CN105988006A - Use of urine protein complement component 3 - Google Patents
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- CN105988006A CN105988006A CN201510067728.0A CN201510067728A CN105988006A CN 105988006 A CN105988006 A CN 105988006A CN 201510067728 A CN201510067728 A CN 201510067728A CN 105988006 A CN105988006 A CN 105988006A
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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
Abstract
The invention provides a use of a urine protein complement component 3 (C3) in urine noninvasive detection. A research proves that the urine protein complement component C3 fragment of a normal person can be stably expressed. According to the advantage of urine sample noninvasive acquisition, the urine protein complement component C3 is detected by a random urine sample so that the urine detection bottleneck problem of urine volume-caused influence on urine protein/polypeptide detection is solved and the defects of creatinine as urine related detection internal reference are overcome. The urine protein complement component C3 provides a simple and convenient detection method for disease diagnosis.
Description
Technical field
The present invention relates to the new application of urine protein complement C3, be specifically related to the normal expression internal reference as uroscopy method of urine protein complement C3, unconventionality expression is as the biomarker of medical diagnosis on disease.
Background technology
At present, in routine clinical detection, utilizing most specimen types is blood preparation.But, blood preparation obtains invasive, occurs that some project cannot be detected by the specimen of lipidemia or haemolysis, collecting device mostly is sharp weapon, and this not only needs to do some training very often, veteran blood sampling person, and blood product itself has an increased the risk of occupational exposure.On the other hand, add hypoglycemic risk within least 8 hours, on an empty stomach anemia of pregnant woman, old man and the such patient of child.For needing the disease such as diabetes, hematopathy, chemotherapy of tumors etc. of blood sampling the most throughout one's life also to bring great misery to patient.
Urine is the ultrafiltrate of blood, it it is the end metabolite eventually heavily absorbing, drain and secreting generation through glomerular filtration, renal tubules and collecting tubule, it it is the window of reflection body change, the change of its composition is the specific manifestations of some morbid state, and urine specimen is collected simply, noinvasive, amount are many, can be repeated several times, preserve convenience, patient compliance height, and need not the help of medical personnel.Compared with blood, urine protein composition is relatively easy, be prone to analysis, has and have great advantage in terms of humoral diagnostic disease.The composition of urine, quantity not only carry diseases of urinary system generation, development and the various information of prognosis with the change of character, also reflects the overall metabolism state of body, for research organ function, evaluate fuselage state, metabolic condition, dynamic monitoring progression of disease and judge that the aspects such as curative effect provide various information.In urine in addition to containing a large amount of protein, also comprise more polypeptide fragments.The molecular weight proteins and peptides less than 10kDa can freely filter glomerule, and the polypeptide fragments in urine is likely to the product of proteolysis, analyzes urine and is more likely to be appropriate for the disease of diagnostic system, discloses the pathophysiological features of disease.Therefore it is diagnosis and judges the desirable material that disease classification is in progress.
Complement C_3 is the core protein in complement system, is mainly produced by hepatocyte and macrophage.The gene of encoding complement C3 is positioned at No. 19 chromosomes, is made up of 41 exons, and its cDNA coded sequence total length is about 5052bp, successively encoding leader peptide, β chain and α chain.Complement C_3 is during synthesis, and its single chain precursor forwards to cut 22 amino acid whose guiding peptides during endoplasmic reticulum, gives up alkaline connection peptides, secrets out of cell with the form of two chains.Complement C_3 maturation protein contains 1663 aminoacid, and relative molecular weight is 180kDa.In the various composition of complement, the serum content of C3 is the highest；Functionally, C3 also centrality, it is crossing of several activated pathway, is again the basis of C3b dependency positive feedback loop；Meanwhile, C3 is activated and after cracking, and its serial fragment produced and associated proteins thereof are complicated and various, play a significant role in immune defence, immunoregulation and immunopathogenesis.In addition, non-complement function relevant to cell proliferation and differentiation for C3 also has been reported that, as participated in propagation and the establishment of bastem and lens vesicle, participate in propagation and the growth of external B cell, this may give the credit to complement component effect played in mitogenesis event, and C3 may dedifferente in early days and even also work in the atomization in later stage.Along with going deep into of research, C3 is considered also to play a role in tumor formation, signal transduction, apoptosis.Meanwhile, can play a protective role when anti-oxidation stress.
But, urine volume is easily affected by amount of drinking water and some other physiologic factor, so, the concentration of urine related sign object depends not only on the excretion rate of mark, and depends on uroflow amount.24h urine is the goldstandard of assessment urine markers thing, but this specimen collection mode is loaded down with trivial details, and the compliance of patient is poor.At present research is applied creatinine, urine osmole or specific gravity of urine etc. to calculate the impact of correction urine volume, be most widely used is urine creatine, but these methods not only, the mental status, physiological status, morbid state fat or thin by age, sex, build and diet etc. are affected, and be lack of consistency in different reports and limit cross validation's research, so, the best approach exploring correction urine volume is to solve albumen and the key of polypeptide detection by quantitative in urine.
In order to uroscopy is applied in clinical position, first the present invention has filtered out the stable high-abundance proteins polypeptide occurred in urine.When body is in normal physiological status, this urine protein polypeptide can be stablized at certain level, and being raised and lowered of its level is relevant to some specific disease.
Summary of the invention
It is an object of the invention to provide the application in preparation is used for urine coherence check of a kind of urine protein complement C3 (Complement Component 3).
Preferably, the aminoacid sequence of described urine protein complement C3 is as shown in SEQ ID NO:1:
Preferably, described preparation is urine Complement C_3 protein detection kit.Described test kit is antibody antigen reaction.
Preferably, described antigen antibody reaction is coated by urine Complement C_3 albumen or polypeptide and its antibody or is marked at solid phase or liquid phase carrier.
First inventor have collected the random urine specimen of normal health check-up, takes supernatant, utilize weak cation exchange magnetic beads for purifying and separated urine specimen after being centrifuged.By 1 μ l specimen and the 10 μ l substrate (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) after mixing, take 1 μ l point at Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) on target plate, mass spectral analysis is carried out after specimen ionizing, gather the data in the range of 1000-10000Da, it is thus achieved that the mass spectrum polypeptide figure being made up of the protein peak of different mass-to-charge ratioes.Application ClinProTools2.1 analyzes all mass spectruies of software and is analyzed, and filters out the high-abundance proteins polypeptide of stable expression.Then inventor utilizes Liquid Chromatography-Tandem Mass Spectrometry instrument to identify these protein polypeptides filtered out, Complement C_3 (Complement Component 3, Complement C_3 albumen) albumen is obtained at International Protein Index (IPI human v3.45fasta with 71983entries) database retrieval.
By research, the present invention confirms that Complement C_3 albumen can stable in the urine of the people of normal health check-up occur.Thus propose to detect urine protein complement C3 and can be used for the application in urine coherence check.
The present invention plays urine specimen and obtains noninvasive advantage, utilizes random urine Samples detection Complement C_3 albumen or polypeptide.
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, it is described in detail below.
Accompanying drawing explanation
Fig. 1 be mass-to-charge ratio be between 1000-10000 a little meansigma methods in 30 example normal health check-up specimen.
Fig. 2 be mass-to-charge ratio be the scatterplot expressed o'clock in 30 example normal health check-up specimen of 1726.8.
Fig. 3 is the mass spectrum of Complement C_3 albumen.
Detailed description of the invention
Embodiment 1The collection of urine specimen and process
Collect the random CCMS liquid specimen of the 30 normal health check-ups of example (Beijing Shijitan Hospital, CMU's MEC), in 2h centrifugal (1500rpm, 5min), retain supernatant.After subpackage ,-80 DEG C of refrigerators are frozen.
Embodiment 2Polypeptide in magnetic beads for purifying and separated urine specimen
Taking out urine specimen from-80 DEG C of refrigerators, 4 DEG C melt again, take supernatant standby after centrifugal (3000rpm, 10min).Balance weak cation magnetic bead (MB-WCX) under room temperature, and manually mix bead suspension.Adding 10ul MB-WCX in sample cell and 10ul magnetic bead combines buffer, sample loading gun blows and beats mixing up and down, it is to avoid bubble.Standing 1 minute on magnetic frame after adding 5ul urine supernatant, fully mixing in sample cell, magnetic bead separates with the liquid of suspension.Remove the clear liquid suspended with sample loading gun, rifle head should avoid contact to magnetic bead, it is to avoid siphons away magnetic bead.After adding 100ul magnetic bead cleaning buffer solution, fully mixing in sample cell, sample cell being stood on magnetic frame 1 minute, magnetic bead is adherent, separates with the liquid suspended, and removes the liquid suspended with sample loading gun.It is repeated 3 times, discards suspension.In sample cell, add 5ul magnetic bead elution buffer, repeatedly inhale and make a call to more than 10 times, make magnetic bead and elution buffer mixing, it is to avoid bubble.Sample cell is positioned on magnetic frame, stands 2min, make magnetic bead be sufficiently separated with suspension, supernatant (eluent) is moved into marked new 0.5ml sample cell.Adding 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 substance, 1 μ l eluent and 10 μ l substrate (alpha-cyano-4-hydroxycinnamic acid of 0.3%, HCCA) are mixed, takes 1 μ l point at Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) on target plate, drying at room temperature.Irradiated by nitrogen laser and carry out mass spectral analysis after making specimen ionizing, gather the data in the range of 1000-10000Da, it is thus achieved that the mass spectrum being made up of the protein peak of different mass-to-charge ratioes.For each MALDI crystalline temperature, 400 laser of concurrent irradiation (8 different positions of each crystalline temperature respectively irradiate 50 times), meansigma methods represents a specimen, thus obtains many peptide mappings of all samples.Application ClinProTools2.1 analyzes software and is analyzed Normal group, type 2 diabetes mellitus without the mass spectrum of complication and complication group and type 2 diabetes mellitus merging albuminuria group, screens diversity polypeptide.Screening conditions: mass range 1000-10000Da, signal to noise ratio (S/N) is more than 5, and Mass Drift is less than 0.1%, and all mass spectruies are normalized according to total ion current.Specifically refer to Fig. 1, it shows in 30 example urine specimens the meansigma methods of the point of all mass-to-charge ratioes between 1000-10000Da；Peak area is as quantitative standard, and Fig. 2 illustrates Complement C_3 expression in all Urine specimens, and as can be seen from the figure the m/z 1726.8 peak area in all specimen is both greater than 600.
Embodiment 4The qualification of protein polypeptide
Magnetic bead eluent in sample cell is rotated and is evaporated, add 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, trap time 3min with the speed of 15 μ l/min.Then entering 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 are shown in Table 1).Analysis time 60min, chromatogram column temperature 35 DEG C, all eluting compositions enter spectrometer analysis.Nano ion source, spray voltage 1.8kV；Mass spectrum pattern is data dependence and dynamically gets rid of, sweep limits 400-2000m/z；One-level scanning (MS) uses Obitrap, and resolution setting is 100000；CID and two grades of scannings 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 BioworksBrowser 3.3.1SP1 carries out SequestTMRetrieval.Searching database is International Protein Index (IPI human v3.45fasta with 71983entries).Parent ion error is set as that 100ppm, fragment ion error are set to 1Da, and enzyme action mode is non-enzyme action, variable be modified to methionine oxidized.Retrieval result parameter is set as deltacn >=0.10, two electric charge Xcorr 2.6, tricharged Xcorr 3.1, the above Xcorr of tricharged 3.5.In data base, retrieval obtains protein complement C3, and the mass spectrum of Complement C_3 albumen refer to Fig. 3.
The program of table 1 analytical column gradient elution
Embodiment 5The preparation of test kit
1. coated antibody and the selection of enzyme labelled antibody working concentration
Operate according to the BCA determination of protein concentration test kit description of Pierce company, measure antibody and the concentration of antigen, then the chessboard assay method of standard is used, it is 10.0ng/ml, 1.0ng/ml and 0.1ng/ml be coated buffer anti-human for rabbit Complement C_3 protein polyclone antibody (Abcam company) being diluted to concentration, it is coated on solid phase elisa plate and liquid phase magnetic bead respectively, each concentration includes three stringers, and 4 DEG C overnight, washs 3 times.Wherein one walk crosswise be coated in hole addition strong positive antigen liquid, another walks crosswise the weak positive antigen liquid of middle addition, and the third line adds negative control.Hatch 2 hours for 37 DEG C, wash 3 times.Add mouse-anti human complement c 3 monoclonal antibody (Abcam company), hatch 1 hour for 37 DEG C, wash 3 times.Adding the two of labelling to resist, hatch 30 minutes for 37 DEG C, wash 4 times, add substrate, room temperature lucifuge is placed 20 minutes, adds and stops liquid, reading.Select coated antibody optium concentration.
2. the preparation of test kit
Being diluted with being coated buffer by anti-human for rabbit Complement C_3 protein polyclone antibody, add it in solid phase microwell plate and liquid phase magnetic bead, 4 DEG C are coated jog overnight.Remove the most coated liquid, wash 3 times, add retardance liquid and stop nonspecific binding site, hatch 1 hour for 37 DEG C, wash 3 times.Put into 4 DEG C to save backup.Test kit subpackage add mouse-anti human complement c 3 monoclonal antibody, labelling two anti-etc..
Embodiment 7The detection of test kit sensitivity
Complement C_3 recombiant protein (OriGene company of Germany) 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 in the above-mentioned ELISA Plate being coated and in liquid phase magnetic bead, hatch 2 hours for 37 DEG C, wash 3 times.Mouse-anti Complement C_3 monoclonal antibody being diluted by 1: 2000, every hole adds 100ul, hatches 1 hour, washes 3 times for 37 DEG C.Add labelling two to resist, hatch 30 minutes, wash 3 times for 37 DEG C.Add substrate room temperature to place 15 minutes, add stop buffer, reading.Detecting minimum Complement C_3 amount, result shows, this reagent can detect the concentration of 0.01ng/ml Complement C_3 albumen, illustrates have higher detection sensitivity.In above-mentioned experiment shows the test kit detection urine specimen of the present invention, the content of Complement C_3, has the highest sensitivity, and lowest detectable limit 0.01ng/ml of sample, the response rate is 90% ± 13%.Needed for this test kit, instrument is less, it is only necessary to microplate reader, agitator, centrifuge, pipettor etc., required low cost.
Embodiment 8The specificity of test kit, the detection of stability
Take normal health check-up (Beijing Shijitan Hospital, CMU) cleaning to be measured stage casing random urine 30-50ml, load the urinary catheter of cleaning, menstrual period should be avoided during women preserving urine sample, vaginal secretions should be prevented to be mixed in urine, under room temperature, 1500rpm is centrifuged 5 minutes, takes supernatant to be checked.
The quantitative Complement C_3 recombiant protein of purification as standard substance, by antigen (Urine specimens after Li Xin) by 1: 3 dilution after, join before in the most coated good ELISA Plate, hatch 2 hours, wash away unconjugated antigen, blot residual liquid for 37 DEG C.Add mouse-anti human complement c 3 protein monoclonal antibody 37 DEG C to hatch 1 hour, wash away unconjugated antibody, blot residual liquid.Add the two of labelling to resist, hatch 30 minutes for 37 DEG C, wash 4 times, blot residual liquid.Adding chromogenic substrate, room temperature is placed 10 minutes, adds stop buffer and terminates reaction, microplate reader reading, calculates the content of Complement C_3 albumen in sample.
With it, inventor have detected Complement C_3 protein content in 100 example normal health check-up random urine, rate of accuracy reached to more than 97%, there is good specificity.
Take the random urine of two normal health check-ups respectively, utilizing said method to carry out ELISA mensuration, every day measures once, is repeated 10 times altogether, calculate between criticizing by the formula coefficient of variation (CV)=S/X × 100% (S is standard deviation, and X is meansigma methods) and variation within batch coefficient.Finally give in criticizing and be respectively 2.81% and 3.26% with interassay coefficient of variation, good stability is described.
Although the present invention discloses as above with preferred embodiment; so it is not limited to the present invention, any person of ordinary skill in the field, without departing from the spirit and scope of the present invention; when making a little change and improvement, therefore protection scope of the present invention is when being as the criterion depending on as defined in claim.
Claims (5)
1. urine Complement C_3 albumen or polypeptide application in preparation is used for urine Non-invasive detection.
Application the most according to claim 1, it is characterised in that described urine Complement C_3 albumen or many
The aminoacid sequence of peptide is as shown in SEQ ID NO:1.
Application the most according to claim 1, it is characterised in that described preparation is urine Complement C_3 egg
White or polypeptide detection kit.
Application the most according to claim 3, it is characterised in that described test kit is antigen antibody reaction.
Application the most according to claim 4, it is characterised in that described reaction is urine Complement C_3 egg
White or polypeptide and its antibody are coated or be marked at solid phase or liquid phase carrier.
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| CN201510067728.0A CN105988006A (en) | 2015-02-10 | 2015-02-10 | Use of urine protein complement component 3 |
| PCT/CN2015/000626 WO2016127278A1 (en) | 2015-02-10 | 2015-09-01 | Application of urine complement component 3 (c3) protein |
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| CN110669123A (en) * | 2019-09-03 | 2020-01-10 | 南京市妇幼保健院 | Secretory endogenous polypeptide PDFF-CO3 and application thereof |
| CN113092769A (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 complement C4-A and polypeptide fragment thereof in allergic diseases |
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| US20140038203A1 (en) * | 2012-07-09 | 2014-02-06 | Musc Foundation For Research Development | Methods for detecting or predicting kidney disease |
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| CN1423130A (en) * | 2002-04-26 | 2003-06-11 | 帕弗瑞生物技术(北京)有限公司 | HLA cross matching method for stream measuring fixed complement antibody and kit thereof |
| CN1444044A (en) * | 2002-06-18 | 2003-09-24 | 帕弗瑞生物技术(北京)有限公司 | HLA complement-dependent cytotoxcity antibody detection method using ELIA as basis and its kit |
| WO2006015616A1 (en) * | 2004-08-13 | 2006-02-16 | Indivumed Gmbh | Use of transthyretin as a biomarker for colorectal adenoma and/or carcinoma; method for detection and test system |
| CN101238373A (en) * | 2005-08-19 | 2008-08-06 | 因迪维姆德有限公司 | Use of an endoplasmin fragment and derivatives thereof as a biomarker for colorectal adenoma and/or carcinoma; method for detection and test system |
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| US20140038203A1 (en) * | 2012-07-09 | 2014-02-06 | Musc Foundation For Research Development | Methods for detecting or predicting kidney disease |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110669123A (en) * | 2019-09-03 | 2020-01-10 | 南京市妇幼保健院 | Secretory endogenous polypeptide PDFF-CO3 and application thereof |
| CN113092769A (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 complement C4-A and polypeptide fragment thereof in allergic diseases |
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