CN106596968A - Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit - Google Patents
Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit Download PDFInfo
- Publication number
- CN106596968A CN106596968A CN201611095552.0A CN201611095552A CN106596968A CN 106596968 A CN106596968 A CN 106596968A CN 201611095552 A CN201611095552 A CN 201611095552A CN 106596968 A CN106596968 A CN 106596968A
- Authority
- CN
- China
- Prior art keywords
- antibody
- human serum
- liquid
- serum albumins
- gold
- 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
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 239000010931 gold Substances 0.000 title claims abstract description 85
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 85
- 210000002700 urine Anatomy 0.000 title claims abstract description 66
- 230000008595 infiltration Effects 0.000 title abstract 7
- 238000001764 infiltration Methods 0.000 title abstract 7
- 239000007788 liquid Substances 0.000 claims abstract description 74
- 238000001514 detection method Methods 0.000 claims abstract description 52
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 239000000243 solution Substances 0.000 claims abstract description 46
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000007983 Tris buffer Substances 0.000 claims abstract description 40
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 30
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 30
- 102000009027 Albumins Human genes 0.000 claims description 98
- 108010088751 Albumins Proteins 0.000 claims description 98
- 210000002966 serum Anatomy 0.000 claims description 91
- 239000000084 colloidal system Substances 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 43
- 238000006243 chemical reaction Methods 0.000 claims description 39
- 239000012153 distilled water Substances 0.000 claims description 39
- 239000008363 phosphate buffer Substances 0.000 claims description 38
- 238000004140 cleaning Methods 0.000 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 28
- 239000000020 Nitrocellulose Substances 0.000 claims description 26
- 229920001220 nitrocellulos Polymers 0.000 claims description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 22
- 239000011248 coating agent Substances 0.000 claims description 21
- 238000000576 coating method Methods 0.000 claims description 21
- 239000012898 sample dilution Substances 0.000 claims description 21
- 238000004587 chromatography analysis Methods 0.000 claims description 18
- 238000000502 dialysis Methods 0.000 claims description 18
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000011026 diafiltration Methods 0.000 claims description 16
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 14
- 239000000463 material Substances 0.000 claims description 13
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 claims description 12
- 239000001263 FEMA 3042 Substances 0.000 claims description 12
- 239000007836 KH2PO4 Substances 0.000 claims description 12
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims description 12
- 239000002250 absorbent Substances 0.000 claims description 12
- 230000002745 absorbent Effects 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 12
- 238000010612 desalination reaction Methods 0.000 claims description 12
- 238000007710 freezing Methods 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims description 12
- 229940033123 tannic acid Drugs 0.000 claims description 12
- 235000015523 tannic acid Nutrition 0.000 claims description 12
- 229920002258 tannic acid Polymers 0.000 claims description 12
- 239000001509 sodium citrate Substances 0.000 claims description 11
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 11
- 229940038773 trisodium citrate Drugs 0.000 claims description 11
- 239000002994 raw material Substances 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 6
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000004044 response Effects 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 229920002160 Celluloid Polymers 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 244000248349 Citrus limon Species 0.000 claims 1
- 235000005979 Citrus limon Nutrition 0.000 claims 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims 1
- ZXPSRPAUXQIYID-UHFFFAOYSA-N [N].[Na] Chemical compound [N].[Na] ZXPSRPAUXQIYID-UHFFFAOYSA-N 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical compound [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 claims 1
- 239000002245 particle Substances 0.000 abstract description 11
- 239000003446 ligand Substances 0.000 abstract description 6
- 239000000427 antigen Substances 0.000 abstract description 5
- 102000036639 antigens Human genes 0.000 abstract description 5
- 108091007433 antigens Proteins 0.000 abstract description 5
- 230000009881 electrostatic interaction Effects 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 3
- 108091006905 Human Serum Albumin Proteins 0.000 abstract description 2
- 102000008100 Human Serum Albumin Human genes 0.000 abstract description 2
- 239000003085 diluting agent Substances 0.000 abstract 2
- 239000008055 phosphate buffer solution Substances 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 38
- 238000002360 preparation method Methods 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000001195 anabolic effect Effects 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000010025 steaming Methods 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- 108010071390 Serum Albumin Proteins 0.000 description 3
- 102000007562 Serum Albumin Human genes 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- MYTMXVHNEWBFAL-UHFFFAOYSA-L dipotassium;carbonate;hydrate Chemical compound O.[K+].[K+].[O-]C([O-])=O MYTMXVHNEWBFAL-UHFFFAOYSA-L 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229910021505 gold(III) hydroxide Inorganic materials 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 206010027525 Microalbuminuria Diseases 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- -1 pH value are 6.2~6.4 Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000000885 nephron Anatomy 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/6827—Total protein determination, e.g. albumin in urine
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a dot gold infiltration kit for detecting urine microalbumin, and application of the dot gold infiltration kit. The dot gold infiltration kit comprises a sample diluent, a washing liquid, a colloidal gold labeled human serum albumin antibody, a urine microalbumin chromatographic device and a calibration card, wherein the sample diluents comprises Triton-100, a phosphate buffer solution and a Tris buffer solution; the washing liquid comprises bovine serum albumin, a phosphate buffer solution and a Tris buffer solution. Compared with the prior art, the dot gold infiltration kit for detecting the urine microalbumin has the advantages that colloidal gold particles are provided with negative charges on the surfaces and can form firm combination with the positive charge groups of urine microalbumin antibody protein molecules due to electrostatic interaction; meanwhile, the gold particles have the characteristic of high electron density, and when the antibody labeled by the gold particles is combined with a corresponding antigen ligand and a large number of the god particles are gathered at the site of the antibody ligand, macroscopic red dots are formed. The dot gold infiltration kit for detecting the urine microalbumin, provided by the invention, has the advantages of short detection time, high precision degree, low requirements on instruments and environment, and convenience in clinical popularization and application.
Description
Technical field
The present invention relates to technical field of medical examination, more particularly to a kind of detection microdose urine protein spot gold diafiltration reagent
Box and its application.
Background technology
Albumin is the normal protein in a kind of blood, but only occurs the white egg of very small amount in urine in physiological conditions
In vain.Microalbuminuria reflects renal abnormality proteinaceous effluent.Under normal circumstances, the albumin in urine is few, tool for body metabolism
Body to every liter of urinary albumin less than 20mg (<20mg/L), so being microalbumin.If found after health check-up micro- in urine
Amount albumin belongs to microalbuminuria in the range of 20mg/L-200mg/L, just.In clinic, commonly used urine micro protein refers to
Mark to monitor the generation of kidney trouble.
The detection of urine micro protein is that early detection ephrosis is most sensitive, one of most reliable diagnosis index.It is micro by urinating
Albuminous numerical value, just can accurately diagnose the state of an illness and judge the course of disease with reference to the statement of incidence, symptom and medical history.
After Canonical management kidney, the nephron is repaired, and albuminous amount recovers normal level in urine, so urine Microalbunin
In vain can be used as kidney trouble prognosis index.
Microdose urine protein detection technique mainly has immunoturbidimetry, colloidal gold method, ELISA etc..Immunoturbidimetry
Relative operation is relatively complicated and easily occurs hook effect in the presence of high concentration antigen, so as to the result for affecting to measure；
Simultaneously it is not suitable for small lot packaging, limits its popularization in basic hospital.The colloidal gold method testing time is longer, precision compared with
Difference, and it is easily influenced by environmental conditions.The operation of enzyme linked immunological rule is extremely loaded down with trivial details and higher to operator's requirement, and detection is time-consuming
Long, its popularization and application in clinic is limited.
The content of the invention
Present invention solves the technical problem that be provide a kind of detection microdose urine protein spot gold diafiltration kit and its
Using, with detection time it is short, precision is good the characteristics of.
In view of this, the invention provides a kind of spot gold of detection microdose urine protein is percolated kit, including：PH value
For cleaning solution that 6.5～6.7 Sample dilution, pH value are 6.2～6.4, colloid gold label human serum albumins antibody, urinate micro-
Amount albumin chromatography device and calibration card, the Sample dilution is slow including Triton-100, phosphate buffer and Tris
Rush liquor；The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
Preferably, the concentration of Triton-100 is 0.1～10% in the Sample dilution, phosphate buffer
Concentration is 0.005～1mol/L, and the concentration of Tris buffer solns is 0.01～1mol/L；Bovine serum albumin in the cleaning solution
White concentration is 0.1～10%, and the concentration of phosphate buffer is 0.01mol/L, and the concentration of Tris buffer solns is
0.01～1mol/L.
Preferably, the phosphate buffer is prepared by following raw material：Na2HPO40.263-0.376 weight portions,
KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, ox blood are pure
The weight portion of albumen 7；The Tris buffer solns are by trishydroxymethylaminomethane 4.6-5.5 weight portions, KH2PO43.0-2.1 weight
Amount part, the weight portions of NaCl 8, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
Preferably, the colloid gold label human serum albumins antibody is prepared as follows：It is 1% by mass fraction
Gold chloride and distilled water by volume 1：79 are made into A liquid；It is 1% by trisodium citrate that mass fraction is 1%, mass fraction
Tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～0.080：15.84～15.93 are made into B
Liquid；By the A liquid and B liquid while being heated to 59～61 DEG C, stirring A liquid in rapidly joining B liquid, continues to stir 1 point in water-bath
Clock, was heated to boiling in 10～13 minutes, obtained collaurum liquid；Using 0.25M K2CO3Adjusting the collaurum liquid pH value is
6.8～7.1；By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, in absorption
Clear liquid；The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing obtains human serum albumins
Antibody；The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the human serum albumins resists
Body is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, sequentially adds 1～30%
Sodium azide and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) are with the volume ratio of the collaurum liquid
500μl：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min, removes supernatant, sinks
Starch is colloid gold label human serum albumins antibody.
Preferably, the trisodium citrate, tannic acid, K2CO3It is 4 with the volume ratio of distilled water：0.08：0.08：15.84, institute
State the colloid gold particle that a diameter of 10 ＜ D ＜ 11nm are formed in collaurum liquid.
Preferably, the reaction box of a diameter of 2.8cm, the reaction box bag are provided with the microdose urine protein chromatography device
Include cassette bottom and lid, the lid top surface central authorities are provided with upper surface a diameter of 0.56cm, a diameter of 0.27cm of lower surface
Small sircle hole；The full absorbent material of pad in the reaction box；Place between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of one 1.18 × 1.18cm.
Preferably, the nitrocellulose filter of the coating human serum albumins antibody is prepared according to following steps：By human blood
Pure albumen coated antibody stoste is at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；Will be described
Supernatant loads bag filter, with distilled water dialysis desalination, bottling freezing after ultrafiltration concentration, obtains human serum albumins and resists
Body；The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, obtains 0.8mg/ml's
Coating buffer；The coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking oven by every diaphragm 0.02ml
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after middle drying.
Preferably, the calibration card detects the corresponding detection of color by human serum albumins gradient mass concentration solution
Signal value.According to the signal value that Test paper shows, compare with calibration card signal value, you can quantitatively judge its mass concentration.
Preferably, the gradient mass concentration is respectively 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 120mg/
L, 200mg/L, and the signal of the gradient mass concentration detection is corresponding with calibration card.
Accordingly, the present invention also provides a kind of application of the spot gold diafiltration kit of detection microdose urine protein, including
Following steps：Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant is extremely
Mix in Sample dilution, obtain sample to be tested；Sample to be detected is accurately drawn using pipettor, the nitre in reaction box circular hole is put
On acid cellulose face；After sample to be detected penetrates into, colloid gold label human serum albumins antibody, institute is added dropwise to reaction box face
The volume ratio that colloid gold label human serum albumins antibody is stated with the detection sample is 1：1；Treat colloid gold label human seralbumin
After protein antibodies penetrate into, cleaning solution is added dropwise on reaction box face, the cleaning solution is 1 with the volume ratio of the detection sample：
1；Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value, by instrument
Detection sample microdose urine protein concentration is calculated with reference to calibration card information, its mass concentration is quantitatively judged.
The invention provides a kind of spot gold diafiltration kit of detection microdose urine protein and its application, including：PH value
For cleaning solution that 6.5～6.7 Sample dilution, pH value are 6.2～6.4, colloid gold label human serum albumins antibody, urinate micro-
Amount albumin chromatography device and calibration card, the Sample dilution is slow including Triton-100, phosphate buffer and Tris
Rush liquor and；The cleaning solution includes bovine serum albumin(BSA) and phosphate buffer and Tris buffer solns.With it is existing
Have technology to compare, due to the surface of colloid gold particle it is negatively charged, can be with the positive electricity of microdose urine protein antibody protein molecule
Lotus group firm combination because electrostatic interaction is formed, while gold grain has the characteristic of high electron density again, when being marked by gold grain
Antibody combine with corresponding antigen ligand, gold grain just forms macroscopic red when antibody ligand site is assembled in a large number
Color spot point.The microdose urine protein spot gold diafiltration kit of the present invention has detection time short, and precision is good, to instrument and ring
The requirement in border is relatively low, the advantages of be easy to clinical application.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described with reference to embodiment, but
It should be appreciated that these descriptions are simply to further illustrate the features and advantages of the present invention, rather than to the claims in the present invention
Limit.
The embodiment of the invention discloses a kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is
6.5～6.7 Sample dilution, the cleaning solution that pH value is 6.2～6.4, colloid gold label human serum albumins antibody, urine are micro
Albumin chromatographs device and calibration card, and the Sample dilution includes Triton-100, phosphate buffer and Tris buffering
Liquor；The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
Dot immunogold filtration assay is to allow sample to be tested vertical penetration to cross microporous barrier (for example：Nitrocellulose filter), envelope
Upper coated capture probe capture, then allow collaurum mark probe to be percolated microporous barrier in the same fashion, tie with captured part
Aggregation after conjunction forms macroscopic punctation.Dot Immunogold Filtration Assay of the present invention based on affinity chromatography principle,
Nitrocellulose filter is adopted for solid phase carrier, to adsorb antibody on celluloid as solid phase, with antigen in human blood and
Antibody labeling collaurum is liquid phase, and while using antibody labeling collaurum as probe and indicator, and set up a kind of new
The immunologic detection method of type.The negatively charged positive electricity with microdose urine protein antibody protein molecule in surface of wherein colloid gold particle
Lotus group firm combination because electrostatic interaction is formed, while gold grain has the characteristic of high electron density again, when being marked by gold grain
Antibody combine with corresponding antigen ligand, gold grain just forms macroscopic red when antibody ligand site is assembled in a large number
Color spot point.
Preferably, the concentration of Triton-100 is 0.1～10% in the Sample dilution, phosphate buffer
The concentration of solution is 0.005～1mol/L, and the concentration of Tris buffer solns is 0.01～1mol/L；Ox blood in the cleaning solution
Clear albuminous concentration is 0.1～10%, and the concentration of phosphate buffer is 0.005～1mol/L, and Tris buffer solutions are molten
The concentration of liquid is 0.01～1mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO4 0.263-
0.376 weight portion, KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight of distilled water 1000
Part, the weight portion of bovine serum albumin(BSA) 7；The Tris buffer solns by trishydroxymethylaminomethane 4.6-5.5 weight portions,
KH2PO43.0-2.1 weight portions, the weight portions of NaCl 8, the weight portions of KCl 0.3, the weight portion of distilled water 1200, bovine serum albumin(BSA) 9
Weight portion is formulated.
Preferably, the colloid gold label human serum albumins antibody is prepared as follows：By quality point
Number for 1% gold chloride and distilled water by volume 1：79 are made into A liquid；By trisodium citrate, quality point that mass fraction is 1%
Number is 1% tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～0.080：15.84～
15.93 it is made into B liquid；The A liquid and B liquid are heated in water-bath 59～61 DEG C simultaneously, A liquid is stirred, in rapidly joining B liquid,
Continue to stir 1 minute, boiling was heated in 10～13 minutes, obtain collaurum liquid；Using 0.25M K2CO3Adjust the glue
Body gold liquid pH value is 6.8～7.1；By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations
60 minutes, Aspirate supernatant；The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is obtained
Human serum albumins antibody；The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the people
Seralbumin antibody is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, according to
1～30% sodium azide of secondary addition and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) and the colloid
The volume ratio of golden liquid is 500 μ l：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min,
Supernatant is removed, sediment is colloid gold label human serum albumins antibody.
Preferably, by trisodium citrate, the tannic acid that mass fraction is 1%, 25mM that mass fraction is 1%
K2CO3With distilled water by volume 4：0.08：0.08：15.84 are made into B liquid, and a diameter of 10 ＜ D ＜ are formed in the collaurum liquid
The colloid gold particle of 11nm.
Preferably, the reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, it is described anti-
Box is answered to include cassette bottom and lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, lower surface is a diameter of
The small sircle hole of 0.27cm；The full absorbent material of pad in the reaction box；Below the small sircle hole and above the absorbent material it
Between place 1.18 × 1.18cm coating human serum albumins antibody nitrocellulose filter, it is anabolic reaction to close tight lid
Box.The non-relevant areas of most of sample to be tested and label probe from beyond spot can be avoided to be lost in, affect detection sensitivity, can
So that the technology is widely used in clinical examination.
Preferably, the nitrocellulose filter of the coating human serum albumins antibody is prepared according to the following steps：Will
Human serum albumins coated antibody stoste is at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；Will
The supernatant loads bag filter, and with distilled water dialysis desalination, bottling freezing, obtains human serum albumins after ultrafiltration concentration
Antibody；The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, 0.8mg/ml is obtained
Coating buffer；The coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.02ml
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after being dried in case.
Preferably, the calibration card detects that color is corresponding by human serum albumins gradient mass concentration solution
Detected signal value.The gradient mass concentration be respectively preferably 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L,
120mg/L、200mg/L。
Accordingly, the present invention also provides a kind of application of detection microdose urine protein spot gold diafiltration kit, including with
Lower step：Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant is to sample
Mix in this dilution, obtain sample to be tested；Sample to be detected is accurately drawn using pipettor, the nitric acid in reaction box circular hole is put
On cellulose face；After sample to be detected penetrates into, colloid gold label human serum albumins antibody is added dropwise to reaction box face, it is described
Colloid gold label human serum albumins antibody is 1 with the volume ratio of the detection sample：1；Treat colloid gold label human seralbumin egg
After Bai Kangti penetrates into, cleaning solution is added dropwise on reaction box face, the cleaning solution is 1 with the volume ratio of the detection sample：1；
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value is tied by instrument
Close calibration card information and calculate detection sample microdose urine protein concentration, quantitatively judge its mass concentration.
From above scheme as can be seen that the present invention is simple and convenient, the color that device shows is chromatographed according to microdose urine protein, with
Correspondence batch targeted message, you can the mass concentration of quantitative material to be detected.
For a further understanding of the present invention, the technical scheme that the present invention is provided is carried out specifically with reference to embodiment
Bright, protection scope of the present invention is not limited by the following examples.
The raw material and chemical reagent that the embodiment of the present invention is adopted is commercial.
Embodiment 1
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.6 Sample dilution, pH
It is worth the cleaning solution for 6.3, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
5.6%, the concentration of phosphate buffer is 0.58mol/L, and the concentration of Tris buffer solns is 0.55mol/L；It is described
The concentration of bovine serum albumin(BSA) is 0.47% in cleaning solution, and the concentration of phosphate buffer is 0.49mol/L, and Tris is buffered
The concentration of liquor is 0.52mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.313 weight
Amount part, KH2PO41.05 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, bovine serum albumin
White 7 weight portion；The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 4.9, KH2PO42.45 weight portions, NaCl
8 weight portions, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
9500r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.055：0.051：15.88 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 60.5 DEG C, stir A liquid, it is quick plus
Enter B liquid, continue to stir 1 minute, boiling was heated in 30 seconds 11 minutes, make the collaurum of particle diameter 10.2nm, the glue
Body gold liquid color by it is black → blue → purple → red when；(2) step (1) collaurum liquid is used in the adjustment of collaurum pH value
0.25M K2CO3Adjustment pH value be 6.98 after it is standby；(3) preparation of human serum albumins antibody and dialysis, by antibody at 4 DEG C
9500r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing is standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum
Albumin antibody 0.288mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes,
Sequentially add the μ l of 10% sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 3.3 DEG C after stirring evenly, under the conditions of 6000r/min from
The heart 30 minutes, remove supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 50min, 3000r/min centrifugation 30min,
The μ L of Aspirate supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：Accurately inhaled with pipettor
25 μ L detection samples are taken, is put on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins is added dropwise
Antibody：After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) treat
After colloid gold label human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record
As a result：Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value passes through
Instrument combines calibration card information and calculates detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Embodiment 2
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.5 Sample dilution, pH
It is worth the cleaning solution for 6.2, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
0.%, the concentration of phosphate buffer is 0.05mol/L, and the concentration of Tris buffer solns is 0.01mol/L；It is described to wash
The concentration for washing bovine serum albumin(BSA) in liquid is 0.1%, and the concentration of phosphate buffer is 0.005mol/L, Tris buffer solutions
The concentration of solution is 0.01mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.263 weight
Part, KH2PO41.0 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, bovine serum albumin(BSA) 7
Weight portion；The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 4.6, KH2PO42.1 weight portions, the weights of NaCl 8
Amount part, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
6000r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.035：0.035：15.84 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 59 DEG C, stir A liquid, rapidly join
B liquid, continues to stir 1 minute, and boiling was heated in 10 minutes, makes the collaurum of particle diameter 8nm, the collaurum liquid color
By it is black → blue → purple → red when；(2) adjustment of collaurum pH value is by step (1) collaurum liquid 0.25M K2CO3Adjustment
PH value be 6.8 after it is standby；(3) preparation of human serum albumins antibody and dialysis, the 6000r/min centrifugations 60 at 4 DEG C by antibody
Minute, Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with distilled water dialysis desalination
Afterwards, bottling freezing, standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum albumins antibody
0.27mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes, sequentially adds 10%
The μ l of sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 2.5 DEG C after stirring evenly, are centrifuged 45 minutes under the conditions of 4600r/min, remove
Supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 15min, 2000r/min centrifugation 45min,
The μ L of Aspirate supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：Accurately inhaled with pipettor
25 μ L detection samples are taken, is put on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins is added dropwise
Antibody：After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) treat
After colloid gold label human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record
As a result：Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value passes through
Instrument combines calibration card information and calculates detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Embodiment 3
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.7 Sample dilution, pH
It is worth the cleaning solution for 6.4, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
10%, the concentration of phosphate buffer is 1mol/L, and the concentration of Tris buffer solns is 1mol/L；In the cleaning solution
The concentration of bovine serum albumin(BSA) is 10%, and the concentration of phosphate buffer is 1mol/L, the concentration of Tris buffer solns
For 1mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.376 weight portion, KH2PO4
1.109 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, the weight portion of bovine serum albumin(BSA) 7；
The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 5.5, KH2PO43.0 weight portions, the weight portions of NaCl 8,
The weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
13000r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.08：0.08：15.93 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 61 DEG C, stir A liquid, rapidly join B
Liquid, continues to stir 1 minute, and boiling was heated in 13 minutes, makes the collaurum of particle diameter 12nm, the collaurum liquid color
By it is black → blue → purple → red when；(2) adjustment of collaurum pH value is by step (1) collaurum liquid 0.25M K2CO3Adjustment
PH value be 7.1 after it is standby；(3) preparation of human serum albumins antibody and dialysis, the 13000r/min centrifugations 60 at 4 DEG C by antibody
Minute, Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with distilled water dialysis desalination
Afterwards, bottling freezing, standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum albumins antibody
0.30mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes, sequentially adds 10%
The μ l of sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 4.0 DEG C after stirring evenly, are centrifuged 19 minutes under the conditions of 8000r/min, remove
Supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 2h, 4000r/min centrifugation 20min, drawn
The μ L of supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：25 μ are accurately drawn with pipettor
L detects sample, puts on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins antibody is added dropwise：
After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) collaurum is treated
After mark human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record result：
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value is tied by instrument
Close calibration card information and calculate detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Upper embodiment and embodiment are both needed to the usage amount that appropriateness as the case may be adjusts temperature and distilled water, this explanation
It is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that for the ordinary skill people of the art
Member for, under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention, these improve and
Modification is also fallen in the protection domain of the claims in the present invention.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or using the present invention.
Various modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The most wide scope for causing.
Claims (10)
1. a kind of spot gold of detection microdose urine protein is percolated kit, it is characterised in that include：
Cleaning solution, colloid gold label human serum albumins that Sample dilution that pH value is 6.5～6.7, pH value are 6.2～6.4
Antibody, microdose urine protein chromatography device and calibration card,
The Sample dilution includes Triton-100, phosphate buffer and Tris buffer solns；
The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
2. spot gold according to claim 1 is percolated kit, it is characterised in that
The concentration of Triton-100 is 0.1～10% in the Sample dilution, and the concentration of phosphate buffer is 0.005
The concentration of～1mol/L, Tris buffer soln is 0.01～1mol/L；
The concentration of bovine serum albumin(BSA) is 0.1～10% in the cleaning solution, the concentration of phosphate buffer is 0.005～
The concentration of 1mol/L, Tris buffer soln is 0.01～1mol/L.
3. spot according to claim 1 gold diafiltration kit, it is characterised in that the phosphate buffer by with
Lower raw material is prepared：Na2HPO40.263-0.376 weight portions, KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6,
KCl0.2 weight portions, the weight portion of distilled water 1000, the weight portion of bovine serum albumin(BSA) 7；The Tris buffer solns are by three hydroxyl first
Base aminomethane 4.6-5.5 weight portions, KH2PO43.0-2.1 weight portions, the weight portions of NaCl 8, the weight portions of KCl 0.3, distilled water
1200 weight portions, the weight portion of bovine serum albumin(BSA) 9 are formulated.
4. spot gold according to claim 1 is percolated kit, it is characterised in that the colloid gold label human seralbumin egg
Bai Kangti is prepared as follows：
By gold chloride that mass fraction is 1% and distilled water by volume 1：79 are made into A liquid；By the lemon that mass fraction is 1%
Sour trisodium, mass fraction are 1% tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～
0.080：15.84～15.93 are made into B liquid；The A liquid and B liquid are heated in water-bath 59～61 DEG C simultaneously, A liquid is stirred, soon
Speed is added in B liquid, continues to stir 1 minute, and boiling was heated in 10～13 minutes, obtains collaurum liquid；
Using 0.25M K2CO3The collaurum liquid pH value is adjusted for 6.8～7.1；
By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；
The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing obtains human serum albumins antibody；
The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the human serum albumins antibody
It is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, sequentially adds 1～30% and changes
Nitrogen sodium and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) are with the volume ratio of the collaurum liquid
500μl：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min, removes supernatant, sinks
Starch is colloid gold label human serum albumins antibody.
5. spot gold according to claim 4 is percolated kit, it is characterised in that the trisodium citrate, tannic acid, K2CO3
It is 4 with the volume ratio of distilled water：0.08：0.08：15.84, the colloid of a diameter of 10 ＜ D ＜ 11nm is formed in the collaurum liquid
Gold grain.
6. spot gold according to claim 1 is percolated kit, it is characterised in that in the microdose urine protein chromatography device
The reaction box of a diameter of 2.8cm is provided with, the reaction box includes cassette bottom and lid, and the lid top surface central authorities are provided with one
The a diameter of 0.56cm in upper surface, the small sircle hole of a diameter of 0.27cm of lower surface；The full absorbent material of pad in the reaction box；Described
The coating human serum albumins antibody of a 1.18 × 1.18cm is placed between small sircle hole lower section and absorbent material top
Nitrocellulose filter.
7. spot gold according to claim 6 is percolated kit, it is characterised in that the coating human serum albumins antibody
Nitrocellulose filter according to following steps prepare：
By human serum albumins coated antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, in absorption
Clear liquid；
The supernatant is loaded into bag filter, with distilled water dialysis desalination, bottling freezing, obtains human serum after ultrafiltration concentration
Albumin antibody；
The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, obtains 0.8mg/ml's
Coating buffer；
The coating buffer is dropped on the nitrocellulose filter for cutting, every diaphragm 0.02ml is placed in diaphragm in baking oven
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after drying.
8. spot gold according to claim 1 is percolated kit, it is characterised in that the calibration card is human serum albumins
Gradient mass concentration solution detects the corresponding detected signal value of color.
9. spot gold according to claim 8 is percolated kit, it is characterised in that the gradient mass concentration is respectively
5mg/L、10mg/L、20mg/L、40mg/L、80mg/L、120mg/L、200mg/L。
10. the application of the spot gold diafiltration kit of a kind of detection microdose urine protein, it is characterised in that comprise the following steps：
Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant to sample is dilute
Release in liquid and mix, obtain sample to be tested；
Sample to be detected is accurately drawn using pipettor, is put on celluloid face in reaction box circular hole；
After sample to be detected penetrates into, colloid gold label human serum albumins antibody, the collaurum mark is added dropwise to reaction box face
Note human serum albumins antibody is 1 with the volume ratio of the detection sample：1；
After colloid gold label human serum albumins antibody penetrates into, be added dropwise cleaning solution on reaction box face, the cleaning solution with
The volume ratio of the detection sample is 1：1；
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value, by instrument
Device combines calibration card information and calculates detection sample microdose urine protein concentration, quantitatively judges its mass concentration.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610707362 | 2016-08-23 | ||
| CN2016107073623 | 2016-08-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106596968A true CN106596968A (en) | 2017-04-26 |
Family
ID=58595452
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611094793.3A Pending CN106771144A (en) | 2016-08-23 | 2016-12-02 | One kind detection C reactive proteins dot immunogold diafiltration kit and quantitative detecting method |
| CN201611094803.3A Pending CN106855573A (en) | 2016-08-23 | 2016-12-02 | Detection PSA spot gold diafiltration kit and its detection method |
| CN201611095552.0A Pending CN106596968A (en) | 2016-08-23 | 2016-12-02 | Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611094793.3A Pending CN106771144A (en) | 2016-08-23 | 2016-12-02 | One kind detection C reactive proteins dot immunogold diafiltration kit and quantitative detecting method |
| CN201611094803.3A Pending CN106855573A (en) | 2016-08-23 | 2016-12-02 | Detection PSA spot gold diafiltration kit and its detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN106771144A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108303550A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of the spot gold diafiltration kit and its quantitative detecting method of detection adiponectin |
| CN113009150A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting urine microalbumin in sweat, detection kit comprising same and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112485449A (en) * | 2020-11-23 | 2021-03-12 | 爱若维生物科技(苏州)有限公司 | Spot immunogold filtration kit for detecting cat SAA and semi-quantitative detection method |
| CN113588610B (en) * | 2021-07-21 | 2024-02-02 | 重庆创芯生物科技有限公司 | Filtering membrane pretreatment liquid for improving immunofluorescence detection sensitivity, and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1664585A (en) * | 2005-02-07 | 2005-09-07 | 浙江省医学科学院 | Domestic animal schistosomiasis antibody gold standard immune rapid detection kit and preparation method |
| CN1700008A (en) * | 2005-06-21 | 2005-11-23 | 浙江省农业科学院 | Diagnosis of domestic animal schistosomiasis spotted gold percolation kit and its preparation and application method |
| CN1866014A (en) * | 2006-05-19 | 2006-11-22 | 浙江省农业科学院 | Dot immunogold filtration assay kit for diagnosing cysticercosis cellulosae, preparation and application thereof |
| CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
| CN102608321A (en) * | 2012-02-29 | 2012-07-25 | 厦门大学 | Echinococcus multilocularis circulating antigen dot immunogold filtration kit and preparation method |
| CN204008663U (en) * | 2014-07-16 | 2014-12-10 | 李娟� | A kind of semi-quantitative gold immunity percolation microdose urine protein detection kit |
| CN105785042A (en) * | 2016-04-06 | 2016-07-20 | 陈秀海 | Detection kit for epidemic hemorrhagic fever virus |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2213997Y (en) * | 1994-08-30 | 1995-11-29 | 徐兵 | Checkout apparatus for spot immunity with one-step percolation method |
| CN1164949C (en) * | 2001-08-17 | 2004-09-01 | 上海数康生物科技有限公司 | Reagent case for synchronous detecting multiple kinds of infectious disease and its preparing method |
| CN101320041B (en) * | 2007-08-09 | 2012-11-14 | 上海奥普生物医药有限公司 | Colloidal gold method for fast quantitative determination of C-reaction protein and its application |
-
2016
- 2016-12-02 CN CN201611094793.3A patent/CN106771144A/en active Pending
- 2016-12-02 CN CN201611094803.3A patent/CN106855573A/en active Pending
- 2016-12-02 CN CN201611095552.0A patent/CN106596968A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1664585A (en) * | 2005-02-07 | 2005-09-07 | 浙江省医学科学院 | Domestic animal schistosomiasis antibody gold standard immune rapid detection kit and preparation method |
| CN1700008A (en) * | 2005-06-21 | 2005-11-23 | 浙江省农业科学院 | Diagnosis of domestic animal schistosomiasis spotted gold percolation kit and its preparation and application method |
| CN1866014A (en) * | 2006-05-19 | 2006-11-22 | 浙江省农业科学院 | Dot immunogold filtration assay kit for diagnosing cysticercosis cellulosae, preparation and application thereof |
| CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
| CN102608321A (en) * | 2012-02-29 | 2012-07-25 | 厦门大学 | Echinococcus multilocularis circulating antigen dot immunogold filtration kit and preparation method |
| CN204008663U (en) * | 2014-07-16 | 2014-12-10 | 李娟� | A kind of semi-quantitative gold immunity percolation microdose urine protein detection kit |
| CN105785042A (en) * | 2016-04-06 | 2016-07-20 | 陈秀海 | Detection kit for epidemic hemorrhagic fever virus |
Non-Patent Citations (1)
| Title |
|---|
| 曹丹如 等。: "快速检测尿微量白蛋白的半定量金免疫渗滤法的研究", 《检验医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108303550A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of the spot gold diafiltration kit and its quantitative detecting method of detection adiponectin |
| CN113009150A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting urine microalbumin in sweat, detection kit comprising same and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106771144A (en) | 2017-05-31 |
| CN106855573A (en) | 2017-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101659529B1 (en) | Paper based diagnostic test | |
| CN105445449B (en) | Saliva uric acid rapid semi-quantitative detection means and preparation method thereof | |
| CN108414748A (en) | A kind of test strip and detection method of THSD7A antibody | |
| CN106596968A (en) | Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit | |
| US20220113322A1 (en) | Rapid measurement of total vitamin d in blood | |
| JP2011502260A (en) | Method and apparatus for detecting occult blood | |
| CN107102150A (en) | A kind of microdose urine protein determines kit and preparation method thereof | |
| CN108627652B (en) | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen | |
| CN102841210A (en) | Retinol detection kit and preparation method thereof | |
| CN106324251B (en) | The preparation method and beta 2-microglobulin detecting kit of small fragment BMG antibody | |
| JP4223163B2 (en) | Immunochromatographic test strip and chromatographic analysis method | |
| CN204330777U (en) | People's myeloperoxidase (MPO) fast quantification immunochromatographytest test kit | |
| CN106153893B (en) | The enzyme-linked immunologic detecting kit of saliva anti-mitochondrial antibody M2 types | |
| CN101738476B (en) | Rapid diagnosis kit for pre-S1 antigens of hepatitis B viruses and method for preparing same | |
| CN106771145A (en) | One kind detection D dimers spot gold diafiltration kit and quantitative detecting method | |
| CN101504417A (en) | Colloid selenium test paper for semi-quantitative determination of urinary trace albumin | |
| CN117110613A (en) | Diagnostic kit and application of LMWK-Fc in preparation of liver fibrosis diagnostic reagent | |
| CN104391124A (en) | Myohemoglobin detection reagent and preparation method thereof | |
| WO2004081568A1 (en) | Method of examining specimen and specimen container to be used in the examinaton method | |
| CN107860930A (en) | The immunoturbidimetry detection reagent and method of a kind of cardic fatty acid binding protein | |
| Datta | Methodological issues in point of care testing devices | |
| JP2010117244A (en) | Method for measuring hemoglobin and kit for measurement | |
| US20090191646A1 (en) | Molybdenum complex & test kit to enhance accuracy of analysis of endogenous analytes in biological fluids | |
| JP2004251851A (en) | Examination method of feces component | |
| WO2001061343A1 (en) | Method for examination of feces occult blood |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170426 |
|
| RJ01 | Rejection of invention patent application after publication |