CN104714020A - Applications of urine Inter-alpha-trypsin inhibitor heavy chain H4 in type 2 diabetes mellitus complicated by coronary heart disease - Google Patents
Applications of urine Inter-alpha-trypsin inhibitor heavy chain H4 in type 2 diabetes mellitus complicated by coronary heart disease Download PDFInfo
- Publication number
- CN104714020A CN104714020A CN201310683375.8A CN201310683375A CN104714020A CN 104714020 A CN104714020 A CN 104714020A CN 201310683375 A CN201310683375 A CN 201310683375A CN 104714020 A CN104714020 A CN 104714020A
- Authority
- CN
- China
- Prior art keywords
- itih4
- urine
- diabetes
- application according
- heart disease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N33/68—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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Optics & Photonics (AREA)
- Biophysics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides applications of a urine Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) protein, specifically applications of the urine ITIH4 in preparation of preparations for diagnosis or detection of type 2 diabetes mellitus complicated by coronary heart disease. According to the present invention, research results confirm that the specific expression of the ITIH4 of the patients in the type 2 diabetes mellitus complicated by coronary heart disease group is down-regulated compared with the normal control group and the simple type 2 diabetes mellitus group, such that the urine ITIH4 can be used for diagnosis and treatment of the type 2 diabetes mellitus complicated by coronary heart disease; and the advantages of non-invasive obtaining, mass re-sampling and convenient storage of the urine sample are provided, and the random urine sample is utilized to detect the ITIH4.
Description
Technical field
The present invention relates to the novelty teabag of urine endogenous α trypsin inhibitor heavy chain H4, be specifically related to the application of urine endogenous α trypsin inhibitor heavy chain H4 in the diagnosis and treatment of diabetes B merging coronary heart disease.
Background technology
Diabetes B (diabetes mellitus type2) is one of important public health problem in the whole world, recent two decades comes, along with the development of China's economic, the life style of people there occurs significant change, and the number of patients of diabetes B is in sharply increasing trend.Due to the trend of Chinese population aging, the diabetes B patient of more than 60 years old close to 50%, and with annual 0.1% speed increase.Diabetes B has become one of important diseases threatening Chinese residents life and health, is great public health problem urgently to be resolved hurrily.
The chronic blood sugar that diabetes B mainly causes due to insulin secretion and (or) effect defect increases, and relates to the carbohydrate of general, lipid and protein metabolism disorderly.Fluctuation is produced, so the continuous Monitoring Blood Glucose of diabetes B needs of patients changes, to adjust drug dose accordingly because blood sugar concentration is easily subject to the impact of many factors (such as excitement, fatigue, flu, fever and infection).Poor blood glucose control and fat, protein metabolism disorder can cause histoorgan and neural chronic progressive external pathology, hypofunction and the exhaustion such as eye, kidney, heart, blood vessel.According to statistics, in the mortality ratio in the whole world, diabetes B account for about 5.2%, and die from merge cardiopathic account for wherein 68%.Therefore need people at highest risk diabetes B being merged to coronary heart disease to carry out early screening and early diagnosis, to carry out early treatment, improve the life quality of patient.
ITI (Inter-alpha-trypsin inhibitor, endogenous α trypsin inhibitor) be the trypsin ihhibitor be present in serum human, by measuring liver mRNAs and sequence clone, find that ITI includes heavy chain and light chain polypeptide, respectively by different gene codes.Up to now, ITI comprises ITIH1, ITIH2, ITIH3, ITIH4 and ITIH5, and light chain bikunin.ITIH4 (Inter-alpha-trypsin inhibitor heavy chain H4, endogenous α trypsin inhibitor heavy chain H4) is a kind of plasma glycoprotein, mainly expresses in liver, in pancreatic tissue, also has expression.ITIH4 is very responsive to plasma kallikrein, is considered to the substrate of plasma kallikrein effect.ITIH4 has different biological functions, generally believes now the main and inflammation of ITIH4 and related to cancer.ITIH4 can be degraded to 35kDa (coo-end) and 85kDa (N-end) by kallikrein in blood, and the latter can also be degraded to the fragment of the fragment of 57kDa and the 28kDa of supposition.In body, the concrete biological function of ITIH4 is not clear.There is the fragment of ITIH4 in research display blood relevant with prostate cancer, carcinoma of urinary bladder, breast cancer, oophoroma and melanoma and cancer of pancreas etc., there is no the relevant report about ITIH4 and diabetes and complication or complication at present.
In order to understand progression of disease, timely complication, diabetes B needs of patients Monitoring Blood Glucose symptom management.In existing clinical examination, gather peripheral blood or the venous blood best sample as blood sugar monitoring, but the acquisition of blood preparation has wound, chylemia or haemolysis can affect the accuracy of blood sugar test, and frequent blood sampling can cause psychological burden to patient, especially the elderly and obese people.Therefore, if a kind of woundless testing method of diabetes B patient being carried out to examination, monitoring can be set up, the early screening of diabetes B and complication or complication, diagnosis, Prevention and Curation are all extremely important.
Summary of the invention
A kind of urine endogenous α trypsin inhibitor heavy chain H4 (Inter-alpha-trypsin inhibitor heavy chain H4, ITIH4) is the object of the present invention is to provide to merge the application in the preparation of coronary heart disease for the preparation of diagnosis or detection diabetes B.
Preferably, the amino acid sequence of described urine endogenous α trypsin inhibitor heavy chain H4 is as SEQ ID NO:Shown in 1, (, MKPPRPVRTC, SKVLVLLSLL, AIHQTTTAEK, NGIDIYSLTV, DSRVSSRFAH, TVVTSRVVNR, ANTVQEATFQ, MELPKKAFIT, NFSMIIDGMT, YPGIIKEKAE, AQAQYSAAVA, KGKSAGLVKA, TGRNMEQFQV, SVSVAPNAKI, TFELVYEELL, KRRLGVYELL, LKVRPQQLVK, HLQMDIHIFE, PQGISFLETE, STFMTNQLVD, ALTTWQNKTK, AHIRFKPTLS, QQQKSPEQQE, TVLDGNLIIR, YDVDRAISGG, SIQIENGYFV, HYFAPEGLTT, MPKNVVFVID, KSGSMSGRKI, QQTREALIKI, LDDLSPRDQF, NLIVFSTEAT, QWRPSLVPAS, AENVNKARSF, AAGIQALGGT, NINDAMLMAV, QLLDSSNQEE, RLPEGSVSLI, ILLTDGDPTV, GETNPRSIQN, NVREAVSGRY, SLFCLGFGFD, VSYAFLEKLA, LDNGGLARRI, HEDSDSALQL, QDFYQEVANP, LLTAVTFEYP, SNAVEEVTQN, NFRLLFKGSE, MVVAGKLQDR, GPDVLTATVS, GKLPTQNITF, QTESSVAEQE, AEFQSPKYIF, HNFMERLWAY, LTIQQLLEQT, VSASDADQQA, LRNQALNLSL, AYSFVTPLTS, MVVTKPDDQE, QSQVAEKPME, GESRNRNVHS, GSTFFKYYLQ, GAKIPKPEAS, FSPRRGWNRQ, AGAAGSRMNF, RPGVLSSRQL, GLPGPPDVPD, HAAYHPFRRL, AILPASAPPA, TSNPDPAVSR, VMNMKIEETT, MTTQTPAPIQ, APSAILPLPG, QSVERLCVDP, RHRQGPVNLL, SDPEQGVEVT, GQYEREKAGF, SWIEVTFKNP, LVWVHASPEH, VVVTRNRRSS, AYKWKETLFS, VMPGLKMTMD, KTGLLLLSDP, DKVTIGLLFW, DGRGEGLRLL, LRDTDRFSSH, VGGTLGQFYQ, EVLWGSPAAS, DDGRRTLRVQ, GNDHSATRER RLDYQEGPPG VEISCWSVEL).
Preferably, described preparation is that diabetes B merges patients with coronary heart disease urine endogenous α trypsin inhibitor heavy chain H4 detection kit.Described kit comprises box body and the ELISA Plate that is arranged in box body and reagent.
Preferably, described ELISA Plate is reacted capillary strips by outer frame support and removable 12 enzyme marks of being placed on it and is formed, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
Preferably, each reacting hole pre-coated goat-anti people ITIH4 polyclonal antibody.
Preferably, described reagent is 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 cover plate film, the recessed bottle position of placing reagent and valve bag.
Preferably, totally 13, described recessed bottle position, is made up of plastic foam.
Preferably, described ELISA Plate is made up of polystyrene.
First inventor have collected diabetes B and merges coronary heart disease, without complication and the diabetes B patient of complication and the random urine specimen of normal control, gets supernatant, utilize weak cation exchange magnetic beads for purifying and separated urine sample after centrifugal.By 1 μ l sample and the 10 μ l matrix (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) after mixing, get 1 μ l point at Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) on target plate, mass spectrophotometry is carried out after sample ionization, gather the data within the scope of 1000-10000Da, obtain the mass spectrum polypeptide figure be made up of the protein peak of different mass-to-charge ratio.Application ClinProTools2.1 analysis software merges CHD group to diabetes B, analyzes without complication and the diabetes B group of complication and all mass spectrograms of Normal group, screens otherness polypeptide.Then inventor carries out Matrix-assisted laser desorption ionization (Matrix-assisted laser desorption ionization time of flight mass spectrometry to the otherness polypeptide with statistical significance, MALDI-TOF-MS) identify, ITIH4 (the Inter-alpha-trypsin inhibitor heavy chain H4 that diabetes B merges CHD group and normal control and express without the diabetes B group difference of complication and complication is obtained at International Protein Index (IPI human v3.45fasta with71983entries) database retrieval, endogenous α trypsin inhibitor heavy chain H4, Q14624) albumen, and compare with the diabetes B group without complication and complication with normal control, ITIH4 albumen merges in the urine of patients with coronary heart disease in low expression at diabetes B.
The present invention confirms to compare with the diabetes B group without complication and complication with normal control by research, endogenous α trypsin inhibitor heavy chain H4 (ITIH4) merges specifically expressing in the urine of CHD group patient at diabetes B and lowers, thus proposition detects the diagnosis and treatment that urine endogenous α trypsin inhibitor heavy chain H4 can be used for diabetes B merging coronary heart disease.
The present invention play urine specimen obtain without wound, can repeated sampling on a large scale, preserve advantage easily, utilize random urine Samples 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.
Accompanying drawing explanation
Fig. 1 is Normal group, diabetes B merges the scatter diagram of expressing in all samples of CHD group without complication and complication group and diabetes B.
Fig. 2 and Fig. 3 average peak area that to be ITIH4 at Normal group, diabetes B merge without complication and complication group and diabetes B in CHD group.Red (arrow 1) represents Normal group, and green (arrow 2) represents diabetes B without complication and complication group, and blue (arrow 3) represents diabetes B and merge CHD group.
Fig. 4 is the mass spectrogram of ITIH4 albumen.
Fig. 5 is the schematic diagram of the ELISA Plate be arranged in the box body of kit of the present invention, and wherein 1 is enzyme mark reaction capillary strip, and 2 is the outer frame support of ELISA Plate, and 3 for being coated with the hole of ITIH4 fusion polyclonal antibody.
Fig. 6 is that Western Blot detects ITIH4 and merges coronary heart disease, without the expression of results in the diabetes B patient of complication and complication and normal control urine at diabetes B.
Embodiment
embodiment 1the collection of urine specimen and process
Collect diabetes B merge patients with coronary heart disease 28 example, without complication and other complication diabetes B patient 30 example (Beijing Shijitan Hospital, CMU's endocrine clinic) CCMS liquid sample at random, centrifugal (1500rpm in 2h, 5min), supernatant is retained.After packing ,-80 DEG C of refrigerators are frozen.Normal group is 29 routine normal persons (Beijing Shijitan Hospital, CMU's MECs).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 melt again, get supernatant for subsequent use after centrifugal (3000rpm, 10min).Equilibrate at room temperature weak cation magnetic bead (MB-WCX), and manually mix bead suspension.In sample hose, add 10ul MB-WCX and 10ul magnetic bead binding buffer liquid, sample loading gun blows and beats mixing up and down, avoids bubbling.In sample hose, add 5ul urine supernatant, fully after mixing, magnetic frame leaves standstill 1 minute, the fluid separation applications of magnetic bead and suspension.With the clear liquid that sample loading gun removing suspends, rifle head should be avoided touching magnetic bead, avoids siphoning away magnetic bead.In sample hose, add 100ul magnetic bead cleaning buffer solution, fully after mixing, sample hose is left standstill 1 minute on magnetic frame, magnetic bead is adherent, with the fluid separation applications suspended, and the liquid suspended with sample loading gun removing.Repeat 3 times, discard suspending liquid.In sample hose, add 5ul magnetic bead elution buffer, repeatedly inhale and make a call to more than 10 times, make magnetic bead and elution buffer mixing, avoid bubbling.Sample hose is positioned on magnetic frame, leaves standstill 2min, magnetic bead is fully separated with suspending liquid, supernatant (eluent) is moved into the new 0.5ml sample hose marked.Add 5ul stabilizing buffer, sample loading gun carefully blows and beats mixing.
embodiment 3the point target of urine specimen and polypeptide spectrum map generalization
After rectifying an instrument with standard items, 1 μ l eluent and 10 μ l matrix (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) are mixed, gets 1 μ l point at Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) on target plate, drying at room temperature.Carry out mass spectrophotometry after making sample ionization by nitrogen laser irradiation, gather the data within the scope of 1000-10000Da, obtain the mass spectrogram be made up of the protein peak of different mass-to-charge ratio.For each MALDI crystallization point, concurrent irradiation 400 laser (position that 8 of each crystallization point are different respectively irradiates 50 times), mean value represents a sample, thus obtains the polypeptide collection of illustrative plates of all samples.Application ClinProTools2.1 analysis software is analyzed without the mass spectrogram of complication and complication group and diabetes B merging CHD group Normal group, diabetes B, screening otherness polypeptide.Screening conditions: mass range 1000-10000Da, signal to noise ratio (S/N ratio) (S/N) is greater than 5, and Mass Drift is no more than 0.1%, and all mass spectrograms are normalized according to total ion current.Specifically please refer to Fig. 1, it illustrates the expression of ITIH4 in all Urine specimens, find to merge the expression that all can detect ITIH4 albumen in the urine of patients with coronary heart disease at normal person, diabetes B without complication and complication patient and diabetes B, and confirm that ITIH4 albumen merges in patients with coronary heart disease urine at diabetes B and all significantly lower.Peak area is as quantitative standard, and the normality of Anderson-Darling check analysis data, the continuous data of t inspection for analyzing normal distribution, Wilcoxon checks the continuous data for analyzing skewed distribution.P < 0.05 thinks to have significant difference.Please refer to Fig. 2 and Fig. 3, ITIH4 is shown at Normal group, diabetes B without the average peak area in complication and complication group and diabetes B merging CHD group, red (arrow 1) represents Normal group, green (arrow 2) represents diabetes B without complication and complication group, and blue (arrow 3) represents diabetes B and merge CHD group.
embodiment 4the qualification of differential peptides
Magnetic bead eluent in sample hose is rotated evaporate to dryness, adds 20ul mobile phase A (5% acetonitrile, 0.1% first aqueous acid) and dissolve, be transferred in sample injection bottle.Sampling volume 18ul, first enters trapping column desalination with the speed of 15 μ l/min, trapping time 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, chromatogram column temperature 35 DEG C, all wash-out compositions enter spectrometer analysis.Nano ion gun, spray voltage 1.8kV; Mass spectrum pattern is data dependence and dynamically gets rid of, sweep limit 400-2000m/z; One-level scanning (MS) uses Obitrap, and resolution setting is 100000; CID and secondary scanning use LTQ; The single isotope choosing 10 the strongest ions of intensity in MS spectrogram carries out MS/MS (single electric charge is got rid of, not as parent ion) as parent ion.Scanning of the mass spectrum time 60min.Application data analysis software BioworksBrowser3.3.1SP1 carries out Sequest
tMretrieval.Searching database is International Protein Index (IPI human v3.45fasta with71983entries).Parent ion error is set as 100ppm, and fragmention error is set to 1Da, and enzyme butt formula is that non-enzymatic is cut, variable be modified to methionine oxidized.Result for retrieval setting parameter is deltacn >=0.10, two electric charge Xcorr2.6, tricharged Xcorr3.1, the above Xcorr3.5 of tricharged.Retrieval obtains ITIH4 (the Inter-alpha-trypsin inhibitor heavy chain H4 that diabetes B merges CHD group, Normal group, diabetes B are expressed without complication and complication group difference in a database, albumen number: Q14624, m/z:2184.9) albumen, and compare without complication and complication group with diabetes B with Normal group, ITIH4 albumen merges in the urine of patients with coronary heart disease in low expression at diabetes B.The mass spectrogram of ITIH4 albumen please refer to Fig. 4.
The program of table 2 analytical column gradient elution
embodiment 5the Western Blot that ITIH4 albumen merges in patients with coronary heart disease urine without complication and complication patient, diabetes B at normal control, diabetes B verifies
1. the extraction of Urine proteins
Get CCMS 50ml in normal control, diabetes B merge patients with coronary heart disease (with embodiment 1) without complication and complication patient, diabetes B fresh 2 hours, be placed in plastic containers, the centrifugal 5min of 1500rpm, gets supernatant, abandons sediment; Supernatant and absolute ethyl alcohol are pressed 1:3 volume ratio mix, after 4 DEG C of standing 30min; 12000rpm15min, goes supernatant to stay precipitation; Be dissolved in sample-loading buffer (urea 9M, CHAPS4%, DTT65mM, 0.2% ampholyte) after precipitation is dried ,-20 DEG C of frozen overnight cracking; After 4 DEG C of centrifugal 20min of 14000rpm, get supernatant, packing, detect protein concentration with Hitachi 7080-ISE Biochemical Analyzer.
2.Western blot
After installing electrophoresis tank inspection impermeability, preparation 1.5mm20ml12% separation gel and 6ml5% concentrate glue respectively, first fill with separation gel, fill with concentrated glue again, loading after 30ug Urine proteins and 2Xloading buffer1:1 being mixed after to be solidified, beginning electrophoresis.Initial voltage is 60V, crosses after concentrated glue until bromophenol blue electrophoresis, and regulation voltage, to 120V, stopping electrophoresis to arriving separation gel 2/3 place, carrying out transferring film after cutting object glue.During transferring film by negative pole to positive pole be followed successively by filter paper, gel, pvdf membrane, filter paper order carry out electricity turn, voltage 90V, transferring film 90min.Take out pvdf membrane after transferring film, be immersed in (PBST+5% skimmed milk power) in confining liquid, shaking table shakes 2h, changes liquid once during 1h.According to 1:2000 confining liquid dilution mouse-anti people ITIH4 polypeptide monoclonal antibody (Sigma-Aldrich), film and antibody seal by hybridization bag, 4 DEG C of overnight incubation.It is each that PBST washes film 20min/, totally 3 times.According to 1:1000 ratio, after diluting sheep anti-mouse igg antibody (ancient cooking vessel state, Beijing) with confining liquid, add in hybridization bag, incubated at room 90min after closed hybridization bag.It is each that PBST washes film 15min/, totally 3 times.Illustrate in darkroom develop the color according to enhancement mode HRP-DAB substrate chromogenic reagent box (sky root, Beijing).Concrete outcome please refer to Fig. 6.Obviously find out from figure, the expression of ITIH4 albumen in diabetes and coronary heart disease group significantly reduces.
embodiment 6the preparation of kit
1. the selection of coated antibody and enzyme labelled antibody working concentration
Operate according to the BCA determination of protein concentration kit instructions of Pierce company, measure the concentration of antibody and antigen, then the chessboard assay method of standard is adopted, (8.4 grams of sodium bicarbonates, 3.56 grams of sodium carbonate are dissolved in 1 liter of deionized water to be buffered liquid with bag, adjust pH to 9.05) goat-anti people ITIH4 fusion polyclonal antibody (Sigma-Aldrich) is diluted to concentration is 20.0ng/ml, 2.0ng/ml and 0.2ng/ml, quilt is wrapped respectively on elisa plate, each concentration comprises three stringers, 4 DEG C are spent the night, and PBST washs 3 times.A bag walked crosswise is added strong positive antigen liquid in hole wherein, and another adds weak positive antigen liquid in walking crosswise, the third line adds negative control.Hatch 2 hours for 37 DEG C, PBST washs 4 times.Add mouse-anti people ITIH4 polypeptide monoclonal antibody (Sigma-Aldrich), hatch 1 hour for 37 DEG C, PBST washs 4 times.Add HRP mark two resist, and hatch 30 minutes for 37 DEG C, PBST washs 4 times, adds OPD substrate, and room temperature lucifuge places 20 minutes, adds and stops liquid (2M sulfuric acid), reading in microplate reader.Select coated antibody optium concentration.
2. the preparation of kit
Goat-anti people ITIH4 fusion polyclonal antibody bag is buffered liquid dilute, is joined 96 hole ELISA Plate, 4 DEG C of bags are spent the night.Remove the liquid not wrapping quilt, wash 3 times with PBST, add 200ul and block liquid prevention nonspecific binding site, hatch 1 hour for 37 DEG C, PBST wash-out 3 times.Put into 4 DEG C to save backup.Kit packing adds mouse-anti people ITIH4 polypeptide monoclonal antibody, HRP marks two resist, other common reagent Tween-20 and OPD.
3. the composition of kit
The ELISA kit of patients with coronary heart disease urine endogenous α trypsin inhibitor heavy chain H4 is merged for detecting diabetes B, comprise the ELISA Plate in one piece of 96 hole in box body and box, 12 bottles of reagent, wherein ELISA Plate is that employing 96 hole agent plate is as solid phase carrier, pre-coated goat-anti people ITIH4 polyclonal antibody in kit micropore, 12 bottles of reagent are respectively 6 bottles of ITIH4 standard solutions, 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 cleaning solution (specifically please refer to Fig. 5).
Further, this kit also comprises a cover plate film and puts recessed bottle position, a valve bag of reagent, and wherein box body 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; The standard solution white PE plastic bottle of red cap, the white PE plastic bottle of ELIAS secondary antibody black caps, the antibody concentrated solution white PE plastic bottle of green cap, the nitrite ion A liquid white PE plastic bottle of white cap, the nitrite ion B liquid black PE plastic bottle of red cap, the stop buffer white PE plastic bottle of yellow cap, the translucent PE plastic bottle of concentrated cleaning solution hyaline cap.Totally 13, recessed bottle position, is made up of plastic foam.
ELISA Plate is reacted capillary strips by outer frame support and removable 12 enzyme marks of being placed on it and is formed, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, each reacting hole pre-coated goat-anti people ITIH4 polyclonal antibody; Cover plate film size and ELISA Plate square section in the same size; Standard solution 6 bottles, 1ml/ bottle; ELIAS secondary antibody 1 bottle, 12ml; Antibody concentrated solution 1 bottle, 1ml; Nitrite ion A liquid 1 bottle, 12ml; Nitrite ion B liquid 1 bottle, 12ml; Stop buffer 1 bottle, 15ml; Concentrated cleaning solution 1 bottle, 50ml.
embodiment 7the detection of kit sensitivity
ITIH4 recombinant protein (German OriGene company) is 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 with PBS, every hole 100ul joins above-mentioned bag by good ELISA Plate, hatches 2 hours for 37 DEG C.3 times are washed with the PBS (PBST) containing 0.1%Tween-20.Mouse-anti ITIH4 polypeptide monoclonal antibody diluted by 1:1000, every hole adds 100ul, and hatch 1 hour for 37 DEG C, PBST washes 4 times.Add HRP mark two to resist, hatch 30 minutes for 37 DEG C, PBST washes 4 times.Add 100ul OPD substrate room temperature and place 15 minutes, add the sulfuric acid of 2M, reading under the microplate reader of 450nm wavelength.Detect the amount of minimum ITIH4, result shows, and this reagent can detect the concentration of 0.01ng/ml ITIH4 albumen, illustrates to have higher detection sensitivity.Show that kit of the present invention detects the content of ITIH4 in diabetes B merging patients with coronary heart disease urine specimen through above-mentioned experiment, have very high sensitivity, the lowest detectable limit 0.01ng/ml of sample, the recovery is 90% ± 13%.Needed for this kit, instrument is less, and only need microplate reader, oscillator, hydro-extractor, pipettor 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 random urine 30-50ml to be measured, load clean urinary catheter, menstrual period should be avoided during women's preserving urine sample, vaginal fluid should be prevented to be mixed in urine, under normal temperature, centrifugal 5 minutes of 1500rpm, gets supernatant to be checked.
The quantitative ITIH4 gene recombinant protein of purifying is as standard items, by antigen (Urine specimens after centrifugal) by after 1:3 dilution, wrap by good ELISA Plate before joining, hatched 2 hours for 37 DEG C, washing away unconjugated antigen with washing plate liquid PBST, blotting residual liquid.Add mouse-anti people ITIH4 polypeptide monoclonal antibody 37 DEG C and hatch 1 hour, PBST washes away unconjugated antibody, blots residual liquid.Add HRP mark two resist, and hatch 30 minutes for 37 DEG C, PBST washs 4 times, blots residual liquid.Add chromogenic substrate, room temperature places 10 minutes, and the yellow of visible each hole display different depth, add 2M sulfuric acid stopped reaction, microplate reader, in 450nm wavelength readings, calculates the content of ITIH4 albumen in sample.
By the method, inventor have detected ITIH4 protein content in patient's random urine of 50 routine diabetes and coronary heart diseases, and rate of accuracy reached, to more than 97%, has good specificity.
Get the random urine that two diabetes Bs merge patients with coronary heart disease respectively, said method is utilized to carry out ELISA mensuration, every day measures once, repeat 10 times altogether, by the formula coefficient of variation (CV)=S/X × 100% (S is standard deviation, and X is mean value) calculate batch between and variation within batch coefficient.Be respectively 3.72% and 4.85% with interassay coefficient of variation in finally obtaining batch, good stability is described.
Although the present invention discloses as above with preferred embodiment; so itself and be not used to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when doing a little change and improvement, therefore protection scope of the present invention is when being as the criterion depending on the claim person of defining.
Sequence table
Claims (10)
1. urine endogenous α trypsin inhibitor heavy chain H4 is merging the application in the preparation of coronary heart disease for the preparation of diagnosis or detection diabetes B.
2. application according to claim 1, is characterized in that, the amino acid sequence of described urine endogenous α trypsin inhibitor heavy chain H4 is as shown in SEQ ID NO:1.
3. application according to claim 1, is characterized in that, described preparation is that diabetes B merges patients with coronary heart disease urine endogenous α trypsin inhibitor heavy chain H4 detection kit.
4. application according to claim 3, is characterized in that, described kit comprises box body and the ELISA Plate that is arranged in box body and reagent.
5. application according to claim 4, is characterized in that, described ELISA Plate is reacted capillary strips by outer frame support and removable 12 enzyme marks of being placed on it and formed, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
6. application according to claim 5, is characterized in that, each reacting hole pre-coated goat-anti people ITIH4 polyclonal antibody.
7. application according to claim 4, is characterized in that, described reagent is ITIH4 standard solution, ELIAS secondary antibody, antibody concentrated solution, nitrite ion A, nitrite ion B, stop buffer and concentrated cleaning solution.
8. application according to claim 3, is characterized in that, described kit also comprises cover plate film, the recessed bottle position of placing reagent and valve bag.
9. application according to claim 8, is characterized in that, totally 13, described recessed bottle position, is made up of plastic foam.
10. application according to claim 3, is characterized in that, described ELISA Plate is made up of polystyrene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310683375.8A CN104714020B (en) | 2013-12-12 | 2013-12-12 | Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310683375.8A CN104714020B (en) | 2013-12-12 | 2013-12-12 | Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104714020A true CN104714020A (en) | 2015-06-17 |
| CN104714020B CN104714020B (en) | 2016-05-25 |
Family
ID=53413528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310683375.8A Active CN104714020B (en) | 2013-12-12 | 2013-12-12 | Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104714020B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112924675A (en) * | 2019-12-05 | 2021-06-08 | 张曼 | Application of urine pancreatic triglyceride lipase protein and polypeptide fragment thereof in normal pregnancy |
| CN113092773A (en) * | 2019-12-23 | 2021-07-09 | 首都医科大学附属北京世纪坛医院 | Application of urine alpha-trypsin inhibitor heavy chain H4 and polypeptide fragment thereof in allergic diseases |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005024429A1 (en) * | 2003-09-05 | 2005-03-17 | Minomic Pty Ltd | Assay for diabetes |
| KR20090052483A (en) * | 2007-11-21 | 2009-05-26 | 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 |
| 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 |
-
2013
- 2013-12-12 CN CN201310683375.8A patent/CN104714020B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005024429A1 (en) * | 2003-09-05 | 2005-03-17 | Minomic Pty Ltd | Assay for diabetes |
| 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 |
| KR20090052483A (en) * | 2007-11-21 | 2009-05-26 | 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 |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| GUANGZHEN FU ET AL.: "The Difference Quantity of Urinary Peptides between Two Groups of Type 2 Diabetic Patients with or without Coronary Artery Disease", 《INTERNATIONAL JOURNAL OF ENDOCRINOLOGY》 * |
| HAO XU ET AL.: "ITIH4: A New Potential Biomarker of "Toxin Syndrome" in Coronary Heart Disease Patient Identified with Proteomic Method", 《EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
| RAJPAL S. KASHYAP ET AL.: "Inter-α-trypsin inhibitor heavy chain 4 is a novel marker of acute ischemic stroke", 《CLINICA CHIMICA ACTA》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112924675A (en) * | 2019-12-05 | 2021-06-08 | 张曼 | Application of urine pancreatic triglyceride lipase protein and polypeptide fragment thereof in normal pregnancy |
| CN112924675B (en) * | 2019-12-05 | 2023-01-13 | 张曼 | Application of urine pancreatic triglyceride lipase protein and polypeptide fragment thereof in normal pregnancy |
| CN113092773A (en) * | 2019-12-23 | 2021-07-09 | 首都医科大学附属北京世纪坛医院 | Application of urine alpha-trypsin inhibitor heavy chain H4 and polypeptide fragment thereof in allergic diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104714020B (en) | 2016-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104422772B (en) | Time-resolved immunochromatography test strip for quantitatively detecting pepsinogen I and preparation method thereof | |
| CN103575913B (en) | The urinalysis test card of microdose urine protein/UCr | |
| CN102207507A (en) | Semi-quantitative detecting test paper of cardiac troponin and preparation method thereof | |
| CN203405464U (en) | Time resolution immunochromatography test strip for quantitatively detecting stomach protein zymogen II and test card using same | |
| CN203405465U (en) | Test strip for detecting propepsin II and reagent card with same | |
| CN111983242A (en) | Electrochemical luminescence immunoassay method and kit | |
| CN104345149A (en) | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof | |
| CN110286234A (en) | The protein markers of gestational diabetes mellitus and its purposes in early diagnosis in urine | |
| CN110487873A (en) | A kind of preparation method of the micro-fluidic optical electro-chemistry sensor for the detection of heart failure marker B-typeNatriuretic Peptide | |
| US20160245804A1 (en) | Methods, Systems and Kits of Fecal NGal Rapid Immunochromatographic Test | |
| CN104569429B (en) | Homogeneous immunometric fluorescent compound set for quickly and quantificationally detecting brain natriuretic peptide (BNP) and preparation method of homogeneous immunometric fluorescent compound set | |
| CN206038696U (en) | Ration calprotectin detects immunochromatographic test strip | |
| CN104714020A (en) | Applications of urine Inter-alpha-trypsin inhibitor heavy chain H4 in type 2 diabetes mellitus complicated by coronary heart disease | |
| US20150203892A1 (en) | Method for determining breast cancer | |
| EP3640644B1 (en) | Target marker gp73 for detecting steatohepatitis and detection application method | |
| CN104714021B (en) | Urine factor albumen merges the application in coronary heart disease at diabetes B | |
| CN105445409B (en) | A kind of method that utilization isotope dilution mass spectrometry measures glycosylated hemoglobin | |
| JP6336957B2 (en) | Breast cancer determination method | |
| CN109633163B (en) | procalcitonin/C reactive protein two-in-one detection kit | |
| CN104714022A (en) | Applications of urine fibrinogen alpha chain in type 2 diabetes mellitus complicated by coronary heart disease | |
| CN104297487A (en) | Application of urine nucleotide triad binding protein 1 | |
| CN108020663A (en) | The application of urine gelsolin and its polypeptide fragment in adenocarcinoma of lung | |
| CN104280552A (en) | Application of urine apolipoprotein J precursor protein | |
| CN108020667A (en) | The application of urine immunoglobulin kappa chain C area's albumen and its polypeptide fragment in adenocarcinoma of lung | |
| CN104297486A (en) | Application of urine glutamoyl-prolyl tRNA synthetic enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |