CN108802375A - A kind of detection kit of antinuclear antibodies spectrum - Google Patents
A kind of detection kit of antinuclear antibodies spectrum Download PDFInfo
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- CN108802375A CN108802375A CN201810883443.8A CN201810883443A CN108802375A CN 108802375 A CN108802375 A CN 108802375A CN 201810883443 A CN201810883443 A CN 201810883443A CN 108802375 A CN108802375 A CN 108802375A
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- microballon
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- detection kit
- antinuclear antibodies
- compounded microbeads
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- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 230000003460 anti-nuclear Effects 0.000 title claims abstract description 14
- 238000001228 spectrum Methods 0.000 title claims abstract description 14
- 239000011325 microbead Substances 0.000 claims abstract description 26
- 239000000725 suspension Substances 0.000 claims abstract description 25
- 239000000427 antigen Substances 0.000 claims abstract description 11
- 102000036639 antigens Human genes 0.000 claims abstract description 11
- 108091007433 antigens Proteins 0.000 claims abstract description 11
- 239000003550 marker Substances 0.000 claims abstract description 9
- 239000012898 sample dilution Substances 0.000 claims abstract description 9
- 108010004729 Phycoerythrin Proteins 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 108010033040 Histones Proteins 0.000 claims description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 5
- 108050006400 Cyclin Proteins 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 4
- 102100026060 Exosome component 10 Human genes 0.000 claims description 4
- 101001055976 Homo sapiens Exosome component 10 Proteins 0.000 claims description 4
- 108010047956 Nucleosomes Proteins 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 claims description 4
- -1 Ribosomal P Proteins 0.000 claims description 4
- 102000004598 Small Nuclear Ribonucleoproteins Human genes 0.000 claims description 4
- 108010003165 Small Nuclear Ribonucleoproteins Proteins 0.000 claims description 4
- 102000018165 U1 Small Nuclear Ribonucleoprotein Human genes 0.000 claims description 4
- 108010091281 U1 Small Nuclear Ribonucleoprotein Proteins 0.000 claims description 4
- 102100022013 U1 small nuclear ribonucleoprotein A Human genes 0.000 claims description 4
- 101710106597 U1 small nuclear ribonucleoprotein A Proteins 0.000 claims description 4
- 102100035136 U1 small nuclear ribonucleoprotein C Human genes 0.000 claims description 4
- 101710106596 U1 small nuclear ribonucleoprotein C Proteins 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 claims description 4
- 210000002230 centromere Anatomy 0.000 claims description 4
- 210000001623 nucleosome Anatomy 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 210000003705 ribosome Anatomy 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 229940033663 thimerosal Drugs 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 239000007836 KH2PO4 Substances 0.000 claims 1
- 239000007983 Tris buffer Substances 0.000 claims 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims 1
- 229910000397 disodium phosphate Inorganic materials 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims 1
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 8
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 208000003250 Mixed connective tissue disease Diseases 0.000 abstract description 5
- 206010039710 Scleroderma Diseases 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 abstract description 3
- 208000011580 syndromic disease Diseases 0.000 abstract description 3
- 230000009885 systemic effect Effects 0.000 abstract description 3
- 208000005987 polymyositis Diseases 0.000 abstract description 2
- 238000005406 washing Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 201000009594 Systemic Scleroderma Diseases 0.000 description 2
- 206010042953 Systemic sclerosis Diseases 0.000 description 2
- 210000003850 cellular structure Anatomy 0.000 description 2
- 208000018631 connective tissue disease Diseases 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 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
- 239000004793 Polystyrene Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of detection kits of antinuclear antibodies spectrum, belong to detection kit technical field.Including compounded microbeads suspension, contain multiple microballons in the compounded microbeads suspension, different types of autoantigen is coated on a portion microballon respectively, the antigen prepared from HEp-2 cells is coated on a part of microballon, a kind of antigen that non-specific antibody can be detected in patient's sample is coated on some microballon, remaining microballon is four kinds of standard control microballons；Fluorescent marker, the fluorescent marker are goat-anti-human IgG of phycoerythrin label（Specific γ chains）；Sample dilution, cleaning solution.The detection of multiple autoantibodies can be done to a patient's sample in same hole of same time, to systemic loupus erythematosus（SLE）, rheumatoid arthritis, scleroderma, polymyositis, mixed connective tissue disease（MCTD）, the multiple systems autoimmune disease such as drug-induced SLE and dry syndrome provides assistance in diagnosis.
Description
Technical field
The present invention relates to a kind of detection kits of antinuclear antibodies spectrum, belong to detection kit technical field.
Background technology
Although the definite cause of disease of current autoimmune disease is unknown, in various autoimmunity patients with connective tissue diseases
It is to determine with the presence of specifically autoantibody.To doubtful all kinds of patients with connective tissue diseases, to combine and often detect SSA
52 kDa, SSA 60 kDa, SSB, Sm, U snRNP B/B’, U1 snRNP 68, U1 snRNP A, U1 snRNP
C, Scl-70, Jo-1, Centromere B, PM Scl, Nucleosome, Ribosomal P, PCNA, dsDNA,
The autoantibodies such as Histone H, Histone HLY and M2, especially these IgG antibodies.In the prior art, pass through reagent
Box is all individually to detect when detecting, inefficiency.
Invention content
Technical problem to be solved by the present invention lies in：A kind of detection kit of antinuclear antibodies spectrum is provided, it is solved
The problem of existing antibody test inefficiency.
The technical problems to be solved by the invention take following technical scheme to realize：
A kind of detection kit of antinuclear antibodies spectrum, the kit include,
Compounded microbeads suspension contains multiple microballons in the compounded microbeads suspension, is coated with respectively on a portion microballon
There is different types of autoantigen, the antigen prepared from HEp-2 cells is coated on a part of microballon, some microballon
On be coated with a kind of antigen that non-specific antibody can be detected in patient's sample, remaining microballon is that four kinds of standard controls are micro-
Pearl；
Fluorescent marker, the fluorescent marker are the goat anti-human igg of phycoerythrin label（Specific γ chains）；
Sample dilution contains phosphate buffer in the Sample dilution；
Cleaning solution contains phosphate buffer in the cleaning solution.
As preferred embodiment, wherein being coated with following autoantigen respectively on branch's microballon：SSA 52 kDa, SSA
60 kDa, SSB, Sm, U snRNP B/B’, U1 snRNP 68, U1 snRNP A, U1 snRNP C, Scl-70,
Jo-1, Centromere B, PM Scl, Nucleosome, Ribosomal P, PCNA, dsDNA, Histone H,
Histone HLY and M2.
As preferred embodiment, the component of compounded microbeads suspension and its is a concentration of,
As preferred embodiment, the lauryl sodium sulfate or 0.5- of the Tween20 0.1-0.5g/L in compounded microbeads suspension
The triton x-100 of 1.5ml/L replaces.
As preferred embodiment, Sodium Azide in compounded microbeads suspension are with thimerosal generation of 0.5g/L-5g/L
It replaces.
As preferred embodiment, the goat anti-human igg of phycoerythrin label（Specific γ chains）Initial concentration be 1.0 ug/
Compounded microbeads suspension dilution calibration is added to concentration 0.6ug/L in mL.
The beneficial effects of the invention are as follows：Multiple autoantibodies can be done to a patient's sample in same hole of same time
Detection, to systemic loupus erythematosus（SLE）, rheumatoid arthritis, scleroderma（And systemic scleroderma）, polymyarian
Inflammation, mixed connective tissue disease（MCTD）, the multiple systems autoimmune disease such as drug-induced SLE and dry syndrome
Provide assistance in diagnosis.To auxiliary diagnosis multiple systems autoimmune disease, it can detect and distinguish a variety of this kit
The detection of Multiple Antibodies can be completed in anti-nucleic acid antibody and cytoplasmic cell components in one-time detection, greatly improves detection
Efficiency.
Specific implementation mode
In order to which the technical means, creative features, achievable purpose and effectiveness to the present invention are easy to understand, with reference to tool
Body embodiment, the present invention is further explained.
A kind of detection kit of antinuclear antibodies spectrum, the kit include,
Compounded microbeads suspension contains multiple microballons in the compounded microbeads suspension, is coated with respectively on a portion microballon
There is different types of autoantigen, the antigen prepared from HEp-2 cells is coated on a part of microballon, some microballon
On be coated with a kind of antigen that non-specific antibody can be detected in patient's sample, remaining microballon is that four kinds of standard controls are micro-
Pearl；
Fluorescent marker, the fluorescent marker are the goat anti-human igg of phycoerythrin label（Specific γ chains）；
Sample dilution contains phosphate buffer in the Sample dilution；
Cleaning solution contains phosphate buffer in the Sample dilution.
In a kind of embodiment wherein, wherein being coated with following autoantigen respectively on branch's microballon：SSA 52
kDa, SSA 60 kDa, SSB, Sm, U snRNP B/B’, U1 snRNP 68, U1 snRNP A, U1 snRNP C,
Scl-70, Jo-1, Centromere B, PM Scl, Nucleosome, Ribosomal P, PCNA, dsDNA,
Histone H, Histone HLY and M2.
In a kind of embodiment wherein, the component of compounded microbeads suspension and its is a concentration of,
In a kind of embodiment wherein, the dodecyl sulphate of the Tween20 0.1-0.5g/L in compounded microbeads suspension
The triton x-100 of sodium or 0.5-1.5ml/L replace.
In a kind of embodiment wherein, the Sodium Azide in compounded microbeads suspension are with 0.5g/L-5g/L's
Thimerosal replaces.
In a kind of embodiment wherein, goat-anti-human IgG of phycoerythrin label（Specific γ chains）Initial concentration
For 1.0 ug/ml, compounded microbeads suspension dilution calibration is added to concentration 0.6ug/L.
To auxiliary diagnosis multiple systems autoimmune disease, it can detect and distinguish a variety of anti-nucleic acid resists this kit
Body and cytoplasmic cell components.Below table lists in ubiquitous system autoimmune disease between autoantibody and disease
Relationship.
Preparation process：
1, compounded microbeads suspension is prepared
Compounded microbeads suspension also serves as sample diluting liquid, washing lotion.The substance dosage needed is calculated according to following concentration proportioning,
The required dosage of reagent preparation.
2, the activation of microballon and coating
（1）It takes 1ml particular number microballons that 1.5ml centrifuge tubes are added, 5-50ul EDC and 5-50ul Sulfo-NHS is added, add
Enter 10-100ug specific antigens（The human IgG of 2-5ug/ml is added in standard control microballon 1, and 6-10ug/ is added in standard control microballon 2
The human IgG of 12-18ug/ml is added in the human IgG of ml, standard control microballon 3, and the anti-human of 8-15ug/ml is added in standard control microballon 4
IgG, then any antigen is added without as blank control using NSC microballons）, vortex mixing 30 seconds, ultrasonic mixing 30 seconds, 2-
8 °C shake up overnight.
If producing antinuclear antibodies spectrum n, need to place n+5 kind antigen-antibodies simultaneously（Bottle）Shaking table, be coated with n respectively
The microballon of+4 kinds of different numbers.
（2）Washing：Using filtering and washing, it is washed after microballon be transferred in compounded microbeads suspension.
（3）Verification quality is come by using Quality Control disk.
（4）A variety of microballons that have been coated with are mixed, washing lower bottle wall with a small amount of compounded microbeads suspension remains microballon, and mixes,
It finally is settled to 120ml with compounded microbeads suspension, after the assay was approved, is distributed into 20 bottles, every bottle of 5.5ml（100 person-portions）.
3, fluorescent marker：
The goat anti-human igg of phycoerythrin label（IgG-PE）:Initial concentration 1ug/ml is diluted to work with compounded microbeads suspension
Make concentration, working concentration 0.6ug/L.
4, concentrated cleaning solution：Microballon preserves 10 times of concentration versions of formula of liquid, i.e., was made into the solution of 1L originally, constant volume is made into
100ml。
Above-mentioned microballon is 5.6 microns of granules of polystyrene.
The present invention can do a patient's sample in same hole of same time the detection of multiple autoantibodies, to right
Systemic loupus erythematosus（SLE）, rheumatoid arthritis, scleroderma（And systemic scleroderma）, polymyositis, mixed type connective group
Knit disease（MCTD）, the multiple systems autoimmune disease such as drug-induced SLE and dry syndrome provides assistance in diagnosis.
To auxiliary diagnosis multiple systems autoimmune disease, it can detect and distinguish a variety of anti-nucleic acid antibodies and cell this kit
The detection of a variety of antigens can be completed in one-time detection, greatly improve detection efficiency for cell plastid component.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, without departing from the spirit and scope of the present invention, this hair
Bright to will also have various changes and improvements, these changes and improvements are both fallen in the scope of protection of present invention.The present invention claims
Protection domain is defined by the appending claims and its equivalent thereof.
Claims (6)
1. a kind of detection kit of antinuclear antibodies spectrum, it is characterised in that：The kit includes,
Compounded microbeads suspension contains multiple microballons in the compounded microbeads suspension, is coated with respectively on a portion microballon
There is different types of autoantigen, the antigen prepared from HEp-2 cells is coated on a part of microballon, some microballon
On be coated with a kind of antigen that non-specific antibody can be detected in patient's sample, remaining microballon is that four kinds of standard controls are micro-
Pearl；
Fluorescent marker, the fluorescent marker are the goat anti-human igg of phycoerythrin label（Specific γ chains）；
Sample dilution contains phosphate buffer in the Sample dilution；
Cleaning solution contains phosphate buffer in the Sample dilution.
2. a kind of detection kit of antinuclear antibodies spectrum according to claim 1, it is characterised in that:Wherein branch's microballon
It is upper to be coated with following autoantigen respectively：SSA 52 kDa, SSA 60 kDa, SSB, Sm, U snRNP B/B', U1
snRNP 68, U1 snRNP A, U1 snRNP C, Scl-70, Jo-1, Centromere B, PM Scl,
Nucleosome, Ribosomal P, PCNA, dsDNA, Histone H, Histone HLY and M2.
3. a kind of detection kit of antinuclear antibodies spectrum according to claim 1, it is characterised in that:Compounded microbeads suspension
Component and its is a concentration of,
。
4. a kind of detection kit of antinuclear antibodies spectrum according to claim 2, it is characterised in that:Compounded microbeads suspension
In Tween20 replaced with the triton x-100 of the lauryl sodium sulfate of 0.1-0.5g/L or 0.5-1.5ml/L.
5. a kind of detection kit of antinuclear antibodies spectrum according to claim 2, it is characterised in that:Compounded microbeads suspend
Sodium Azide in liquid are replaced with the thimerosal of 0.5g/L-5g/L.
6. a kind of detection kit of antinuclear antibodies spectrum according to claim 1, it is characterised in that:Phycoerythrin marks
Goat-anti-human IgG（Specific γ chains）Initial concentration be 1.0 ug/mL, the dilution calibration of compounded microbeads suspension is added to dense
Spend 0.6ug/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810883443.8A CN108802375A (en) | 2018-08-06 | 2018-08-06 | A kind of detection kit of antinuclear antibodies spectrum |
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| CN201810883443.8A CN108802375A (en) | 2018-08-06 | 2018-08-06 | A kind of detection kit of antinuclear antibodies spectrum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113917161A (en) * | 2021-10-18 | 2022-01-11 | 北京和杰创新生物医学科技有限公司 | Detection method of anti-perinuclear factor antibody immunoglobulin G |
| CN114814203A (en) * | 2022-04-20 | 2022-07-29 | 北京胡曼智造科技有限责任公司 | Combined detection kit for antinuclear antibodies |
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| CN114814203A (en) * | 2022-04-20 | 2022-07-29 | 北京胡曼智造科技有限责任公司 | Combined detection kit for antinuclear antibodies |
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