CN106885902A - Gold-labeled kit with NCAM 1 as Testing index and its preparation method and application - Google Patents
Gold-labeled kit with NCAM 1 as Testing index and its preparation method and application Download PDFInfo
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- CN106885902A CN106885902A CN201710123596.8A CN201710123596A CN106885902A CN 106885902 A CN106885902 A CN 106885902A CN 201710123596 A CN201710123596 A CN 201710123596A CN 106885902 A CN106885902 A CN 106885902A
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 147
- 238000012360 testing method Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims description 13
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 title abstract description 7
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 title abstract 6
- 238000001514 detection method Methods 0.000 claims abstract description 79
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 44
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 44
- 208000005777 Lupus Nephritis Diseases 0.000 claims abstract description 42
- 230000000274 adsorptive effect Effects 0.000 claims abstract description 25
- 241000283707 Capra Species 0.000 claims abstract description 20
- 229940027941 immunoglobulin g Drugs 0.000 claims abstract description 20
- 239000011248 coating agent Substances 0.000 claims abstract description 15
- 238000000576 coating method Methods 0.000 claims abstract description 15
- 210000002700 urine Anatomy 0.000 claims abstract description 11
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
- 238000005859 coupling reaction Methods 0.000 claims abstract description 3
- 108050003738 Neural cell adhesion molecule 1 Proteins 0.000 claims description 119
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 119
- 239000000243 solution Substances 0.000 claims description 96
- 239000010931 gold Substances 0.000 claims description 46
- 229910052737 gold Inorganic materials 0.000 claims description 46
- 239000007853 buffer solution Substances 0.000 claims description 40
- 239000007788 liquid Substances 0.000 claims description 25
- 238000007792 addition Methods 0.000 claims description 21
- 239000000463 material Substances 0.000 claims description 20
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 19
- 229940098773 bovine serum albumin Drugs 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 18
- 239000002250 absorbent Substances 0.000 claims description 16
- 230000002745 absorbent Effects 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 16
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 11
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 229940127121 immunoconjugate Drugs 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical class OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000004026 adhesive bonding Methods 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 239000012467 final product Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- -1 trihydroxy methyl amino Chemical group 0.000 claims 2
- 241000283725 Bos Species 0.000 claims 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical class NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims 1
- 108010071390 Serum Albumin Proteins 0.000 claims 1
- 102000007562 Serum Albumin Human genes 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 34
- 239000007864 aqueous solution Substances 0.000 description 8
- 206010013786 Dry skin Diseases 0.000 description 6
- 239000003365 glass fiber Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 4
- 238000005374 membrane filtration Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- 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 description 4
- 229940038773 trisodium citrate Drugs 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000004745 nonwoven fabric Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 239000011260 aqueous acid Substances 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000012073 inactive phase Substances 0.000 description 2
- 206010025135 lupus erythematosus Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 101000581982 Mus musculus Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 102100023616 Neural cell adhesion molecule L1-like protein Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical class CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical class [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000011862 kidney biopsy Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000009603 uroscopy Methods 0.000 description 1
- 230000002792 vascular 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
- G01N33/558—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
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
Abstract
The present invention is using NCAM 1 as Testing index, it is applied to gold-labeled kit, the kit of lupus nephritis liveness must be can detect, including base plate, adsorptive pads, nitrocellulose filter, gold standard pad and sample pad, sample pad, gold standard pad, nitrocellulose filter and adsorptive pads connect be covered on base plate successively, and gold standard pad is covered by sample pad part；Be provided with the nitrocellulose filter by mouse it is anti-human/detection line that is formed of the monoclonal antibodies of rabbit-anti people NCAM 1, any one coating in rabbit-anti people/polyclonal antibodies of goat-anti people NCAM 1；By any one control line that is formed of coating in goat anti-mouse immunoglobulin G and goat anti-rabbit immunoglobulin G polyclonal antibodies, gold standard pad be coated with collaurum it is anti-human with mouse/monoclonal antibodies of rabbit-anti people NCAM 1 in any one coupling formation conjugate.The concentration of NCAM 1 in kit detection urine specimen, so as to judge lupus nephritis liveness, realizes quick, Non-invasive detection.
Description
Technical field
The invention belongs to medical detection instrument, be related to the detection instrument of lupus nephritis liveness, it is more particularly to a kind of with
NCAM-1 is gold-labeled kit of Testing index and its preparation method and application.
Background technology
Lupus nephritis is one of most common complication of systemic loupus erythematosus, and the whole latter stage kidney caused by lupus nephritis
Disease is the principal element for causing lupus erythematosus patients dead.And it is topmost to develop into ESRD from lupus nephritis
Reason result in treatment not in time just because the lupus nephritis of active period is not found in time, and kidney triggers irreversible
Glomerulosclerosis and interstitial vascular fibrosis.For the judgement of lupus nephritis liveness, " goldstandard " is by renal puncture
Biopsy carries out pathological examination to renal tissue, is diagnosed according to pathological examination result.And renal biopsy is
Invasive inspection, contraindication is more, it is impossible to repeatedly conventional to carry out, therefore clinically lacks noninvasive, accurate, quick, safe at present
Detection method and instrument.
NCAM-1（Neural cell adhesion molecule~1, Chinese name：N-CAM~1）It is
A kind of non-Ca-dependent adhesion factor, all has having certain effect at the aspect such as cell signal identification and conduction, nerve regneration.
The content of the invention
Inventor has found that concentration of the NCAM-1 in active period patients with lupus nephritis urine is obvious in long-term research
Higher than inactive phase patients with lupus nephritis and do not merge concentration in the Patients with SLE urine of lupus nephritis, because
This, based on this result of study, NCAM-1 can be used as the diagnosis index of kidneys with lupus nephritis inflammation liveness.
It is first it is an object of the invention to overcome the shortcomings of that prior art detects lupus nephritis liveness by percutaneous biopsy
It is secondary to be specifically applied in gold-labeled kit by Testing index of NCAM-1, in obtaining gold-labeled kit by detecting urine specimen
NCAM-1 concentration is Testing index, so as to judge lupus nephritis liveness, realizes quick, Non-invasive detection.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions；
NCAM-1 is application of the Testing index in the gold-labeled kit of detection lupus nephritis liveness.The present invention first will
NCAM-1 is applied to gold-labeled kit as Testing index, prepares and can be used for the gold mark examination for detecting lupus nephritis liveness
Agent box, the gold-labeled kit by detecting urine specimen in NCAM-1 concentration be used as index, so as to judge lupus nephritis liveness.
The gold-labeled kit of above-mentioned detection lupus nephritis liveness, including base plate 7, adsorptive pads 6, nitrocellulose filter 3,
Gold standard pad 2 and sample pad 1, the sample pad, gold standard pad, nitrocellulose filter 3 and adsorptive pads 6 connect be covered in base plate successively
On, and gold standard pad covered by sample pad part；Be provided with the nitrocellulose filter by the anti-human NCAM-1 monoclonal antibodies of mouse,
Rabbit-anti people's NCAM-1 monoclonal antibodies, it is any in rabbit-anti people NCAM-1 polyclonal antibodies and goat-anti people's NCAM-1 polyclonal antibodies
It is a kind of to be coated with the detection line for being formed；By any in goat anti-mouse immunoglobulin G and goat anti-rabbit immunoglobulin G polyclonal antibodies
A kind of to be coated with the control line for being formed, the gold standard pad is coated with collaurum and the anti-human NCAM-1 monoclonal antibodies of mouse and rabbit-anti people
The conjugate that any one coupling in NCAM-1 monoclonal antibodies is formed.
Using detection instrument of the present invention, when visible one of Quality Control line position clearly red line, it was demonstrated that institute of the present invention
State fast detecting tool effective；In the case of fast detecting tool of the present invention is effective, when visible one of line position of detection
Clearly red line, this result judgement is the positive（I.e. the patient is in lupus nephritis active period）；If detection line position does not occur
Red line, then be judged to feminine gender（I.e. the patient is in the lupus nephritis inactive phase）.
The detection line, the width of control line are respectively controlled to 0.5 mm~0.8 mm, between the detection line and control line
Spacing be controlled to 5 mm~10 mm.
The gold-labeled kit of detection lupus nephritis liveness of the invention is shaped as a rectangular strip.The sample pad be by
The pad that glass fibre with absorbent function, polyester fiber film, cellulosic filter paper or non-woven fabrics make.Gold standard pad is water-absorption material
Material makes, and absorbent material is including glass fibre, polyester fiber film, non-woven fabrics or cellulosic filter paper etc..The adsorptive pads 6 are by inhaling
The pad that water paper makes.
The preparation of the kit comprises the steps：
（1）Detection line, the coating of nature controlling line：
With the anti-human NCAM-1 monoclonal antibodies of mouse, rabbit-anti people's NCAM-1 monoclonal antibodies, rabbit-anti people NCAM-1 polyclonal antibodies and
In goat-anti people's NCAM-1 polyclonal antibodies any one be solute, additions cushioning liquid be solvent, be configured to concentration be 1~
The detection line solution of 4mg/mL；With goat anti-mouse immunoglobulin G polyclonal antibodies or goat anti-rabbit immunoglobulin G Anti-TNF-αs
Body is solute, and addition cushioning liquid is solvent, is configured to the nature controlling line solution that concentration is 0.5~1 mg/mL；Will by Film-cutting machine
The control line solution for preparing, detection line solution are coated on nitrocellulose filter, and the nitrocellulose filter is attached to base plate
On, the nitrocellulose filter-floor combination body after coating will be completed in 37 DEG C of dryings 12~16 hours；
（2）Prepare gold standard pad：
1. with the anti-human NCAM-1 monoclonal antibodies of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies as solute, it is molten to add buffer solution
Agent, compound concentration is the anti-human NCAM-1 monoclonal antibody solutions of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies of 0.5~2mg/mL
Solution；
2. by step 1. in the anti-human NCAM-1 monoclonal antibody solutions of mouse that prepare or rabbit-anti people's NCAM-1 monoclonal antibodies
Solution mixes with appropriate colloidal gold solution, and the anti-human NCAM-1 monoclonal antibodies of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies is obtained
Concentration is the mixed liquor of 10~20 μ g/mL；
3. mixed liquor shaking table mark 20min~30 min for 2. step being prepared, is subsequently adding concentration for 1 mg/mL ox bloods
Pure protein solution simultaneously continues shaking table mark 20min~30 min, and mark is centrifuged after terminating, and it is anti-human that the precipitation of acquisition is mouse
NCAM-1 monoclonal antibodies conjugate or rabbit-anti people's NCAM-1 monoclonal antibody conjugates；With 8000rpm~12 during centrifugation,
000rpm is centrifuged 30min~40min；
4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, the gold mark mouse for being configured to the mg/mL of concentration 0.2~0.4 resists
People NCAM-1 monoclonal antibodies or gold mark rabbit-anti people's NCAM-1 monoclonal antibody conjugate solution,
5. the gold mark anti-human NCAM-1 monoclonal antibodies of mouse or gold mark rabbit-anti people NCAM-1 monoclonal antibodies are combined by metal spraying machine
Thing solution is sprayed on the absorbent material for making gold standard pad, and gold standard pad is obtained within 12~16 hours in 37 DEG C of dryings.
In the present invention, colloidal gold solution is prepared as follows：Take gold chloride to be dissolved in tri-distilled water, form gold chloride dense
It is the aqueous solution of chloraurate of 0.1~0.2g/100 mL to spend, and be heated to solution boiling, under agitation by concentration be 0.9~
The trisodium citrate aqueous solution of 1.1g/100mL is added in the aqueous solution of chloraurate of boiling, and trisodium citrate aqueous solution is golden with chlorine
The volume ratio of aqueous acid is 1.35~1.5:50, heating is stopped when the mixed liquor is presented transparent claret, allow it certainly
K is used after being so cooled to room temperature2CO3Solution adjusts pH to 6~7, that is, obtain colloidal gold solution；
（3）Combination
By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body,
Obtain final product the gold-labeled kit.
In step（1）In, buffer solution is phosphate buffer, carbonate buffer solution, boric acid when preparing detection line solution
Any one in salt buffer, trishydroxymethylaminomethane salt buffer；It is phosphate to prepare nature controlling line solution buffer solution
Any one in buffer solution, carbonate buffer solution, borate buffer solution；Step 1. in, the buffer solution is slow for phosphate
Any one in fliud flushing, carbonate buffer solution, borate buffer solution, trishydroxymethylaminomethane salt buffer.
Step 3. in, the concentration of the addition of the bovine serum albumin solution with bovine serum albumin(BSA) in mixed liquor
0.1g/100 mL are reached to be defined.
Step 4. in, the gold mark redissolve liquid be Tris, bovine serum albumin(BSA), Sodium azide are dissolved in tri-distilled water and
Into, the gold mark is redissolved in liquid, and the concentration of Tris is 0.01~0.02 mol/L, the concentration of bovine serum albumin(BSA) for 0.1~
0.5g/100mL, the concentration of Sodium azide is 0.2g/100mL；
Step 5. in, the gold mark anti-human NCAM-1 monoclonal antibodies of mouse or gold mark rabbit-anti people NCAM-1 monoclonal antibodies are combined
The discharge rate of thing is 2~4 μ l/cm2。
PH=7.2~7.4 of the phosphate buffer, concentration is 0.01mol/L, the pH=of the carbonate buffer solution
9.2~10.7, concentration is 0.05mol/L, and pH=8.5~10.0 of the borate buffer solution, concentration is 0.05mol/L, institute
PH=7.0~9.2 of trishydroxymethylaminomethane salt buffer are stated, concentration is 0.05mol/L.
The invention has the advantages that：
1st, it is of the invention for patients with lupus nephritis detection ephritis liveness provides a kind of new detection work using new Testing index
Tool.
2nd, it is non-to clinical definite lupus nephritis active period and clinical definite lupus nephritis using detection instrument of the present invention
Active period patient carries out sample detecting, and testing result shows：The sensitivity of the detection instrument reaches as high as 90% or so, specificity
Highest is also up to 90% or so.
3rd, simple to operate using detection instrument of the present invention, one sample of detection is only needed 3~6 minutes, and clinically existing
There is detection method（Goldstandard, common detection methods）Compare, detection speed is increased substantially.Because detection sample is the urine of patient
Liquid sample, thus be a kind of Non-invasive detection.
4th, detection tool construction of the present invention is simple, it is easy to processing and fabricating, low production cost, is easy to industrialized production.
Brief description of the drawings
1st, Fig. 1 is gold-labeled kit structural representation of the present invention.
Reference in above-mentioned accompanying drawing：1 sample pad, 2 gold standard pads, 3 nitrocellulose filters, 4 detection lines, 5 control lines, 6
Adsorptive pads, 7 base plates.
Specific embodiment
The present invention is described in detail with reference to specific embodiment.
In following embodiments：
The anti-human NCAM-1 monoclonal antibodies of mouse, rabbit-anti people's NCAM-1 monoclonal antibodies, rabbit-anti people NCAM-1 polyclonal antibodies,
Goat-anti people NCAM-1 polyclonal antibodies are purchased from bio-techne companies of the U.S., Abcam companies of the U.S. respectively；
Goat anti-mouse immunoglobulin G and goat anti-rabbit immunoglobulin G polyclonal antibodies are purchased from Wuhan doctor's moral company.
Phosphate buffer, carbonate buffer solution, borate buffer solution, trishydroxymethylaminomethane salt buffer can be certainly
Make, also the commercially available commercially available each buffer solution for meeting above-mentioned requirements.
It is prepared by phosphate buffer：Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphates and 0.24g di(2-ethylhexyl)phosphates
Hydrogen potassium is dissolved in 900mL distilled waters, with hydrochloric acid conditioning solution pH value to 7.2, is eventually adding distilled water and is settled to 1L, and solution passes through
It is stored in after 0.45 μm of membrane filtration in 4 DEG C of refrigerators.
It is prepared by borate buffer solution：Weigh 19.07g boraxs to be dissolved in 900mL distilled waters, pH value of solution is adjusted with NaOH
Value is eventually adding distilled water and is settled to 1L to 9.0, and solution is in being stored in 4 DEG C of refrigerators after 0.45 μm of membrane filtration.
It is prepared by carbonate buffer solution：Weigh 0.16g sodium carbonate and 0.3g sodium acid carbonates are dissolved in 1L distilled waters and are pH and are
9.6 carbonate buffer solutions, solution is in being stored in 4 DEG C of refrigerators after 0.45 μm of membrane filtration.
It is prepared by trishydroxymethylaminomethane salt buffer：Weigh 6.06g trishydroxymethylaminomethanes and be dissolved in 800mL distilled waters
In, with hydrochloride adjusted solution pH value to 8.0, it is eventually adding distilled water and is settled to 1L, solution after 0.45 μm of membrane filtration by protecting
It is stored in 4 DEG C of refrigerators.
Bovine serum albumin(BSA), Sodium azide are purchased from Suo Laibao companies.
Embodiment 1
Application using NCAM-1 as the gold-labeled kit of Testing index in lupus nephritis liveness is detected, described gold mark examination
Agent box by detecting urine specimen in NCAM-1 concentration be used as index.
The gold-labeled kit of the above-mentioned detection lupus nephritis liveness using NCAM-1 as Testing index, including base plate 7,
Adsorptive pads 6, nitrocellulose filter 3, gold standard pad and sample pad 1, the sample pad 1, gold standard pad 2, nitrocellulose filter 3 and water suction
Pad 6 connects be covered on base plate 7 successively, and the pad of gold mark 2 is by the covering of the pad part of sample 1；It is provided with the nitrocellulose filter 3
By being coated with the detection line 4 for being formed in goat-anti people's NCAM-1 polyclonal antibodies；It is coated with by goat anti-mouse immunoglobulin G polyclonal antibodies
The control line 5 of formation, the gold standard pad 2 is coated with collaurum and the anti-human NCAM-1 monoclonal antibodies of mouse are coupled the combination to be formed
Thing.
In the present embodiment, the detection line 4, the width of control line 5 are respectively controlled to 0.5mm, the detection line 4 and control line
Spacing between 5 is controlled to 10mm.
In the present embodiment, the gold-labeled kit of described detection lupus nephritis liveness is shaped as a rectangular strip.It is described
Sample pad 1 is the pad made by the glass fibre with absorbent function.Gold standard pad 2 makes for absorbent material, and absorbent material is
Glass fibre.The adsorptive pads 6 are the pads made by blotting paper.
In order to preferably implement the present invention, the present embodiment further provides the gold mark examination using NCAM-1 as Testing index
The preparation method of agent box, the preparation of the kit comprises the steps：
（1）Detection line, the coating of nature controlling line：
Goat-anti people NCAM-1 polyclonal antibodies are solute, and addition PBS is solvent, are configured to concentration for 1mg/mL
Detection line solution；With goat anti-mouse immunoglobulin G polyclonal antibodies as solute, addition PBS is solvent, is matched somebody with somebody
It is made the nature controlling line solution that concentration is 0.5mg/mL；Control line solution, the detection line solution bag that will be prepared by Film-cutting machine
By on nitrocellulose filter, the nitrocellulose filter is attached on base plate, will complete the nitrocellulose filter-base plate after coating
Assembly was in 37 DEG C of dryings 12 hours；
（2）Prepare gold standard pad：
1. with the anti-human NCAM-1 monoclonal antibodies of mouse as solute, addition phosphate buffer is solvent, and compound concentration is 1mg/mL
The anti-human NCAM-1 monoclonal antibody solutions of mouse；
2. by step 1. in the anti-human NCAM-1 monoclonal antibody solutions of mouse that prepare mix with appropriate colloidal gold solution, be obtained
The concentration of the anti-human NCAM-1 monoclonal antibodies of mouse is the mixed liquor of 10 μ g/mL；
3. the mixed liquor shaking table mark 20min for 2. step being prepared, is subsequently adding concentration for 1 mg/mL bovine serum albumin(BSA)s
Solution simultaneously continues shaking table mark 20min, and mark is centrifuged after terminating, and the precipitation of acquisition is the anti-human NCAM-1 monoclonal antibodies knot of mouse
Compound；30~40 min are centrifuged with 9000rpm during centrifugation；
4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, the gold mark mouse for being configured to concentration 0.3mg/mL is anti-human
NCAM-1 monoclonal antibody conjugate solution；
5. the gold mark anti-human NCAM-1 monoclonal antibodies conjugate solution of mouse is sprayed on by the water-absorption material for making gold standard pad by metal spraying machine
On material, gold standard pad is obtained within 12 hours in 37 DEG C of dryings.
In the present embodiment, colloidal gold solution is prepared as follows：Take gold chloride to be dissolved in tri-distilled water, form gold chloride
Concentration is the aqueous solution of chloraurate of 0.1~0.2g/100 mL, and be heated to solution boiling, under agitation by concentration be 0.9~
The trisodium citrate aqueous solution of 1.1g/100mL is added in the aqueous solution of chloraurate of boiling, and trisodium citrate aqueous solution is golden with chlorine
The volume ratio of aqueous acid is 1.35~1.5:50, heating is stopped when the mixed liquor is presented transparent claret, allow it certainly
K is used after being so cooled to room temperature2CO3Solution adjusts pH to 6~7, that is, obtain colloidal gold solution；
（3）Combination
By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body,
Obtain final product the gold-labeled kit.
Embodiment 2
Using NCAM-1 as the gold-labeled kit of Testing index, including base plate 7, adsorptive pads 6, nitrocellulose filter 3, gold standard pad 2
With sample pad 1, the sample pad 1, gold standard pad 2, nitrocellulose filter 3 and adsorptive pads 6 connect be covered on base plate 7 successively, and
Gold standard pad 2 is covered by the part of sample pad 1；It is provided with the nitrocellulose filter 3 by rabbit-anti people's NCAM-1 monoclonal antibodies
It is coated with the detection line 4 for being formed；By goat anti-mouse immunoglobulin G）The control line 5 for being formed is coated with, the gold standard pad 2 is coated with colloid
Gold is coupled the conjugate to be formed with the anti-human NCAM-1 monoclonal antibodies of mouse.
In the present embodiment, the detection line 4, the width of control line 5 are respectively controlled to 0.5mm, the detection line 4 and control line
Spacing between 5 is controlled to 10mm.
In the present embodiment, the gold-labeled kit of described detection lupus nephritis liveness is shaped as a rectangular strip.It is described
Sample pad 1 is the pad made by the glass fibre with absorbent function.Gold standard pad 2 makes for absorbent material, and absorbent material is
Glass fibre.The adsorptive pads 6 are the pads made by blotting paper.
In order to preferably implement the present invention, the present embodiment further provides the gold mark examination using NCAM-1 as Testing index
The preparation method of agent box, the preparation method of the present embodiment comprises the steps：
（1）Detection line, the coating of nature controlling line：
With rabbit-anti people's NCAM-1 monoclonal antibodies as solute, addition cushioning liquid is solvent, is configured to the inspection that concentration is 1mg/mL
Survey line solution；With goat anti-mouse immunoglobulin G polyclonal antibodies as solute, addition cushioning liquid is solvent, and being configured to concentration is
The nature controlling line solution of 0.5mg/mL；It is fine that the control line solution that will be prepared by Film-cutting machine, detection line solution are coated on nitric acid
On the plain film of dimension, the nitrocellulose filter is attached on base plate, will complete the nitrocellulose filter-floor combination body after coating 37
DEG C drying 14 hours；
（2）Prepare gold standard pad：
1. with the anti-human NCAM-1 monoclonal antibodies of mouse as solute, addition buffer solution is solvent, and compound concentration is that the mouse of 1 mg/mL resists
People's NCAM-1 monoclonal antibody solutions；
2. by step 1. in the anti-human NCAM-1 monoclonal antibody solutions of mouse that prepare mix with appropriate colloidal gold solution, be obtained
The concentration of the anti-human NCAM-1 monoclonal antibodies of mouse is the mixed liquor of 10 μ g/mL；
3. the min of mixed liquor shaking table mark 30 for 2. step being prepared, is subsequently adding concentration for 1mg/mL bovine serum albumin(BSA)s
Solution simultaneously continues the min of shaking table mark 30, and mark is centrifuged after terminating, and the precipitation of acquisition is the anti-human NCAM-1 monoclonal antibodies of mouse
Conjugate；30 min are centrifuged with 12,000 rpm during centrifugation；
4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, the gold mark mouse for being configured to concentration 0.2mg/mL is anti-human
NCAM-1 monoclonal antibodies or conjugate solution,
5. the gold mark anti-human NCAM-1 monoclonal antibodies body conjugate solution of mouse is sprayed on by the water suction for making gold standard pad by metal spraying machine
On material, gold standard pad is obtained within 16 hours in 37 DEG C of dryings.
（3）Combination
By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body,
Obtain final product the gold-labeled kit.
In the present embodiment, in step（1）In, prepare detection line solution when buffer solution be phosphate buffer,；Prepare matter
Control line solution buffer solution is phosphate buffer；Step 1. in, the buffer solution be phosphate buffer.
In the present embodiment, step 3. described in bovine serum albumin solution addition with bovine serum albumin(BSA) in mixed liquor
In concentration reach 0.1g/100 mL and be defined.
Step 2. described in colloidal gold solution by the method for embodiment 1 prepare.
Step 4. described in gold mark redissolve liquid it is in the same manner as in Example 1.
Step 5. described in gold mark the anti-human NCAM-1 monoclonal antibodies of mouse discharge rate be 4 μ L/cm2。
Embodiment 3
Using NCAM-1 as the gold-labeled kit of Testing index, including base plate 7, adsorptive pads 6, nitrocellulose filter, gold standard pad and
Sample pad, the sample pad 1, gold standard pad 2, nitrocellulose filter 3 and adsorptive pads 6 connect be covered on base plate 7 successively, and gold mark
Pad 2 is covered by the part of sample pad 1；It is provided with the nitrocellulose filter 3 and shape is coated with by rabbit-anti people NCAM-1 polyclonal antibodies
Into detection line 4；The control line 5 for being formed is coated with by goat anti-mouse immunoglobulin G, the gold standard pad 2 is coated with collaurum and mouse
Anti-human NCAM-1 monoclonal antibodies are coupled the conjugate to be formed.
In the present embodiment, the detection line 4, the width of control line 5 are respectively controlled to 0.5mm, the detection line 4 and control line
Spacing between 5 is controlled to 10mm.
In the present embodiment, the gold-labeled kit of described detection lupus nephritis liveness is shaped as a rectangular strip.It is described
Sample pad 1 is the pad made by the cellulosic filter paper with absorbent function.Gold standard pad 2 makes for absorbent material, absorbent material
It is cellulosic filter paper.The adsorptive pads 6 are the pads made by blotting paper.
In order to preferably implement the present invention, the present embodiment further provides the gold mark examination using NCAM-1 as Testing index
The preparation method of agent box, the preparation method of the present embodiment comprises the steps：
（1）Detection line, the coating of nature controlling line：
With rabbit-anti people's NCAM-1 polyclonal antibodies as solute, addition cushioning liquid is solvent, is configured to the inspection that concentration is 1mg/mL
Survey line solution；With goat anti-mouse immunoglobulin G polyclonal antibodies as solute, addition cushioning liquid is solvent, and being configured to concentration is
The nature controlling line solution of 0.5mg/mL；It is fine that the control line solution that will be prepared by Film-cutting machine, detection line solution are coated on nitric acid
On the plain film of dimension, the nitrocellulose filter is attached on base plate, will complete the nitrocellulose filter-floor combination body after coating 37
DEG C drying 13 hours；
（2）Prepare gold standard pad：
1. with the anti-human NCAM-1 monoclonal antibodies of mouse as solute, addition buffer solution is solvent, and compound concentration is that the mouse of 1 mg/mL resists
People's NCAM-1 monoclonal antibody solutions；The buffer solution is phosphate buffer.
2. by step 1. in the anti-human NCAM-1 monoclonal antibody solutions of mouse that prepare mix with appropriate colloidal gold solution,
The concentration that the anti-human NCAM-1 monoclonal antibodies of mouse are obtained is the mixed liquor of 10 μ g/mL；
3. the mixed liquor shaking table mark 30min for 2. step being prepared, is subsequently adding concentration for 1 mg/mL bovine serum albumin(BSA)s
Solution simultaneously continues shaking table mark 30min, and mark is centrifuged after terminating, and the precipitation of acquisition is the anti-human NCAM-1 monoclonal antibodies knot of mouse
Compound；40min is centrifuged with 10,000rpm during centrifugation；
4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, the gold mark mouse for being configured to concentration 0.4mg/mL is anti-human
NCAM-1 monoclonal antibody conjugate solution；
5. the gold mark anti-human NCAM-1 monoclonal antibodies conjugate solution of mouse is sprayed on by the water-absorption material for making gold standard pad by metal spraying machine
On material, dry 12h at 37 DEG C and obtain gold standard pad.
（3）Combination
By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body,
Obtain final product the gold-labeled kit.
In the present embodiment, in step（1）In, prepare detection line solution when buffer solution be phosphate buffer,；Prepare matter
Control line solution buffer solution is phosphate buffer.
In the present embodiment, step 3. described in bovine serum albumin solution addition with bovine serum albumin(BSA) in mixed liquor
In concentration reach 0.1g/100 mL and be defined.
Step 2. described in colloidal gold solution by the method for embodiment 1 prepare.
Step 4. described in gold mark redissolve liquid it is in the same manner as in Example 1.
Step 5. described in gold mark the anti-human NCAM-1 monoclonal antibodies conjugate of mouse discharge rate be 4 μ L/cm2。
Embodiment 4
Using NCAM-1 as the gold-labeled kit of Testing index, including base plate 7, adsorptive pads 6, nitrocellulose filter, gold standard pad and
Sample pad, the sample pad 1, gold standard pad 2, nitrocellulose filter 3 and adsorptive pads 6 connect be covered on base plate 7 successively, and gold mark
Pad 2 is covered by the part of sample pad 1；It is provided with the nitrocellulose filter 3 and shape is coated with by goat-anti people NCAM-1 polyclonal antibodies
Into detection line 4；The control line 5 for being formed is coated with by goat anti-rabbit immunoglobulin G polyclonal antibodies, the gold standard pad 2 is coated with
Collaurum is coupled the conjugate to be formed with rabbit-anti people's NCAM-1 monoclonal antibodies.
In the present embodiment, the detection line 4, the width of control line 5 are respectively controlled to 0.5mm, the detection line 4 and control line
Spacing between 5 is controlled to 10mm.
In the present embodiment, the gold-labeled kit of described detection lupus nephritis liveness is shaped as a rectangular strip.It is described
Sample pad 1 is the pad made by the non-woven fabrics with absorbent function.Gold standard pad 2 makes for absorbent material, and absorbent material is nothing
Spin cloth.The adsorptive pads 6 are the pads made by blotting paper.
In order to preferably implement the present invention, the present embodiment further provide the present embodiment using NCAM-1 as gold mark examination
The preparation method of agent box, the preparation method of the present embodiment comprises the steps：
（1）Detection line, the coating of nature controlling line：
With goat-anti people's NCAM-1 polyclonal antibodies as solute, addition cushioning liquid is solvent, is configured to the inspection that concentration is 1mg/mL
Survey line solution；With goat anti-rabbit immunoglobulin G polyclonal antibodies as solute, addition cushioning liquid is solvent, and being configured to concentration is
The nature controlling line solution of 1mg/mL；The control line solution that to be prepared by Film-cutting machine, detection line solution are coated on cellulose nitrate
On plain film, the nitrocellulose filter is attached on base plate, will complete the nitrocellulose filter-floor combination body after coating at 37 DEG C
Dry 14 hours；
（2）Prepare gold standard pad：
1. with rabbit-anti people's NCAM-1 monoclonal antibodies as solute, addition buffer solution is solvent, and compound concentration is the rabbit-anti of 2 mg/mL
People's NCAM-1 monoclonal antibody solutions；
2. by step 1. in the rabbit-anti people NCAM-1 monoclonal antibody solutions that prepare mix with appropriate colloidal gold solution, be obtained
The concentration of rabbit-anti people's NCAM-1 monoclonal antibodies is the mixed liquor of 20 μ g/mL；
3. the mixed liquor shaking table mark 30min for 2. step being prepared, is subsequently adding the mg/mL bovine serum albumin(BSA)s of concentration 1 molten
Liquid simultaneously continues shaking table mark 30min, and mark is centrifuged after terminating, and the precipitation of acquisition is the combination of rabbit-anti people NCAM-1 monoclonal antibodies
Thing；30min is centrifuged with 8000rpm during centrifugation；
4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, is configured to the gold mark rabbit-anti people of the mg/mL of concentration 0.4
NCAM-1 monoclonal antibody conjugate solution,
5. gold mark rabbit-anti people's NCAM-1 monoclonal antibody conjugate solution is sprayed on by the water-absorption material for making gold standard pad by metal spraying machine
On material, gold standard pad is obtained within 14 hours in 37 DEG C of dryings.
（3）Combination
By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body,
Obtain final product the gold-labeled kit.
In the present embodiment, in step（1）In, prepare detection line solution when buffer solution be phosphate buffer,；Prepare matter
Control line solution buffer solution is phosphate buffer；Step 1. in, the buffer solution be phosphate buffer.
In the present embodiment, step 3. described in bovine serum albumin solution addition with bovine serum albumin(BSA) in mixed liquor
In concentration reach 0.1g/100 mL and be defined.
Step 2. described in colloidal gold solution by the method for embodiment 1 prepare.
Step 4. described in gold mark redissolve liquid it is in the same manner as in Example 1.
Step 5. described in gold mark rabbit-anti people's NCAM-1 monoclonal antibody conjugates discharge rate be 4 μ L/cm2。
In above-described embodiment 1-4, pH=7.2~7.4 of the phosphate buffer, concentration is 0.01mol/L.
Above-described embodiment 1-4 kits are used to detect the clinical activity lupus nephritis made a definite diagnosis and inactive lupus
The urine specimen of nephritis patient, and by testing result sensitivity and specificity statistics in table 1 below；
Table 1
From the data of upper table 1, kit of the present invention detects that its sensitivity or specificity reach as high as 90% for lupus nephritis,
It is woundless testing compared with existing goldstandard-biopsy, repeatedly can be routinely detected, compared to existing conventional inspection
Survey（Uroscopy shows Urine proteins>0.5g/ days or Urine proteins>=3+, or have cellular cast）, kit detection of the present invention is more
Fast, safety.
Claims (10)
- Applications of the 1.NCAM-1 as Testing index in the gold-labeled kit of detection lupus nephritis liveness.
- 2. it is a kind of it is as claimed in claim 1 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：Described gold Labeled kit by detecting urine specimen in NCAM-1 be used as Testing index.
- 3. it is according to claim 2 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：The gold mark examination Agent box includes base plate（7）, adsorptive pads（6）, nitrocellulose filter（3）, gold standard pad（2）And sample pad（1）, the sample pad（1）、 Gold standard pad（2）, nitrocellulose filter（3）And adsorptive pads（6）Connect successively and be covered in base plate（7）On, and gold standard pad（2）By sample Pad（1）Part covers；The nitrocellulose filter（3）On be provided with by the anti-human NCAM-1 monoclonal antibodies of mouse, rabbit-anti people NCAM- 1 monoclonal antibody, any one the coating shape in rabbit-anti people NCAM-1 polyclonal antibodies and goat-anti people's NCAM-1 polyclonal antibodies Into detection line（4）；By any one bag in goat anti-mouse immunoglobulin G and goat anti-rabbit immunoglobulin G polyclonal antibodies The control line being formed（5）, the gold standard pad（2）It is coated with collaurum and the anti-human NCAM-1 monoclonal antibodies of mouse and rabbit-anti people The conjugate that any one coupling in NCAM-1 monoclonal antibodies is formed.
- 4. it is according to claim 3 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：The detection line （4）, control line（5）Width be respectively controlled to 0.5 mm~0.8 mm, the detection line（4）With control line（5）Between spacing It is controlled to 5 mm~10 mm.
- 5. it is according to claim 3 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：The kit Preparation comprise the steps：（1）Detection line, the coating of nature controlling line：With the anti-human NCAM-1 monoclonal antibodies of mouse, rabbit-anti people NCAM-1 monoclonal antibodies, rabbit-anti people NCAM-1 polyclonal antibodies and In goat-anti people's NCAM-1 polyclonal antibodies any one be solute, additions cushioning liquid be solvent, be configured to concentration be 1~ The detection line solution of 4mg/mL；With goat anti-mouse immunoglobulin G polyclonal antibodies or goat anti-rabbit immunoglobulin G Anti-TNF-αs Body is solute, and addition cushioning liquid is solvent, is configured to the nature controlling line solution that concentration is 0.5~1 mg/mL；Will by Film-cutting machine The control line solution for preparing, detection line solution are coated on nitrocellulose filter, and the nitrocellulose filter is attached to base plate On, the nitrocellulose filter-floor combination body after coating will be completed in 37 DEG C of 12~16h of drying；（2）Prepare gold standard pad：1. with the anti-human NCAM-1 monoclonal antibodies of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies as solute, it is molten to add buffer solution Agent, compound concentration is the anti-human NCAM-1 monoclonal antibody solutions of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies of 0.5~2 mg/mL Solution；2. by step 1. in the anti-human NCAM-1 monoclonal antibody solutions of mouse that prepare or rabbit-anti people's NCAM-1 monoclonal antibodies Solution mixes with appropriate colloidal gold solution, and the anti-human NCAM-1 monoclonal antibodies of mouse or rabbit-anti people's NCAM-1 monoclonal antibodies is obtained Concentration is the mixed liquor of 10~20 μ g/mL；3. min~30 min of mixed liquor shaking table mark 20 for 2. step being prepared, is subsequently adding concentration for 1 mg/mL oxen Serum albumin solution simultaneously continues min~30 min of shaking table mark 20, and mark is centrifuged after terminating, and the precipitation of acquisition is mouse and resists People's NCAM-1 monoclonal antibodies conjugate or rabbit-anti people's NCAM-1 monoclonal antibody conjugates；4. the conjugate that 3. step obtains is dissolved in gold mark redissolution liquid, the gold mark mouse for being configured to the mg/mL of concentration 0.2~0.4 resists People NCAM-1 monoclonal antibodies or gold mark rabbit-anti people's NCAM-1 monoclonal antibody conjugate solution,5. the gold mark anti-human NCAM-1 monoclonal antibodies of mouse or gold mark rabbit-anti people NCAM-1 monoclonal antibodies are combined by metal spraying machine Thing solution is sprayed on the absorbent material for making gold standard pad, and gold standard pad is obtained in 37 DEG C of 12~16h of drying；（3）Combination：By adsorptive pads, gold standard pad and sample pad gluing steps（1）On the base plate of the nitrocellulose filter for obtaining-floor combination body, Obtain final product the gold-labeled kit.
- 6. it is according to claim 5 it is a kind of detect lupus nephritis liveness gold-labeled kit, it is characterised in that：In step （1）In, buffer solution is phosphate buffer, carbonate buffer solution, borate buffer solution, three hydroxyl first when preparing detection line solution Any one in base aminomethane salt buffer；Prepare nature controlling line solution buffer solution be phosphate buffer, carbonate delay Any one in fliud flushing, borate buffer solution；Step 1. in, the buffer solution be phosphate buffer, carbonate buffer Any one in liquid, borate buffer solution, trishydroxymethylaminomethane salt buffer.
- 7. it is according to claim 5 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：In step 3. In, the concentration of the addition of the bovine serum albumin solution with bovine serum albumin(BSA) in mixed liquor reaches 0.1 g/100 mL It is defined.
- 8. it is according to claim 5 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：In step 4. In, it is that Tris, bovine serum albumin(BSA), Sodium azide are dissolved in tri-distilled water to form that the gold mark redissolves liquid, and the concentration of Tris is 0.01mol/L~0.02 mol/L, the concentration of bovine serum albumin(BSA) is 0.1 g/100mL~0.5 g/100mL, Sodium azide it is dense It is 0.2 g/100mL to spend.
- 9. it is according to claim 5 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：In step 5. In, the discharge rate of the gold mark anti-human NCAM-1 monoclonal antibodies of mouse or gold mark rabbit-anti people's NCAM-1 monoclonal antibody conjugates is 2 ~4 μ l/cm2。
- 10. it is according to claim 6 detection lupus nephritis liveness gold-labeled kit, it is characterised in that：The phosphoric acid PH=7.2~7.4 of salt buffer, concentration is 0.01mol/L, pH=9.2~10.7 of the carbonate buffer solution, and concentration is 0.05mol/L, pH=8.5~10.0 of the borate buffer solution, concentration is 0.05mol/L, the trihydroxy methyl amino first PH=7.0~9.2 of alkane salt buffer, concentration is 0.05mol/L.
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| CN109900897A (en) * | 2019-01-16 | 2019-06-18 | 安徽科技学院 | A kind of optimization method that the lateral flow biosensor based on golden platinum nano flower tests and analyzes |
| CN117074668A (en) * | 2022-10-31 | 2023-11-17 | 安徽艾赛尔智能科技有限公司 | HPV virus typing detection kit and preparation method thereof |
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