant is sample to be tested.
2. the processing of ELISA Plate: by coating oxidized form Genetyping poA-1 polyclonal antibody 0.1M glycine buffer
Liquid, PH=9.0；Each hole of ELISA Plate, every hole 100ul are added after dilution, absorption overnight, washes version with 0.1M glycine buffer；With
Confining liquid (skim milk) closing is overnight to get the oxidized form Genetyping poA-1 polyclonal antibody coated elisa plate.
3. be added oxidized form Genetyping poA-1 standard items (concentration is respectively 0.25ng/ml, 0.5ng/ml after dilution,
1ng/ml, 2ng/ml, 4ng/ml, 8ng/ml) each 50ul in reacting hole, sample to be tested 50ul is added in reacting hole.Immediately plus
Enter the oxidized form Genetyping poA-1 of the biotin labeling of 50ul.Diaphragm plate is covered, gently oscillation mixes, and 37 DEG C incubate 1 hour.
4. getting rid of liquid in hole, cleaning solution (1M Na is filled it up in every hole2HPO4+1M NaH2PO4), it vibrates 30 seconds, gets rid of and wash
Liquid is patted dry with blotting paper.Repeat this operation 3 times.If washed with board-washing machine washing, washing times increase primary.
5. the affine chain enzyme-HRP of 80ul is added in every hole, gently oscillation is mixed, and 37 DEG C incubate 30 minutes.
6. getting rid of liquid in hole, cleaning solution is filled it up in every hole, is vibrated 30 seconds, is got rid of cleaning solution, patted dry with blotting paper.Repeat this
Operation 3 times.If washed with board-washing machine washing, washing times increase primary.
7. substrate A (1M H2O2), B (3,3', 5,5'- tetramethyl benzidine TMB of 1M) each 50ul is added in every hole, gently shake
Mixing is swung, 37 DEG C incubate 10 minutes.Avoid illumination.
8. taking out ELISA Plate, it is rapidly added 50ul terminate liquid (2M H2SO4), being added after terminate liquid should measurement result immediately.
9. measuring the OD value in each hole at 450nm wavelength.
Experimental result and data processing: using absorbance OD value as ordinate (Y), corresponding standard concentration is abscissa
(X), it is made corresponding curve, the test substance content of sample can converse corresponding concentration by standard curve according to its OD value,
Calibration curve equation is y=2.32X2+ 3.15X-4.25, R=0.975 > 0.95, it was demonstrated that this method data are reliable, can be used for
The experiment of next step.
Embodiment 2
Count example
Collect 10 Healthy Peoples, the peripheral blood of 10 HBV infection persons and 10 early liver cancer patients, with the side of embodiment 1
Method measures the expression quantity (ng/ml) of the oxidized form Genetyping poA-1 in peripheral blood, as a result as shown in the following table 1 and attached drawing 2,
1 10 Healthy Peoples of table, 10 HBV infection persons and 10 early liver cancer peripheral blood in patients oxidized form apolipoproteins
The expression quantity (ng/ml) of apoA-1
| Healthy People | HBV infection person | Early liver cancer patient |
| 4.12 | 7.97 | 8.65 |
| 5.63 | 6.58 | 7.68 |
| 7.12 | 8.25 | 7.58 |
| 3.24 | 6.25 | 5.69 |
| 5.14 | 7.25 | 8.64 |
| 3.20 | 5.25 | 7.69 |
| 1.25 | 6.45 | 7.98 |
| 2.65 | 7.58 | 4.89 |
| 4.84 | 7.69 | 9.25 |
| 6.57 | 8.95 | 7.58 |
Pass through the data of table 1 and the figure of attached drawing 2, it can be seen that oxidized form Genetyping poA-1 is in normal person and HBV
There is more apparent difference among the infected or early liver cancer patient, this method is for distinguishing Healthy People from the point of view of angle of statistics
There is greater significance with HBV infection person or difference Healthy People and early liver cancer patient.
In summary it tests: present invention firstly discovers that the oxidized form Genetyping poA-1 content in blood can be used as HBV
The biomarker of infection and the diagnosis of early liver cancer knot, according to above-mentioned experiment content it is disclosed by the invention for HBV infection or
The detection kit of person's liver cancer early detection, in terms of HBV infection and liver cancer with good application prospect.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, this technology neck
The variations, modifications, additions or substitutions that the technical staff in domain is made within the essential scope of the present invention also should belong to of the invention
Protection scope.
Claims (10)
1. oxidized form Genetyping poA-1 is as HBV infection or the purposes of the molecule marker of hepatocarcinoma early diagnosis.
2. a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that including at least one kind for detecting
The reagent of oxidized form Genetyping poA-1.
3. according to claim 2 a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that
The kit forms of the diagnosis are as follows:
(1) for carrying out the reagent that pretreatment obtains sample to sample to be tested；
(2) for the detection reagent that oxidized form Genetyping poA-1 expression quantity is detected in sample；
(3) label or operation instructions, the label or operation instructions indicate the kit for diagnosing HBV or early stage
Liver cancer.
4. according to claim 3 a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that
For being biotin to the detection reagent that oxidized form Genetyping poA-1 expression quantity is detected in sample in the step (2)
The oxidized form Genetyping poA-1 monoclonal antibody of label.
5. according to claim 4 a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that
It further include having standard items, cleaning solution, substrate solution, developing solution, terminate liquid and oxidized form Genetyping poA-1 polyclonal antibody packet
By ELISA Plate.
6. according to claim 5 a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that
The oxidized form Genetyping poA-1 polyclonal antibody coated elisa plate is made of following steps: coating oxidized form is carried rouge
Albumen apoA-1 polyclonal antibody 0.1M glycine buffer, PH9.0；Addition each hole of ELISA Plate after dilution, every hole 100ul,
Absorption overnight, washes version with 0.1M glycine buffer；With confining liquid closing overnight to get the oxidized form Genetyping poA-1
Polyclonal antibody coated elisa plate.
7. it is described in any item a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis according to claim 3-6, it is special
Sign is that the sample to be tested is blood, serum or plasma sample.
8. according to claim 7 a kind of for HBV infection or the kit of hepatocarcinoma early diagnosis, which is characterized in that
The sample to be tested is human peripheral.
9. it is a kind of using as claimed in claim 8 a kind of for HBV infection or the method for the kit of hepatocarcinoma early diagnosis,
The following steps are included:
(1) venous collection peripheral blood；
(2) collected peripheral blood is pre-processed；
(3) oxidized form Genetyping poA-1 antibody incubation is added；
(4) Incubating Solution is added in ELISA Plate together with comparison liquid and is analysed and compared.
10. it is according to claim 9 a kind of using HBV infection or the method for the kit of hepatocarcinoma early diagnosis, it is special
Sign is, in the step (2) to collected peripheral blood carry out pretreated method be after acquisition in 30 minutes in 1000 × g
Centrifugation 15 minutes, takes supernatant.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910705480.4A CN110333351A (en) | 2019-08-01 | 2019-08-01 | It is a kind of for HBV infection or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis |
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| CN201910705480.4A CN110333351A (en) | 2019-08-01 | 2019-08-01 | It is a kind of for HBV infection or the oxidized form apoA-1 detection kit of hepatocarcinoma early diagnosis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110780071A (en) * | 2019-11-11 | 2020-02-11 | 彭涛 | Hepatitis B-related hepatocellular carcinoma prognosis detection kit |
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| CN1777682A (en) * | 2003-04-25 | 2006-05-24 | 香港大学 | Serum biomarkers of hepatitis b virus infected liver and methods for detection thereof |
| CN201707337U (en) * | 2010-06-17 | 2011-01-12 | 武汉优尔生科技股份有限公司 | Human carried lipoprotein A1 enzyme linked immunoabsorbent assay kit |
| CN201707338U (en) * | 2010-06-24 | 2011-01-12 | 武汉优尔生科技股份有限公司 | Human apolipoprotein B100 (Apo-B100) ELISA kit |
| CN103505720A (en) * | 2012-06-30 | 2014-01-15 | 复旦大学 | Application of apolipoprotein A-I in preparation of medicine used for preventing and treating liver disease metabolic syndrome |
-
2019
- 2019-08-01 CN CN201910705480.4A patent/CN110333351A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1777682A (en) * | 2003-04-25 | 2006-05-24 | 香港大学 | Serum biomarkers of hepatitis b virus infected liver and methods for detection thereof |
| CN201707337U (en) * | 2010-06-17 | 2011-01-12 | 武汉优尔生科技股份有限公司 | Human carried lipoprotein A1 enzyme linked immunoabsorbent assay kit |
| CN201707338U (en) * | 2010-06-24 | 2011-01-12 | 武汉优尔生科技股份有限公司 | Human apolipoprotein B100 (Apo-B100) ELISA kit |
| CN103505720A (en) * | 2012-06-30 | 2014-01-15 | 复旦大学 | Application of apolipoprotein A-I in preparation of medicine used for preventing and treating liver disease metabolic syndrome |
Non-Patent Citations (3)
| Title |
|---|
| JOKIN FERNÁNDEZ-IRIGOYEN等: "Oxidation of specific methionine and tryptophan residues of apolipoprotein A-I in hepatocarcinogenesis", 《PROTEOMICS》 * |
| MASASHI UEDA等: "Establishment and evaluation of 2 monoclonal antibodies against oxidized apolipoprotein A-I (apoA-I) and its application to determine blood oxidized apoA-I levels", 《CLINICA CHIMICA ACTA》 * |
| 胡智祥: "《医院临床检验技术操作规范与实（化）验室管理全书 1卷》", 31 August 2004, 银声音像出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110780071A (en) * | 2019-11-11 | 2020-02-11 | 彭涛 | Hepatitis B-related hepatocellular carcinoma prognosis detection kit |
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Application publication date: 20191015 |