CN106556703A - A kind of chronic kidney disease mark suPAR detection kit and preparation method - Google Patents
A kind of chronic kidney disease mark suPAR detection kit and preparation method Download PDFInfo
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- CN106556703A CN106556703A CN201610908391.6A CN201610908391A CN106556703A CN 106556703 A CN106556703 A CN 106556703A CN 201610908391 A CN201610908391 A CN 201610908391A CN 106556703 A CN106556703 A CN 106556703A
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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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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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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
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
The present invention relates to a kind of chronic kidney disease mark suPAR detection kit and preparation method.This test kit includes PVC base plates, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；The sample pad, nitrocellulose filter, fluorescence pad, adsorptive pads are fixed on PVC base plates by horizontal direction.There are the anti-human 1 nanometer fluorescent microspheres complex of suPAR monoclonal antibodies of coated Mus, chicken IgY nanometer fluorescent microspheres complex on the fluorescence pad；The nature controlling line for having the detection line and rabbit-anti chicken IgY antibody of the anti-human composition of suPAR monoclonal antibodies 2 of Mus to constitute on described nitrocellulose filter.The present invention detects suPAR using nanometer fluorescent microspheres, and its background value is low, detection time is short, specificity and sensitivity height, testing result are accurate, is that chronic kidney disease early clinical diagnosis and generaI investigation provide relatively reliable foundation.
Description
Technical field
The present invention relates to a kind of chronic kidney disease mark suPAR detection kit and preparation method, the invention belongs to cure
Treat product scope.
Background technology
Nephropathy (KD）It is the chronic Renal Structure and dysfunction caused by a variety of causes, the species of nephropathy is various,
Relevant pyelonephritis of the more typical acute glomerulonephritis for having immunity injury to cause and bacterium infection etc., in addition diabetes, high blood
Pressure and the patient such as systemic lupus erythematosus also Chang Bingfa nephropathy.Over nearly more than 10 year, interior chronic kidney disease at the international level
（CKD）Research become wide concerned focus.The national survey of the developed countries such as the U.S., Germany, Norway shows that CKD is normal
The chronic disease seen, in Adult Groups, the prevalence of CKD is 10.2% ~ 13.0%[1-5], and be 10.8 in Chinese Adult prevalence
%[6-7].The features such as chronic kidney disease has high prevalence, high mortality and low awareness, it has also become most normal in world wide
The morning seen sends out the risk factor of disease and death.Diagnosis is made to KD rapidly and accurately for correctly effectively treatment extremely can closing
Key, but the diagnosis of KD faces many difficulties, current diagnosis KD relies primarily on renal biopsy with gun-biopsy, as KD is focal, sections
Sexually transmitted disease (STD) become, when Renal biospy draw materials it is not good, especially do not get skin marrow have a common boundary tissue when, possible mistaken diagnosis.There is no the biochemical diagnosis of standard
Method, and renal biopsy with gun-biopsy contraindication and complication are more and body is adversely affected, and are difficult to be accepted by patients, cause
One of major reason of KD difficult diagnosiss.
Human urokinase type Plasminogen activation receptor（uPAR）It is a kind of protein of high glycosylation, in being fibrinolytic system
A multifunctional receptor, with 3 homeodomains such as D1, D2 and D3, by disulfide bond.In neutrophilic granulocyte, gulp down
Express in the cell such as phagocyte, activity T cells, endotheliocyte and cancerous cell, can be used as urokinase and some other transmembrane protein
Receptor, by various lifes such as activation, signal transmission, cell adhesion and the cell transfer of plasminogen are mediated with its ligand binding
Reason function.When uPAR come off from cell surface discharge into become in Peripheral Circulation soluble urokinase type plasminogen swash
Being receptor（suPAR）, suPAR is the specific receptor of uPA, be widely present in internal various hemocytees, vascular endothelial cell,
On smooth muscle cell and tumor cell membrane.UPAR by being combined with uPA, with cell local fibrinolytic function, in various physiology and
Play a role under pathologic condition, including Plasminogen activation, improve cell adhesion and migration, inflammatory reaction, immunne response, thrombosis
Formation and the growth of tumor cell, invasion and attack and transfer etc.[8-10].SuPAR is the soluble form of uPAR, be present in Healthy People and
In the body fluid such as the blood of various Diseases, blood plasma, cerebrospinal fluid, urine, the activation levels of body immune system are reflected[11]。
Denmark scholar is divided for diagnosis or prognosis streptococcus pneumoniae and tuberculosis by setting up uPAR or suPAR ELISA detection kits
The infection of branch bacillus[12], the method complex operation, and this article does not embody the correlational study with regard to chronic kidney disease.Grind in the U.S.
Study carefully the suPAR concentration that personnel have detected this 3683 experimenters, and to wherein 2292 people's in research beginning and follow-up follow-up
Renal function is assessed, and as a result shows that baseline suPAR concentration is higher higher relevant with decreased renal function amplitude later.Grind
Study carefully show detection suPAR haemoconcentrations can before disease initially causes tissue injury in 5 years reliable prediction kidney disease morbidity
Risk, early discovery, prevention and monitoring that this will likely improve kidney disease[13].Therefore suPAR can be used as chronic kidney disease
Early diagnosis marker.
Fluorescent microsphere nano-particle has tremendous potential in marker detection field.It has the characteristics that：1st, sensitivity
It is high：Not by extraneous ambient interferences；2nd, stability is high：Avoid the quenching phenomenon that sample corrosion or itself decay cause；3rd, spirit
It is active high：Diversified spectrum can be combined；4th, it is safe：For tester, detection sample and environment are all safe from harm；5th, side
Just it is simple：Sample can directly be detected, without the need for special handling；6th, above feature determines which is applied in terms of biomarker
Prospect is broader, especially the detection field by the bed.By the anti-human SUPAR monoclonal antis of fluorescent microsphere nanoparticle label Mus
Body, set up a kind of chronic kidney disease mark suPAR detection kit and preparation method have in Clinical detection field it is important
Using value.
The content of the invention
For the problem with present on, the invention provides a kind of chronic kidney disease mark suPAR detection kit and
Preparation method.It is characteristic of the invention that：With reference to dry type immuno-fluorescence assay people suPAR, by specific monoclonal antibody and sample
SuPAR antigens form double-antibody sandwich structure, and its accuracy and specificity are higher；Using dry type determination of immunofluorescence method, its back of the body
Scape value is low, and it is convenient, accurate, simple to operate to measure.
It is an object of the invention to provide a kind of chronic kidney disease mark suPAR detection kit and preparation method.
Test kit of the present invention includes PVC base plates, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；The sample
Product pad, nitrocellulose filter, fluorescence pad, adsorptive pads are fixed on PVC base plates by horizontal direction.On the fluorescence pad
There are the anti-human suPAR monoclonal antibodies 1- fluorescent microsphere nano-particle complex of coated Mus, chicken IgY- fluorescent microsphere nano-particle
Complex；There are the detection line and rabbit-anti chicken IgY of the anti-human composition of SUPAR monoclonal antibodies 2 of Mus anti-on described nitrocellulose filter
The nature controlling line that body is constituted；Described nano-particle footpath is 100 ~ 200nm.
The technical scheme is that：
1）Nitrocellulose filter（NC films）The preparation of-PVC base plates：Nitrocellulose filter is cut into into 25mm × 300mm, PVC is attached to
On base plate.Mus anti-human suPAR monoclonal antibodies 2 are rule on nitrocellulose filter and obtains detection line；By rabbit-anti chicken IgY in cellulose nitrate
Line on plain film obtains nature controlling line.Then by nitrocellulose filter（NC films）- PVC base plates are placed in drying baker drying；
2）Using the anti-human suPAR monoclonal antibodies 1 of nanometer fluorescent microspheres particle marker Mus and chicken IgY, save backup.
3）The preparation of pad：Pad is cut into into 9mm × 300mm slices, will be Mus anti-human suPAR monoclonal antibodies 1- glimmering
Light microsphere nano particle composites, chicken IgY- fluorescent microsphere nano-particle complexes mix according to a certain percentage, are uniformly layered on knot
Close on pad, be put into stove-drying.
4）Sample pad：Glass fibre membrane is cut into into 17mm × 300mm slices.
5）Absorption pad：17mm × 300mm slices are cut into in absorbent paper.
7）Assembling：Above-mentioned adsorptive pads, sample pad, fluorescence pad are attached on PVC base plates successively, by the intermedium for posting
The reagent strip of one fixed width is cut into cutting machine.
Description of the drawings
Fig. 1 dry type Immunofluorescence test system structure diagrams, wherein 1 is sample pad；2 is fluorescence pad；3 is nitric acid
Cellulose membrane；4 is adsorptive pads；5 is PVC base plates；6 is target antigen in sample；7 is that the anti-human suPAR of fluorescent microsphere labelling Mus is mono-
Anti- 1；8 is fluorescent microsphere labelling chicken IgY；9 is detection line：The anti-human suPAR monoclonal antibodies of Mus 2；10 is nature controlling line：Rabbit-anti chicken IgY.
Specific embodiment
Embodiment 1 prepares the dry type immunofluorescent reagent box of chronic kidney disease suPAR of the present invention
Test kit of the present invention includes PVC base plates, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；The sample
Pad, nitrocellulose filter, fluorescence pad, adsorptive pads are fixed on PVC base plates by horizontal direction.
First, nitrocellulose filter（NC films）The preparation of-PVC base plates
1st, draw film：By celluloid enzyme action into 25mm × 300mm, it is attached on PVC base plates.Rabbit-anti chicken IgY is diluted to PBS
Concentration is 1mg/ml, used as C line coating buffers；It is 1mg/ml that the Mus anti-human PBS of SUPAR monoclonal antibodies 2 is diluted to concentration, used as T lines.
By spraying film instrument, T lines coating buffer and C line coating buffers are rule on nitrocellulose filter and obtains detection line and nature controlling line, 37 degree
Dry, you can obtain monoclonal antibody coating test strip.
2nd, the preparation of pad
1st, traget antibody：Nanometer fluorescent microspheres granule and the anti-human suPAR monoclonal antibodies coupling method of Mus are by nanometer fluorescent microspheres
Aldehyde radical be condensed by schiff base base under mild alkaline conditions with the amino on antibody protein, formed nanometer fluorescent microspheres-CH-
NH2-suPAR antibody complexes；The same manner forms nanometer fluorescent microspheres-CH-NH2- chicken IgY antibody complexes.Fluorescent microsphere
Nano-particle and the anti-human suPAR monoclonal antibodies of Mus or chicken IgY are according to 1:1（ul：ug）Hybrid reaction 2h, prepares the anti-human suPAR of Mus
Monoclonal antibody 1- fluorescent microsphere nano-complex or chicken IgY- fluorescent microsphere nano-particle complexes.
2nd, the preparation of pad：Pad is cut into into 9mm × 300mm slices, will be Mus anti-human suPAR monoclonal antibodies 1- glimmering
Mix homogeneously is layered on knot according to a certain percentage for light microsphere nano particle composites or chicken IgY- fluorescent microspheres nano-particle complex
Close on pad, be put into the lower baking 2h of 37 degree of baking oven.
3rd, prepared by other auxiliary materials
1st, glass fibre membrane is cut into into 17mm × 300mm slices.
2nd, absorption pad：17mm × 300mm slices are cut into in absorbent paper.
4th, assemble
Assembling test strips：Nitrocellulose filter, absorbent paper, fluorescence pad, sample pad are pasted on PVC base plates successively, is used
Cutting machine is cut into the reagent strip of one fixed width.
The using method of 2 test kit of the present invention of embodiment
1st, pipette 50ul serum, blood plasma, CSF sample with pipettor to be added on the sample-adding pad of test strips；
2nd, it is stored at room temperature 10min；
3rd, 10min terminates, and test strips is put in immunofluorescence quantitative analysis instrument and reads data；
4th, immunofluorescence quantitative analysis instrument is measured to optical signalling and is analyzed and processed, and quantitatively draws the concentration of measured matter；
5th, yin and yang attribute is judged according to reference value.
3 test kit of the present invention of embodiment is compared with ELISA test kits
The test kit of the present invention detects the Patients with Chronic Kidney Disease serum 101 of clinical definite simultaneously with ELISA test kits, just
Ordinary person's control serum 96, testing result see the table below 1：
The test kit of 1 present invention of table and ELISA test kit testing results
As it can be seen from table 1 the test kit of the present invention to the positive rate of Patients with Chronic Kidney Disease apparently higher than ELISA reagents
Box, is feminine gender to normal person's Virus monitory, it is seen that the test kit of the present invention is more more sensitive than ELISA kit, special, accurate.
Illustrate that test kit of the present invention has higher clinical coincidence rate, can provide more accurate, reliable for the detection of current chronic kidney disease
Value.
List of references
[1] US Renal Data System: USRDS Annual data report 2011. Volumeone:
Atlasofend stage renal disease in the United States [S].
[2] Ou Yanglingxia, Zhou Pan, land-sea Na. chronic kidney disease Epidemiology [J]. international transplanting and blood
Purification magazine, 2012,10 (3): 1-3.
[3] Paul E, Marije Ron TG, et al. Screening for Chronic Kidney Disease:
Where Does Europe Go [J]. Clin J Am Soc Nephrol, 2008, 3(2): 616-623.
[4] Coresh J, Selvin E, Stevens LA, et a1. Prevalence of chronic kidney
disease in the United States. JAMA, 2007, 298: 2038-2047.
[5] Hallan SI, Coresh J, Astor BC, et a1. International comparison of the
relationship of chronic kidney disease prevalence and ESRD risk. J Am Soc
Nephro1. 2006, 17: 2275-2284.
[6] Zhang L, Wang F, Wang L, et a1. Prevalence of chronic kidney disease
in China: a cross-sectional survey. Lancet, 2012, 379: 815-822.
[7] Zhang Luxia, Wang Fang, Wang Li, etc. the cross-section survey of Chinese chronic kidney disease prevalence. Chinese internal medicine is miscellaneous
Will, 2012,51:570.
[8] Jalkanen V, Yang R, Linko R, et a1. SuPAR and Pal-1 in crtically ill,
mechanically ventilated patients[J]. Intensive Care Med, 2013, 39(3): 489-
496.
[9] Donadello K, Scolletta S, Covajes C, et a1. suPAR as a prognostic bin
marker in sepsis[J]. BMC Med, 2012, 10: 2.
[10] Haupt TH, Petersen J, Ellekilde G, et a1. Plasma suPAR levels are
associated with mortality, admission time, and Charlson Comorbidity Index in
the acutely admitted medical patient: a prospective observational study[J].
Crit Care, 2012, 16(4): 130.
[11] Thunp M, Macho B, Eugen, Olsen J. suPAR: the molecular crystal ball
[J]. Dis Markers, 2009, 27(3): 157-172.
[12] J. You Gen-Mancur Olson. the test kit of experimenter's condition of assessment infection respiratory tract antibacterial. profit ZL
02812214.8
[13] Jorgensen M, Hlatky M, Kober L, et al.β-blocker associated risks in
patients with uncomplicated hypertension undergoing noncardiac surgery [J].
JAMA Intern Med, 2015, 175(12):1923-31。
Claims (15)
1. a kind of chronic kidney disease mark suPAR detection kit and preparation method, its special disease is that this test kit includes
PVC base plates, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；The sample pad, fluorescence pad, cellulose nitrate
Plain film, adsorptive pads are fixed on PVC base plates by horizontal direction；The knot fluorescence to be closed and have the anti-human MCM5 monoclonals of coated Mus on pad
Antibody 1- nanometer fluorescent microspheres complex, chicken IgY- nanometer fluorescent microspheres complex；There are Mus to resist on described nitrocellulose filter
The nature controlling line that the detection line and rabbit-anti chicken IgY antibody that people MCM5 monoclonal antibodies 2 are constituted is constituted.
2. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
It is the red fluorescent microsphere of aldehyde radicalization in, described nanometer fluorescent microspheres, nano particle diameter is 100 ~ 200nm, excitation wavelength is
365nm, launch wavelength are 612 ± 5nm, and there is its content of aldehyde groups on surface for 0.1 ~ 0.5mMol/g.
3. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
In capture antibody being can be with the monoclonal antibody of target antigen specific binding.
4. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
In binding antibody being can be with the monoclonal antibody of target antigen specific binding.
5. a kind of chronic kidney disease mark suPAR detection kit and preparation method as described in claim 3,4, its special disease
It is that capture antibody and binding antibody are mouse monoclonal antibody, two different locis on identification MCM5 protein moleculars.
6. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
In test kit be dry type immunofluorescence quantitative method test kit.
7. a kind of chronic kidney disease mark suPAR detection kit and preparation method as described in claim 1,3,4, which is special
Disease is to detect people's suPAR albumen using double antibody sandwich method Quick kit.
8. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
Short the time required to detection, detection only needs 10min.
9. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease exist
Simple to operate in the method, test strips are put into immunofluorescence fixed without the need for special handling after being directly added into sample 10min by sample
Amount analyser in read data, immunofluorescence quantitative analysis instrument is measured to optical signalling and is analyzed and processed, quantitatively draw by
Survey the concentration of material.
10. a kind of chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is the method detection sensitivity height, high specificity, accuracy height, nanometer fluorescent microspheres are not disturbed by extraneous background.
A kind of 11. chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is that the method stability is high, nanometer fluorescent microspheres are not because of being quenched that sample corrosion or itself decay cause.
A kind of 12. chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is the method range of linearity width, the range of linearity can reach up to thousand times of Radix Achyranthis Bidentatae.
A kind of 13. chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is that detection sample is serum, blood plasma, whole blood, cerebrospinal fluid, urine, ascites.
A kind of 14. chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is with chronic kidney disease mark suPAR as target antigen.
A kind of 15. chronic kidney disease mark suPAR detection kit as claimed in claim 1 and preparation method, its special disease
It is that clinical diagnosis disease is chronic kidney disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108226497A (en) * | 2017-12-29 | 2018-06-29 | 无锡壹闪生物科技有限公司 | Soluble urokinase type Plasminogen activator receptor detection kit and detection method |
| WO2019019471A1 (en) * | 2017-07-24 | 2019-01-31 | Wwhs Biotech, Inc | Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof |
| CN112180103A (en) * | 2020-10-23 | 2021-01-05 | 福州大学 | Kit for clinically detecting active urokinase receptor in plasma of new coronary pneumonia patient |
| US11026625B2 (en) | 2017-08-08 | 2021-06-08 | Fresenius Medical Care Holdings, Inc. | Systems and methods for treating and estimating progression of chronic kidney disease |
| WO2022095227A1 (en) * | 2020-11-03 | 2022-05-12 | 浙江大学 | Plasma-soluble urokinase plasminogen activator receptor and application thereof |
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| CN105807067A (en) * | 2016-04-13 | 2016-07-27 | 上海凯璟生物科技有限公司 | Dry-type immunofluorescence kit for detecting NCAM2 (neural cell adhesion molecules 2) of patient suffering from alzheimer's syndromes and preparation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019019471A1 (en) * | 2017-07-24 | 2019-01-31 | Wwhs Biotech, Inc | Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof |
| US11026625B2 (en) | 2017-08-08 | 2021-06-08 | Fresenius Medical Care Holdings, Inc. | Systems and methods for treating and estimating progression of chronic kidney disease |
| CN108226497A (en) * | 2017-12-29 | 2018-06-29 | 无锡壹闪生物科技有限公司 | Soluble urokinase type Plasminogen activator receptor detection kit and detection method |
| CN112180103A (en) * | 2020-10-23 | 2021-01-05 | 福州大学 | Kit for clinically detecting active urokinase receptor in plasma of new coronary pneumonia patient |
| WO2022095227A1 (en) * | 2020-11-03 | 2022-05-12 | 浙江大学 | Plasma-soluble urokinase plasminogen activator receptor and application thereof |
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Application publication date: 20170405 |