CN106066401B - Biomarker VWF and ADAMTS13 and its purposes in liver cirrhosis diagnosis reagent - Google Patents

Biomarker VWF and ADAMTS13 and its purposes in liver cirrhosis diagnosis reagent Download PDF

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CN106066401B
CN106066401B CN201610203495.7A CN201610203495A CN106066401B CN 106066401 B CN106066401 B CN 106066401B CN 201610203495 A CN201610203495 A CN 201610203495A CN 106066401 B CN106066401 B CN 106066401B
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cirrhosis
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adamts13
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高平
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CHONGQING ZEARLY BIO-TECH Co.,Ltd.
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Chongqing Seven Days Biological Polytron Technologies Inc
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    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

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Abstract

The present invention relates to disease biomarkers purposes fields, disclose cirrhosis biomarker VWF and ADAMTS13 and its purposes in liver cirrhosis diagnosis reagent.By amino acid sequence, the accession number in ncbi database is that the protein of GI 317373549 forms to VWF of the present invention, and by amino acid sequence, the accession number in ncbi database is that the protein of GI 74749836 forms to the ADAMTS13.Liver cirrhosis diagnosis is carried out to subject with biomarker VWF and ADAMTS13, has many advantages, such as that simple and easy, diagnosis process safety hurtless measure is easy to be received by patient, diagnostic criteria unification is influenced by factor and individual subjective factor, and smaller, diagnostic result is specific and sensitivity is high, is easy to carry out a large amount of screenings selects.

Description

Biomarker VWF and ADAMTS13 and its purposes in liver cirrhosis diagnosis reagent
Technical field
The present invention relates to disease biomarkers purposes field, be related specifically to cirrhosis biomarker VWF and ADAMTS13 and its purposes in liver cirrhosis diagnosis reagent.
Background technique
Cirrhosis is chronic progressive hepatopathy, by the diffusivity liver damage that one or more causes of disease are long-term or repeated action is formed Evil.It is posthepatitic cirrhosis in most of China, small part is alcoholic cirrhosis and Cirrhosis In Schistosomiasis.Histopathology On have extensive necrosis of liver cells, regeneration of remaining liver cell nodules, connective tissue proliferation and fiber every formation, lead to lobuli hepatis Structure is destroyed and pseudolobuli is formed, and liver gradually deforms, is hardened and develops as cirrhosis.Early stage is stronger due to liver compensation No obvious symptom, stage is main performance with hepatic disorder and portal hypertension, and has multisystem involvement, and advanced stage often occurs The complication such as upper gastrointestinal bleeding, hepatic encephalopathy, secondary infection, hypersplenia, ascites, canceration.
In China, the case fatality rate of cirrhosis is only second to malignant tumour, has 154 people to die of liver every about 100,000 populations every year Hardening.Worldwide, cirrhosis also increasingly becomes clinical common one of disease, and cirrhosis has become the world according to statistics One of three big fatal causes of disease.How convenient, fast, timely, accurate detection diagnoses cirrhosis, and controls patient's cirrhosis Therapeutic effect tracing and monitoring has become cirrhosis health field problem in the urgent need to address.
Earliest cirrhosis detection method uses needle biopsy of liver method, and the diagnostic result of this method is that cirrhosis is examined Break accurate liver cirrhosis diagnosis method recognized within the industry.However, easily there is significant limitation in this method.Firstly, should Method be it is traumatic, be not easy to be received by patient；Secondly, the possibility for having complication to occur when operation, should not carry out frequently repeatedly Biopsy；Third, needle biopsy of liver method puncture sample is small, only accounts for the 1/50000 of liver mass, there are biggish sampling errors.
Currently, China's diagnosis cirrhosis using it is more be B ultrasound.But B ultrasound be see liver form and pathology simultaneously It is not directly relevant to, accurately cirrhosis cannot be analyzed, can not judge cirrhosis disease progression, and to instrument and operator Member has higher requirement.
CT and nuclear-magnetism detection method are helpful to the diagnosis of cirrhosis, but this equipment is costly, and conventional development is very tired Difficulty is not suitable for China's actual conditions.FibroScan technology starts to walk in China, but many factors influence the accuracy of FibroScan, Such as ascites, liver inflammation is fat, fatty liver etc..Quite a few patient can not carry out the inspection of FibroScan.
Thus low in cost, quick, the easy and invasive minimum cirrhosis for being easy to receive for patient is badly in need of in this field Diagnostic method.
VWF be English von Willebrand factor acronym, Chinese can be translated into von Willebrand disease because Son,Www.uniprot.org website log number is P04275Entry its amino acid sequence is had been disclosed.VWF is a kind of Macro-molecular protein polymer is mainly used for the VWD i.e. diagnosis of von Willebrand disease.Meanwhile publication number CN102614513A Chinese patent " application of the area anti-angiogenic property christmas factor A3 bi-functional monoclonal antibody " also discloses VWF and is preparing anti-blood Purposes in bolt drug.
ADAMTS13 is English Adisintegrin and metalloproteinase with thrombospondin The abbreviation of motifs 13 is referred to as.Other substitution titles there are also von Willebrand factor-cleaving protease, Chinese can be translated into vWF ELISA catabolic enzyme.Www.uniprot.org website log number isThe entry of Q76LX8 is to it Amino acid sequence has been disclosed, and shares 3 subunit sequences.
Summary of the invention
In order to overcome various shortcoming existing for prior art liver cirrhosis diagnosis, the present invention provides two kinds of cirrhosis biological markers Object VWF and ADAMTS13.The VWF protein that the accession number in ncbi database is GI 317373549 by amino acid sequence Composition, by amino acid sequence, the accession number in ncbi database is that the protein of GI 74749836 forms to the ADAMTS13.
It was found by the inventors of the present invention that can be identified in some time before there are apparent cirrhosis disease symptoms Relevant biomarker VWF and ADAMTS13 in serum or other body fluid.And human serum whether cirrhosis illness The content difference of middle VWF or ADAMTS13 is very big, it is possible thereby to consider by human serum biomarker VWF and The content of ADAMTS13 is used as liver cirrhosis diagnosis tool, and realizes according to the diagnostic result of these diagnostic tools to liver cirrhosis patient Personalized medicine.
Based on above-mentioned discovery, the present invention provides biomarker VWF and/or ADAMTS13 to examine in preparation diagnosis cirrhosis Purposes in disconnected reagent.
Terms used herein is generally defined as：
VWF：The albumen that the protein that accession number is GI 317373549 in ncbi database by amino acid sequence forms Matter；
ADAMTS13：The protein that accession number is GI 74749836 in ncbi database by amino acid sequence forms Protein；
First reference value：The content of VWF in patients with chronic liver subject's serum of cirrhosis is not developed into；
Second reference value：The content of ADAMTS13 in patients with chronic liver subject's serum of cirrhosis is not developed into；
Third reference value：VWF content and ADAMTS13 in patients with chronic liver subject's serum of cirrhosis are not developed into The ratio of content.
All terms not being defined herein are all identical as the meaning that those of ordinary skill in the art are generally understood.
Specifically, one of scheme：The diagnostic reagent is joined by detecting the content of VWF in subject's serum with first Value is examined compared to relatively to diagnose cirrhosis；The two of scheme：The diagnostic reagent is contained by ADAMTS13 in detection subject serum Amount, and relatively cirrhosis is diagnosed compared with the second reference value；The three of scheme：The diagnostic reagent passes through while detecting subject's blood The content of VWF and ADAMTS13 in clear, and relatively cirrhosis is diagnosed compared with third reference value with the ratio of the two.
In more specific scheme, electrophoresis can be used, immuno-chemical method such as radiommunoassay measurement is immunized Fluoremetry, enzyme linked immunosorbent assay (ELISA), chemiluminescence, electrochemical luminescence, colloidal gold (immunochromatography measurement), immunoturbidimetry are surveyed Fixed, specific antibody combined techniques such as direct competition method, ELISA method, RIA method, Flow cytometry, is immunized indirect competitive Chromatography etc. commonly uses detection method to detect the content of VWF and/or ADAMTS13 in subject's serum.Preferably, pass through colloid Golden immunochromatographic method detects the content of VWF and/or ADAMTS13 in subject's serum.
Further, subject's serum described herein can with subject's whole blood, blood plasma, haemocyte, ascites, lymph, Saliva, sputum, sweat, urine, mucus, interstitial fluid or tissue biopsy substitution.
The present invention also provides biomarker VWF and/or ADAMTS13 and its specificity junction mixture in preparation for leading to It crosses the contents level of VWF and/or ADAMTS13 in detection subject's serum and cirrhosis screening is carried out to patients with chronic liver Purposes in kit.
The present invention also provides biomarker VWF and/or ADAMTS13 and its specificity junction mixture in preparation for leading to The examination crossed the contents level of VWF and/or ADAMTS13 in detection subject's serum and the prognosis of liver cirrhosis patient is judged Purposes in agent box.
The present invention also provides biomarker VWF and/or ADAMTS13 and its specificity junction mixture in preparation for leading to It crosses the contents level of VWF and/or ADAMTS13 in detection subject's serum and judges operation or drug to liver cirrhosis patient and control Whether treatment is effective and/or when decision stops the purposes in the kit for the treatment of.
For the ease of application, the present invention also provides a kind of for diagnosing the diagnostic kit of cirrhosis, which includes Bottom plate, sample pad, gold-labelled pad, NC film and absorption pad；Be provided on the bottom plate sequentially mutually overlapped sample pad, gold-labelled pad, NC film and absorption pad, the gold-labelled pad are coated with the colloidal gold solution comprising antibody one, are provided with detection line and matter on the NC film Line is controlled, is coated respectively with antibody two and antibody three at the detection line and nature controlling line.The antibody one, antibody two and antibody three are all The specific binding antibody or antibody one, antibody two and antibody three of VWF is all the specific antibody of ADAMTS13.The antibody One is monoclonal antibody.
Further, the specificity that antibody one, antibody two and antibody three are VWF or ADAMTS13 in the diagnostic kit Antibody fragment.
It is beneficial with protruding as follows that diagnosis is carried out to cirrhosis disease using biomarker VWF and ADAMTS13 of the present invention Effect：Operation is simple for diagnosis process, diagnosis process safety hurtless measure is easy to be received by patient, diagnostic criteria is unified by a People's subjective factor influences the advantages that smaller, diagnostic result is specific and sensitivity is high, is easy to carry out a large amount of screenings selects etc..Tentatively Experimental result by detecting content of the biomarker VWF and/or ADAMTS13 of the present invention in human serum, and carries out liver For the high sensitivity of cirrhosis-diagnostic up to 80% or more, specificity is up to 90% or more.
The present invention is described in detail：
Based on the VWF the inventors discovered that two kinds of new cirrhosis biomarkers, that is, being present in human serum and ADAMTS13。
The present invention separates two kinds of protein, the first protein, that is, VWF is logged in ncbi database by amino acid sequence The protein number protein for being GI 317373549 composition or be GI 317373549 including accession number in ncbi database；The By amino acid sequence, the accession number in ncbi database is that the protein of GI 74749836 forms to two kinds of protein, that is, ADAMTS13 Or including accession number in ncbi database be GI 317373549 protein.
The present inventor carries out detection and analysis discovery by the blood to hundreds liver cirrhosis patient, the VWF in serum and The contents level of ADAMTS13 and cirrhosis lesion degree are closely related.Therefore the VWF in serum and ADAMTS13 can be used as New cirrhosis marker is used for the diagnosis and prognosis of cirrhosis.Particularly, the inventors discovered that, VWF in serum and Diagnosis and prognostic indicator of the ratio of the contents level of ADAMTS13 as cirrhosis have more preferable sensitivity and specificity.
The invention further relates to the reagents of present protein (i.e. VWF and ADAMTS13) contents level in detection serum Box.Kit of the invention includes protein VWF and/or ADAMTS13 and its specificity junction mixture of the invention.Of the invention Kit can be used for carrying out the diagnosis of cirrhosis by the contents level of VWF and/or ADAMTS13 in detection serum.
The invention further relates to protein of the invention and its specificity junction mixture in preparing diagnostic kit for liver cirrhosis Purposes, the kit for example can be used for diagnosing cirrhosis by the level of VWF and/or ADAMTS13 in detection serum In the presence of；For by VWF in detection serum and/or ADAMTS13 is horizontal carries out cirrhosis screening to patients with chronic liver； For by VWF in detection serum and/or ADAMTS13 is horizontal judges the prognosis of liver cirrhosis patient；Or for leading to The VWF and/or ADAMTS13 crossed in detection serum is horizontal and judges whether operation to patient with liver cirrhosis or drug therapy are effective And/or determine when stop treating.
In the present invention, " specificity junction mixture " of present protein refers to high-affinity combination present protein Molecule.Particularly, including with the molecule of high-affinity combination VWF and/or ADAMTS13." specificity junction mixture " is preferred The specific antibody or antibody fragment of VWF and/or ADAMTS13, more preferably, the specific antibody is monoclonal antibody Or antibody fragment.
The present invention also relates to the level by the present protein in detection serum, and diagnoses to cirrhosis Method.The content of VWF and/or ADAMTS13 in serum can be detected by any suitable method.The method includes direct Or level of the indirect determination present protein in serum, to help to diagnose cirrhosis.
Direct method for measuring includes detecting the present invention using the specificity junction mixture of the VWF and/or ADAMTS13 Protein.The specificity junction mixture can be polyclonal antibody, can also be monoclonal antibody, to the animal kind in its source There is no limit for class.In addition, antibody includes the antibody and partial antibody being made of immunoglobulin overall length.Partial antibody refer to containing Antigen-binding site, the antibody fragment with antigen-binding activity.In addition, in the case where with mark substance labelled antibody, as Mark substance refers to for example：Fluorescent material (such as：FITC, rhodamine, phallotoxin), golden isocolloid particle, Fluorescent microsphere, the heavy metal (such as gold, platinum etc.), chromoprotein matter (example of Luminex (registered trademark, Luminex company) etc. Such as, phycoerythrin, phycocyanin etc.), radioactive isotope (for example, 3H, 14C, 32P, 35S, 125I, 131I etc.), (example such as enzyme Such as, peroxidase, alkaline phosphatase etc.), biotin, the substances such as Streptavidin, but not limited to this.Specifically, for example making ELISA detection is carried out with the specific antibody of identification present protein.Firstly, the corresponding specificity of biomarker is anti- Body (antibody 1) is fixed in the solid phases such as microwell plate.Biomarker and antibody knot when adding serum to the solid phase, in serum It closes, forms immune complex.After remaining serum is removed, addition identification and 1 different epitopes of antibody, marked with marker substances Antibody (antibody 2), in conjunction with biomarker.Remaining antibody 2 is cleaned after removing, measures mark remaining on microwell plate Remember the amount of substance.The calibration for making the relationship of amount for showing the mark object amount added to microwell plate and remaining mark substance in advance is bent Line can calculate the mark object amount in blood using the calibration curve.
The method of indirect determination include for example by detect VWF and/or ADAMTS13 activity come reflect VWF and/or The concentration of ADAMTS13.
The present inventor confirms through preliminary research, VWF and ADAMTS13 in the patients with chronic liver serum of non-suffering from liver cirrhosis Content range be 5704355~28972720ng/ml and 318.4~768.3ng/ml respectively, focus more on 8579371~ 26303400ng/ml and 429.9~625.0ng/ml；Patients with chronic liver the serum VWF's and ADAMTS13 of non-suffering from liver cirrhosis Content ratio range is 9054.532~91004.04, focuses more on 13536.9~50563.2.In the cirrhosis compensatory phase In the serum of patient VWF and ADAMTS13 content range be respectively 12075480~46999200ng/ml and 238.9~ 660.2ng/ml focuses more on 18460920~36234580ng/ml and 350.1~549.3ng/ml；It is compensatory in cirrhosis VWF and ADAMTS13 content ratio range is 34501.71~126663.3 in the serum of the patient of phase, focuses more on 53940.3 ~899741.VWF and ADAMTS13 content range is 31456240 respectively in the serum of patient in cirrhosis patients in decompensation ~70655840ng/ml and 104.8~560.0ng/ml, focus more on 37005190~65421470ng/ml and 185.3~ 469.8ng/ml；The ratio range of VWF and ADAMTS13 content is in the serum of patient in cirrhosis patients in decompensation 118746.6~231246.7, focus more on 128448.6~168945.8.
By above data, hence it is evident that it can be concluded that, in human body in serum VWF content：The patient of cirrhosis patients in decompensation is bright The aobvious patient higher than the cirrhosis compensatory phase, the patient of cirrhosis compensatory phase are apparently higher than the patients with chronic liver of non-suffering from liver cirrhosis； In human body in serum ADAMTS13 content：The patient of cirrhosis patients in decompensation is significantly lower than the patient of cirrhosis compensatory phase, liver The patient for hardening the compensatory phase is significantly lower than the patients with chronic liver of non-suffering from liver cirrhosis.Edge this, VWF in human serum and ADAMTS13 level can be used as new cirrhosis marker, for judging that cirrhosis whether there is.Preferably, it can examine simultaneously The content of VWF and ADAMTS13 is surveyed, and cirrhosis is relatively diagnosed compared with normal level by the ratio of VWF and ADAMTS13. Tentative confirmation is tested through the present inventor, using VWF and ADAMTS13 ratio as diagnosis index, there is more excellent diagnosis knot Fruit.When VWF and ADAMTS13 cirrhosis ratio critical value is set to 64747.76, liver cirrhosis diagnosis sensitivity is 96.6%, special The opposite sex is 81.4%；Using VWF and ADAMTS13 ratio as diagnosis index, when VWF and ADAMTS13 Decompensated liver cirrhosis ratio When critical value is set to 79928.59, Decompensated liver cirrhosis diagnostic sensitivity is 83.1%, and specificity is 94.9%.
In specific implementation, the present inventor's preliminary advice can be by the cirrhosis critical value of VWF and ADAMST13 content ratio It is set as 64747.76, the patients with chronic liver of insufficient critical value is that cirrhosis group, Chronic Liver more than critical value do not occur Cirrhosis has occurred in patient.Can be by the Decompensated liver cirrhosis critical value setting of the content ratio of VWF and ADAMST13 79928.59, the patients with chronic liver of insufficient critical value is that Decompensated liver cirrhosis group, Chronic Liver more than critical value do not occur Decompensated liver cirrhosis occurs for patient.After cirrhosis patients in decompensation patient treatment, by measuring serum collected before and after treatment In VWF and ADAMST13 content ratio, the patient outcomes can be predicted.After treatment, under VWF and ADAMST13 content ratio The patient of drop, can determine whether for therapeutic effect it is good, VWF and ADAMST13 content ratio be lower than decompensation critical value 79928.59 when, The patient reenters the cirrhosis compensatory phase.
In a concrete scheme, kit of the invention or method can be used for determining individual with the presence or absence of cirrhosis.For This, can be used VWF in the blood serum sample of kit of the invention or method measurement from cirrhosis suspected patient and/or ADAMTS13 is horizontal, and is optionally compared with normal control, then according to the VWF and/or ADAMTS13 level in sample Judge that a possibility that cirrhosis occurs in the patient.
In another concrete scheme, kit of the invention or method can be used for through VWF in detection serum and/or ADAMTS13 is horizontal and carries out cirrhosis screening to patients with chronic liver.It is surveyed for this purpose, kit or method of the invention can be used VWF and/or ADAMTS13 in the fixed blood serum sample from people at highest risk is horizontal, and is optionally compared with normal control, Then judge which individual may have already appeared cirrhosis in the crowd according to VWF the and/or ADAMTS13 level in sample.
In another concrete scheme, kit of the invention or method can be used for through VWF in detection serum and/or ADAMTS13 is horizontal and judges the prognosis of liver cirrhosis patient.For this purpose, kit or method measurement of the invention can be used VWF in blood serum sample from liver cirrhosis patient and/or ADAMTS13 is horizontal, and optionally with normal control or the patient with Past serum VWF and/or ADAMTS13 level is compared, and is then judged according to VWF the and/or ADAMTS13 level in sample The prognosis of the liver cirrhosis patient.VWF and/or ADAMTS13 maintains high level or VWF and/or the horizontal further raising of ADAMTS13 It may be related to unfavorable prognosis.Accordingly, doctor can be reminded to carry out closer observation to the patient, and change mesh when necessary Preceding therapeutic scheme.
In another concrete scheme, kit of the invention or method can be used for through VWF in detection serum and/or Effectively and/or when decision stops treating whether ADAMTS13 is horizontal and judge operation or drug therapy to patient with liver cirrhosis. For this purpose, can be used VWF in the blood serum sample of kit of the invention or method measurement from liver cirrhosis patient and/or ADAMTS13 is horizontal, and optionally previous serum VWF and/or ADAMTS13 level compares with normal control or the patient Compared with then judging whether are operation to the patient with liver cirrhosis or drug therapy according to VWF the and/or ADAMTS13 level in sample Effectively and/or determine when stop treating.
Detailed description of the invention
Attached drawing 1 is using Gold standard kit to normal person, patients with chronic liver, the compensatory phase patient of cirrhosis and cirrhosis The comparative result figure that VWF is detected in the serum of Decompensated stage patient；
By attached drawing 1 it is found that along with the cirrhosis state of an illness development, the content of VWF gradually significantly increases.
Attached drawing 2 is using Gold standard kit to normal person, patients with chronic liver, the compensatory phase patient of cirrhosis and cirrhosis The comparative result figure that ADAMTS13 is detected in the serum of Decompensated stage patient；
By attached drawing 2 it is found that along with the cirrhosis state of an illness development, the content of ADAMTS13 gradually significantly reduces.
Attached drawing 3 be patients with chronic liver, in cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum VWF quantitative inspection Survey result；
The quantitative detection of VWF is shown, patients with chronic liver, cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum The mean value of middle VWF contents level respectively may be about 1.7 × 107ng/ml、2.8×107ng/ml、4.7×107ng/ml。
Attached drawing 4 is patients with chronic liver, ADAMTS13 in cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum Quantitative detection result；
The quantitative detection of ADAMTS13 is shown, patients with chronic liver, cirrhosis compensatory phase and cirrhosis patients in decompensation are suffered from The mean value of ADAMTS13 contents level respectively may be about 560ng/ml, 400ng/ml, 350ng/ml in person's serum.
Attached drawing 5 be patients with chronic liver, in cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum VWF with The ratio of ADAMTS13 quantifies distribution map；
The ratio quantitative detection of VWF and ADAMTS13 shows that patients with chronic liver, cirrhosis compensatory phase and cirrhosis lose generation The mean value for repaying the ratio of VWF and ADAMTS13 in phase patients serum respectively may be about 30000 times, 70000 times, 150000 times, exist Significant difference.
Attached drawing 6 is the structural schematic diagram of Gold standard kit of the present invention；
In attached drawing 6,1 is sample pad, and 2 be golden labelled antibody glass fibre membrane, and 3 be nitrocellulose coated film, and 4 be water suction Paper, 5 be PVC bottom plate.
Specific embodiment
The purpose of the present invention is further elaborated below in conjunction with the drawings and specific embodiments, listed embodiment is for side Just those skilled in the art more fully understand inventive concept of the invention, are not construed as to protection scope of the present invention Limitation.Under the premise of without prejudice to inventive concept of the present invention, several difference variations can be still made, can not be carried out here one by one Exhaustion, these are not paid creative simple change and should all be included within the scope of the present invention.
Used reagent medicament is all ordinary commercial products in embodiment described herein.Used instrument is also logical for industry Use equipment and instrument.The corresponding antibody 1 of used VWF and ADAMTS13, antibody 2 and resist for ordinary commercial products more, antibody Preparation method can be used conventional preparation method for antibody and obtain, and details are not described herein again.
Embodiment 1：The preparation of Gold standard kit and its application in liver cirrhosis diagnosis.
One, the preparation of kit：
The antibody that this kit preparation process uses is the specific antibody of VWF or ADAMTS13.Note that same reagent box In preparation process：If the antibody that monoclonal antibody 1 used in golden labelled antibody glass fibre membrane is VWF, corresponds to nitrocellulose packet Monoclonal antibody 2 used in envelope preparation process and more anti-antibody that should also be as using VWF；If making in golden labelled antibody glass fibre membrane Monoclonal antibody 1 is the antibody of ADAMTS13, then corresponds to monoclonal antibody 2 used in nitrocellulose coated film preparation process and resist more The antibody of ADAMTS13 should be used.
1, the preparation of colloidal gold solution：Ultrapure water is set and is heated with stirring to boiling on magnetic stirring apparatus, by whole mass concentration It is rapidly added the chlorauric acid solution that mass concentration is 1% for the amount of a ten thousandth, is boiled 5 minutes；It is molten by gold chloride be added again The citric acid three sodium solution that mass concentration is 1% is added in liquid equal volume amounts, boils after ten minutes, is cooled to room temperature, uses ultrapure water It is settled to the final concentration of a ten thousandth of gold chloride, room temperature is kept in dark place spare.
2, the preparation of golden labelled antibody glass fibre membrane：With 2mol/L solution of potassium carbonate adjust colloidal gold solution pH value to 7.0.Monoclonal antibody 1 is added by 3 μ g antibody/milliliter colloidal gold solution proportional concentration, mixes, stands 10 minutes；2% volume is pressed again The BSA solution that concentration is 20% is added, mixes, stands 10 minutes；13000rpm is centrifuged 15 minutes, abandons supernatant, precipitating label Cleaning solution washed once, and supernatant be abandoned after centrifugation, it is molten that the gold labeling antibody of 1/20th initial colloid gold volume of precipitating saves liquid Then solution is coated on glass fibre membrane by the amount that every milliliter of solution spreads 15 square centimeters, after vacuum drying 2 hours, set and be equipped with It is saved backup in the hermetic bag of desiccant.
The concrete component proportion that above-mentioned 20%BSA solution, label cleaning solution and gold labeling antibody save liquid is as follows：
A.20%BSA solution：200g containing bovine serum albumin(BSA) in every 1000mL ultrapure water.
B. cleaning solution is marked：Bovine serum albumin(BSA) containing 2g in every 1000mL ultrapure water, 1g PEG 20000,5g sucrose, 1.211g Tris, and pH is adjusted to 7.6.
C. gold labeling antibody saves liquid：Bovine serum albumin(BSA) containing 10g in every 1000mL ultrapure water, 1g PEG 20000, 10.2g Tris, 1g sodium hydroxide, and pH is adjusted to 7.6.
3, the preparation of nitrocellulose coated film：It sprays monoclonal antibody 2 respectively on nitrocellulose filter and resists more, and be in two Line is arranged in parallel, is respectively formed detection line and nature controlling line, 37 DEG C drying and processing 3 hours, set in the hermetic bag equipped with desiccant and protect It deposits spare.
Detection line：Debugging spray film instrument, spouting liquid are 1.5 μ l/cm, dilute monoclonal antibody 2, concentration 2mg/ with coating buffer ML, with spray film instrument spraying scribing line.
Nature controlling line：Debugging spray film instrument, spouting liquid are 1.5 μ l/cm, mostly anti-with coating buffer dilution, concentration 2mg/mL, With spray film instrument spraying scribing line.
The concrete component of above-mentioned coating buffer matches：0.01g containing bovine serum albumin(BSA) in every 1000mL ultrapure water, ten Two hypophosphite monohydrate disodium hydrogen 30.072g, potassium dihydrogen phosphate 2.176g.
Drawn two-lines answer fine uniform on nitrocellulose coated film, and adjacent line-to-line is every 0.8cm.
4, big board group item：
Big board group each component specification (long × wide)：PVC bottom plate：30cm×8cm；Sample pad：30cm×4.0cm；Jin Biao Remember antibody glass fibre membrane：30cm×0.5cm；Nitrocellulose coated film：30cm×2.5cm；Blotting paper：30cm×3cm.
Each component carries out a group item as follows：The bottom surface of nitrocellulose coated film is pasted on PVC bottom plate, at this Golden labelled antibody glass fibre membrane and blotting paper are pasted in the upper surface of coated film both ends respectively, on golden labelled antibody glass fibre membrane Paste sample pad in face；Sample pad, golden labelled antibody glass fibre membrane, nitric acid are sequentially mutually pasted to overlap joint i.e. on PVC bottom plate Cellulose coated film and blotting paper form big plate.The humidity of composing room will control below 30%.
5, it cuts：
Big plate is cut into single with cutting machine, every width is 4 millimeters, and random inspection, sensitivity can detect Internal Quality Control Product, nothing but specific band.
6, envelope and group box：
Test strips that 1 part has cut, one are responsible for a task until it is completed drying prescription and a plastic dropper in aluminium foil bag, with sealing secret Envelope, is put into kit after sealing, and air drying saves.So far kit and the detection of detection VWF are respectively obtained The kit of ADAMTS13.
Two, the diagnosis of cirrhosis is carried out using mentioned reagent box：
The whole blood of normal person, patients with chronic liver, cirrhosis compensatory phase patient and cirrhosis patients in decompensation patient are taken respectively, (4 DEG C or so) are sent to laboratory within 24 hours, under cryogenic conditions, must avoid haemolysis, sample is in case of haemolysis, then again Acquisition.It is centrifuged twice in 4 DEG C, 6000g, takes supernatant.Using the kit detection VWF and ADAMTS13 being prepared as above in serum Content.It will test result to be compareed with clinical diagnosis, to verify the content of VWF and ADAMTS13 and cirrhosis in serum Correlation.Testing result is as shown shown in Figure 1 and Figure 2.
By the testing result of attached drawing 1 and attached drawing 2 it is found that along with the cirrhosis state of an illness development, the content of VWF is gradually significant It increases, the content of ADAMTS13 gradually significantly reduces.
Embodiment 2：Sensitivity and specificity research of the VWF and ADAMTS13 as cirrhosis biomarker.
Sensitivity and specificity as described herein are defined：
Sensitivity：Sensitivity described herein is that the practical patients with chronic liver with cirrhosis is correctly determined as kidney-Yang The ratio of property；Or the ratio for cirrhosis patients in decompensation patient will be actually entered correctly being determined as true positives.
Specificity：Specificity described herein be the patients with chronic liver of practical non-suffering from liver cirrhosis is correctly determined as it is Kidney-Yin The ratio of property；Or the patient for being practically in the cirrhosis compensatory phase is correctly determined as to the ratio of true negative.
VWF and ADAMTS13 cirrhosis ratio critical value：The content ratio number of a VWF and ADAMTS13 as defined in this paper According to it is to determine that the patients with chronic liver is hard with liver that the content ratio of VWF and ADAMTS13, which is higher than the data, in subject's serum Change.
VWF and ADAMTS13 Decompensated liver cirrhosis ratio critical value：The content of a VWF and ADAMTS13 as defined in this paper Ratio data, the content ratio of VWF and ADAMTS13 is higher than the data and determines that the patient enters cirrhosis in subject's serum Decompensated stage.
VWF the and ADAMTS13 cirrhosis ratio critical value and VWF and ADAMTS13 cirrhosis that the present embodiment is taken lose generation Repaying ratio critical value respectively is previously described 64747.76 and 79928.59,
The present inventor is to 300 compensatory phase patients of patients with chronic liver and cirrhosis and cirrhosis patients in decompensation patient VWF and ADAMTS13 content ratio data are analyzed using Receiver operating curve (i.e. ROC curve), and ROC curve analysis is aobvious Show that VWF and ADAMST13 ratio can be used for diagnosing the generation of cirrhosis and Decompensated liver cirrhosis, VWF and ADAMTS13 cirrhosis Ratio critical value (i.e. cirrhosis threshold value) and VWF and ADAMTS13 Decompensated liver cirrhosis ratio critical value (i.e. Decompensated liver cirrhosis Threshold value) it is previously described 64747.76 and 79928.59 respectively.
Using previously described ELISA method to VWF in the 236 patients with chronic liver case sample serum randomly selected and The content of ADAMTS13 carries out quantitative detection.It will test result and use routine clinical detection means (CT, B ultrasound, needle biopsy of liver Method etc.) corresponding confirmed result is listed in Tables 1 and 2 respectively.In Tables 1 and 2：The unit of VWF and ADAMTS13 is all ng/ml.
Table 1
Case number vWF/ADAM vWF ADAMTS 13 Conventional means are made a definite diagnosis Case number vWF/ADAM vWF ADAMTS 13 Conventional means are made a definite diagnosis
1 345265 122544389 354.9285 Cirrhosis 60 100677.1 27740771.3 275.5421 Cirrhosis
2 204156 59763403 292.734 Cirrhosis 61 86218.3 42042023 487.623 Non- cirrhosis
3 25995.1 12269687 472 Non- cirrhosis 62 122570.8 49520844.9 404.0185 Cirrhosis
4 87701.84 31846678 363.1244 Cirrhosis 63 64747.76 20467350 316.109 Non- cirrhosis
5 71857.17 21763223 302.8678 Cirrhosis 64 25975.1 16896248.5 650.4787 Non- cirrhosis
6 135015 28908426 214.1127 Cirrhosis 65 72533.29 17326288.1 238.8736 Cirrhosis
7 16427.05 7269960.2 442.5603 Non- cirrhosis 66 358944.9 52722293.5 146.8813 Cirrhosis
8 16652.12 10490836 630 Non- cirrhosis 67 73453.57 45551473.2 620.1397 Non- cirrhosis
9 104999 38960552 371.0563 Cirrhosis 68 85128.53 30635297.7 359.8711 Cirrhosis
10 145554.4 68379841 469.7889 Cirrhosis 69 109928.6 50552981.6 459.8711 Cirrhosis
11 109115.1 31800965 291.4443 Cirrhosis 70 25985.1 15155585.1 583.2414 Non- cirrhosis
12 189535.3 23941704 126.3179 Cirrhosis 71 144870.7 65727909 453.7005 Cirrhosis
13 17175.54 5468132 318.3674 Non- cirrhosis 72 69340.9 25763278.6 371.5452 Cirrhosis
14 84339.31 62697843 743.4 Non- cirrhosis 73 17145.54 10836280.3 632.0174 Non- cirrhosis
15 113056.5 40940337 362.1229 Cirrhosis 74 61801.5 31358081.1 507.4 Non- cirrhosis
16 130591.3 46033881 352.5035 Cirrhosis 75 17155.54 13358414.6 778.6648 Non- cirrhosis
17 16417.05 5491359.6 334.4913 Non- cirrhosis 76 36848.57 19035352.2 516.5832 Non- cirrhosis
18 407864.3 143963022 352.9679 Cirrhosis 77 36838.57 20920411.5 567.8942 Non- cirrhosis
19 16622.12 12411637 746.694 Non- cirrhosis 78 58414.21 28112790.7 481.2663 Non- cirrhosis
20 70344.22 28329193 402.7224 Cirrhosis 79 73463.57 21277857.6 289.6382 Non- cirrhosis
21 103680.9 35446165 341.8775 Cirrhosis 80 46888.22 30170508.1 643.456 Non- cirrhosis
22 61811.5 27093767 438.3289 Non- cirrhosis 81 17212.93 8059804.72 468.2413 Non- cirrhosis
23 49889 17460979 349.9966 Cirrhosis 82 84349.31 35660878.2 422.7762 Non- cirrhosis
24 91891.3 26439370 287.7244 Cirrhosis 83 73433.57 50422490.6 686.6409 Non- cirrhosis
25 93211.84 48792297 523.456 Cirrhosis 84 383191.7 59872336.7 156.2464 Cirrhosis
26 86198.3 29323809 340.1901 Non- cirrhosis 85 578643.4 249404260 431.0155 Cirrhosis
27 420638.3 77930985 185.2684 Cirrhosis 86 84329.31 53286241 631.8828 Non- cirrhosis
28 61791.5 40449658 654.6152 Non- cirrhosis 87 226598.9 125309192 553 Cirrhosis
29 140422.2 25512172 181.6819 Cirrhosis 88 140397.6 34367586.2 244.7876 Cirrhosis
30 35327.79 33806995 956.9519 Non- cirrhosis 89 199905.3 52829885.2 264.2746 Cirrhosis
31 402754.2 178360681 442.8524 Cirrhosis 90 116114.4 28908798.1 248.9682 Cirrhosis
32 209706.6 33412725 159.3308 Cirrhosis 91 17165.54 13187697.3 768.2658 Non- cirrhosis
33 46898.22 23791088 507.2919 Non- cirrhosis 92 35317.79 34254895.5 969.9048 Non- cirrhosis
34 161376.1 38240682 236.9662 Cirrhosis 93 16397.05 12665835.4 772.446 Non- cirrhosis
35 15310.69 6124276 400 Non- cirrhosis 94 121303.4 12711227.7 104.7887 Cirrhosis
36 26005.1 10746342 413.2398 Non- cirrhosis 95 58404.21 32269770.6 552.5247 Non- cirrhosis
37 78017.56 28987050 371.5452 Cirrhosis 96 17182.93 14190671.5 825.8587 Non- cirrhosis
38 293474.2 100960042 344.0168 Cirrhosis 97 58394.21 45823571.6 784.728 Non- cirrhosis
39 58384.21 53633487 918.63 Non- cirrhosis 98 15300.69 5748064.25 375.6735 Non- cirrhosis
40 428898.6 220859061 514.9447 Cirrhosis 99 73443.57 34367107.1 467.939 Non- cirrhosis
41 142269.5 38325444 269.3862 Cirrhosis 100 146422.8 44995140.7 307.296 Cirrhosis
42 64717.76 47016510 726.4854 Non- cirrhosis 101 46878.22 33160202.3 707.3691 Non- cirrhosis
43 64727.76 38054952 587.9232 Non- cirrhosis 102 194416.4 74381047.2 382.5863 Cirrhosis
44 16407.05 11482970 699.8802 Non- cirrhosis 103 158605.9 107852012 680 Cirrhosis
45 80136.75 44074940 549.9966 Cirrhosis 104 84098.54 28356520.2 337.1821 Cirrhosis
46 69838.4 24650716 352.9679 Cirrhosis 105 228857.1 76288854 333.3471 Cirrhosis
47 85811.79 21603225 251.7512 Cirrhosis 106 86228.3 17070270.5 197.966 Non- cirrhosis
48 35347.79 18576752 525.5421 Non- cirrhosis 107 61781.5 46909341.2 759.2781 Non- cirrhosis
49 36818.57 34093224 925.979 Non- cirrhosis 108 328530.5 129930247 395.4891 Cirrhosis
50 63942.98 22437061 350.8917 Cirrhosis 109 17202.93 5983733.96 347.8323 Non- cirrhosis
51 17192.93 13384696 778.5 Non- cirrhosis 110 36828.57 30271363 821.9533 Non- cirrhosis
52 131040.8 73382826 560 Cirrhosis 111 196250.6 61473498.7 313.2398 Cirrhosis
53 474189.7 148083271 312.287 Cirrhosis 112 35337.79 21540792.2 609.5682 Non- cirrhosis
54 16642.12 12273564 737.5 Non- cirrhosis 113 79014.82 24240443 306.7835 Cirrhosis
55 86208.3 51678885 599.4653 Non- cirrhosis 114 91738.15 26112044.3 284.6367 Cirrhosis
56 15280.69 9844727 644.2593 Non- cirrhosis 115 64737.76 42922478.8 663.0208 Non- cirrhosis
57 15290.69 5429040.4 355.0553 Non- cirrhosis 116 46908.22 20170534.6 430 Non- cirrhosis
58 83000.2 27597298 332.4968 Cirrhosis 117 415614.2 200159799 481.6 Cirrhosis
59 167824.1 79813785 475.58 Cirrhosis 118 16632.12 10102069.7 607.3832 Non- cirrhosis
Known to analytical table 1：VWF and ADAMTS13 content detection and clinical definite discovery to 118 patients with chronic liver, 59 are diagnosed as the patients with chronic liver of non-suffering from liver cirrhosis, wherein have 48 VWF and ADAMTS13 content ratios lower than VWF and ADAMTS13 cirrhosis ratio critical value 64747.76, clinical specificity 81.4%.59 are diagnosed as the patient of cirrhosis, In 57 VWF and ADAMTS13 content ratios be higher than VWF and ADAMTS13 cirrhosis ratio critical value 64747.76, clinical spirit Sensitivity is 96.6%.
Table 2：
Known to analytical table 2：118 liver cirrhosis patient VWF and ADAMTS13 content detections and clinical definite are found, 59 It is diagnosed as the compensatory phase patient of cirrhosis, wherein there are 56 VWF and ADAMTS13 content ratios lower than VWF and ADAMTS13 cirrhosis Decompensation ratio critical value 79928.59, clinical specificity 94.9%.59 are diagnosed as the patient of Decompensated liver cirrhosis, In 49 VWF and ADAMTS13 content ratios be higher than VWF and ADAMTS13 cirrhosis ratio critical value 79928.59, it is clinical sensitive Degree is 83.1%.
It can be concluded that, use VWF and/or ADAMTS13 as cirrhosis biology by the above specificity and sensitivity study Marker diagnoses patients with chronic liver, has very strong specific and very high sensitivity.
It is worth noting that, the determination of cirrhosis threshold value and Decompensated liver cirrhosis threshold value is not fixation, to bigger VWF and ADAMTS13 content ratio test sample carries out ROC curve analysis, will obtain more accurate reasonable threshold value.With this Threshold value is also more reasonable accurate as the diagnosis that reference pair subject makees cirrhosis.

Claims (5)

  1. The purposes of 1.VWF and/or ADAMTS13 and its specificity junction mixture in the diagnostic reagent of preparation diagnosis cirrhosis, it is described Diagnostic reagent passes through the ratio of VWF and ADAMTS13 in detection subject's serum, and hard compared with to diagnose liver compared with normal level Change, by amino acid sequence, the accession number in ncbi database is that the protein of GI 317373549 forms to the VWF；It is described By amino acid sequence, the accession number in ncbi database is that the protein of GI 74749836 forms to ADAMTS13.
  2. 2.VWF and/or ADAMTS13 and its specificity junction mixture preparation for by VWF in detection subject's serum with The ratio of ADAMTS13 carries out the purposes in the kit of cirrhosis screening compared with normal level to patients with chronic liver, By amino acid sequence, the accession number in ncbi database is that the protein of GI 317373549 forms to the VWF；It is described By amino acid sequence, the accession number in ncbi database is that the protein of GI 74749836 forms to ADAMTS13.
  3. 3.VWF and/or ADAMTS13 and its specificity junction mixture preparation for by VWF in detection subject's serum with The ratio of ADAMTS13 judge compared with normal level operation to liver cirrhosis patient or drug therapy whether effectively and/or Determine when to stop the purposes in the kit for the treatment of, the accession number in ncbi database is the VWF by amino acid sequence The protein of GI317373549 forms；By amino acid sequence, the accession number in ncbi database is the ADAMTS13 The protein of GI74749836 forms.
  4. 4. purposes described in claim 2~3 any claim, is characterized in that, the specificity junction mixture be VWF or The specific Ab fragments of ADAMTS13.
  5. 5. purposes described in claim 2~3 any claim, wherein pass through electrophoresis, immuno-chemical method, direct competitive Method, indirect competitive, ELISA method, RIA method, Flow cytometry or immunochromatographic method detect the VWF in subject's serum And/or the content of ADAMTS13.
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