CN104280552A - Application of urine apolipoprotein J precursor protein - Google Patents
Application of urine apolipoprotein J precursor protein Download PDFInfo
- Publication number
- CN104280552A CN104280552A CN201310294304.9A CN201310294304A CN104280552A CN 104280552 A CN104280552 A CN 104280552A CN 201310294304 A CN201310294304 A CN 201310294304A CN 104280552 A CN104280552 A CN 104280552A
- Authority
- CN
- China
- Prior art keywords
- urine
- application according
- diabetes
- precursor protein
- clu
- 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.)
- Pending
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Optics & Photonics (AREA)
- Biophysics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides an application of a urine apolipoprotein J precursor protein, in particular to an application of the urine apolipoprotein J precursor protein in preparing preparations for diagnosing or detecting type-2 diabetes. Compared with normal control, the specific expression of the urine apolipoprotein J precursor protein in the urine of a type-2 diabetes patient is proved to be lowered in the research, so that the urine apolipoprotein J precursor protein can be used for diagnosing the type-2 diabetes. The advantages that a urine specimen can be noninvasively acquired, the urine can be repeatedly sampled in a large scale, and the urine specimen is convenient to store are exerted, and the apolipoprotein J precursor protein is detected by utilizing a random urine specimen.
Description
Technical field
The present invention relates to the novelty teabag of urine SP-40 precursor protein, be specifically related to the application of urine SP-40 precursor protein in diabetes B diagnosis and treatment.
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.Therefore diabetes B being carried out to examination and the early diagnosis of people at highest risk, to carry out early treatment, improve the life quality of patient, is the important topic of diabetes B research.
SP-40 precursor (clusterin precursor protein, CLU) is the glycoprotein that a kind of disulfide bond of high conservative connects, and wide expression is in the tissue relevant with pathogenesis to multiple physiology.Secreting type CLU(sCLU) there is cytoprotection, kytoplasm/cell caryogram shows apoptosis characteristic.Research find, the effect of sCLU is similar to the molecular chaperones of small heat shock protein, Cell protection from ROS(Reactive Oxygen Species, active oxygen) murder by poisoning.Under normal circumstances, when cell is subject to a large amount of ROS stimulation, CLU gene expression is raised and is played cytoprotection.Find in addition, leptin resistance is a key factor of insulin resistance.The compound of CLU and leptin can maintain Leptin signaling and transduce, and make leptin produce biological effect, and the shortage of CLU makes the leptin content of the abiology effect of free form in circulation increase, and then causes hyperinsulinemia and insulin resistance.In addition, have increasing evidence to show, CLU is a kind of growth factor-like molecule, take part in the process that pancreatic stem cells is regenerated as pancreatic beta cell.Therefore, the impaired oxidation resistance that may cause of CLU function declines and Pancreatic beta cells function obstacle.Therefore, the reduction of CLU concentration and insulin resistance and/or islet beta cell function obstacle closely related.
In order to understand progression of disease, timely complication, diabetes B needs of patients Monitoring Blood Glucose controls situation.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, diagnosis, Prevention and Curation are all extremely important.
Summary of the invention
The object of the present invention is to provide the application of a kind of urine SP-40 precursor protein (clusterin precursor protein, CLU) in the preparation for the preparation of diagnosis or detection diabetes B.
Preferably, the amino acid sequence of described urine SP-40 precursor protein is as shown in SEQ ID NO:1.
Preferably, described preparation is diabetes B Urine in Patients SP-40 precursor protein 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, the anti-human CLU polyclonal antibody of the pre-coated rabbit of each reacting hole.
Preferably, described reagent is LU 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 without complication and the diabetes B patient of other 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.The all mass spectrograms of application ClinProTools2.1 analysis software to diabetes B group and Normal group are analyzed, screening 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, at International Protein Index(IPI human v3.45fasta with71983entries) database retrieval obtains the CLU(clusterin precursor protein of differential expression between diabetes B group and Normal group, albumen number: IPI00291262.3, m/z:2123.5) albumen, and compared with normal control, CLU albumen is low expression in the urine of diabetes B patient.
The present invention is confirmed compared with Normal group by research, and SP-40 precursor protein (CLU) specifically expressing in the urine of diabetes B group patient is lowered, thus proposition detects the diagnosis and treatment that urine SP-40 precursor protein can be used for diabetes B.
The present invention play urine specimen obtain without wound, can repeated sampling on a large scale, preserve advantage easily, utilize random urine Samples detection SP-40 precursor protein.
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 the urine polypeptide spectrum of diabetes B group and all samples of Normal group.Red (red (arrow 1)) represents diabetes B group, and green (green (arrow 2)) represents Normal group, and x-axis represents m/z, and y-axis represents relative intensity.
Fig. 2 and Fig. 3 is the expression of CLU in all urine specimens.Red (arrow 1) represents diabetes B group, and green (arrow 2) represents Normal group.
Fig. 4 and Fig. 5 is the average peak area of CLU in normal control and diabetes B group.Red (arrow 1) represents diabetes B group, and green (arrow 2) represents Normal group.
Fig. 6 is the mass spectrogram of CLU albumen.
Fig. 7 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 CLU fusion polyclonal antibody.
Fig. 8 is that Western Blot detects the expression of results of CLU in normal control and Urine of Patients with Diabetes Mellitus.
Embodiment
embodiment 1the collection of urine specimen and process
Collect diabetes B patient 28 example (Beijing Shijitan Hospital, CMU's endocrine clinic) the CCMS liquid sample at random without the medical history more than 5 years of complication and other complication, in 2h centrifugal (1500rpm, 5min), reservation supernatant.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 1:2 patients with type Ⅰ DM group and Normal 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 10 μ l 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 5 μ l urine supernatants, 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 100 μ l magnetic bead cleaning buffer solutions, 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 5 μ l magnetic bead elution buffers, 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 5 μ l stabilizing buffers, 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, get 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 obtain the polypeptide collection of illustrative plates of all samples, specifically please refer to Fig. 1, red (arrow 1) represents diabetes B group, and green (arrow 2) represents Normal group, x-axis represents m/z, and y-axis represents relative intensity.The mass spectrogram of application ClinProTools2.1 analysis software to diabetes B group and Normal group is analyzed, 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. 2 and Fig. 3, it illustrates the expression of CLU in all Urine specimens, and red (arrow 1) represents diabetes B group, and green (arrow 2) represents Normal group; Find the expression that all can detect CLU albumen in normal person and diabetes B Urine in Patients, and confirm that CLU albumen is all significantly lowered in diabetes B Urine in Patients.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. 4 and Fig. 5, the average peak area of CLU in normal control and diabetes B group is shown, red (arrow 1) represents diabetes B group, and green (arrow 2) represents Normal group.
embodiment 4the qualification of differential peptides
Magnetic bead eluent in sample hose is rotated evaporate to dryness, adds 20 μ l mobile phase A (5% acetonitrile, 0.1% first aqueous acid) and dissolve, be transferred in sample injection bottle.Sampling volume 18 μ l, 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, and 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 the mono-electric charge of MS/MS(as parent ion and gets rid of, not 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.At International Protein Index(IPI human v3.45fasta with71983entries) database retrieval obtains the CLU(clusterin precursor protein of differential expression between diabetes B group and Normal group, albumen number: IPI00291262.3, m/z:2123.5) albumen, and compared with normal control, CLU albumen is low expression in the urine of diabetes B patient.The mass spectrogram of CLU albumen please refer to Fig. 6.
The program of table 2 analytical column gradient elution
embodiment 5the Western Blot of CLU albumen in normal control and Urine of Patients with Diabetes Mellitus verifies
1. the extraction of Urine proteins
CCMS 50ml in fresh 2 hours that get normal control and diabetic's (with embodiment 1), 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 CLU 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. 8.Obviously find out from figure, the expression of CLU albumen in diabetes 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) anti-human for rabbit CLU fusion polyclonal antibody (Sigma-Aldrich) is diluted to concentration is 20.0ug/ml, 2.0ug/ml and 0.2ug/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 (the CLU-GST fusion of 10ug/ml) in hole wherein, another adds weak positive antigen liquid (the CLU-GST fusion of 0.001ug/ml) in walking crosswise, the third line adds negative control.Hatch 2 hours for 37 DEG C, PBST washs 4 times.Add mouse-anti people CLU 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
Anti-human for rabbit CLU 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 CLU polypeptide monoclonal antibody, HRP marks two resist, other common reagent Tween-20 and OPD.
3. the composition of kit
For detecting the ELISA kit of diabetes B Urine in Patients SP-40 precursor protein, 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, the anti-human CLU polyclonal antibody of pre-coated rabbit in kit micropore, 12 bottles of reagent are respectively 6 bottles of CLU 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. 7).
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, the anti-human CLU polyclonal antibody of the pre-coated rabbit of each reacting hole; 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
CLU recombinant protein (German OriGene company) is diluted to 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 10ug/ml, 2ug/ml, 0.5ug/ml, 0.05ug/ml, 0.01ug/ml, 0ug/ml with PBS, every hole 100 μ l 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 CLU polypeptide monoclonal antibody diluted by 1: 1000, every hole adds 100 μ l, 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 100 μ l OPD substrate room temperatures and place 15 minutes, add the sulfuric acid of 2M, reading under the microplate reader of 450nm wavelength.Detect the amount of minimum CLU, result shows, and this reagent can detect the concentration of 0.01ug/ml CLU albumen, illustrates to have higher detection sensitivity.Show that kit of the present invention detects the content of CLU in diabetes B Urine in Patients sample through above-mentioned experiment, have very high sensitivity, the lowest detectable limit 200ug/ml of sample, the recovery is 90% ± 15%.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 patient (Beijing Shijitan Hospital, CMU) clean stage casing random urine 30-50ml to be measured, load clean urinary catheter, should menstrual period 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 CLU 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 CLU 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 CLU albumen in sample.
By the method, inventor have detected CLU protein content in 50 routine glycemic control diabetic's random urine not up to standard, and rate of accuracy reached, to more than 97%, has good specificity.
Get the random urine of two diabetes B patients respectively, utilize said method to carry out ELISA mensuration, every day measures once, repeats 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 3.63% and 4.75% 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.
Claims (10)
1. the application of urine SP-40 precursor protein in the preparation 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 SP-40 precursor protein is as shown in SEQ ID NO:1.
3. application according to claim 1, is characterized in that, described preparation is diabetes B Urine in Patients SP-40 precursor protein 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, the anti-human CLU polyclonal antibody of the pre-coated rabbit of each reacting hole.
7. application according to claim 4, is characterized in that, described reagent is CLU 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 |
|---|---|---|---|
| CN201310294304.9A CN104280552A (en) | 2013-07-12 | 2013-07-12 | Application of urine apolipoprotein J precursor protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310294304.9A CN104280552A (en) | 2013-07-12 | 2013-07-12 | Application of urine apolipoprotein J precursor protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104280552A true CN104280552A (en) | 2015-01-14 |
Family
ID=52255638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310294304.9A Pending CN104280552A (en) | 2013-07-12 | 2013-07-12 | Application of urine apolipoprotein J precursor protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104280552A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107957498A (en) * | 2016-10-18 | 2018-04-24 | 厦门德馨尚品医疗科技有限公司 | A kind of kit containing Apolipoprotein J and its application in clinical diagnosis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GR20020100196A (en) * | 2002-04-23 | 2003-12-22 | Ευσταθιος Γκονος | Method and kit for the quantitave measurement of serum levels of clusterin/apolipoprotein j |
| CN1791422A (en) * | 2003-03-28 | 2006-06-21 | 应用研究系统Ars股份公司 | Use of clusterin for the treatment and/or prevention of peripheral neurological diseases |
| CN101672850A (en) * | 2009-01-05 | 2010-03-17 | 中山大学 | Kit for distinguishing and diagnosing hepatocirrhosis and early liver cancer and application of serum clusterin in preparing same |
| CN202939176U (en) * | 2012-11-28 | 2013-05-15 | 昆明理工大学 | ELISA (Enzyme Linked Immunosorbent Assay) test kit of walnut protein |
-
2013
- 2013-07-12 CN CN201310294304.9A patent/CN104280552A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GR20020100196A (en) * | 2002-04-23 | 2003-12-22 | Ευσταθιος Γκονος | Method and kit for the quantitave measurement of serum levels of clusterin/apolipoprotein j |
| CN1791422A (en) * | 2003-03-28 | 2006-06-21 | 应用研究系统Ars股份公司 | Use of clusterin for the treatment and/or prevention of peripheral neurological diseases |
| CN101672850A (en) * | 2009-01-05 | 2010-03-17 | 中山大学 | Kit for distinguishing and diagnosing hepatocirrhosis and early liver cancer and application of serum clusterin in preparing same |
| CN202939176U (en) * | 2012-11-28 | 2013-05-15 | 昆明理工大学 | ELISA (Enzyme Linked Immunosorbent Assay) test kit of walnut protein |
Non-Patent Citations (2)
| Title |
|---|
| LINA CHU ET AL: "Identification of urinary biomarkers for type 2 diabetes using bead-based proteomic approach", 《DIABETES RESEARCH AND CLINICAL PRACTICE》 * |
| LINA CHU ET AL: "Identification of urinary biomarkers for type 2 diabetes using bead-based proteomic approach", 《DIABETES RESEARCH AND CLINICAL PRACTICE》, vol. 101, no. 2, 13 June 2013 (2013-06-13) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107957498A (en) * | 2016-10-18 | 2018-04-24 | 厦门德馨尚品医疗科技有限公司 | A kind of kit containing Apolipoprotein J and its application in clinical diagnosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2863798B1 (en) | Quick test device and method | |
| 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 | |
| CN104422772A (en) | Time resolution immunochromatographic test strip for quantitatively detecting pepsinogen I as well as preparation method of time resolution immunochromatographic test strip | |
| CN111781350A (en) | Colloidal gold immunochromatographic test strip for detecting novel coronavirus and preparation method thereof | |
| CN111983242A (en) | Electrochemical luminescence immunoassay method and kit | |
| CN104345149A (en) | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof | |
| US20160245804A1 (en) | Methods, Systems and Kits of Fecal NGal Rapid Immunochromatographic Test | |
| CN206038696U (en) | Ration calprotectin detects immunochromatographic test strip | |
| CN104714020B (en) | Urine endogenous α trypsin inhibitor heavy chain H4 merges the application in coronary heart disease at diabetes B | |
| US11067579B2 (en) | Target marker GP73 for detecting steatohepatitis and detection application method | |
| CN106771144A (en) | One kind detection C reactive proteins dot immunogold diafiltration kit and quantitative detecting method | |
| CN104280552A (en) | Application of urine apolipoprotein J precursor protein | |
| CN111557298B (en) | Novel coronavirus N protein extraction preservation solution and preparation method and application thereof | |
| CN104714022B (en) | The application in type 2 diabetes mellitus merges coronary heart disease of the urine Fibrinogen α chain | |
| CN104714021B (en) | Urine factor albumen merges the application in coronary heart disease at diabetes B | |
| CN104297487A (en) | Application of urine nucleotide triad binding protein 1 | |
| CN108982868B (en) | Application of nucleosomal protein SP110 and kit containing same in preparation of alcoholic cardiomyopathy early diagnosis reagent | |
| CN109633163B (en) | procalcitonin/C reactive protein two-in-one detection kit | |
| CN114994306B (en) | Application of protein PKNOX1 in preparation of reagent for diagnosing alcoholic cardiomyopathy and diagnostic kit | |
| CN104297486A (en) | Application of urine glutamoyl-prolyl tRNA synthetic enzyme | |
| CN203365440U (en) | Kit for testing HINT1 (Histidine Triad Nucleotide-binding Protein 1) in urine of patients suffering from diabetes mellitus type 2 | |
| CN108020663A (en) | The application of urine gelsolin and its polypeptide fragment in adenocarcinoma of lung | |
| CN208060540U (en) | A kind of multi objective colloidal gold kit for pyemia Quantitative detection | |
| WO2014067032A1 (en) | Method and kit for detecting haemoglobin in rectal exudate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150114 |