CN106066401A - Biomarker VWF and ADAMTS13 and the purposes in liver cirrhosis diagnosis reagent thereof - Google Patents

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

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CN106066401A
CN106066401A CN201610203495.7A CN201610203495A CN106066401A CN 106066401 A CN106066401 A CN 106066401A CN 201610203495 A CN201610203495 A CN 201610203495A CN 106066401 A CN106066401 A CN 106066401A
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vwf
liver cirrhosis
adamts13
antibody
gold
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CN106066401B (en
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高平
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CHONGQING ZEARLY BIO-TECH Co.,Ltd.
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高平
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Abstract

The present invention relates to disease biomarkers purposes field, disclose liver cirrhosis biomarker VWF and ADAMTS13 and the purposes in liver cirrhosis diagnosis reagent thereof.VWF of the present invention is made up of the protein that aminoacid sequence accession number in ncbi database is GI 317373549, and described ADAMTS13 is made up of the protein that aminoacid sequence accession number in ncbi database is GI 74749836.With biomarker VWF and ADAMTS13, subjects is carried out liver cirrhosis diagnosis, have simple, diagnosis process safety hurtless measure is prone to be accepted by patient, diagnostic criteria unification is affected less, diagnostic result specificity and susceptiveness height, the advantage such as be prone to carry out that a large amount of examination is selected by factor and individual subjective factor.

Description

Biomarker VWF and ADAMTS13 and the purposes in liver cirrhosis diagnosis reagent thereof
Technical field
The present invention relates to disease biomarkers purposes field, be related specifically to liver cirrhosis biomarker VWF and ADAMTS13 and the purposes in liver cirrhosis diagnosis reagent thereof.
Background technology
Liver cirrhosis is chronic progressive external hepatopathy, and the diffusivity liver that or repeated action long-term by one or more causes of disease is formed damages Evil.Being posthepatitic cirrhosis at China's great majority, small part is alcoholic cirrhosis and Cirrhosis In Schistosomiasis.Histopathology On have hepatic necrosis, remaining liver cell nodules regeneration, connective tissue proliferation and fiber widely every formation, to cause lobules of liver Structural deterioration and pseudolobuli are formed, and liver gradually deforms, hardening and develop into liver cirrhosis.Stronger due to liver compensation in early days Can non-evident sympton, stage is with liver function injury and portal hypertension for main performance, and has multisystem to get involved, and late period often occurs The complication such as upper gastrointestinal hemorrhage, hepatic encephalopathy, secondary infection, hypersplenism, ascites, canceration.
Case fatality rate at China's liver cirrhosis is only second to malignant tumor, i.e. has 154 people to die from liver every about 100,000 populations every year Hardening.Worldwide, liver cirrhosis also increasingly becomes one of clinical common disease, and liver cirrhosis has become the world according to statistics One of three big fatal causes of disease.How can convenient, fast, in time, checkout and diagnosis liver cirrhosis accurately, and patient's liver cirrhosis is controlled Therapeutic effect tracing and monitoring, has become as the problem that liver cirrhosis health field is in the urgent need to address.
Liver cirrhosis detection method the earliest uses needle biopsy of liver method, the diagnostic result of this method to be that liver cirrhosis is examined Disconnected accurate liver cirrhosis diagnosis method recognized within the industry.But, easily there is significant limitation in this method.First, should Method is traumatic, is difficult to be accepted by patient；Secondly, there is the possibility that complication occurs during operation, should not carry out the most repeatedly Biopsy；3rd, it is little that needle biopsy of liver method punctures sample, only accounts for the 1/50000 of liver mass, there is bigger error of sampling.
At present, what the application of China's diagnosis liver cirrhosis was more is B ultrasonic.But B ultrasonic simply sees the form of liver, and pathology is also It is not directly relevant to, it is impossible to accurately liver cirrhosis is analyzed, it is impossible to judge liver cirrhosis disease progression, and to instrument and operator Member has higher requirement.
CT and nuclear-magnetism detection method are helpful to the diagnosis of liver cirrhosis, but this equipment is costly, and routine is carried out the most tired Difficulty, is not suitable for China's actual conditions.FibroScan technology starts to walk in China, but many factors affects the accuracy of FibroScan, Such as ascites, inflammation, obesity, fatty liver etc..Quite a few patient cannot be carried out the inspection of FibroScan.
Thus this area is badly in need of with low cost, quick, the easy and minimum liver cirrhosis being prone to accept of invasive for patient Diagnostic method.
VWF is the acronym of English von Willebrand factor, Chinese can be translated into von Willebrand because of Son,Www.uniprot.org website log number is P04275Entry its aminoacid sequence is had been disclosed.VWF is a kind of Macro-molecular protein polymer, is mainly used in the VWD i.e. diagnosis of von Willebrand.Meanwhile, publication number CN102614513A Chinese patent " application of anti-angiogenic property christmas factor A3 district bi-functional monoclonal antibody " also discloses VWF and is preparing anti-blood Purposes in bolt medicine.
ADAMTS13 is English Adisintegrin and metalloproteinase with thrombospondin The abbreviation of motifs 13 is called for short.Other substitute title and also have 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 Aminoacid sequence has been disclosed, and has 3 subunit sequence.
Summary of the invention
For the various shortcoming overcoming prior art liver cirrhosis diagnosis to exist, the present invention provides two kinds of liver cirrhosis biological markers Thing VWF and ADAMTS13.Described VWF is the protein of GI 317373549 by aminoacid sequence accession number in ncbi database Composition, described ADAMTS13 is made up of the protein that aminoacid sequence accession number in ncbi database is GI 74749836.
It was found by the inventors of the present invention that just can identify in some time before obvious liver cirrhosis disease symptoms occurs Biomarker VWF and ADAMTS13 relevant in serum or other body fluid.And at the human serum that whether ill liver cirrhosis is The content difference of middle VWF or ADAMTS13 is very big, thus can consider the biomarker VWF in human serum and The content of ADAMTS13 is used as liver cirrhosis diagnosis instrument, and realizes liver cirrhosis patient according to the diagnostic result of these diagnostic tools Personalized medicine.
Based on above-mentioned discovery, the invention provides biomarker VWF and/or ADAMTS13 and examine at preparation diagnosis liver cirrhosis Purposes in disconnected reagent.
Terms used herein is generally defined as:
VWF: the albumen being made up of the protein that aminoacid sequence accession number in ncbi database is GI 317373549 Matter；
ADAMTS13: be made up of the protein that aminoacid sequence accession number in ncbi database is GI 74749836 Protein；
First reference value: do not develop into the content of VWF in patients with chronic liver subjects's serum of liver cirrhosis；
Second reference value: do not develop into the content of ADAMTS13 in patients with chronic liver subjects's serum of liver cirrhosis；
3rd reference value: do not develop into VWF content and ADAMTS13 in patients with chronic liver subjects's serum of liver cirrhosis The ratio of content.
The implication that all terms not being defined herein are all generally understood with those of ordinary skill in the art is identical.
Concrete, one of scheme: described diagnostic reagent is by the content of VWF in detection subjects's serum, and joins with first Examine value to compare and relatively diagnose liver cirrhosis；The two of scheme: described diagnostic reagent is by the containing of ADAMTS13 in detection subjects's serum Amount, and relatively diagnose liver cirrhosis compared with the second reference value；The three of scheme: described diagnostic reagent is by detection subjects's blood simultaneously The content of VWF and ADAMTS13 in clear, and the ratio and the 3rd reference value with both compares and relatively diagnoses liver cirrhosis.
In the most specific scheme, it is possible to use electrophoresis method, immuno-chemical method such as radioimmunoassay measures, immunity Fluoremetry, enzyme-linked immunosorbent assay, chemiluminescence, electrochemiluminescence, gold colloidal (immunochromatography measurement), immunoturbidimetry are surveyed Fixed, specific antibody combined techniques such as direct competition method, indirect competitive, ELISA method, RIA method, Flow cytometry, immunity The conventional detection method such as chromatography detects the content of VWF and/or ADAMTS13 in subjects's serum.Preferably, colloid is passed through Gold immunochromatographic method detects the content of VWF and/or ADAMTS13 in subjects's serum.
Further, subjects's serum described herein can use subjects's whole blood, blood plasma, hemocyte, ascites, lymph fluid, Saliva, sputum, perspiration, urine, mucus, interstitial fluid or biopsy substitute.
Present invention also offers biomarker VWF and/or ADAMTS13 and specificity junction mixture thereof in preparation for leading to Cross the contents level of VWF and/or ADAMTS13 in detection subjects's serum and patients with chronic liver is carried out liver cirrhosis examination Purposes in test kit.
Present invention also offers biomarker VWF and/or ADAMTS13 and specificity junction mixture thereof in preparation for leading to The examination crossing the contents level of VWF and/or ADAMTS13 in detection subjects's serum and the prognosis of liver cirrhosis patient is judged Purposes in agent box.
Present invention also offers biomarker VWF and/or ADAMTS13 and specificity junction mixture thereof in preparation for leading to Cross the contents level of VWF and/or ADAMTS13 in detection subjects's serum and judge that the operation to liver cirrhosis patient or medicine are controlled Treat the most effectively and/or determine the purposes when stopping in the test kit for the treatment of.
For the ease of application, the present invention also provides for a kind of diagnostic kit for diagnosing liver cirrhosis, and this test kit includes Base plate, sample pad, gold mark pad, NC film and absorption pad；Be provided with on described base plate the sample pad of sequentially mutually overlap joint, gold mark pad, NC film and absorption pad, described gold mark pad scribbles the colloidal gold solution comprising antibody one, described NC film is provided with detection line and matter Control line, is coated respectively with antibody two and antibody three at described detection line and nature controlling line.Described antibody one, antibody two and antibody three are all The specific binding antibody of VWF or antibody one, antibody two and antibody three are all the specific antibody of ADAMTS13.Described antibody One is monoclonal antibody.
Further, antibody one, antibody two and specificity that antibody three is VWF or ADAMTS13 in described diagnostic kit Antibody fragment.
Use biomarker VWF and ADAMTS13 of the present invention that liver cirrhosis disease carries out diagnosis and have following prominent useful Effect: diagnosis process operation is simple, diagnosis process safety hurtless measure is prone to be accepted by patient, diagnostic criteria is unified by individual People's subjective factors affects that less, diagnostic result specificity and susceptiveness are high, be prone to carry out a large amount of examination selects etc. advantage.Tentatively Experimental result, by detection biomarker VWF and/or ADAMTS13 of the present invention content in human serum, and carries out liver The sensitivity of cirrhosis-diagnostic is up to more than 80%, and specificity is up to more than 90%.
The present invention describes in detail:
Based on having the inventors discovered that two kinds of new liver cirrhosis biomarkers, the VWF being i.e. present in human serum and ADAMTS13。
The present invention separates two kinds of protein, and the first protein i.e. VWF is logged in ncbi database by aminoacid sequence Number for GI 317373549 protein composition or include that in ncbi database, accession number is the protein of GI 317373549；The Two kinds of i.e. ADAMTS13 of protein are made up of the protein that aminoacid sequence accession number in ncbi database is GI 74749836 Or include that in ncbi database, accession number is the protein of GI 317373549.
The present inventor by the blood of hundreds liver cirrhosis patient is carried out detection analyze find, the VWF in serum and The contents level of ADAMTS13 is closely related with liver cirrhosis lesion degree.Therefore VWF and ADAMTS13 in serum can conduct New liver cirrhosis mark is used for diagnosis and the prognosis of liver cirrhosis.Particularly, the inventors discovered that, the VWF in serum and The ratio of the contents level of ADAMTS13 has more preferable susceptiveness and specificity as diagnosis and the prognostic indicator of liver cirrhosis.
The invention still further relates to detect the reagent of present protein (i.e. VWF and the ADAMTS13) contents level in serum Box.The test kit of the present invention includes protein VWF and/or ADAMTS13 and the specificity junction mixture thereof of the present invention.The present invention's Test kit can be used for carrying out the diagnosis of liver cirrhosis by detecting the contents level of VWF and/or ADAMTS13 in serum.
The invention still further relates to the protein of the present invention and specificity junction mixture thereof in preparing diagnostic kit for liver cirrhosis Purposes, described test kit such as can be used for the level by VWF and/or ADAMTS13 in detection serum and diagnoses liver cirrhosis Exist；For patients with chronic liver being carried out liver cirrhosis examination by VWF and/or the ADAMTS13 level in detection serum； For by VWF and/or the ADAMTS13 level in detection serum, prognosis to liver cirrhosis patient judges；Or be used for leading to Cross VWF and/or the ADAMTS13 level in detection serum and judge that the operation to patient with liver cirrhosis or Drug therapy are the most effective And/or determine when stop treatment.
In the present invention, " specificity junction mixture " of present protein refers to combine present protein with high-affinity Molecule.Particularly, including the molecule combining VWF and/or ADAMTS13 with high-affinity.Described " specificity junction mixture " is preferred The specific antibody of VWF and/or ADAMTS13 or antibody fragment, be more highly preferred to, and described 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 liver cirrhosis Method.Can be by the content of VWF and/or ADAMTS13 in any suitable method detection serum.Described method includes directly Or the level that indirect determination present protein is in serum, in order to contribute to diagnosing liver cirrhosis.
Directly method for measuring includes that the specificity junction mixture using described VWF and/or ADAMTS13 is to detect the present invention Protein.Described specificity junction mixture can be polyclonal antibody, it is possible to for monoclonal antibody, the animal kind being originated it Class does not limit.Additionally, antibody includes antibody and the partial antibody being made up of immunoglobulin total length.Partial antibody refer to containing Antigen-binding site, there is the antibody fragment of antigen-binding activity.Additionally, with in the case of mark substance traget antibody, as Mark substance, refers to such as: fluorescent material (such as: FITC, rhodamine, phallotoxin), gold isocolloid particle, The fluorescent microsphere of Luminex (registered trade mark, Luminex company) etc., heavy metal (such as gold, platinum etc.), chromoprotein matter (example As, phycoerythrin, phycocyanin etc.), radiosiotope (such as, 3H, 14C, 32P, 35S, 125I, 131I etc.), (example such as enzyme As, peroxidase, alkali phosphatase etc.), biotin, the material such as Streptavidin, but be not limited to this.Concrete, such as make ELISA detection is carried out by the specific antibody identifying present protein.First, specificity corresponding for biomarker is resisted Body (antibody 1) is fixed in the solid phases such as microwell plate.When this solid phase is added serum, the biomarker in serum is tied with antibody Close, form immune complex.By remaining serum remove after, add identify epi-positions different from antibody 1, use marker substances labelling Antibody (antibody 2), be combined with biomarker.Remaining antibody 2 is cleaned after removing, measure the mark of remaining on microwell plate The amount of note material.Make in advance and illustrate that the mark amount added to microwell plate is bent with the calibration of the relation of the amount of remaining mark substance Line, uses this calibration curve, can calculate the mark amount in blood.
The method of indirect determination include such as by detection VWF and/or ADAMTS13 activity 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 serum VWF's and ADAMTS13 of non-suffering from liver cirrhosis Content ratio scope is 9054.532～91004.04, focuses more on 13536.9～50563.2.It is in the liver 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 in liver cirrhosis compensatory In the serum of the patient of phase, VWF and ADAMTS13 content ratio scope is 34501.71～126663.3, focuses more on 53940.3 ～899741.In the serum of the patient being in cirrhosis patients in decompensation, VWF and ADAMTS13 content range is 31456240 respectively ～70655840ng/ml and 104.8～560.0ng/ml, focus more on 37005190～65421470ng/ml and 185.3～ 469.8ng/ml；In the serum of the patient being in cirrhosis patients in decompensation, the ratio range of VWF and ADAMTS13 content is 118746.6～231246.7, focus more on 128448.6～168945.8.
By data above, hence it is evident that can draw, the content of the middle clearly VWF of blood in human body in: the patient of cirrhosis patients in decompensation is bright The aobvious patient higher than the liver cirrhosis compensatory phase, the patient of liver cirrhosis compensatory phase is apparently higher than the patients with chronic liver of non-suffering from liver cirrhosis； The content of the middle clearly ADAMTS13 of blood in human body in: the patient of cirrhosis patients in decompensation is significantly lower than the patient of liver cirrhosis compensatory phase, liver Harden the patient of compensatory phase significantly lower than the patients with chronic liver of non-suffering from liver cirrhosis.Edge this, the VWF in human serum and ADAMTS13 level can be used for judging whether liver cirrhosis exists as new liver cirrhosis mark.Preferably, can examine simultaneously Survey the content of VWF and ADAMTS13, and relatively diagnose liver cirrhosis compared with normal level by the ratio of VWF with ADAMTS13. Test tentative confirmation through the present inventor, using VWF and ADAMTS13 ratio as diagnosis index, there is the most excellent diagnosis knot Really.When VWF and ADAMTS13 liver cirrhosis ratio marginal 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 marginal value is set to 79928.59, Decompensated liver cirrhosis diagnostic sensitivity is 83.1%, and specificity is 94.9%.
In being embodied as, the present inventor's preliminary advice can be by the liver cirrhosis marginal value of VWF and ADAMST13 content ratio It is set as 64747.76, the patients with chronic liver of the not enough marginal value Chronic Liver for there is not liver cirrhosis colony, more than marginal value Patient there occurs liver cirrhosis.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 the not enough marginal value Chronic Liver for there is not Decompensated liver cirrhosis colony, more than marginal value There is Decompensated liver cirrhosis in patient.After cirrhosis patients in decompensation patient treatment, the serum gathered before and after being treated by mensuration In VWF and ADAMST13 content ratio, this patient outcomes measurable.After treatment, under VWF and ADAMST13 content ratio The patient of fall, it is good to can determine whether as therapeutic effect, when VWF and ADAMST13 content ratio is less than mistake compensatory marginal value 79928.59, This patient reenters the liver cirrhosis compensatory phase.
In a concrete scheme, the test kit of the present invention or method can be used for determining whether individuality exists liver cirrhosis.For This, the test kit of the present invention or method can be used to measure from the VWF in the blood serum sample of liver cirrhosis suspected patient and/or ADAMTS13 level, and optionally compare with normal control, then according to VWF and/or the ADAMTS13 level in sample Judge that the probability of liver cirrhosis occurs in this patient.
In another concrete scheme, the test kit of the present invention or method can be used for by detection serum in VWF and/or ADAMTS13 level and patients with chronic liver is carried out liver cirrhosis examination.To this end, the test kit of the present invention or method can be used to survey Fixed from VWF and/or the ADAMTS13 level in the blood serum sample of high-risk group, and optionally compare with normal control, Then judge in this crowd, liver cirrhosis may occur in which individuality according to VWF and/or the ADAMTS13 level in sample.
In another concrete scheme, the test kit of the present invention or method can be used for by detection serum in VWF and/or ADAMTS13 level and prognosis to liver cirrhosis patient judge.To this end, the test kit of the present invention or method can be used to measure From VWF and/or the ADAMTS13 level in the blood serum sample of liver cirrhosis patient, and optionally with normal control or this patient with Past serum VWF and/or ADAMTS13 level compare, and then judge according to VWF and/or the ADAMTS13 level in sample The prognosis of this liver cirrhosis patient.VWF and/or ADAMTS13 maintains high level or VWF and/or ADAMTS13 level to raise further May be relevant to disadvantageous prognosis.Accordingly, can remind doctor that this patient carries out closer observation, and change mesh if desired Front therapeutic scheme.
In another concrete scheme, the test kit of the present invention or method can be used for by detection serum in VWF and/or ADAMTS13 level and judge the operation to patient with liver cirrhosis or Drug therapy the most effectively and/or determine when stop treatment. To this end, the test kit of the present invention or method can be used to measure from the VWF in the blood serum sample of liver cirrhosis patient and/or ADAMTS13 level, and optionally conventional with normal control or this patient serum VWF and/or ADAMTS13 level compare Relatively, then judge whether are the operation to this patient with liver cirrhosis or Drug therapy according to VWF and/or the ADAMTS13 level in sample When effective and/or decision stops treatment.
Accompanying drawing explanation
Accompanying drawing 1 is for using Gold standard test kit to normal person, patients with chronic liver, liver cirrhosis compensatory phase patient and liver cirrhosis In the serum of Decompensated stage patient, VWF carries out the Comparative result figure detected；
From accompanying drawing 1, along with the development of the liver cirrhosis state of an illness, the content of VWF significantly raises.
Accompanying drawing 2 is for using Gold standard test kit to normal person, patients with chronic liver, liver cirrhosis compensatory phase patient and liver cirrhosis In the serum of Decompensated stage patient, ADAMTS13 carries out the Comparative result figure detected；
From accompanying drawing 2, along with the development of the liver cirrhosis state of an illness, the content of ADAMTS13 gradually significantly reduces.
Accompanying drawing 3 is the quantitative inspection of VWF in patients with chronic liver, liver cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum Survey result；
The detection by quantitative of VWF is shown, patients with chronic liver, liver cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum The average of middle VWF contents level respectively may be about 1.7 × 107ng/ml、2.8×107ng/ml、4.7×107ng/ml。
Accompanying drawing 4 is ADAMTS13 in patients with chronic liver, liver cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum Detection by quantitative result；
Showing the detection by quantitative of ADAMTS13, patients with chronic liver, liver cirrhosis compensatory phase and cirrhosis patients in decompensation are suffered from In person's serum, the average of ADAMTS13 contents level respectively may be about 560ng/ml, 400ng/ml, 350ng/ml.
Accompanying drawing 5 be in patients with chronic liver, liver cirrhosis compensatory phase and cirrhosis patients in decompensation patients serum VWF with The ratio quantitative distribution figure of ADAMTS13；
The ratio detection by quantitative of VWF Yu ADAMTS13 shows, patients with chronic liver, liver cirrhosis compensatory phase and liver cirrhosis lose generation Repay the average of the ratio of VWF Yu ADAMTS13 in phase patients serum and respectively may be about 30000 times, 70000 times, 150000 times, exist Significant difference.
Accompanying drawing 6 is the structural representation of Gold standard test kit of the present invention；
In accompanying drawing 6,1 is sample pad, and 2 is gold traget antibody glass fibre membrane, and 3 is celluloid coated film, and 4 is water suction Paper, 5 is PVC base plate.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated, and listed embodiment purpose is for side Just those skilled in the art are more fully understood that the inventive concept of the present invention, and it is not construed as protection scope of the present invention Limit.On the premise of inventive concept of the present invention, still can make several different changes, cannot be carried out here one by one Exhaustive, these simple change not paying creativeness all should contain within protection scope of the present invention.
Reagent medicament used in embodiment described herein is all common commercially available prod.Institute's use instrument also leads to for industry Use equipment and instrument.The antibody 1 corresponding for VWF with ADAMTS13, antibody 2 and the multi-resistance that are used are common commercially available prod, antibody Preparation method can use the preparation method for antibody of routine to obtain, and here is omitted.
Embodiment 1: the preparation of Gold standard test kit and the application in liver cirrhosis diagnosis thereof.
One, the preparation of test kit:
The specific antibody that antibody is VWF or ADAMTS13 that this test kit preparation process uses.Note, same reagent box In preparation process: if the monoclonal antibody 1 used in gold traget antibody glass fibre membrane is the antibody of VWF, then corresponding celluloid bag The monoclonal antibody 2 used in tunicle preparation process and multi-resistance should also be as using the antibody of VWF；If gold traget antibody glass fibre membrane makes Monoclonal antibody 1 be the antibody of ADAMTS13, then the monoclonal antibody 2 used in corresponding celluloid coated film preparation process and multi-resistance are also The antibody of ADAMTS13 should be used.
1, the preparation of colloidal gold solution: put by ultra-pure water and be heated with stirring to boiling on magnetic stirring apparatus, by whole mass concentration Be ten thousand/ amount be rapidly added the chlorauric acid solution that mass concentration is 1%, boil 5 minutes；Molten by added gold chloride again It is the citric acid three sodium solution of 1% that liquid equal volume amounts adds mass concentration, after boiling 10 minutes, is cooled to room temperature, uses ultra-pure water Be settled to gold chloride final concentration of ten thousand/, room temperature keeps in Dark Place standby.
2, the preparation of gold traget antibody glass fibre membrane: with 2mol/L solution of potassium carbonate regulation colloidal gold solution pH value extremely 7.0.Add monoclonal antibody 1, mixing by the proportional concentration of 3 μ g antibody/milliliter colloidal gold solution, stand 10 minutes；Again by 2% volume Add the BSA solution that concentration is 20%, mixing, stand 10 minutes；13000rpm is centrifuged 15 minutes, abandons supernatant, precipitation labelling Cleaning mixture washed once, and abandons supernatant after being centrifuged, and precipitates with the golden labeling antibody preservation liquid of 1/20th initial colloid gold volume molten Solve, then spread the amount of 15 square centimeters by every milliliter of solution and be coated on glass fibre membrane, after being vacuum dried 2 hours, put equipped with The sealing bag of desiccant saves backup.
The concrete component proportion that above-mentioned 20%BSA solution, labelling cleaning mixture and gold labeling antibody preserve liquid is as follows:
A.20%BSA solution: the 200g Han bovine serum albumin in every 1000mL ultra-pure water.
B. labelling cleaning mixture: the bovine serum albumin Han 2g in every 1000mL ultra-pure water, 1g PEG 20000,5g sucrose, 1.211g Tris, and regulate pH to 7.6.
C. gold labeling antibody preservation liquid: the bovine serum albumin Han 10g in every 1000mL ultra-pure water, 1g PEG 20000, 10.2g Tris, 1g sodium hydroxide, and regulate pH to 7.6.
3, the preparation of celluloid coated film: spray monoclonal antibody 2 and multi-resistance on nitrocellulose filter respectively, and in two Line is arranged in parallel, forms detection line and nature controlling line respectively, 37 DEG C of drying and processings 3 hours, puts and protects equipped with in the sealing bag of desiccant Deposit standby.
Detection line: debugging spray film instrument, spouting liquid is 1.5 μ l/cm, and with being coated buffer dilution monoclonal antibody 2, concentration is 2mg/ ML, with spray film instrument spraying line.
Nature controlling line: debugging spray film instrument, spouting liquid is 1.5 μ l/cm, and with being coated buffer dilution multi-resistance, concentration is 2mg/mL, With spray film instrument spraying line.
Above-mentioned be coated buffer concrete component proportion be: the 0.01g Han bovine serum albumin in every 1000mL ultra-pure water, ten Two hypophosphite monohydrate disodium hydrogen 30.072g, potassium dihydrogen phosphate 2.176g.
On celluloid coated film, drawn two-lines answers fine uniform, and adjacent line-to-line is every 0.8cm.
4, big plate group bar:
Big plate group bar each component specification (long × wide): PVC base plate: 30cm × 8cm；Sample pad: 30cm × 4.0cm；Jin Biao Note antibody glass fibre membrane: 30cm × 0.5cm；Celluloid coated film: 30cm × 2.5cm；Absorbent paper: 30cm × 3cm.
Each component carries out organizing bar as follows: be pasted onto on PVC base plate the bottom surface of celluloid coated film, at this The above of coated film two ends pastes gold traget antibody glass fibre membrane and absorbent paper respectively, on gold traget antibody glass fibre membrane Sample pad is pasted in face；On PVC base plate, i.e. sequentially paste sample pad, gold traget antibody glass fibre membrane, nitric acid mutually overlap joint Cellulose coated film and absorbent paper, form big plate.The humidity of composing room to control below 30%.
5, cutting:
With cutting cutter, big plate being cut into wall scroll, every width is 4 millimeters, inspects by random samples at random, and sensitivity can detect Internal Quality Control Product, nothing but specific band.
6, envelope and group box:
By 1 part of test strips cut, one it is responsible for a task until it is completed drying prescription and a plastic dropper is contained in aluminium foil bag, by sealing secret Envelope, puts into after sealing in test kit, and normal temperature drying preserves.The most i.e. respectively obtain test kit and the detection of detection VWF The test kit of ADAMTS13.
Two, mentioned reagent box is used to carry out the diagnosis of liver cirrhosis:
Take normal person, patients with chronic liver, liver cirrhosis compensatory phase patient and the whole blood of cirrhosis patients in decompensation patient respectively, Within 24 hours, under cryogenic conditions, (about 4 DEG C) deliver to laboratory, must avoid haemolysis, and sample is if it occur that haemolysis, the most again Gather.In 4 DEG C, 6000g be centrifuged twice, take supernatant.Use test kit detection VWF and ADAMTS13 prepared as above in serum Content.Testing result is compareed with clinical diagnosis, to verify the content of VWF and ADAMTS13 and liver cirrhosis in serum Dependency.Testing result is as shown in Figures 1 and 2.
From accompanying drawing 1 and the testing result of accompanying drawing 2, along with the development of the liver cirrhosis state of an illness, the content of VWF is the most notable Raising, the content of ADAMTS13 gradually significantly reduces.
Embodiment 2:VWF and ADAMTS13 are as the sensitivity of liver cirrhosis biomarker and specificity research.
Sensitivity as herein described and specificity are defined:
Sensitivity: sensitivity described herein is that the actual patients with chronic liver suffering from liver cirrhosis is correctly judged to kidney-Yang The ratio of property；Or cirrhosis patients in decompensation patient will be actually entered correctly it is judged to the ratio of true positives.
Specificity: specificity described herein is correctly to be judged to Kidney-Yin by the patients with chronic liver of non-for reality suffering from liver cirrhosis The ratio of property；Or the patient being practically in the liver cirrhosis compensatory phase is correctly judged to the ratio of true negative.
VWF and ADAMTS13 liver cirrhosis ratio marginal value: the content ratio number of VWF and ADAMTS13 specified herein According to, in subjects's serum, i.e. to judge that this patients with chronic liver suffers from liver hard higher than these data for the content ratio of VWF and ADAMTS13 Change.
VWF and ADAMTS13 Decompensated liver cirrhosis ratio marginal value: the content of VWF and ADAMTS13 specified herein Ratio data, in subjects's serum, higher than these data, the content ratio of VWF and ADAMTS13 i.e. judges that this patient enters liver cirrhosis Decompensated stage.
VWF and ADAMTS13 liver cirrhosis ratio marginal value and VWF and ADAMTS13 liver cirrhosis that the present embodiment is taken lose generation Repaying ratio marginal value is previously described 64747.76 and 79928.59 respectively,
The present inventor is to the patients with chronic liver of 300 examples and liver cirrhosis compensatory phase patient and cirrhosis patients in decompensation patient VWF and ADAMTS13 content ratio data acquisition is analyzed with Receiver operating curve's (i.e. ROC curve), and ROC curve analysis shows Show that VWF Yu ADAMST13 ratio can be used for diagnosing liver cirrhosis and the generation of Decompensated liver cirrhosis, VWF and ADAMTS13 liver cirrhosis Ratio marginal value (i.e. liver cirrhosis threshold value) and VWF and ADAMTS13 Decompensated liver cirrhosis ratio marginal value (i.e. Decompensated liver cirrhosis Threshold value) it is previously described 64747.76 and 79928.59 respectively.
Use in the previously described ELISA method 236 example patients with chronic liver case sample serum to randomly drawing VWF and The content of ADAMTS13 carries out detection by quantitative.By testing result and use routine clinical detection means (CT, B ultrasonic, 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 is made a definite diagnosis Case number vWF/ADAM vWF ADAMTS 13 Conventional means is made a definite diagnosis
1 345265 122544389 354.9285 Liver cirrhosis 60 100677.1 27740771.3 275.5421 Liver cirrhosis
2 204156 59763403 292.734 Liver cirrhosis 61 86218.3 42042023 487.623 Non-liver cirrhosis
3 25995.1 12269687 472 Non-liver cirrhosis 62 122570.8 49520844.9 404.0185 Liver cirrhosis
4 87701.84 31846678 363.1244 Liver cirrhosis 63 64747.76 20467350 316.109 Non-liver cirrhosis
5 71857.17 21763223 302.8678 Liver cirrhosis 64 25975.1 16896248.5 650.4787 Non-liver cirrhosis
6 135015 28908426 214.1127 Liver cirrhosis 65 72533.29 17326288.1 238.8736 Liver cirrhosis
7 16427.05 7269960.2 442.5603 Non-liver cirrhosis 66 358944.9 52722293.5 146.8813 Liver cirrhosis
8 16652.12 10490836 630 Non-liver cirrhosis 67 73453.57 45551473.2 620.1397 Non-liver cirrhosis
9 104999 38960552 371.0563 Liver cirrhosis 68 85128.53 30635297.7 359.8711 Liver cirrhosis
10 145554.4 68379841 469.7889 Liver cirrhosis 69 109928.6 50552981.6 459.8711 Liver cirrhosis
11 109115.1 31800965 291.4443 Liver cirrhosis 70 25985.1 15155585.1 583.2414 Non-liver cirrhosis
12 189535.3 23941704 126.3179 Liver cirrhosis 71 144870.7 65727909 453.7005 Liver cirrhosis
13 17175.54 5468132 318.3674 Non-liver cirrhosis 72 69340.9 25763278.6 371.5452 Liver cirrhosis
14 84339.31 62697843 743.4 Non-liver cirrhosis 73 17145.54 10836280.3 632.0174 Non-liver cirrhosis
15 113056.5 40940337 362.1229 Liver cirrhosis 74 61801.5 31358081.1 507.4 Non-liver cirrhosis
16 130591.3 46033881 352.5035 Liver cirrhosis 75 17155.54 13358414.6 778.6648 Non-liver cirrhosis
17 16417.05 5491359.6 334.4913 Non-liver cirrhosis 76 36848.57 19035352.2 516.5832 Non-liver cirrhosis
18 407864.3 143963022 352.9679 Liver cirrhosis 77 36838.57 20920411.5 567.8942 Non-liver cirrhosis
19 16622.12 12411637 746.694 Non-liver cirrhosis 78 58414.21 28112790.7 481.2663 Non-liver cirrhosis
20 70344.22 28329193 402.7224 Liver cirrhosis 79 73463.57 21277857.6 289.6382 Non-liver cirrhosis
21 103680.9 35446165 341.8775 Liver cirrhosis 80 46888.22 30170508.1 643.456 Non-liver cirrhosis
22 61811.5 27093767 438.3289 Non-liver cirrhosis 81 17212.93 8059804.72 468.2413 Non-liver cirrhosis
23 49889 17460979 349.9966 Liver cirrhosis 82 84349.31 35660878.2 422.7762 Non-liver cirrhosis
24 91891.3 26439370 287.7244 Liver cirrhosis 83 73433.57 50422490.6 686.6409 Non-liver cirrhosis
25 93211.84 48792297 523.456 Liver cirrhosis 84 383191.7 59872336.7 156.2464 Liver cirrhosis
26 86198.3 29323809 340.1901 Non-liver cirrhosis 85 578643.4 249404260 431.0155 Liver cirrhosis
27 420638.3 77930985 185.2684 Liver cirrhosis 86 84329.31 53286241 631.8828 Non-liver cirrhosis
28 61791.5 40449658 654.6152 Non-liver cirrhosis 87 226598.9 125309192 553 Liver cirrhosis
29 140422.2 25512172 181.6819 Liver cirrhosis 88 140397.6 34367586.2 244.7876 Liver cirrhosis
30 35327.79 33806995 956.9519 Non-liver cirrhosis 89 199905.3 52829885.2 264.2746 Liver cirrhosis
31 402754.2 178360681 442.8524 Liver cirrhosis 90 116114.4 28908798.1 248.9682 Liver cirrhosis
32 209706.6 33412725 159.3308 Liver cirrhosis 91 17165.54 13187697.3 768.2658 Non-liver cirrhosis
33 46898.22 23791088 507.2919 Non-liver cirrhosis 92 35317.79 34254895.5 969.9048 Non-liver cirrhosis
34 161376.1 38240682 236.9662 Liver cirrhosis 93 16397.05 12665835.4 772.446 Non-liver cirrhosis
35 15310.69 6124276 400 Non-liver cirrhosis 94 121303.4 12711227.7 104.7887 Liver cirrhosis
36 26005.1 10746342 413.2398 Non-liver cirrhosis 95 58404.21 32269770.6 552.5247 Non-liver cirrhosis
37 78017.56 28987050 371.5452 Liver cirrhosis 96 17182.93 14190671.5 825.8587 Non-liver cirrhosis
38 293474.2 100960042 344.0168 Liver cirrhosis 97 58394.21 45823571.6 784.728 Non-liver cirrhosis
39 58384.21 53633487 918.63 Non-liver cirrhosis 98 15300.69 5748064.25 375.6735 Non-liver cirrhosis
40 428898.6 220859061 514.9447 Liver cirrhosis 99 73443.57 34367107.1 467.939 Non-liver cirrhosis
41 142269.5 38325444 269.3862 Liver cirrhosis 100 146422.8 44995140.7 307.296 Liver cirrhosis
42 64717.76 47016510 726.4854 Non-liver cirrhosis 101 46878.22 33160202.3 707.3691 Non-liver cirrhosis
43 64727.76 38054952 587.9232 Non-liver cirrhosis 102 194416.4 74381047.2 382.5863 Liver cirrhosis
44 16407.05 11482970 699.8802 Non-liver cirrhosis 103 158605.9 107852012 680 Liver cirrhosis
45 80136.75 44074940 549.9966 Liver cirrhosis 104 84098.54 28356520.2 337.1821 Liver cirrhosis
46 69838.4 24650716 352.9679 Liver cirrhosis 105 228857.1 76288854 333.3471 Liver cirrhosis
47 85811.79 21603225 251.7512 Liver cirrhosis 106 86228.3 17070270.5 197.966 Non-liver cirrhosis
48 35347.79 18576752 525.5421 Non-liver cirrhosis 107 61781.5 46909341.2 759.2781 Non-liver cirrhosis
49 36818.57 34093224 925.979 Non-liver cirrhosis 108 328530.5 129930247 395.4891 Liver cirrhosis
50 63942.98 22437061 350.8917 Liver cirrhosis 109 17202.93 5983733.96 347.8323 Non-liver cirrhosis
51 17192.93 13384696 778.5 Non-liver cirrhosis 110 36828.57 30271363 821.9533 Non-liver cirrhosis
52 131040.8 73382826 560 Liver cirrhosis 111 196250.6 61473498.7 313.2398 Liver cirrhosis
53 474189.7 148083271 312.287 Liver cirrhosis 112 35337.79 21540792.2 609.5682 Non-liver cirrhosis
54 16642.12 12273564 737.5 Non-liver cirrhosis 113 79014.82 24240443 306.7835 Liver cirrhosis
55 86208.3 51678885 599.4653 Non-liver cirrhosis 114 91738.15 26112044.3 284.6367 Liver cirrhosis
56 15280.69 9844727 644.2593 Non-liver cirrhosis 115 64737.76 42922478.8 663.0208 Non-liver cirrhosis
57 15290.69 5429040.4 355.0553 Non-liver cirrhosis 116 46908.22 20170534.6 430 Non-liver cirrhosis
58 83000.2 27597298 332.4968 Liver cirrhosis 117 415614.2 200159799 481.6 Liver cirrhosis
59 167824.1 79813785 475.58 Liver cirrhosis 118 16632.12 10102069.7 607.3832 Non-liver cirrhosis
Analytical table 1 understands: VWF and ADAMTS13 content detection and clinical definite to 118 example patients with chronic liver find, 59 examples are diagnosed as the patients with chronic liver of non-suffering from liver cirrhosis, wherein have 48 examples VWF and ADAMTS13 content ratio less than VWF and ADAMTS13 liver cirrhosis ratio marginal value 64747.76, clinical specificity is 81.4%.59 examples are diagnosed as the patient of liver cirrhosis, its In 57 examples VWF and ADAMTS13 content ratio higher than VWF and ADAMTS13 liver cirrhosis ratio marginal value 64747.76, clinical spirit Sensitivity is 96.6%.
Table 2:
Analytical table 2 understands: find 118 example liver cirrhosis patient VWF and ADAMTS13 content detection and clinical definite, 59 examples It is diagnosed as liver cirrhosis compensatory phase patient, wherein has 56 examples VWF and ADAMTS13 content ratio less than VWF and ADAMTS13 liver cirrhosis Losing compensatory ratio marginal value 79928.59, clinical specificity is 94.9%.59 examples are diagnosed as the patient of Decompensated liver cirrhosis, its In 49 examples VWF and ADAMTS13 content ratio higher than VWF and ADAMTS13 liver cirrhosis ratio marginal value 79928.59, clinical sensitive Degree is 83.1%.
Can be drawn by above specificity and sensitivity study, use VWF and/or ADAMTS13 biological as liver cirrhosis Patients with chronic liver is diagnosed by mark, has the strongest specificity and the highest sensitivity.
The determination that it should be noted that liver cirrhosis threshold value and Decompensated liver cirrhosis threshold value is not fixing, to bigger VWF and ADAMTS13 content ratio test sample carries out ROC curve analysis, can obtain the most accurate reasonably threshold value.With this Threshold value is as the most reasonable accurately with reference to the diagnosis that subjects makees liver cirrhosis.

Claims (10)

1.VWF and/or ADAMTS13 and specificity junction mixture purposes in the diagnostic reagent of preparation diagnosis liver cirrhosis thereof, described Diagnostic reagent is by the content of VWF and/or ADAMTS13 in detection subjects's serum, and relatively diagnoses compared with normal level Liver cirrhosis, described VWF is made up of the protein that aminoacid sequence accession number in ncbi database is GI 317373549；Described ADAMTS13 is made up of the protein that aminoacid sequence accession number in ncbi database is GI 74749836.
2.VWF and/or ADAMTS13 and specificity junction mixture thereof preparation in by detection subjects's serum VWF and/or The contents level of ADAMTS13 and patients with chronic liver is carried out the purposes in the test kit of liver cirrhosis examination, described VWF is by ammonia Base acid sequence accession number in ncbi database is the protein composition of GI 317373549；Described ADAMTS13 is by aminoacid sequence It is listed in the protein composition that accession number in ncbi database is GI 74749836.
3.VWF and/or ADAMTS13 and specificity junction mixture thereof preparation in by detection subjects's serum VWF and/or The contents level of ADAMTS13 and purposes in the test kit that judges the prognosis of liver cirrhosis patient, described VWF is by amino Acid sequence accession number in ncbi database is the protein composition of GI 317373549；Described ADAMTS13 is by aminoacid sequence In ncbi database, accession number is the protein composition of GI 74749836.
4.VWF and/or ADAMTS13 and specificity junction mixture thereof preparation in by detection subjects's serum VWF and/or The contents level of ADAMTS13 and judge the operation to liver cirrhosis patient or Drug therapy the most effectively and/or determine when stop Purposes in the test kit for the treatment of, described VWF is the egg of GI317373549 by aminoacid sequence accession number in ncbi database White matter forms；Described ADAMTS13 is by the protein group that aminoacid sequence accession number in ncbi database is GI74749836 Become.
5. the purposes described in claim 2～4 any claim, is characterised by, described specificity junction mixture be VWF or The specific Ab fragments of ADAMTS13.
6. the purposes described in claim 2～4 any claim, wherein, by electrophoresis method, immuno-chemical method, direct competitive Method, indirect competitive, ELISA method, RIA method, Flow cytometry or immunochromatographic method detect the VWF in subjects's serum And/or the content of ADAMTS13.
7. the purposes described in claim 2～4 any claim, is characterised by, by VWF in subjects's serum with The ratio of ADAMTS13 relatively diagnoses liver cirrhosis compared with normal level.
8. diagnosing the diagnostic kit of liver cirrhosis, described test kit includes base plate (5), sample pad (1), gold mark pad (2), NC film And absorption pad (4) (3)；Be provided with on described base plate (5) sample pad (1) of sequentially mutually overlap joint, gold mark pad (2), NC film (3) and Absorption pad (4), described gold mark pad (2) scribble the colloidal gold solution comprising antibody one, described NC film (3) is provided with detection line and Nature controlling line, is coated respectively with antibody two and antibody three at described detection line and nature controlling line, described antibody one, antibody two and antibody three are all It is all the specific antibody of ADAMTS13 for the specific binding antibody of VWF or antibody one, antibody two and antibody three, described anti- Body one is monoclonal antibody.
Diagnostic kit the most according to claim 8, it is characterised in that described antibody one, antibody two and antibody three are VWF Or the specific Ab fragments of ADAMTS13.
10. the preparation method of diagnostic kit described in claim 8, is characterised by, comprises the following steps:
The monoclonal antibody 1 used in following steps is the antibody of VWF, and the monoclonal antibody 2 of use and multi-resistance are also the antibody of VWF；
1), the preparation of colloidal gold solution: ultra-pure water is put and is heated with stirring to boiling on magnetic stirring apparatus, be ten thousand by whole mass concentration The amount of/mono-is rapidly added the chlorauric acid solution that mass concentration is 1%, boils 5 minutes；Again by added chlorauric acid solution etc. It is the citric acid three sodium solution of 1% that volume adds mass concentration, after boiling 10 minutes, is cooled to room temperature, uses ultra-pure water constant volume To gold chloride final concentration of ten thousand/, room temperature keeps in Dark Place standby；
2), the preparation of gold traget antibody glass fibre membrane: with 2mol/L solution of potassium carbonate regulation colloidal gold solution pH value to 7.0； Add monoclonal antibody 1, mixing by the proportional concentration of 3 μ g antibody/milliliter colloidal gold solution, stand 10 minutes；Add by 2% volume again Concentration is the BSA solution of 20%, mixing, stands 10 minutes；13000rpm is centrifuged 15 minutes, abandons supernatant, and precipitation labelling washs Liquid washed once, and abandons supernatant after being centrifuged, and precipitates and preserves liquid dissolving, so with the golden labeling antibody of 1/20th initial colloid gold volume It is coated on glass fibre membrane by the amount of every milliliter of solution paving 15 square centimeters afterwards, after being vacuum dried 2 hours, puts equipped with desiccant Sealing bag in save backup；
The concrete component proportion that above-mentioned 20%BSA solution, labelling cleaning mixture and gold labeling antibody preserve liquid is as follows:
A.20%BSA solution: the 200g Han bovine serum albumin in every 1000mL ultra-pure water；
B. labelling cleaning mixture: the bovine serum albumin Han 2g in every 1000mL ultra-pure water, 1g PEG 20000,5g sucrose, 1.211gTris, and regulate pH to 7.6；
C. gold labeling antibody preservation liquid: the bovine serum albumin Han 10g in every 1000mL ultra-pure water, 1g PEG 20000, 10.2gTris, 1g sodium hydroxide, and regulate pH to 7.6；
3), the preparation of celluloid coated film: spray monoclonal antibody 2 and multi-resistance on nitrocellulose filter respectively, and in two-lines Arranged in parallel, form detection line and nature controlling line respectively, 37 DEG C of drying and processings 3 hours, put and preserve equipped with in the sealing bag of desiccant Standby；
Detection line: debugging spray film instrument, spouting liquid is 1.5 μ l/cm, and with being coated buffer dilution monoclonal antibody 2, concentration is 2mg/mL, uses Spray film instrument spraying line；
Nature controlling line: debugging spray film instrument, spouting liquid is 1.5 μ l/cm, and with being coated buffer dilution multi-resistance, concentration is 2mg/mL, with spray The spraying line of film instrument；
Above-mentioned be coated buffer concrete component proportion be: the 0.01g Han bovine serum albumin, Shi Ershui in every 1000mL ultra-pure water Close disodium hydrogen phosphate 30.072g, potassium dihydrogen phosphate 2.176g；
On celluloid coated film, drawn two-lines answers fine uniform, and adjacent line-to-line is every 0.8cm；
4), big plate group bar:
Big plate group bar each component specification (long × wide): PVC base plate: 30cm × 8cm；Sample pad: 30cm × 4.0cm；Gold labelling resists Body glass fibre membrane: 30cm × 0.5cm；Celluloid coated film: 30cm × 2.5cm；Absorbent paper: 30cm × 3cm；
Each component carries out organizing bar as follows: is pasted onto on PVC base plate the bottom surface of celluloid coated film, is coated at this The above of film two ends pastes gold traget antibody glass fibre membrane and absorbent paper respectively, viscous on gold traget antibody glass fibre membrane Patch sample pad；On PVC base plate, i.e. sequentially paste sample pad, gold traget antibody glass fibre membrane, cellulose nitrate mutually overlap joint Element coated film and absorbent paper, form big plate and i.e. obtain the test kit of detection VWF；The humidity of composing room to control below 30%.
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