CN102213722A - Application of kit for detecting protein expression level in preparation of kit for diagnosing hepatocellular carcinoma - Google Patents
Application of kit for detecting protein expression level in preparation of kit for diagnosing hepatocellular carcinoma Download PDFInfo
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Abstract
The invention discloses application of a kit for detecting the vitronectin protein expression level in preparation of a kit for diagnosing hepatocellular carcinoma. The kit for detecting the vitronectin protein expression level is an enzymelinked immunosorbent kit. The kit for diagnosing liver cancer, which is prepared from a kit for detecting the vitronectin protein concentration, has remarkable application value on the clinical examination aspect of AFP (alpha fetoprotein) negative hepatocellular carcinoma.
Description
Technical field
The present invention relates to detect the application of kit in the kit of preparation diagnosing hepatocellular carcinoma of vitronectin expression, the kit that particularly detects the vitronectin expression is used for the application of the enzyme-linked immunosorbent assay kit that the differentiation of hepatocellular carcinoma and liver cirrhosis patient diagnoses in preparation.
Background technology
(hepatocellular carcinoma HCC) is one of worldwide malignant tumour to hepatocellular carcinoma, and higher M ﹠ M is arranged, and annual about 650,000 people die from HCC, and its incidence of disease has the trend that increases.This disease is mainly in Southeast Asia and Africa, wherein has approximately to betide China more than 50%, occupies second of China's cancer mortality.
Hepatocellular carcinoma a situation arises be one multifactor, the multistage, complicated process, (hepatitisB virus, HBV) chronic infection is important high risk factor to hepatitis type B virus.Tumor suppression, hepatotomy and transplanting are the main methods of treatments of present stage liver cancer, but about patient of 60% to 100% understands postoperative recurrence.Therefore, the people at highest risk particularly cirrhosis (liver cirrhosis, LC) examination early liver cancer patient among the patient in time takes the treatment measure, can improve the survival rate and the quality of life of liver cancer patient.
But the HCC early diagnosis is difficult, and disease progression is fast, and prognostic level is low.HCC people at highest risk's such as hepatitis, cirrhosis examination and HCC clinical diagnosis and state of illness monitoring etc. still mainly depend on imaging examination associating blood serum designated object alpha-fetoprotein (alpha-fetoprotein, AFP) detections of level such as B ultrasonic at present.And AFP is as HCC diagnosis at present and topmost biomarker of state of illness monitoring, and its sensitivity and specificity are still undesirable, particularly has negatively above 30% patient HCC AFP in clinical, can't play a role for this part patient AFP.Though Des-gamma carboxy-prothrombin (DCP) and lectin-bound AFP (AFP-L3) are considered to very potential HCC diagnosis marker, but the result shows recently, AFP still is better than DCP and AFP-L3 (Marrero for the effect of HCC early diagnosis, J.A.et al.alpha-Fetoprotein, Des-gamma Carboxyprothrombin, and Lectin-Bound alpha-Fetoprotein in Early Hepatocellular Carcinoma.Gastroenterology (2009)).
Seek responsive liver cancer biomarker, extremely important for early detection and diagnosing hepatocellular carcinoma.In recent years, there are many laboratories carrying out the relevant proteomics research of hepatocarcinoma mark thing.The liver cancer tumor tissues is carried out iTRAQ to be analyzed, three kinds of differential protein fibroleukin have been obtained through the SABC checking, myeloid associateddifferentiation marker and ornithine carbamoyl transferase (Chaerkady, R.et al. A quantitativeproteomic approach for identification of potential biomarkers in hepatocellular carcin
In sum, the research of proteomics aspect liver cancer marker in recent years, major part still rests on the discovery stage, and in the checking of mark, particularly the progress that obtains in the checking of serum levels is also few.Therefore, set up a kind of detection method, help the clinical diagnosis of tumour at the serum proteins that have significant variation in the liver cell carcinogenesis process.
Vitronectin (vitronectin) is multi-functional adhesion glycoprotein and the invasin that is present in blood and some tissues, can be integrated the special member identification of plain family, in adhering to, cell-matrix plays a role, multiple physiology such as the variation of vitronectin level and blood coagulation, immune correlated disease and pathologic process are closely related in the human body, but still unmatchful its carries out the report that serum ELISA detection is used for diagnosis of hepatoma.(Vitronectin:UniProtKB/Swiss-Prot storehouse albumen P04004, GenBank number: X05006)
Enzyme linked immunosorbent assay (ELISA) (enzyme-linked immunosorbent assay is called for short ELISA) is a kind of novel immunoassay that grows up on immunoenzyme technics (immunoenzymatic techniques), the ELISA process comprises: antigen is adsorbed on the solid phase carrier, this process is called the bag quilt, add antibody to be measured, add corresponding enzymic-labelled antibody again, generate antigen--antibody to be measured--compound of enzymic-labelled antibody, the substrate reactions with this enzyme generates coloured product again.Amount by spectrophotometric light absorption calculating antibody.The amount of antibody to be measured is directly proportional with coloured product.In like manner but also coated antibody is measured antigenic content.Four kinds of methods that ELISA is the most frequently used: direct method is measured antigen; Indirect method is measured antibody; Double antibody sandwich method is measured antigen; Competition law is measured antigen.
(1) direct method is measured the step of antigen
A. antigen is adsorbed on carrier surface;
B. add enzyme labelled antibody, form antigen-antibody complex;
C. add substrate.
In this method, the degradation amount of substrate=antigen amount.
(2) indirect method is measured antibody
A. antigen is adsorbed in surface of solid phase carriers;
B. add antibody, form antigen-antibody complex;
C. add enzyme labelled antibody;
D. add substrate.
In this method, measure the degradation amount=antibody amount of substrate.
(3) double antibody sandwich method is measured antigen
A. antibody is adsorbed in solid phase surface;
B. add antigen, form antigen-antibody complex;
C. add enzyme labelled antibody;
E. add substrate.
In this method, the degradation amount of substrate=antigen amount.
(4) competition law is measured antigen
A. antibody is adsorbed on surface of solid phase carriers;
B. add enzyme-labelled antigen and determined antigen, the competition binding antibody; Contrast only adds enzyme-labelled antigen;
C. add substrate.
In this method, the poor=unknown antigen amount of control wells and sample well degradation of substrates amount.
Summary of the invention
The purpose of this invention is to provide the application of kit in the kit of preparation diagnosing hepatocellular carcinoma that detects vitronectin (Vitronectin) expression.
The kit of above-mentioned detection vitronectin (Vitronectin) expression all can as long as satisfy the requirement of vitronectin (Vitronectin) concentration in can detection architecture, enzyme linked immunological kit particularly, as American DiagnosticaInc (ADI) company, the enzyme linked immunological kit of article No. 803 (
Vitronectin ELISA).
The kit of above-mentioned detection vitronectin (Vitronectin) expression also can obtain with vitronectin and/or its Antibody Preparation.The kit of the diagnosing hepatocellular carcinoma of the kit preparation of this detection vitronectin (Vitronectin) expression does not rely on the blood serum designated object alpha-fetoprotein, and (alpha-fetoprotein, AFP) expression promptly can diagnosing liver cancer.
Reason same as described above, utilize vitronectin and/or its antibody to make to be easy to detect, with a high credibility, do not rely on blood serum designated object alpha-fetoprotein (alpha-fetoprotein, AFP) enzyme linked immunological kit of the diagnosing hepatocellular carcinoma of expression.
The enzyme linked immunological kit of this diagnosing hepatocellular carcinoma can be the enzyme linked immunological kit of conventional detected vitronectin (Vitronectin) expression, generally include solid phase carrier, label and can with the substrate of described label generation chromogenic reaction; Described label is enzyme labeling vitronectin albumen, enzyme labeling vitronectin protein antibodies, biotin labeling vitronectin albumen or biotin labeling vitronectin protein antibodies.Described label can be made by vitronectin albumen or vitronectin protein antibodies.
Above-mentioned vitronectin protein antibodies can be monoclonal antibody or polyclonal antibody, and wherein monoclonal antibody can be available from Santa Cruz company (article No. sc-52752), and polyclonal antibody can be available from Proteintech company (article No. 15833-1-AP).
The mentioned reagent box can be used for the expression that enzyme linked immunological absorption detects vitronectin albumen, specifically can be direct method and measures antigen, double antibody sandwich method mensuration antigen and competition law mensuration antigen.When using double antibody sandwich method to measure antigen and competition law mensuration antigen, described enzyme linked immunological kit also comprises coating antigen, and described coating antigen is the vitronectin protein antibodies.
The kit that direct method is used, described label are enzyme labeling vitronectin protein antibodies or biotin labeling vitronectin protein antibodies.
The kit that double antibody sandwich method is used, described coating antigen are the vitronectin protein antibodies, and described label is enzyme labeling vitronectin protein antibodies or biotin labeling vitronectin protein antibodies.
Competition law is measured the used kit of antigen, and described coating antigen is the vitronectin protein antibodies, and described label is enzyme labeling vitronectin albumen or biotin labeling vitronectin albumen.
The used marker enzyme of described label is horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase.
The material of described solid phase carrier is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of solid phase carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.; Wherein be preferably the micro-reaction plate shrinkage pool that polystyrene is made.Polystyrene has the performance of stronger adsorbed proteins, still keeps original immunocompetence after antigen adsorbs on it, and blank value is low, and transparency height at the bottom of the hole is between each plate, performance is close between each hole of same plate, so solid phase carrier is preferably polystyrene.
Above-mentioned described vitronectin protein antibodies is preferably one and resists, and can be with vitronectin albumen is the monoclonal antibody or the polyclonal antibody of antigen; Described vitronectin protein antibodies can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, is preferably mouse source antibody; When using two sandwich method, need the antibody that uses two kinds different antigenic determinants or different plant species source are arranged, wherein the preferred mouse of coating antigen source antibody.
Described kit also comprises the vitronectin protein standard substance.The vitronectin protein standard substance can be enrichment or expression, synthetic people vitronectin holoprotein or the peptide section that comprises vitronectin proteantigen determinant;
When label is horseradish peroxidase, to form by substrate A and substrate B with the substrate of described enzyme labeling thing generation chromogenic reaction, described substrate A is hydrogen peroxide or urea peroxide, described substrate B be o-phenylenediamine (O-phenylenediamine, OPD), 3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine (3 ', 3 ', 5 ', 5 ' ,-tetramethylbenzidine, TMB) or ABTS[2,2 '-azino-di-(3-ethylbenziazobine sulfonate-6)]; When label was alkaline phosphatase, substrate was a 4-nitrophenols phosphate buffer; When label was biotin, substrate A was an enzyme mark Avidin, and substrate B is the substrate of enzyme.
Also comprise in the described kit concentrating sample dilution, antibody diluent, washing lotion, chromogenic reagent, stop buffer etc., these reagent can prepare with conventional method.
The inventor finds the protein vitronectin of up-regulated expression among the hepatocellular carcinoma patients serum by a large amount of experiments, and provide and to have utilized the kit that detects this kind protein foundation as the kit of diagnosing hepatocellular carcinoma, and the decision threshold standard of utilizing this kit diagnosing hepatocellular carcinoma, promptly utilize its characteristic to set up the kit of diagnosing liver cancer, this kit can be used for the particularly clinical detection of the negative hepatocellular carcinoma of AFP of hepatocellular carcinoma.Experiment conclusion shows, vitronectin albumen has obvious high expressed than liver cirrhosis patient in the hepatocellular carcinoma patients serum, and expression does not rely on blood serum designated object alpha-fetoprotein (alpha-fetoprotein, AFP) expression, therefore of the present invention particularly have outstanding using value aspect the negative hepatocellular carcinoma clinical examination of AFP by detecting kit that the vitronectin protein concentration comes diagnosing liver cancer in hepatocellular carcinoma.
Application provided by the present invention, the protein vitronectin of utilization up-regulated expression in the hepatocellular carcinoma patients serum is as the preparation or the direct kit as diagnosing hepatocellular carcinoma of diagnosis marker kit, it has highly sensitive, the characteristics that accuracy is high, and do not rely on the blood serum designated object alpha-fetoprotein (alpha-fetoprotein that conventional method must rely on, AFP) express, be applicable in biology, medical science and pharmaceutical field hepatocellular carcinoma is carried out clinical detection research and pathogenesis research, have practicality widely.Purposes includes but not limited to: carry out various hepatocellular carcinoma coherent detections and research in fields such as biology, medical science and pharmacy.
Description of drawings
Fig. 1 .vitronectin level infects the Western blot testing result of (every group is the mixing of 30 routine samples) among liver cirrhosis group (LC) relevant hepatocellular carcinoma group with HBV (HCC) patients serum in normal control group (Normal), HBV.
The ELISA testing result of Fig. 2 .vitronectin level in the serum sample of normal control 30 examples (N), HBV infection liver cirrhosis patient 50 examples (LC), relevant hepatocellular carcinoma patient 52 examples (HCC, 42 routine AFP feminine genders, the 10 routine AFP positives) of HBV and other Infusion in Patients with Digestive 15 examples (T).
Fig. 3. the ROC tracing analysis result of vitronectin protein level in liver cirrhosis group and the hepatocellular carcinoma group serum.
The ELISA testing result of Fig. 4 .vitronectin level in 42 routine AFP feminine genders and the positive hepatocellular carcinoma patients serums of 10 routine AFP.
Fig. 5. the ROC tracing analysis result of vitronectin protein level among the negative hepatocellular carcinoma group of liver cirrhosis group and the AFP patients serum.
Embodiment
The following examples will be further explained the present invention, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
The design of the kit of embodiment 1, diagnosing hepatocellular carcinoma
The present inventor found through experiments, and the expression of vitronectin albumen in the hepatocellular carcinoma patients serum raises, and further further verified this conclusion by Western blot, and concrete experimental technique is as follows:
To normal control group (healthy people), clinical identification is that HBV infection liver cirrhosis group is (to get 30 routine sample mixed in equal amounts for every group in the relevant hepatocellular carcinoma group of the HBV serum with clinical identification, mixing can be subdued individual difference, embody average level, making the result more representative) the vitronectin level carried out Western blot and detected: at first serum sample is carried out conventional SDS-PAGE electrophoresis, after forwarding on the nitrocellulose filter, puts into electricity confining liquid (TBST 5% skimmed milk) sealing 1h, in 1: 1500 dilution vitronectin one anti-(Proteintech company, 4 ℃ of overnight incubation 15833-1-AP), TBST washes film 5 times, add 1: 5000 dilution goat-anti rabbit two anti-(KPL company, 074-1516) hatch 1h under the room temperature, the TBST damping fluid is washed film 5 times, ECL kit (Pierce company) colour developing.
The result as shown in Figure 1, the result shows, finds that the vitronectin protein level descends in liver cirrhosis group, the hepatocellular carcinoma group is gone up.This result shows and can come diagnosing hepatocellular carcinoma by detecting the vitronectin protein expression level.
Therefore, the present inventor is foundation with the The above results, can use vitronectin albumen and/or its antibody and set up by detecting the ELISA detection kit that the vitronectin protein expression level comes diagnosing hepatocellular carcinoma, it comprises: solid phase carrier, label and can with the substrate of described label generation chromogenic reaction; Label is enzyme labeling vitronectin albumen, enzyme labeling vitronectin protein antibodies, biotin labeling vitronectin albumen or biotin labeling vitronectin protein antibodies.Concrete kit can be made up of following reagent:
1) ELISA Plate (solid phase carrier);
2) people vitronectin standard protein;
3) label;
4) sample dilution;
5) antibody diluent;
6) washing lotion;
7) chromogenic reagent;
8) stop buffer.
It is known to those skilled in the art that above component only is schematically, described ELISA Plate can be an anti-bag by (being used for double fastener heart ELISA method, competition law) that cross or bag by (being used for direct ELISA method) that cross; People vitronectin standard protein can be enrichment or expression, synthetic holoprotein or the peptide section that comprises antigenic determinant; Label can be horseradish peroxidase-labeled or biotin labeling; Chromogenic reagent can be TMB, OPD or ABTS etc.The ELISA method can be two sandwich methods, direct method or competition law.Use for convenient, this kit also can comprise one or more in the following component: negative control, positive control can contain the operation instruction of state administrative organs's approval etc. in addition.
The using method of this kit is as follows:
1) ELISA Plate is with the vitronectin antibody sandwich or do not wrap quilt;
2) use sample diluted people vitronectin standard protein to variable concentrations, add in the ELISA Plate and hatch, be used for the production standard curve, with sample diluted human serum sample to finite concentration, add in the ELISA Plate and hatch, be used for pattern detection, hatch the back and wash with washing lotion;
3) the label diluted is certain working fluid, adds in the ELISA Plate to hatch, and hatches the back and washes with washing lotion;
4) add the zymolyte colour developing;
5) add the stop buffer cessation reaction;
6) OD pH-value determination pH and result calculate;
The all ingredients that agents useful for same is well known to those skilled in the art.For example, ELISA Plate can or not wrapped by (directly ELISA method) with vitronectin antibody sandwich (double fastener heart ELISA method) in the step 1); Institute's usage flag thing can adopt horseradish peroxidase or biotin labeling in the step 3); Use in the step 4) enzyme chromogenic substrate can be 3 ', 3 ', 5 ', 5 ',-tetramethyl benzidine (3 ', 3 ', 5 ', 5 ',-tetramethylbenzidine, TMB), o-phenylenediamine (O-phenylenediamine, OPD) or ABTS[2,2 '-azino-di-(3-ethylbenziazobine sulfonate-6)] etc.; Used wavelength is corresponding with used chromogenic substrate in the step 4) in the step 6); The concrete dilute concentration of antibody, albumen or sample step 1), 2), 3) can be done suitable adjustment.But vitronectin antibody commodity in use product.The ELISA method can be two sandwich methods, direct method or competition law.
With following specific embodiment principle of the present invention is described, the effect of checking kit of the present invention.
Embodiment 2, utilization detect the compliance test result of the enzyme linked immunological kit of vitronectin protein expression level as the kit of diagnosing hepatocellular carcinoma
One, the composition of ELISA kit (double fastener heart ELISA method)
The enzyme linked immunological kit of the detection vitronectin albumen that the present invention utilizes (double fastener heart ELISA method, AmericanDiagnostica Inc (ADI) company, article No. 803,
Vitronectin ELISA), comprise following reagent:
1) ELISA Plate (solid phase carrier has wrapped the antibody by vitronectin);
2) people vitronectin protein standard solution: vitronectin concentration is the human normal plasma of 110 μ g/ml, is the vitronectin albumen total length of GenBank X05006.
3) the people vitronectin antibody of HRP mark;
4) sample dilution;
5) washing lotion;
6) chromogenic substrate reagent (substrate is TMB);
7) stop buffer.
Two, detect the effect experiment of the enzyme linked immunological kit diagnosing liver cancer of vitronectin albumen
Vitronectin level in the serum sample of normal control 30 examples, HBV infection liver cirrhosis patient 50 examples, relevant hepatocellular carcinoma patient 52 examples (42 routine AFP feminine genders, the 10 routine AFP positives) of HBV and other Infusion in Patients with Digestive 15 examples is carried out ELISA detect, concrete grammar is as follows:
1, get the ELISA Plate that vitronectin standard protein 0ng/ml, 6.88ng/ml, 13.75ng/ml, 27.5ng/ml, 55ng/ml and 110ng/ml solution 100 μ l add coated antibody respectively, each gradient is done 2 parallel holes, is used for the production standard curve.
2, normal control group (healthy people's 30 examples; N), (clinical identification is the 50 routine patients that HBV infects liver cirrhosis patient to liver cirrhosis group; LC), (clinical identification is the patient of HBV infected liver cell cancer to the hepatocellular carcinoma group, wherein negative 42 examples of AFP, positive 10 examples of AFP; HCC) and other tumor in digestive tract group (clinical identification is a tumor in digestive tract and do not suffer from patient's 15 examples of liver cancer; T) serum sample, respectively get 2 μ l with the sample diluted after (extension rate is normal control group 1: 5000, liver cirrhosis group 1: 4000, hepatocellular carcinoma group 1: 4000), every routine sample is got the ELISA Plate that 100 μ l add coated antibody, and each sample is done 2 parallel holes.With the hole that only adds the sample dilution as negative control.
3, after above-mentioned sample room temperature (18-25 ℃) horizontal shaking table (250rpm) was hatched 1h, every hole added 350 μ l washing lotions washing 4 times, each 3 minutes.
4, every hole adds the people vitronectin antibody 100 μ l of HRP mark, and after room temperature (18-25 ℃) horizontal shaking table (250rpm) was hatched 1h, every hole added 350ul washing lotion washing 4 times, each 3 minutes.
5, add chromogenic substrate reagent 100 μ l/ holes, room temperature (18-25 ℃) colour developing 5-10 minute; Add stop buffer 50 μ l/ holes, the 450nm wavelength is measured the OD value of each hole sample.
6, by standard items concentration and OD value drawing standard curve,, calculate the content of vitronectin albumen in each serum sample by the measured OD value of each hole serum sample.
The result as shown in Figure 2, the result shows, the vitronectin protein level descends in liver cirrhosis group, the hepatocellular carcinoma group is gone up, wherein the concentration mean value of normal control group vitronectin albumen is 128.21 μ g/ml, liver cirrhosis group is 56.19 μ g/ml, the hepatocellular carcinoma group is 104.07 μ g/ml, and vitronectin protein content in each group sample is carried out statistical analysis, finds that liver cirrhosis group and hepatocellular carcinoma group serum vitronectin protein level have significant difference.
7, liver cirrhosis group and hepatocellular carcinoma group serum vitronectin protein level are carried out the ROC tracing analysis
By GraphPad Prism software liver cirrhosis group and hepatocellular carcinoma group serum vitronectin protein level being carried out the ROC tracing analysis, is that control group, hepatocellular carcinoma group are disease group with the liver cirrhosis group, AUC=0.8781 (as shown in Figure 3).According to youden index (sensitivity+specificity-1) calculating optimum working point, when serving as when judging the threshold value of hepatocellular carcinoma with vitronectin protein concentration＞79 μ g/ml, sensitivity is 88%, specificity is 80%.The ELISA checking of above-mentioned 147 routine human serum samples has very high credibility.The above results explanation vitronectin is the potential mark that can be used for hepatocellular carcinoma and liver cirrhosis patient differentiation diagnosis.
8, positive hepatocellular carcinoma group serum vitronectin protein level of AFP feminine gender and AFP and the contrast of ROC curve thereof
AFP feminine gender (40 example) and the AFP positive (10 example) hepatocellular carcinoma group serum vitronectin protein level data that contrast step 3 obtains, there was no significant difference (as shown in Figure 4).Liver cirrhosis group and the negative hepatocellular carcinoma group of AFP serum vitronectin protein level are carried out the ROC tracing analysis, are that the negative hepatocellular carcinoma group of control group, AFP is a disease group with the liver cirrhosis group, AUC=0.88 (as shown in Figure 5).The above results explanation vitronectin can be used for the differentiation diagnosis of negative hepatocellular carcinoma of AFP and liver cirrhosis patient.
To sum up experiment conclusion shows, vitronectin albumen has obvious high expressed than liver cirrhosis patient in the hepatocellular carcinoma patients serum, and expression does not rely on AFP, therefore the present invention utilizes the kit of the enzyme linked immunological kit of detection vitronectin albumen as diagnosing liver cancer, perhaps directly utilize the preparation of vitronectin albumen and/or vitronectin protein antibodies by detecting the kit that the vitronectin protein concentration comes diagnosing liver cancer, particularly have using value aspect the negative hepatocellular carcinoma clinical examination of AFP in hepatocellular carcinoma.
Claims (9)
1. detect the application of kit in the kit of preparation diagnosing hepatocellular carcinoma of vitronectin protein expression level.
2. application according to claim 1 is characterized in that: the kit of described detection vitronectin protein expression level is an enzyme linked immunological kit.
3. application according to claim 2 is characterized in that: the kit of described detection vitronectin protein expression level comprises the label of vitronectin albumen and/or vitronectin protein antibodies and/or vitronectin albumen and/or the label of vitronectin protein antibodies.
4. application according to claim 3 is characterized in that: the kit of described detection vitronectin protein expression level is that the article No. of American Diagnostica Inc company production is 803 enzyme linked immunological kit, and the kit name is called
Vitronectin ELISA.
5. according to any described application among the claim 1-4, it is characterized in that: described hepatocellular carcinoma is that HBV infects the hepatocellular carcinoma that causes.
6. according to any described application among the claim 1-5, it is characterized in that: described hepatocellular carcinoma is the hepatocellular carcinoma of AFP feminine gender.
7.vitronectin the application of protein antibodies in the kit of preparation diagnosing hepatocellular carcinoma.
8. application according to claim 7, it is characterized in that: described vitronectin protein antibodies is monoclonal antibody or polyclonal antibody, described monoclonal antibody is the antibody of in vitro culture hybridoma cell line acquisition or the antibody of commercial sources acquisition, and described polyclonal antibody is the antibody that commercial sources obtains.
9.vitronectin the application of albumen in the kit of preparation diagnosing hepatocellular carcinoma.
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| CN103743903A (en) * | 2014-01-08 | 2014-04-23 | 江苏省苏北人民医院 | Detection method and kit of predictive marker Capn4 of postoperative recurrence of liver cancer |
| CN106198769A (en) * | 2015-05-06 | 2016-12-07 | 眭维国 | Hepatocarcinoma phosphoprotemics model and construction method thereof and application |
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| CN103743903A (en) * | 2014-01-08 | 2014-04-23 | 江苏省苏北人民医院 | Detection method and kit of predictive marker Capn4 of postoperative recurrence of liver cancer |
| CN106198769A (en) * | 2015-05-06 | 2016-12-07 | 眭维国 | Hepatocarcinoma phosphoprotemics model and construction method thereof and application |
| CN106198769B (en) * | 2015-05-06 | 2019-06-21 | 眭维国 | Liver cancer phosphoprotemics model and its construction method and application |
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