CN104714020B - Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B - Google Patents
Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B Download PDFInfo
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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
- G01N33/6893—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
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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
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
Do you the invention provides a kind of urine endogenous α trypsin inhibitor heavy chain H4 (Inter-alpha-trypsin? inhibitor? heavy? chain? H4, ITIH4) application of albumen, is specially the reagent that detects urine endogenous α trypsin inhibitor heavy chain H4 for the preparation of diagnosis or detect the application in the preparation that diabetes B merges coronary heart disease. The present invention compares with simple diabetes B group by studies confirm that with normal control, endogenous α trypsin inhibitor heavy chain H4 specifically expressing in diabetes B merging CHD group patient's urine is lowered, thereby can be used in detection or the diagnosis that diabetes B merges coronary heart disease. The present invention brings into play urine specimen and obtains without wound, repeated sampling on a large scale, preserves advantage easily, utilizes random urine Samples detection endogenous α trypsin inhibitor heavy chain H4.
Description
Technical field
The new purposes that the present invention relates to urine endogenous α trypsin inhibitor heavy chain H4, is specifically related to urine endogenous αTrypsin inhibitor heavy chain H4 merges the application in coronary heart disease diagnosis and treatment at diabetes B.
Background technology
Diabetes B (diabetesmellitustype2) is one of important public health problem in the whole world, nearly 20Nian Lai, along with the development of China's economic, there is significant change in people's life style, and the number of patients of diabetes B isSharply increase trend. Due to the trend of Chinese population aging, within 60 years old, above diabetes B patient has approached 50%, and withAnnual 0.1% speed increase. Diabetes B has become one of important diseases threatening Chinese residents life and health, is urgentlyGreat public health problem to be solved.
Diabetes B is mainly to increase due to insulin secretion and (or) the chronic blood sugar that causes of effect defect, relates to completeCarbohydrate, lipid and the protein metabolism disorder of body. For example, because blood sugar concentration is easily subject to many factors (excited, tired, senseEmit, have a fever and infect) impact and produce fluctuation, so the continuous Monitoring Blood Glucose of diabetes B needs of patients change, with correspondingAdjustment drug dose. Poor blood glucose control and fat, protein metabolism disorder can cause the tissues such as eye, kidney, heart, blood vesselOrgan and neural chronic progressive external pathology, hypofunction and exhaustion. According to statistics, in the global death rate, 2 type glycosuriasIt is about 5.2% that disease has accounted for, and die from merge cardiopathic accounted for wherein 68%. Therefore need diabetes B to merge hatThe people at highest risk of worry carries out early screening and early diagnosis, to carry out early treatment, improves patient's life quality.
ITI (Inter-alpha-trypsininhibitor, endogenous α trypsin inhibitor) is to be present in the mankindTrypsin ihhibitor in serum, by liver mRNAs and sequence clone are measured, finds that ITI includes heavy chain and light chainPolypeptide, respectively by different gene codes. Up to now, ITI comprises ITIH1, ITIH2, and ITIH3, ITIH4 and ITIH5, andLight chain bikunin. ITIH4 (Inter-alpha-trypsininhibitorheavychainH4, endogenous α tryptoseEnzyme inhibitor heavy chain H4) be a kind of plasma glycoprotein, mainly in liver, express, in pancreatic tissue, also there is expression. ITIH4 is to bloodSlurry kallikrein is very responsive, is considered to the substrate of plasma kallikrein effect. ITIH4 has different biology meritsCan, generally believe now ITIH4 mainly and inflammation and related to cancer. ITIH4 can be degraded to by kallikrein in blood35kDa (coo-end) and 85kDa (N-end), the latter can also be degraded to the sheet of the fragment of 57kDa and the 28kDa of suppositionSection. In body, the concrete biological function of ITIH4 is not clear. Have and studies show that the fragment of ITIH4 in blood and prostate cancer, bladderCancer, breast cancer, oophoroma and melanoma and cancer of pancreas etc. are relevant, there is no at present about ITIH4 and diabetes and concurrentThe relevant report of disease or complication.
In order to understand PD, complication in time, the diabetes B needs of patients Monitoring Blood Glucose control state of an illness. ?In existing clinical examination, gather peripheral blood or the venous blood best sample as blood sugar monitoring, but having obtained of blood preparationWound, chylemia or haemolysis can affect the accuracy of blood sugar test, and frequently blood sampling meeting causes psychological burden to patient, especiallyThe elderly and obese people. Therefore, if can set up a kind of non-invasive inspection of diabetes B patient being carried out to examination, monitoringMethod, early screening, diagnosis, prevention and treatment to diabetes B and complication or complication all have extremely importantMeaning.
Summary of the invention
The object of the present invention is to provide a kind of urine endogenous α trypsin inhibitor heavy chain H4 (Inter-alpha-TrypsininhibitorheavychainH4, ITIH4) merging coronary disease for the preparation of diagnosis or detection diabetes BApplication in sick preparation.
Preferably, the amino acid sequence of described urine endogenous α trypsin inhibitor heavy chain H4 is as SEQIDNO:1 instituteShow (MKPPRPVRTCSKVLVLLSLLAIHQTTTAEKNGIDIYSLTVDSRVSSRFAHTVVTSR VVNRANTVQEATFQMELPKKAFITNFSMIIDGMTYPGIIKEKAEAQAQYSAAVAKGKSAGLVKATGRNMEQFQVSVSVAPNAKITFELVYEELLKRRLGVYELLLKVRPQQLVKHLQMDIHIFEPQGISFLETESTFMTNQLVDALTTWQNKTKAHIRFKPTLSQQQKSPEQQETVLDGNLIIRYDVDRAISGGSIQIENGYFVHYFAPEGLTTMPKNVVFVIDKSGSMSGRKIQQTREALIKILDDLSPRDQFNLIVFSTEATQWRPSLVPASAENVNKARSFAAGIQALGGTNINDAMLMAVQLLDSSNQEERLPEGSVSLIILLTDGDPTVGETNPRSIQNNVREAVSGRYSLFCLGFGFDVSYAFLEKLALDNGGLARRIHEDSDSALQLQDFYQEVANPLLTAVTFEYPSNAVEEVTQNNFRLLFKGSEMVVAGKLQDRGPDVLTATVSGKLPTQNITFQTESSVAEQEAEFQSPKYIFHNFMERLWAYLTIQQLLEQTVSASDADQQALRNQALNLSLAYSFVTPLTSMVVTKPDDQEQSQVAEKPMEGESRNRNVHSGSTFFKYYLQGAKIPKPEASFSPRRGWNRQAGAAGSRMNFRPGVLSSRQLGLPGPPDVPDHAAYHPFRRLAILPASAPPATSNPDPAVSRVMNMKIEETTMTTQTPAPIQAPSAILPLPGQSVERLCVDPRHRQGPVNLLSDPEQGVEVTGQYEREKAGFSWIEVTFKNPLVWVHASPEHVVVTRNRRSSAYKWKETLFSVMPGLKMTMDKTGLLLLSDPDKVTIGLLFWDGRGEGLRLLLRDTDRFSSHVGGTLGQFYQEVLWGSPAASDDGRRTLRVQGNDHSATRERRLDYQEGPPGVEISCWSVEL)。
Preferably, described preparation is that diabetes B merges patients with coronary heart disease urine endogenous α trypsin inhibitor heavy chainH4 detection kit. Described kit comprises box body and is arranged on ELISA Plate and the reagent in box body.
Preferably, described ELISA Plate is by outer frame support and removable 12 enzyme marks reaction capillary strip groups of being placed on itBecome, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
Preferably, the pre-coated goat-anti people of each reacting hole ITIH4 polyclonal antibody.
Preferably, described reagent be ITIH4 standard solution, ELIAS secondary antibody, antibody concentrated solution, nitrite ion A, nitrite ion B,Stop buffer and concentrated cleaning solution.
Preferably, described kit also comprises recessed bottle position and the valve bag of cover plate film, placement reagent.
Preferably, totally 13 of described recessed bottle positions, are made up of plastic foam.
Preferably, described ELISA Plate is made up of polystyrene.
Inventor first collected diabetes B merge coronary heart disease, without the diabetes B patient of complication and complication andThe random urine specimen of normal control, gets supernatant after centrifugal, utilizes weak cation exchange magnetic beads for purifying and separated urine sample. WillAfter 1 μ l sample and 10 μ l matrix (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) mix, get 1 μ l point at AnchorchipOn (AutoflexMALDITOF, Bruker-Dalton) target plate, after sample ionization, carry out mass spectral analysis, gather 1000-Data within the scope of 10000Da, obtain the mass spectrum polypeptide figure being made up of the protein peak of different mass-to-charge ratioes. ApplicationClinProTools2.1 analysis software merges CHD group, diabetes B group without complication and complication to diabetes BAnalyze screening otherness polypeptide with all mass spectrograms of Normal group. Then inventor is to having statistical significanceOtherness polypeptide carries out substance assistant laser desorpted ionized flight time mass spectrum (Matrix-assistedlaserDesorptionionizationtimeofflightmassspectrometry, MALDI-TOF-MS) qualification,InternationalProteinIndex (IPIhumanv3.45fastawith71983entries) database retrievalObtaining diabetes B merges CHD group and normal control and expresses without the diabetes B group difference of complication and complicationITIH4 (Inter-alpha-trypsininhibitorheavychainH4, endogenous α trypsin inhibitor weightChain H4, Q14624) albumen, and compare ITIH4 albumen with the diabetes B group without complication and complication with normal controlIn the urine of diabetes B merging patients with coronary heart disease, be low expression.
The present invention compares with the diabetes B group without complication and complication by studies confirm that with normal control, endogenousProperty α trypsin inhibitor heavy chain H4 (ITIH4) merge in CHD group patient's urine under specifically expressing at diabetes BAdjust, can be used for examining of diabetes B merging coronary heart disease thereby propose to detect urine endogenous α trypsin inhibitor heavy chain H4Control.
The present invention brings into play urine specimen and obtains without wound, repeated sampling on a large scale, preserves advantage easily, utilizes random urineSamples detection endogenous α trypsin inhibitor heavy chain H4.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly,And coordinate accompanying drawing, be described in detail below.
Brief description of the drawings
Fig. 1 is that Normal group, diabetes B are without complication and complication group and diabetes B merging CHD group instituteThere is the scatter diagram of expressing in sample.
To be ITIH4 close without complication and complication group and diabetes B at Normal group, diabetes B for Fig. 2 and Fig. 3And average peak area in CHD group. Red (arrow 1) represents Normal group, green (arrow 2) represent diabetes B withoutComplication and complication group, blue (arrow 3) represents that diabetes B merges CHD group.
Fig. 4 is the mass spectrogram of ITIH4 albumen.
Fig. 5 is arranged on the schematic diagram of the ELISA Plate in the box body of kit of the present invention, and wherein 1 is the reaction of enzyme markCapillary strip, the 2 outer frame supports that are ELISA Plate, 3 for being coated with the hole of ITIH4 fusion polyclonal antibody.
Fig. 6 is that WesternBlot detects ITIH4 and merges coronary heart disease, 2 types without complication and complication at diabetes BExpression of results in diabetic and normal control urine.
Detailed description of the invention
Embodiment 1The collection of urine specimen and processing
Collect diabetes B merge patients with coronary heart disease 28 examples, without complication and other complication diabetes B patientThe random CCMS liquid sample of 30 examples (Beijing Shijitan Hospital, CMU's endocrine outpatient service), centrifugal in 2h(1500rpm, 5min), retains supernatant. After packing ,-80 DEG C of refrigerators are frozen. Normal group is that (capital medical courses in general are large for 29 routine normal personsLearn attached Beijing Shijitan Hospital MEC). Specifically please refer to following table 1:
The clinical data of table 1:3 group
Embodiment 2Polypeptide in magnetic beads for purifying and separated urine sample
Take out urine specimen from-80 DEG C of refrigerators, 4 DEG C of multiple melting, get supernatant after centrifugal (3000rpm, 10min) for subsequent use. Room temperatureLower balance weak cation magnetic bead (MB-WCX), and manually mix bead suspension. In sample cell, add 10ulMB-WCX and10ul magnetic bead binding buffer liquid, sample loading gun is blown and beaten and is mixed up and down, avoids bubbling. In sample cell, add 5ul urine supernatant, fullyMix on rear magnetic frame and leave standstill 1 minute, the fluid separation applications of magnetic bead and suspension. Remove the clear liquid of suspension with sample loading gun, rifle headShould avoid touching magnetic bead, avoid siphoning away magnetic bead. In sample cell, add 100ul magnetic bead cleaning buffer solution, will after fully mixingSample cell leaves standstill 1 minute on magnetic frame, and magnetic bead is adherent, with the fluid separation applications suspending, removes the liquid of suspension with sample loading gun. HeavyMultiple 3 times, discard suspension. In sample cell, add 5ul magnetic bead elution buffer, repeatedly inhale and make a call to more than 10 times, make magnetic bead and washDe-buffer solution mixes, and avoids bubbling. Sample cell is positioned on magnetic frame, leaves standstill 2min, magnetic bead is fully separated with suspension,Supernatant (eluent) is moved into the new 0.5ml sample cell of mark. Add 5ul stabilizing buffer, sample loading gun is is carefully blown and beatenMix.
Embodiment 3The point target of urine specimen and polypeptide spectrum map generalization
After rectifying an instrument with standard items, by 1 μ l eluent and 10 μ l matrix (alpha-cyano-4-hydroxycinnamic acid of 0.3%,HCCA) mix, get 1 μ l point on Anchorchip (AutoflexMALDITOF, Bruker-Dalton) target plate, room temperature is dryDry. Irradiate and make to carry out mass spectral analysis after sample ionization by nitrogen laser, gather the data within the scope of 1000-10000Da, obtainThe mass spectrogram that must be formed by the protein peak of different mass-to-charge ratioes. For each MALDI crystalline temperature, 400 laser of concurrent irradiation(the each irradiation 50 times in 8 different positions of each crystalline temperature), mean value represents a sample, thereby obtains the many of all samplesPeptide mapping. Application ClinProTools2.1 analysis software to Normal group, diabetes B without complication and complication group and 2The mass spectrogram of type diabetes and coronary heart disease group is analyzed, screening otherness polypeptide. Screening conditions: mass range 1000-10000Da, signal to noise ratio (S/N) is greater than 5, and Mass Drift is no more than 0.1%, and all mass spectrograms carry out normalizing according to total ion currentChange. Specifically please refer to Fig. 1, it illustrates the expression of ITIH4 in all urine samples, finds at normal person, diabetes B without concurrentIn the urine of disease and complication patient and diabetes B merging patients with coronary heart disease, all can detect the expression of ITIH4 albumen, andConfirm that ITIH4 albumen merges in patients with coronary heart disease urine and all significantly lowers at diabetes B. Peak area is as quantitative standard,The normality of Anderson-Darling check analysis data, t inspection is for analyzing the continuous data of normal distribution, WilcoxonInspection is for analyzing the continuous data of Non-Gaussian Distribution. P < 0.05 thinks to have significant difference. Please refer to Fig. 2 and Fig. 3, showGo out ITIH4 and merge flat in CHD group at Normal group, diabetes B without complication and complication group and diabetes BAll peak areas, red (arrow 1) represents Normal group, green (arrow 2) represents that diabetes B is without complication and complicationGroup, blue (arrow 3) represents that diabetes B merges CHD group.
Embodiment 4The qualification of differential peptides
By magnetic bead eluent rotation evaporate to dryness in sample cell, add 20ul mobile phase A (5% acetonitrile, 0.1% formic acid water-solubleLiquid) dissolve, be transferred in sample injection bottle. Sampling volume 18ul, first enters trapping column desalination with the speed of 15 μ l/min, when trappingBetween 3min. Then enter analytical column with the flow velocity of 400nl/min and carry out gradient elution, gradient is 5%B-50%B-80%B-80%B-50%B-5%B (Mobile phase B: 95% acetonitrile, 0.1% first aqueous acid, in table 2). Analysis time 60min, look35 DEG C of spectrum column temperature, all wash-out compositions enter spectrometer analysis. Nano ion gun, spray voltage 1.8kV; Mass spectrum pattern is numberAccording to relying on and dynamically getting rid of sweep limits 400-2000m/z; One-level scanning (MS) is used Obitrap, and resolution setting is100000; CID and secondary scanning are used LTQ; In MS spectrogram, choose the single isotope conduct of 10 ions that intensity is the strongestParent ion carries out MS/MS (single electric charge is got rid of, not as parent ion). Scanning of the mass spectrum time 60min. Application data analysis softwareBioworksBrowser3.3.1SP1 carries out SequestTMRetrieval. Searching database is InternationalProteinIndex (IPIhumanv3.45fastawith71983entries). Parent ion error is set as 100ppm, fragment ionError is made as 1Da, and enzyme butt formula is that non-enzyme is cut, and the variable methionine that is modified to is oxidized. Result for retrieval setting parameter is deltacn>=0.10, two electric charge Xcorr2.6, tricharged Xcorr3.1, the above Xcorr3.5 of tricharged. In database, retrieval obtains 2 typesThe ITIH4 that diabetes and coronary heart disease group, Normal group, diabetes B are expressed without complication and complication group difference(Inter-alpha-trypsininhibitorheavychainH4, albumen number: Q14624, m/z:2184.9) albumen,And compare without complication and complication group with diabetes B with Normal group, ITIH4 albumen merges hat at diabetes BIn cardiaopath's urine, be low expression. The mass spectrogram of ITIH4 albumen please refer to Fig. 4.
The program of table 2 analytical column gradient elution
Embodiment 5ITIH4 albumen closes without complication and complication patient, diabetes B at normal control, diabetes BAnd WesternBlot in patients with coronary heart disease urine checking
1. the extraction of urine albumen
Get normal control, diabetes B and merge patients with coronary heart disease (with real without complication and complication patient, diabetes BExecute example 1) fresh 2 hours in CCMS 50ml, be placed in plastic containers, the centrifugal 5min of 1500rpm, gets supernatant, it is heavy to abandonSlag; Supernatant and absolute ethyl alcohol are mixed by 1:3 volume ratio, after 4 DEG C of standing 30min; 12000rpm15min, goes supernatant to stay precipitation;After being dried, precipitation is dissolved in sample-loading buffer (urea 9M, CHAPS4%, DTT65mM, 0.2% ampholytes)-20 DEG C of mistakesFrozen cracking at night; After 4 DEG C of centrifugal 20min of 14000rpm, get supernatant, packing, with the 7080-ISE of Hitachi Biochemical Analyzer detection eggWhite concentration.
2.Westernblot
Electrophoresis tank is installed and is checked after air-tightness, prepare respectively the concentrated glue of 1.5mm20ml12% separation gel and 6ml5%, first fill withSeparation gel is filled with concentrated glue after solidifying again, and 30ug is urinated to albumen with 2Xloadingbuffer1:1 mixes rear loading, starts electricSwimming. Initial voltage is 60V, and after bromophenol blue electrophoresis is crossed concentrated glue, regulation voltage, to 120V, stops to arriving separation gel 2/3 placeElectrophoresis, carries out transferring film after cutting object glue. When transferring film, be followed successively by the order of filter paper, gel, pvdf membrane, filter paper to positive pole by negative poleCarry out electricity and turn, voltage 90V, transferring film 90min. After transferring film, take out pvdf membrane, be immersed in (PBST+5% skimmed milk power) in confining liquid,Shaking table shakes 2h, changes liquid once when 1h. According to the confining liquid dilution mouse-anti people ITIH4 polypeptide monoclonal antibody (U.S. for 1:2000Sigma-Aldrich company), hybridization bag is by film and antibody sealing, 4 DEG C of overnight incubation. It is each that PBST washes film 20min/, totally 3 times.According to 1:1000 ratio, dilute after sheep anti-mouse igg antibody (ancient cooking vessel state, Beijing) with confining liquid, add in hybridization bag sealing hybridization bagRear incubated at room 90min. It is each that PBST washes film 15min/, totally 3 times. In darkroom according to enhancement mode HRP-DAB substrate colour reagentBox (day root, Beijing) illustrates and develops the color. Concrete outcome please refer to Fig. 6. From figure, obviously find out, ITIH4 albumen is in diabetesThe expression merging in CHD group significantly reduces.
Embodiment 6The preparation of kit
1. the selection of coated antibody and enzyme labelled antibody working concentration
According to the BCA determination of protein concentration kit description operation of Pierce company, measure the concentration of antibody and antigen,Then adopt the chessboard assay method of standard, with coated buffer solution (8.4 grams of sodium acid carbonates, 3.56 grams of sodium carbonate be dissolved in 1 liter go fromIn sub-water, adjust pH to 9.05) by rare goat-anti people ITIH4 fusion polyclonal antibody (Sigma-Aldrich company of the U.S.)Release to concentration be 20.0ng/ml, 2.0ng/ml and 0.2ng/ml, coated on elisa plate respectively, each concentration comprise three verticalOK, 4 DEG C are spent the night, PBST washing 3 times. In the coated hole that is walked crosswise therein, add strong positive antigen liquid, another adds in walking crosswiseEnter weak positive antigen liquid, the third line adds negative control. Hatch 2 hours PBST washing 4 times for 37 DEG C. Add mouse-anti people ITIH4 manyPeptide monoclonal antibody (Sigma-Aldrich company of the U.S.), hatches 1 hour for 37 DEG C, PBST washing 4 times. Add two of HRP markAnti-, to hatch 30 minutes for 37 DEG C, PBST washing 4 times, adds OPD substrate, and room temperature lucifuge is placed 20 minutes, adds and ends liquid (2M sulphurAcid), reading on ELIASA. Select coated antibody optium concentration.
2. the preparation of kit
Goat-anti people ITIH4 fusion polyclonal antibody is diluted with coated buffer solution, joined 96 hole enzymesTarget, 4 DEG C of coated spending the night. Remove the liquid not being coated with, with PBST washing 3 times, add 200ul retardance liquid to stop non-specific knotCo-bit point, hatches 1 hour PBST wash-out 3 times for 37 DEG C. Putting into 4 DEG C saves backup. Kit packing adds mouse-anti people ITIH4 manyTwo of peptide monoclonal antibody, HRP mark resists, other common reagent Tween-20 and OPD.
3. the composition of kit
Merge the ELISA of patients with coronary heart disease urine endogenous α trypsin inhibitor heavy chain H4 for detection of diabetes BKit, comprises the ELISA Plate in 96 holes in box body and box, 12 bottles of reagent, and wherein ELISA Plate is to adopt 96 hole agent plate to doFor solid phase carrier, pre-coated goat-anti people ITIH4 polyclonal antibody in kit micropore, 12 bottles of reagent are respectively 6 bottles of ITIH4 marksAccurate product solution, 1 bottle of ELIAS secondary antibody, 1 bottle of antibody concentrated solution, 1 bottle of nitrite ion A, 1 bottle of nitrite ion B, 1 bottle of stop buffer, 1 bottle of concentrated washingWash liquid (specifically please refer to Fig. 5).
Further, this kit also comprises a cover plate film and puts the recessed bottle position of reagent, valve bag, wherein a boxBody is carton box; 96 hole agent plate are polystyrene ELISA Plate, are put in vacuum aluminium foil bag; Cover plate film is plastic hard membrane; StandardThe white PE plastic bottle of red cap for product solution, the white PE plastic bottle of black caps for ELIAS secondary antibody, antibody concentrated solution is with greenThe white PE plastic bottle of cap, the white PE plastic bottle of white cap for nitrite ion A liquid, nitrite ion B liquid is moulded with the black PE of red capMaterial bottle, the white PE plastic bottle of yellow cap for stop buffer, the translucent PE plastic bottle of hyaline cap for concentrated cleaning solution. Recessed bottle positionTotally 13, made by plastic foam.
ELISA Plate is made up of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, each removableDress enzyme mark reaction capillary strip has 8 reacting holes, the pre-coated goat-anti people of each reacting hole ITIH4 polyclonal antibody; Cover plate film sizeIn the same size with ELISA Plate cross section; 6 bottles of standard solutions, 1ml/ bottle; 1 bottle of ELIAS secondary antibody, 12ml; 1 bottle of antibody concentrated solution,1ml; 1 bottle of nitrite ion A liquid, 12ml; 1 bottle of nitrite ion B liquid, 12ml; 1 bottle of stop buffer, 15ml; 1 bottle of concentrated cleaning solution, 50ml.
Embodiment 7The detection of kit sensitivity
By ITIH4 recombinant protein (German OriGene company) with PBS be diluted to 200ng/ml, 100ng/ml, 50ng/ml,25ng/ml, 10ng/ml, 2ng/ml, 0.5ng/ml, 0.05ng/ml, 0.01ng/ml, 0ng/ml, every hole 100ul joinsState in the ELISA Plate being coated with, hatch 2 hours for 37 DEG C. With PBS (PBST) washing containing 0.1%Tween-20 3 times. Press 1:1000By the dilution of mouse-anti ITIH4 polypeptide monoclonal antibody, every hole adds 100ul, hatches 1 hour for 37 DEG C, and PBST washes 4 times. Add HRP markNote two is anti-, hatches 30 minutes for 37 DEG C, and PBST washes 4 times. Add 100ulOPD substrate room temperature to place 15 minutes, add the sulfuric acid of 2M,Reading under the ELIASA of 450nm wavelength. Detect the amount of minimum ITIH4, result demonstration, this reagent can detect 0.01ng/mlThe concentration of ITIH4 albumen, illustrates and has higher detection sensitivity. Show that through above-mentioned experiment kit of the present invention detects 2 typesIn diabetes and coronary heart disease Urine in Patients sample, the content of ITIH4, has very high sensitivity, the LDL of sample0.01ng/ml, the rate of recovery is 90% ± 13%. The required instrument of this kit is less, only need ELIASA, oscillator, centrifuge,Pipettors etc., required cost is low.
Embodiment 8The specificity of kit, the detection of stability
Get diabetes B and merge patients with coronary heart disease (Beijing Shijitan Hospital, CMU) clean stage casing to be measuredRandom urine 30-50ml, packs clean urinary catheter into, when women's preserving urine sample, should avoid menstrual period, should prevent that vaginal fluid from sneaking intoIn urine, under normal temperature, centrifugal 5 minutes of 1500rpm, gets supernatant to be checked.
The quantitative ITIH4 gene recombinant protein of purifying, as standard items, is pressed 1:3 dilution by antigen (the urine sample after centrifugal)After, join in the ELISA Plate being coated with above, hatch 2 hours for 37 DEG C, wash away unconjugated antigen with washing plate liquid PBST,Blot residual liquid. Add 37 DEG C of mouse-anti people ITIH4 polypeptide monoclonal antibodies to hatch 1 hour, PBST washes away unconjugated antibody,Blot residual liquid. Add two of HRP mark to resist, hatch 30 minutes for 37 DEG C, PBST washing 4 times, blots residual liquid. Add aobviousLook substrate, room temperature is placed 10 minutes, and visible each hole shows the yellow of different depth, adds 2M sulfuric acid stopped reaction, and ELIASA exists450nm wavelength readings, the content of ITIH4 albumen in calculating sample.
By the method, inventor has detected ITIH4 albumen in patient's random urine of 50 routine diabetes and coronary heart diseases and has containedAmount, more than rate of accuracy reached to 97%, has good specificity.
Get respectively the random urine that two diabetes Bs merge patients with coronary heart disease, utilize said method to carry out ELISA surveyFixed, measure once every day, repeats altogether 10 times, and by the formula coefficient of variation (CV)=S/X × 100%, (S is standard deviation, and X is averageValue) calculate batch between and variation within batch coefficient. In finally obtaining criticizing, be respectively 3.72% and 4.85%, explanation with interassay coefficient of variationGood stability.
Although the present invention discloses as above with preferred embodiment, so it is not in order to limit the present invention, any affiliated technologyThe technical staff in field, without departing from the spirit and scope of the present invention, when doing a little change and improvement, therefore the present inventionProtection domain when being as the criterion depending on the claim person of defining.
Claims (9)
1. the reagent that detects urine endogenous α trypsin inhibitor heavy chain H4 is for the preparation of detecting or diagnosed type 2 diabeticApplication in the preparation of merging coronary heart disease; Described preparation is that diabetes B merges patients with coronary heart disease urine endogenous α trypsaseInhibitor heavy chain H4 protein detection kit.
2. application according to claim 1, is characterized in that, described urine endogenous α trypsin inhibitor heavy chain H4Amino acid sequence as shown in SEQIDNO:1.
3. application according to claim 1, is characterized in that, described kit comprises box body and is arranged in box bodyELISA Plate and reagent.
4. application according to claim 3, is characterized in that, described ELISA Plate by outer frame support and be placed on it can12 enzyme mark reaction capillary strip compositions tearing open, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
5. application according to claim 4, is characterized in that, the human endogenous α tryptose of the pre-coated goat-anti of each reacting holeEnzyme inhibitor heavy chain H4 polyclonal antibody.
6. application according to claim 3, is characterized in that, described reagent is endogenous α trypsin inhibitor heavy chainH4 standard solution, ELIAS secondary antibody, antibody concentrated solution, nitrite ion A, nitrite ion B, stop buffer and concentrated cleaning solution.
7. application according to claim 1, is characterized in that, described kit also comprise cover plate film, place reagent underRecessed bottle position and valve bag.
8. application according to claim 7, is characterized in that, totally 13 of described recessed bottle positions, are made up of plastic foam.
9. application according to claim 3, is characterized in that, described ELISA Plate is made up of polystyrene.
Priority Applications (1)
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| CN101568833A (en) * | 2006-11-08 | 2009-10-28 | 布拉姆斯股份公司 | Diagnosis and risk assessment of pancreatic diabetes using MR-proADM |
| EP2333552A1 (en) * | 2008-09-19 | 2011-06-15 | Mcbi Inc. | Novel biomarkers for nonalcoholic fatty liver disease and methods for detecting nonalcoholic fatty liver disease using biomarker |
| CN103154272A (en) * | 2010-08-25 | 2013-06-12 | 香港中文大学 | Methods and kits for predicting the risk of diabetes associated complications using genetic markers and arrays |
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| KR101040321B1 (en) * | 2007-11-21 | 2011-06-10 | 고려대학교 산학협력단 | Bio-marker for diagnosing Cardiovascular disease |
| WO2009108073A1 (en) * | 2008-02-28 | 2009-09-03 | Auckland Uniservices Limited | Biomarkers for prediction of preeclampsia and/or cardiovascular disease |
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| CN101568833A (en) * | 2006-11-08 | 2009-10-28 | 布拉姆斯股份公司 | Diagnosis and risk assessment of pancreatic diabetes using MR-proADM |
| EP2333552A1 (en) * | 2008-09-19 | 2011-06-15 | Mcbi Inc. | Novel biomarkers for nonalcoholic fatty liver disease and methods for detecting nonalcoholic fatty liver disease using biomarker |
| CN103154272A (en) * | 2010-08-25 | 2013-06-12 | 香港中文大学 | Methods and kits for predicting the risk of diabetes associated complications using genetic markers and arrays |
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| Inter-α-trypsin inhibitor heavy chain 4 is a novel marker of acute ischemic stroke;Rajpal S. Kashyap et al.;《Clinica Chimica Acta》;20090122;第160-163页 * |
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| The Difference Quantity of Urinary Peptides between Two Groups of Type 2 Diabetic Patients with or without Coronary Artery Disease;Guangzhen Fu et al.;《International Journal of Endocrinology》;20151231;第1-8页 * |
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