CN104714021B - Urine factor albumen merges the application in coronary heart disease at diabetes B - Google Patents
Urine factor albumen 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
- 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
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Abstract
The invention provides the application of a kind of urine factor albumen (prothrombin), be specially the reagent that detects urine factor albumen 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, and factor albumen specifically expressing in diabetes B merging CHD group patient's urine raises, 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 factor albumen.
Description
Technical field
The present invention relates to the new purposes of urine factor albumen, be specifically related to urine factor albumen and merge 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, recent two decades comes, 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 is sharply increases trend. Due to the trend of Chinese population aging, within 60 years old, above diabetes B patient has approached 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.
Diabetes B is mainly to increase due to insulin secretion and (or) the chronic blood sugar that causes of effect defect, relates to carbohydrate, lipid and the protein metabolism disorder of general. For example, because being easily subject to the impact of many factors (excitement, fatigue, flu, fever and infection), blood sugar concentration produces fluctuation, thus the continuous Monitoring Blood Glucose variation of diabetes B needs of patients, to adjust accordingly drug dose. 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 global death rate, it is about 5.2% that diabetes B has accounted for, and die from merge cardiopathic accounted for wherein 68%. Therefore need the people at highest risk who diabetes B is merged to coronary heart disease to carry out early screening and early diagnosis, to carry out early treatment, improve patient's life quality.
Factor (Prothrombin) is one of clotting factor of the vitamin k-dependent that synthesized by liver, its encoding gene is positioned at chromosome No. 11, full length gene is 21kb, there are 14 extrons and 13 intrones, mRNA is 2kb, the peptide chain of synthetic 622 amino acid composition, and molecular weight is 72KD, isoelectric point is pH=4.2, and in blood plasma, content is 10～15mg/dl. Factor plays central role in clotting mechanism, and under the condition existing at the factor V activating and the phosphatide that provided by blood platelet or other cells, the factor X being activated activates factor and forms fibrin ferment, makes fibrinogen be transformed into fibrin. Fibrin ferment is a kind of proteolytic enzyme, and multiple clotting factor is had to hydrolysis, also has in addition: (1) induced platelet is assembled; (2) activate the XIII factor; (3) make plasminogen be transformed into fibrinolysin, thereby activate fibrinolytic system; (4) activate the inhibitors of fibrinolysis by thrombin activation; (5) activity factor V, VIII, XI, generates more fibrin ferment; (6) activated protein c system; (7) effect such as stimulating wound healing. In the time there is fat metabolic disorder in diabetes B patient simultaneously, lipid synthesis increases further stimulates endarterium smooth muscle cell proliferation, thereby cause basement membrane of blood vessel to thicken, finally cause microcirculation disorder, easily cause tissue ischemia, anoxic, make local a large amount of active oxygens, the arterial intima of producing, make diabetes B patient in pathologic blood coagulation and fibrinolytic state, in urine, factor protein level increases and can indicate that the danger of vascular complication occurs diabetic.
In order to understand PD, prevention and 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 blood preparation obtained wound, chylemia or haemolysis can affect the accuracy of blood sugar test, and frequent blood sampling meeting causes psychological burden to patient, especially the elderly and obese people. Therefore,, if can set up a kind of non-invasive inspection method of diabetes B patient being carried out to examination, monitoring, early screening, diagnosis, prevention and treatment to diabetes B and complication or complication are all extremely important.
Summary of the invention
The object of the present invention is to provide the application of a kind of urine factor albumen in the preparation for the preparation of diagnosis or detection diabetes B merging coronary heart disease.
Optimizing, described in the urine of prothrombin protein amino acid sequence as SEQIDNO: 1 ( MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAIEGRTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGE ) 。
Preferably, described preparation is that diabetes B merges patients with coronary heart disease urine factor protein detection kit. Described kit comprises box body and is arranged on ELISA Plate and the reagent in box body.
Preferably, described ELISA Plate is made up of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
Preferably, the anti-human factor protein polyclone antibody of the pre-coated rabbit of each reacting hole.
Preferably, described reagent is factor protein standard substance solution, antibody concentrated solution, ELIAS secondary antibody, 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 patients with coronary heart disease, without the diabetes B patient of complication and complication and the random urine specimen of normal control, get supernatant after centrifugal, utilize weak cation exchange magnetic beads for purifying and separated urine sample. by 1 μ 1 sample and 10 μ 1 matrix (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) after mixing, get 1 μ l point at Anchorchip (AutoflexMALDITOF, Bruker-Dalton) on target plate, after sample ionization, carry out mass spectral analysis, gather the data within the scope of 1000-10000Da, obtain the mass spectrum polypeptide figure being formed by the protein peak of different mass-to-charge ratioes. application ClinProTools2.1 analysis software merges CHD group, analyzes without the diabetes B group of complication and complication and all mass spectrograms of Normal group diabetes B, screening otherness polypeptide. then inventor carries out substance assistant laser desorpted ionized flight time mass spectrum (Matrix-assistedlaserdesorptionionizationtimeofflightmass spectrometry to having the otherness polypeptide of statistical significance, MALDI-TOF-MS) qualification, obtain diabetes B at InternationalProteinIndex (IPIhumanV3.45fastawith71983entries) database retrieval and merge CHD group and normal control and the factor albumen (prothrombin without the diabetes B group difference expression of complication and complication, albumen number: IPI00019568, m/z:3223.2) albumen, and compare with the diabetes B group without complication and complication with normal control, factor albumen is high expressed in the urine of diabetes B merging patients with coronary heart disease.
The present invention compares with the diabetes B group without complication and complication by studies confirm that with normal control, factor albumen specifically expressing in diabetes B merging CHD group patient's urine raises, and can be used for thereby propose to detect urine factor albumen the diagnosis and treatment 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 factor albumen.
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
The scatter diagram that to be factor albumen merge coronary heart disease at diabetes B to Fig. 1, express in without the diabetes B group of complication and complication and all samples of Normal group.
Fig. 2 and Fig. 3 are that factor albumen merges the average peak area in CHD group at normal control, diabetes B without complication and complication group, diabetes B. Red (arrow 1) represents Normal group, and green (arrow 2) represents that diabetes B is without complication and complication group, and blue (arrow 3) represents that diabetes B merges CHD group.
Fig. 4 is the mass spectrogram of factor 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 enzyme mark reaction capillary strip, the 2 outer frame supports that be ELISA Plate, and 3 is the hole that is coated with factor fusion polyclonal antibody.
Fig. 6 is that WesternBlot detection factor albumen merges the expression of results in patients with coronary heart disease urine at normal control, diabetes B without complication and complication group, diabetes B.
Detailed description of the invention
Embodiment 1The collection of urine specimen and processing
Collect diabetes B and merge patients with coronary heart disease 28 examples, the random CCMS liquid sample of diabetes B patient 30 examples (Beijing Shijitan Hospital, CMU's endocrine outpatient service) without complication and other complication, centrifugal (1500rpm in 2h, 5min), retain supernatant. After packing ,-80 DEG C of refrigerators are frozen. Normal group is 29 routine healthy volunteers (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 of multiple melting, get supernatant after centrifugal (3000rpm, 10min) for subsequent use. Balance weak cation magnetic bead (MB-WCX) under room temperature, and manually mix bead suspension. In sample cell, add 10ulMB-WCX and 10ul 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, fully mix 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 head should avoid touching magnetic bead, avoids siphoning away magnetic bead. In sample cell, add 100ul magnetic bead cleaning buffer solution, after fully mixing, sample cell is left standstill to 1 minute on magnetic frame, magnetic bead is adherent, with the fluid separation applications suspending, removes the liquid of suspension with sample loading gun. Repeat 3 times, discard suspension. In sample cell, add 5ul magnetic bead elution buffer, repeatedly inhale and make a call to more than 10 times, magnetic bead and elution buffer are mixed, avoid 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, the careful piping and druming of sample loading gun mixes.
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, get 1 μ l point at Anchorchip (AutoflexMALDITOF, Bruker-Dalton) on target plate, drying at room temperature. Irradiate and make to carry out mass spectral analysis after sample ionization by nitrogen laser, gather the data within the scope of 1000-10000Da, obtain the mass spectrogram being 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 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) 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 factor albumen in all urine samples, find all can detect the expression of factor albumen in normal person, the urine of diabetes B without complication and complication patient and diabetes B merging patients with coronary heart disease, and confirm that factor albumen merges in patients with coronary heart disease urine and all significantly raises at diabetes B. Peak area is as quantitative standard, the normality of Anderson-Darling check analysis data, and t inspection is for analyzing the continuous data of normal distribution, and Wilcoxon inspection 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, illustrate that factor albumen merges the average peak area in CHD group at Normal group, diabetes B without complication and complication group and diabetes B, red (arrow 1) represents Normal group, green (arrow 2) represents that diabetes B is without complication and complication group, and 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% first aqueous acid) to 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, 35 DEG C of chromatogram column temperatures, 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 limits 400-2000m/z; One-level scanning (MS) is used Obitrap, and resolution setting is 100000; CID and secondary scanning are used LTQ; The single isotope of choosing 10 ions that intensity is the strongest 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 BioworksBrowser3.3.1SPl carries out SequestTMRetrieval. Searching database is InternationalProteinIndex (IPIhumanV3.45fastawith71983entries). Parent ion error is set as 100ppm, and fragment ion error 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 diabetes B merging CHD group and Normal group and the diabetes B factor albumen (albumen number: IPI00019568 without complication and the expression of complication group difference, m/z:3223.2), and compare without complication and complication group with diabetes B with Normal group, factor albumen is high expressed in the urine of diabetes B merging patients with coronary heart disease. The mass spectrogram of factor albumen please refer to Fig. 4.
The program of table 2 analytical column gradient elution
Embodiment 5Factor albumen merges the WesternBlot checking in patients with coronary heart disease urine at normal control, diabetes B without complication and complication patient, diabetes B
1. the extraction of urine albumen
Get normal control, diabetes B and merge CCMS 50ml in fresh 2 hours of patients with coronary heart disease (with embodiment 1) without complication and complication patient, diabetes B, be placed in plastic containers, the centrifugal 5min of 1500rpm, gets supernatant, abandons sediment; 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 frozen cracking of spending the night; After 4 DEG C of centrifugal 20min of 14000rpm, get supernatant, packing, with the 7080-ISE of Hitachi Biochemical Analyzer detection protein concentration.
2.Westernb1ot
Electrophoresis tank is installed and is checked after air-tightness, prepare respectively the concentrated glue of 1.5mm20ml12% separation gel and 6ml5%, first fill with separation gel, after solidifying, fill with again concentrated glue, 30ug is urinated to albumen and 2Xloadingbuffer1: 1 mixes rear loading, start electrophoresis. Initial voltage is 60V, and after bromophenol blue electrophoresis is crossed concentrated glue, regulation voltage, to 120V, stops electrophoresis to arriving separation gel 2/3 place, carries out transferring film after cutting object glue. When transferring film, carry out electricity by negative pole to the anodal order that is followed successively by filter paper, gel, pvdf membrane, filter paper 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 1: 750 use confining liquid dilution former polypeptide monoclonal antibody of mouse-anti human thrombin (Bioworld company of the U.S.), hybridization bag was 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 incubated at room 90min after sealing hybridization bag. It is each that PBST washes film 15min/, totally 3 times. Illustrate and develop the color according to enhancement mode HRP-DAB substrate colour reagent box (day root, Beijing) in darkroom. Concrete outcome please refer to Fig. 6. From figure, obviously find out, the expression of factor albumen in diabetes and coronary heart disease group significantly raised.
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 are dissolved in 1 liter of deionized water, adjust pH to 9.05) anti-human rabbit factor fusion polyclonal antibody (Abcam company of the U.S.) is diluted to concentration is 20.0ng/ml, 2.0ng/ml and 0.2ng/m1, coated on elisa plate respectively, each concentration comprises three stringers, 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 weak positive antigen liquid in walking crosswise, and the third line adds negative control. Hatch 2 hours PBST washing 4 times for 37 DEG C. Add the former polypeptide monoclonal antibody of mouse-anti human thrombin (Sigma-A1drich company of the U.S.), hatch 1 hour for 37 DEG C, PBST washing 4 times. Add two of HRP mark to resist, 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 sulfuric acid), reading on ELIASA. Select coated antibody optium concentration.
2. the preparation of kit
Anti-human rabbit factor fusion polyclonal antibody is diluted with coated buffer solution, joined 96 hole ELISA Plates, 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 nonspecific binding site, hatch 1 hour PBST wash-out 3 times for 37 DEG C. Putting into 4 DEG C saves backup. Kit packing adds two of the former polypeptide monoclonal antibody of mouse-anti human thrombin, HRP mark to resist, other common reagent Tween-20 and OPD.
3. the composition of kit
Merge the ELISA kit of patients with coronary heart disease urine factor albumen for detection of diabetes B, comprise the ELISA Plate in 96 holes in box body and box, 12 bottles of reagent, wherein ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the anti-human factor polyclonal antibody of pre-coated rabbit in kit micropore, 12 bottles of reagent is respectively 6 bottles of factor 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 recessed bottle, a valve bag putting 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 white PE plastic bottle of red cap for standard solution, the white PE plastic bottle of black caps for ELIAS secondary antibody, the white PE plastic bottle of green cap for antibody concentrated solution, the white PE plastic bottle of white cap for nitrite ion A liquid, the black PE plastic bottle of red cap for nitrite ion B liquid, the white PE plastic bottle of yellow cap for stop buffer, the translucent PE plastic bottle of hyaline cap for concentrated cleaning solution. Totally 13 of recessed bottle positions, are made up of plastic foam.
ELISA Plate is made up of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, the anti-human factor protein polyclone antibody of the pre-coated rabbit of each reacting hole; Cover plate film size is in 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
Factor protein recombinant protein (German 0riGene company) is diluted to 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 10ng/ml, 2ng/ml, 0.5ng/ml, 0.05ng/m1,0.01ng/ml, 0ng/ml with PBS, every hole 100ul joins in the above-mentioned ELISA Plate being coated with, and hatches 2 hours for 37 DEG C. With PBS (PBST) washing containing 0.1%Tween-20 3 times. By 1: 1000, by the dilution of mouse-anti factor polypeptide monoclonal antibody, every hole added 100ul, hatches 1 hour for 37 DEG C, and PBST washes 4 times. Add HRP mark two anti-, hatch 30 minutes for 37 DEG C, 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 factor albumen, result demonstration, this reagent can detect the concentration of 0.01ng/m1 factor protein, illustrates and has higher detection sensitivity. Show that through above-mentioned experiment kit of the present invention detects the content that diabetes B merges factor albumen in patients with coronary heart disease urine specimen, there is very high sensitivity, the LDL 0.01ng/ml of sample, the rate of recovery is 92% ± 13%. The required instrument of this kit is less, only needs ELIASA, oscillator, centrifuge, pipettor etc., and 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, pack clean urinary catheter into, when women's preserving urine sample, should avoid menstrual period, should prevent that vaginal fluid from sneaking in urine, under normal temperature, centrifugal 5 minutes of 1500rpm, gets supernatant to be checked.
The quantitative factor GFP recombinant protein of purifying is as standard items, after antigen (the urine sample after centrifugal) was diluted by 1: 3, 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 the former polypeptide monoclonal antibodies of mouse-anti human thrombin to hatch 1 hour, PBST washes away unconjugated antibody, blots residual liquid. Add two of HRP mark to resist, hatch 30 minutes for 37 DEG C, PBST washing 4 times, blots residual liquid. Add chromogenic 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, in 450nm wavelength readings, calculates the content of factor albumen in sample.
By the method, inventor has detected factor protein content in patient's random urine of 50 routine diabetes and coronary heart diseases, 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 mensuration, measure once every day, repeat altogether 10 times, 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. In finally obtaining criticizing, be respectively 3.87% and 4.96% with interassay coefficient of variation, good stability is described.
Although the present invention discloses as above with preferred embodiment; so it is not in order 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.
Claims (9)
1. detect the reagent of urine factor albumen at the preparation for the preparation of detection or diagnosed type 2 diabetic merging coronary heart diseaseIn application; Described preparation is that diabetes B merges patients with coronary heart disease urine factor protein detection kit.
2. application according to claim 1, is characterized in that, the amino acid sequence of described urine factor albumen asShown 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 is by outer frame support and be placed on it12 removable enzyme mark reaction capillary strip compositions, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes.
5. application according to claim 4, is characterized in that, the anti-human factor albumen of the pre-coated rabbit of each reacting holePolyclonal antibody.
6. application according to claim 3, is characterized in that, described reagent be factor protein standard substance solution,Antibody concentrated solution, ELIAS secondary antibody, 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 comprises cover plate film, places reagentRecessed bottle position and valve bag.
8. application according to claim 7, is characterized in that, totally 13 of described recessed bottle positions, by plastic foam systemBecome.
9. application according to claim 3, is characterized in that, described ELISA Plate is made up of polystyrene.
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| CN118501454B (en) * | 2024-05-20 | 2025-02-25 | 香港大学深圳医院 | Application of biotinidase in the preparation of reagents for the detection of heart failure with preserved ejection fraction in diabetic patients |
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