CN106932593A - The double-antibody sandwich enzyme-linked immunologic adsorption detection kit as Testing index and its application with NCAM 1 - Google Patents
The double-antibody sandwich enzyme-linked immunologic adsorption detection kit as Testing index and its application with NCAM 1 Download PDFInfo
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- CN106932593A CN106932593A CN201710123440.XA CN201710123440A CN106932593A CN 106932593 A CN106932593 A CN 106932593A CN 201710123440 A CN201710123440 A CN 201710123440A CN 106932593 A CN106932593 A CN 106932593A
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- ncam
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- biotin labeling
- liveness
- polyclonal antibodies
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- 238000001514 detection method Methods 0.000 title claims abstract description 48
- 238000012360 testing method Methods 0.000 title claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 title abstract description 11
- 108090000790 Enzymes Proteins 0.000 title abstract description 11
- 230000001900 immune effect Effects 0.000 title abstract description 10
- 238000001179 sorption measurement Methods 0.000 title abstract description 10
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 title abstract 6
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 title abstract 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 87
- 239000011616 biotin Substances 0.000 claims abstract description 50
- 229960002685 biotin Drugs 0.000 claims abstract description 50
- 235000020958 biotin Nutrition 0.000 claims abstract description 50
- 238000002372 labelling Methods 0.000 claims abstract description 37
- 208000005777 Lupus Nephritis Diseases 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 28
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 8
- 210000002700 urine Anatomy 0.000 claims abstract description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 5
- PMJHNEFCWLUZBC-UHFFFAOYSA-N 4-(4-amino-3-methylphenyl)-2,6,6-trimethylcyclohexa-1,3-dien-1-amine Chemical class CC1=C(N)C(C)(C)CC(C=2C=C(C)C(N)=CC=2)=C1 PMJHNEFCWLUZBC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 108050003738 Neural cell adhesion molecule 1 Proteins 0.000 claims description 96
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 95
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 22
- 229940098773 bovine serum albumin Drugs 0.000 claims description 22
- 239000008363 phosphate buffer Substances 0.000 claims description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 16
- -1 biotin N-hydroxy-succinamide ester Chemical class 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000002965 ELISA Methods 0.000 claims description 10
- 239000006193 liquid solution Substances 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 7
- 239000012224 working solution Substances 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 239000002808 molecular sieve Substances 0.000 claims description 3
- 102000013415 peroxidase activity proteins Human genes 0.000 claims description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- 102000009027 Albumins Human genes 0.000 claims description 2
- 108010088751 Albumins Proteins 0.000 claims description 2
- 241000283690 Bos taurus Species 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 238000011010 flushing procedure Methods 0.000 claims 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 2
- 239000010452 phosphate Substances 0.000 claims 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 239000000872 buffer Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 239000003593 chromogenic compound Substances 0.000 abstract description 3
- 108090001008 Avidin Proteins 0.000 abstract 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 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
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 241000282461 Canis lupus Species 0.000 description 1
- 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
- 102000013928 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000020832 chronic kidney disease Diseases 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
- 238000003745 diagnosis Methods 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
- 230000000694 effects Effects 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 239000012073 inactive phase Substances 0.000 description 1
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- 230000002427 irreversible effect Effects 0.000 description 1
- 238000011862 kidney biopsy Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- 230000002792 vascular Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- 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
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70525—ICAM molecules, e.g. CD50, CD54, CD102
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
Abstract
The present invention provides a kind of double-antibody sandwich enzyme-linked immunologic adsorption detection kit for detecting lupus nephritis liveness, the kit is coated with the porous plate of the monoclonal antibodies of NCAM 1, the polyclonal antibodies of the NCAM 1 detection liquid of biotin labeling is combined with the polyclonal antibodies of NCAM 1 of biotin labeling Avidin horseradish peroxidase, chromogenic substrate 3 ', 3 ', 5,5 ' tetramethyl benzidines, terminate liquid sulfuric acid and the recombinant proteins of NCAM 1.The kit, so as to judge lupus nephritis liveness, realizes quick, Non-invasive detection by detecting that NCAM 1 is Testing index in urine specimen.
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
Double-antibody sandwich enzyme-linked immunologic adsorption detection kits and its application of the NCAM-1 for Testing index.
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.
The principle of Double antibody sandwich-ELISA is to be attached to specific antibody to form solid phase on solid phase carrier
Antibody, then combines to form immune complex with the corresponding antigens in serum to be checked, enzyme-added labelled antibody again after washing, and immune
Antigen binding forms enzyme labelled antibody-antigen-insolubilized antibody compound, plus substrate colour developing in compound, judges antigenic content.
NCAM-1（Neural cell adhesion molecule-1, Chinese name：N-CAM -1）It is one
Non- Ca-dependent adhesion factor is planted, 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 applied to detection examination as the diagnosis index of kidneys with lupus nephritis inflammation liveness
In agent box.
It is an object of the invention to overcome the shortcomings of that prior art detects lupus nephritis liveness by percutaneous biopsy, carry
For a kind of new double-antibody sandwich enzyme-linked immunologic adsorption detection kit that can be used to detect lupus nephritis liveness, the reagent
Box is by detecting that NCAM-1 is Testing index in urine specimen, so as to judge lupus nephritis liveness, realizes quick, noninvasive inspection
Survey.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
The present invention is specifically applied to double antibody sandwich enzyme-linked immunosorbent assay reagent first using NCAM-1 as Testing index
In box, obtain double-antibody sandwich enzyme-linked immunologic adsorption detection kit and judge wolf by detecting that the NCAM-1 in urine specimen is used as
The foundation of sore ephritis liveness.
The double-antibody sandwich enzyme-linked immunologic adsorption detection kit of detection lupus nephritis liveness described above, its feature
It is：The kit is coated with the NCAM-1 Anti-TNF-αs of the porous plate of NCAM-1 monoclonal antibodies, biotin labeling
Avidin-horseradish peroxidase, the chromogenic substrate that liquid is combined with the NCAM-1 polyclonal antibodies of biotin labeling are surveyed in physical examination
3 ', 3 ', 5,5 '-tetramethyl benzidine, terminate liquid sulfuric acid and NCAM-1 recombinant proteins.
It is preferred that, the porous plate for being coated with NCAM-1 monoclonal antibodies, its coating NCAM-1 monoclonal antibody 0.1 per hole
~0.2 μ g.
Further, the NCAM-1 polyclonal antibodies detection liquid of the biotin labeling includes the NCAM-1 of biotin labeling
Polyclonal antibody, bovine serum albumin(BSA) and phosphate buffer；In detection liquid, the NCAM-1 Anti-TNF-αs of the biotin labeling
The ultimate density of body is 0.1~0.2 μ g/mL, and the ultimate density of bovine serum albumin(BSA) is 1g/100mL.
Further, the NCAM-1 polyclonal antibodies of the biotin labeling by biotin N-hydroxy-succinamide ester and
NCAM-1 polyclonal antibodies are coupled and formed, the molecule of the biotin N-hydroxy-succinamide ester and NCAM-1 polyclonal antibodies
Ratio is 4: 1.
Further, the porous plate for being coated with NCAM-1 monoclonal antibodies is prepared as follows：First use cow's serum
NCAM-1 monoclonal antibodies are diluted to albumin concentration the working solution of the μ g/mL of concentration 1 for the phosphate buffer of 1g/100mL,
Then the NCAM-1 monoclonal antibodies working solution is added in each hole of porous plate by the amount in 100~200 μ l/ holes, at 20 DEG C
~25 DEG C are coated with 12~16 hours；After the completion of coating, the phosphate buffer containing bovine serum albumin(BSA) 1g/100mL is added many
Each hole on orifice plate, addition is 200 μ l/ holes, and capping is carried out at 20 DEG C~25 DEG C, and the reaction time is 1~4 hour；
After capping terminates, the unreacted reactant that porous plate is removed on porous plate is washed with the phosphate buffer containing 0.05% polysorbas20,
The porous plate for being coated with NCAM-1 monoclonal antibodies is obtained, in 4 DEG C of preservations.
NCAM-1 polyclonal antibodies invention further provides the biotin labeling detect the preparation method of liquid, institute
The method of stating comprises the steps：
（1）With dimethyl sulfoxide (DMSO) as solvent, biotin N-hydroxy-succinamide ester is the life that solute compound concentration is 1 μ g/ μ l
Thing element N-hydroxy-succinamide ester solution；With carbonate buffer solution as solvent, NCAM-1 polyclonal antibodies are that solute preparation is dense
The NCAM-1 Anti-TNF-α liquid solutions for 1mg/mL are spent,
（2）By step（1）The biotin N-hydroxy-succinamide ester solution and NCAM-1 Anti-TNF-α liquid solutions for preparing
Mixing, and the mixed liquor that will be obtained obtains many grams of the NCAM-1 of biotin labeling under agitation in room temperature reaction 2~4 hours
Grand antibody；
（3）By step（2）The NCAM-1 Anti-TNF-α liquid solutions containing biotin labeling for obtaining are fitted into bag filter, use phosphate
Buffer solution is dialysed at 4 DEG C, and dialysis time is 12~16 hours；
（4）By step（3）The liquid that dialysis is obtained is added in the molecular sieve column of 1mL, is slowly washed with 3mL phosphate buffers
It is de-, appropriate bovine serum albumin(BSA) is added in the eluent being collected into so that the final concentration of 1g/100mL of bovine serum albumin(BSA), so
50% glycerine of bovine serum albumin and eluent cumulative volume is added afterwards, that is, form the NCAM-1 Anti-TNF-α physical examinations of biotin labeling
Liquid is surveyed, -20 DEG C of preservations are put.
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, simple to operate using detection kit of the invention, one sample of detection is only needed 3~6 minutes, and clinically existing
There is detection method to compare, detection efficiency is increased substantially.Because detection sample is the urine specimen of patient, thus it is a kind of noninvasive
Detection.
3rd, 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
Fig. 1 is the standard curve of the double-antibody sandwich enzyme-linked immunologic adsorption detection kit of embodiment 3.
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, goat-anti people NCAM-1 polyclonal antibodies and NCAM-1 recombinant proteins are purchased from U.S. bio-
Techne companies.
Glycerine, dimethyl sulfoxide (DMSO), biotin N-hydroxy-succinamide ester, bovine serum albumin(BSA) are purchased from sigma- respectively
Aldrich companies and Suo Laibao companies.
Phosphate buffer and carbonate buffer solution can be made by oneself, and preparation method is as follows：
1. phosphate buffer：Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphates and 0.24g potassium dihydrogen phosphates molten
In 900mL distilled waters, with hydrochloric acid conditioning solution pH value to 7.2, it is eventually adding distilled water and is settled to 1L, solution passes through 0.45 μm
It is stored in after membrane filtration in 4 DEG C of refrigerators.
2. carbonate buffer solution：Weigh 63.6g sodium carbonate and 33.6g sodium acid carbonates are dissolved in 1L distilled waters, adjusted with hydrochloric acid
Section solution ph is 8.0 carbonate buffer solution, and solution is in being stored in 4 DEG C of refrigerators after 0.45 μm of membrane filtration.
Embodiment 1
The double-antibody sandwich enzyme-linked immunologic adsorption detection kit of lupus nephritis liveness is detected, the kit is coated with
The porous plate of NCAM-1 monoclonal antibodies, the NCAM-1 polyclonal antibodies detection liquid of biotin labeling and biotin labeling
Avidin-horseradish peroxidase, chromogenic substrate 3 ', 3 ', 5 that NCAM-1 polyclonal antibodies are combined, 5 '-tetramethyl benzidine,
Terminate liquid sulfuric acid and NCAM-1 recombinant proteins.
In the present embodiment, the porous plate for being coated with NCAM-1 monoclonal antibodies, it is coated with NCAM-1 monoclonals per hole
The μ g of antibody 0.1.
In the present embodiment, the NCAM-1 polyclonal antibodies detection liquid of the biotin labeling includes biotin labeling
NCAM-1 polyclonal antibodies, bovine serum albumin(BSA) and phosphate buffer, in the detection liquid for obtaining, biotin labeling
The concentration of NCAM-1 polyclonal antibodies and bovine serum albumin(BSA) distinguishes 0.1 μ g/mL and 1g/100mL.
In the present embodiment, the NCAM-1 polyclonal antibodies of the biotin labeling are by biotin N-hydroxy-succinamide ester
Coupled with NCAM-1 polyclonal antibodies and formed, the biotin N-hydroxy-succinamide ester is divided with NCAM-1 polyclonal antibodies
Sub- ratio is 4: 1.
Embodiment 2
The difference of the present embodiment and embodiment 1：
In the present embodiment, the porous plate for being coated with NCAM-1 monoclonal antibodies, it is coated with NCAM-1 monoclonal antibodies per hole
0.2μg.In the detection liquid for obtaining, the concentration of the NCAM-1 polyclonal antibodies of biotin labeling distinguishes 0.2 μ g/mL.
Embodiment 3
The present embodiment is on the basis of embodiment 1, it is further provided be coated with many of NCAM-1 monoclonal antibodies in embodiment 1
The preparation method of orifice plate and the NCAM-1 polyclonal antibodies of biotin labeling detect the preparation method of liquid；
Wherein, it is coated with the preparation method of the porous plate of NCAM-1 monoclonal antibodies：First use 1g/100mL containing bovine serum albumin(BSA)
Phosphate buffer NCAM-1 monoclonal antibodies are diluted to the working solution of the μ g/mL of concentration 1, then by the NCAM-1 Dan Ke
Grand antibody working solution is added in each hole of porous plate by the amount in 100 μ L/ holes, is coated with 12 hours at 20 DEG C；After the completion of coating, will contain
The phosphate buffer of bovine serum albumin(BSA) 1g/100mL adds each hole on porous plate, and addition is 200 μ l/ holes, and 20
Capping DEG C is carried out, the reaction time is 1 hour；After capping terminates, washed with the phosphate buffer containing 0.05% polysorbas20
The unreacted reactant that porous plate is removed on porous plate is washed, that is, obtains the porous plate for being coated with NCAM-1 monoclonal antibodies, in 4 DEG C of guarantors
Deposit.
The NCAM-1 polyclonal antibodies of biotin labeling detect the preparation method of liquid：
（1）With dimethyl sulfoxide (DMSO) as solvent, biotin N-hydroxy-succinamide ester is the life that solute compound concentration is 1 μ g/ μ l
Thing element N-hydroxy-succinamide ester solution；With carbonate buffer solution as solvent, NCAM-1 polyclonal antibodies are that solute preparation is dense
The NCAM-1 Anti-TNF-α liquid solutions for 1mg/mL are spent,
（2）By step（1）The biotin N-hydroxy-succinamide ester solution and NCAM-1 Anti-TNF-α liquid solutions for preparing
Mixing, and the mixed liquor that will be obtained obtains the NCAM-1 Anti-TNF-αs of biotin labeling under agitation in room temperature reaction 2 hours
Body；
（3）By step（2）The NCAM-1 Anti-TNF-α liquid solutions containing biotin labeling for obtaining are fitted into bag filter, use phosphate
Buffer solution is dialysed at 4 DEG C, and dialysis time is 12 hours；
（4）By step（3）The liquid that obtains of dialysis is added to volume in 1mL molecular sieve columns, slow with 3mL phosphate buffers
Wash-out, adds appropriate bovine serum albumin(BSA) so that the final concentration of 1g/100mL of bovine serum albumin(BSA) in the eluent being collected into,
50% glycerine of bovine serum albumin and eluent cumulative volume is subsequently adding, that is, forms the NCAM-1 polyclonal antibodies of biotin labeling
Detection liquid, puts -20 DEG C of preservations.
Embodiment 4
The present embodiment is on the basis of embodiment 2, it is further provided be coated with many of NCAM-1 monoclonal antibodies in embodiment 2
The preparation method of orifice plate and the NCAM-1 polyclonal antibodies of biotin labeling detect the preparation method of liquid；
In the preparation of porous plate, its method is with the difference of embodiment 3：The NCAM-1 monoclonal antibodies working solution is pressed
The amount in 200 μ L/ holes is added in each hole of porous plate, is coated with 16 hours at 25 DEG C；After the completion of coating, add and contain bovine serum albumin(BSA)
Capping is carried out after the phosphate buffer of 1g/100mL at 25 DEG C, the reaction time is 4 hours；
In prepared by the NCAM-1 polyclonal antibodies detection liquid of biotin labeling, its preparation method is with the difference of embodiment 3：
Step（2）In, mixed liquor is under agitation 4 hours in the room temperature reaction time；
Step（3）In, dialysis time is 16 hours.
In above-described embodiment 1-4, NCAM-1 monoclonal antibodies select the anti-human NCAM-1 monoclonal antibodies of mouse, and NCAM-1 is more
Clonal antibody selects goat-anti people's NCAM-1 polyclonal antibodies.
The use of double-antibody sandwich enzyme-linked immunologic adsorption detection kit of the invention is consistent with prior art application method.
It is non-to clinical definite lupus nephritis active period and clinical definite lupus nephritis using the kit obtained in embodiment of the present invention 1-4
Active period patient carries out sample detecting, adds terminate liquid to become yellow into positive（Lupus nephritis active period）, and testing result is united
In table 1 below, testing result shows meter：, up to 81% or so, specificity is up to 80% or so for the sensitivity of the detection instrument.
Table 1
The kit of embodiment 3 is drawn standard curve, its standard curve as shown in figure 1, understanding this by standard curve by the present invention
Invention kit is working properly, as a result more reliable.
Claims (9)
1.NCAM-1 is detecting the double antibody sandwich enzyme-linked immunosorbent assay reagent of lupus nephritis liveness as Testing index
Application in box.
2. it is a kind of it is as claimed in claim 1 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The kit by detecting urine specimen in NCAM-1 be used as Testing index.
3. it is according to claim 2 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The kit is coated with the NCAM-1 of the porous plate of NCAM-1 monoclonal antibodies, biotin labeling
Polyclonal antibody detection liquid is combined with the NCAM-1 polyclonal antibodies of biotin labeling Avidin-horseradish peroxidase, show
Color substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine, terminate liquid sulfuric acid and NCAM-1 recombinant proteins.
4. it is according to claim 3 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The porous plate for being coated with NCAM-1 monoclonal antibodies, it is coated with NCAM-1 monoclonal antibodies per hole
0.1~0.2 μ g.
5. it is according to claim 3 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The NCAM-1 polyclonal antibodies detection liquid of the biotin labeling includes that bovine serum albumin(BSA), phosphate delay
The NCAM-1 polyclonal antibodies of fliud flushing and biotin labeling.
6. it is according to claim 3 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The NCAM-1 polyclonal antibodies of the biotin labeling by biotin N-hydroxy-succinamide ester and
NCAM-1 polyclonal antibodies are coupled and formed, the molecular ratio of biotin N-hydroxy-succinamide ester and NCAM-1 polyclonal antibodies
It is 4: 1.
7. it is according to claim 3 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, the porous plate for being coated with NCAM-1 monoclonal antibodies is prepared as follows：First with the phosphoric acid containing bovine serum albumin(BSA)
NCAM-1 monoclonal antibodies are diluted to salt buffer the working solution of the μ g/mL of concentration 1, then by the NCAM-1 monoclonals after dilution
Antibody working solution is added in each hole of porous plate, is coated with 12~16 hours at 20 DEG C -25 DEG C；After the completion of coating, cow's serum will be contained
The phosphate buffer of albumin adds each hole on porous plate, and capping is carried out at 20 DEG C -25 DEG C, and the reaction time is 1
~4 hours；After capping terminates, wash porous plate with the phosphate buffer containing 0.05% polysorbas20 and remove on porous plate
Unreacted reactant, that is, obtain the porous plate for being coated with NCAM-1 monoclonal antibodies, in 4 DEG C of preservations；It is described containing bovine serum albumin(BSA)
In phosphate buffer, the content of bovine serum albumin(BSA) is 1g/100mL.
8. it is according to claim 5 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：The NCAM-1 polyclonal antibodies detection liquid of the biotin labeling is prepared according to the following steps：
（1）With dimethyl sulfoxide (DMSO) as solvent, biotin N-hydroxy-succinamide ester is the life that solute compound concentration is 1 μ g/ μ l
Thing element N-hydroxy-succinamide ester solution；With carbonate buffer solution as solvent, NCAM-1 polyclonal antibodies are that solute preparation is dense
The NCAM-1 Anti-TNF-α liquid solutions for 1mg/mL are spent,
（2）By step（1）The biotin N-hydroxy-succinamide ester solution and NCAM-1 Anti-TNF-α liquid solutions for preparing
Mixing, and the mixed liquor that will be obtained obtains many grams of the NCAM-1 of biotin labeling under agitation in room temperature reaction 2~4 hours
Grand antibody；
（3）By step（2）The NCAM-1 Anti-TNF-α liquid solutions of the biotin labeling for obtaining are fitted into bag filter, slow with phosphate
Fliud flushing is dialysed at 4 DEG C, and dialysis time is 12~16 hours；
（4）By step（3）The liquid that dialysis is obtained is added in the molecular sieve column of 1mL, is slowly eluted with 3mL phosphate buffers,
Appropriate bovine serum albumin(BSA) is added in the eluent being collected into, makes the final concentration of 1g/100mL of the bovine serum albumin(BSA), so
After add 50% glycerine, that is, formed biotin labeling NCAM-1 polyclonal antibodies detection liquid, put -20 DEG C of preservations.
9. it is according to claim 5 detection lupus nephritis liveness double antibody sandwich enzyme-linked immunosorbent assay reagent
Box, it is characterised in that：In the NCAM-1 polyclonal antibodies detection liquid of the biotin labeling, the NCAM- of the biotin labeling
The concentration of 1 polyclonal antibody is 0.1~0.2 μ g/mL, and the concentration of the bovine serum albumin(BSA) is 1g/100mL.
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| CN110672857A (en) * | 2019-10-10 | 2020-01-10 | 四川大学华西第二医院 | TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof |
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| WO2007022436A2 (en) * | 2005-08-17 | 2007-02-22 | Quest Diagnostics Investments Incorporated | Hematopoietic cell phenotyping using circulating cell-free markers |
| CN101846684A (en) * | 2010-02-25 | 2010-09-29 | 四川大学 | Double antibody sandwich enzyme-linked immunosorbent assay kit for detecting AIB1 and preparation method thereof |
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