CN114113590A - Mycoplasma pneumoniae and chlamydia antibody detection kit based on magnetic particle luminescence method - Google Patents
Mycoplasma pneumoniae and chlamydia antibody detection kit based on magnetic particle luminescence method Download PDFInfo
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- CN114113590A CN114113590A CN202111240285.2A CN202111240285A CN114113590A CN 114113590 A CN114113590 A CN 114113590A CN 202111240285 A CN202111240285 A CN 202111240285A CN 114113590 A CN114113590 A CN 114113590A
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- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 241000606161 Chlamydia Species 0.000 title claims abstract description 14
- 241000202934 Mycoplasma pneumoniae Species 0.000 title claims abstract description 14
- 238000002796 luminescence method Methods 0.000 title claims abstract description 12
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 33
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 19
- 239000004005 microsphere Substances 0.000 claims abstract description 19
- 239000011534 wash buffer Substances 0.000 claims abstract description 11
- 238000002474 experimental method Methods 0.000 claims abstract description 9
- 239000012224 working solution Substances 0.000 claims abstract description 9
- 230000003213 activating effect Effects 0.000 claims abstract description 6
- 239000011248 coating agent Substances 0.000 claims abstract description 6
- 238000000576 coating method Methods 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 4
- 239000011325 microbead Substances 0.000 claims description 20
- 230000004913 activation Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 239000004793 Polystyrene Substances 0.000 claims description 6
- 229920002223 polystyrene Polymers 0.000 claims description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 3
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
- 108010004729 Phycoerythrin Proteins 0.000 claims 1
- 108010090804 Streptavidin Proteins 0.000 claims 1
- 239000011324 bead Substances 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 claims 1
- 239000003550 marker Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 238000002331 protein detection Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 239000011550 stock solution Substances 0.000 description 24
- 230000000087 stabilizing effect Effects 0.000 description 12
- 229920001213 Polysorbate 20 Polymers 0.000 description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 10
- 239000012535 impurity Substances 0.000 description 8
- 239000008363 phosphate buffer Substances 0.000 description 8
- 238000007865 diluting Methods 0.000 description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000005406 washing Methods 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000011146 sterile filtration Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
-
- 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
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
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- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
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- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a mycoplasma pneumoniae and chlamydia antibody detection kit based on a magnetic particle luminescence method, and belongs to the technical field of detection reagents. The method comprises a capture microsphere mixed solution, a detection antibody, SA-PE, an experiment buffer solution, a washing buffer solution and a working solution, wherein the production process comprises the steps of activating and coating prepared microspheres, preparing the detection antibody, preparing the SA-PE, preparing the experiment buffer solution, preparing the washing buffer solution and the working solution, so that the problem that four detection reagents are required to be independently used for respectively detecting MP-IgG, MP-IgM, CP-IgG and CP-IgM proteins when detecting the MP-IgG, MP-IgM, CP-IgG and CP-IgM proteins in a sample is solved, the detection time is shortened, and the detection efficiency is improved.
Description
Technical Field
The invention relates to a mycoplasma pneumoniae and chlamydia antibody detection kit based on a magnetic particle luminescence method, belongs to the technical field of detection reagents, and discloses a detection kit capable of simultaneously determining four proteins, namely MP-IgG, MP-IgM, CP-IgG and CP-IgM, in a sample in a micropore.
Background
MP-IgG, MP-IgM, CP-IgG and CP-IgM are common antibodies in mycoplasma pneumoniae and chlamydia, however, with the progress of medical level, MP-IgG, MP-IgM, CP-IgG and CP-IgM can be detected by the existing detection reagent, and then further treatment is carried out, however, when MP-IgG, MP-IgM, CP-IgG and CP-IgM in a sample are detected, four detection reagents are required to be used for separately detecting MP-IgG, MP-IgM, CP-IgG and CP-IgM, so that the detection time is increased, and the detection efficiency is reduced.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the utility model provides a mycoplasma pneumoniae and chlamydia antibody detection kit based on a magnetic particle luminescence method, which solves the problem that when MP-IgG, MP-IgM, CP-IgG and CP-IgM in a sample are detected, four detection reagents are needed to be used separately to detect MP-IgG, MP-IgM, CP-IgG and CP-IgM respectively.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a detection kit for antibodies of mycoplasma pneumoniae and chlamydia based on a magnetic particle luminescence method comprises a capture microsphere mixed solution, a detection antibody, SA-PE, an experiment buffer solution, a washing buffer solution and a working solution,
(1) capturing microsphere mixed liquor: the suspension contains identifiable polystyrene microbeads, and the polystyrene microbeads are respectively coated with the following antibodies: anti-human MP-IgG antibody, anti-human MP-IgM antibody, anti-human CP-IgG antibody, anti-human CP-IgM antibody;
(2) detecting an antibody: diluting the stock solution with protein stabilizing solution 62.5 times under the condition that the concentration of the stock solution is 0.5mg/mL to ensure that the concentration is 0.8 mu g/mL;
(3) and SA-PE: diluting the stock solution with protein stabilizing solution 50 times under the condition that the concentration of the stock solution is 1mg/mL to ensure that the concentration is 2 mug/mL;
(4) working fluid: diluting the stock solution with protein stabilizing solution 6.7 times under the condition that the concentration of the stock solution is 40.6mg/mL to ensure that the concentration is 0.6 mg/mL;
(5) experiment buffer solution: adding 50g BSA, 1mL Tween-20, 3mL Proclin300 and 0.5mL RPE2520 into 1000mL of phosphate buffer, stirring for more than 30min, and performing sterile filtration with the diameter of at least 0.45 μm after the solution is clear and free of impurities;
(6) washing buffer solution: 5mL Tween-20, 3mL Proclin300 and 0.3mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
As a preferred example, the production process of the kit mainly comprises the following steps:
preparing an activation buffer solution, wherein the main components of the activation buffer solution comprise: namely 8.0g/L NaCl reagent, 2.9g/L Na2HPO4 & 12H2O reagent, 2.4g/L KCl reagent, 2.4g/L KH2PO4 reagent, 0.5g/L Tris reagent, 0.5mL/L Tween20 reagent and 0.5g/L SodiumAzide reagent.
② activation and coating of microspheres, adding 1mL of microspheres with specific numbers into a 1.5mL centrifuge tube, adding 5-50uLEDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 5-50uLSuLfo-NHS (N-hydroxy thiosuccinimide), adding 10-100ug of specific antibody, vortex mixing for 30 seconds, ultrasonic mixing for 30 seconds, and shaking for overnight at 2-8 ℃.
Preparing a detection antibody: when the concentration of the stock solution was 0.5mg/mL, the stock solution was diluted 62.5-fold with the protein stabilizing solution to a concentration of 0.8. mu.g/mL.
Preparing SA-PE: when the concentration of the stock solution was 1mg/mL, the stock solution was diluted 50-fold with the protein stabilizing solution to a concentration of 2. mu.g/mL.
Preparing working solution: when the concentration of the stock solution was 40.6mg/mL, the stock solution was diluted 6.7-fold with the protein stabilizing solution to a concentration of 0.6 mg/mL.
Preparing an experimental buffer solution: 50g BSA, 1mL Tween-20, 3mL Proclin300 and 0.5mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
Preparing a washing buffer solution: 5mL Tween-20, 3mL Proclin300 and 0.3mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
The invention has the beneficial effects that: the method comprises the steps of capturing microsphere mixed liquor, detecting antibodies, SA-PE, experiment buffer solution, washing buffer solution and working solution, wherein the production process comprises the steps of preparing activation buffer solution, activating and coating microspheres, preparing detection antibodies, preparing SA-PE, preparing working solution, preparing experiment buffer solution and preparing washing buffer solution, the problem that when MP-IgG, MP-IgM, CP-IgG and CP-IgM in a sample are detected, four detection reagents are required to be used for separately detecting MP-IgG, MP-IgM, CP-IgG and CP-IgM is solved, the detection time is shortened, and the detection efficiency is improved.
Detailed Description
In order to make the technical means, the original characteristics, the achieved purpose and the efficacy of the invention easily understood, the invention is further described with reference to the following embodiments.
A detection kit for antibodies of mycoplasma pneumoniae and chlamydia based on a magnetic particle luminescence method comprises a capture microsphere mixed solution, a detection antibody, SA-PE, an experiment buffer solution, a washing buffer solution and a working solution,
(1) capturing microsphere mixed liquor: the suspension contains identifiable polystyrene microbeads, and the polystyrene microbeads are respectively coated with the following antibodies: anti-human MP-IgG antibody, anti-human MP-IgM antibody, anti-human CP-IgG antibody, anti-human CP-IgM antibody;
(2) detecting an antibody: diluting the stock solution with protein stabilizing solution 62.5 times under the condition that the concentration of the stock solution is 0.5mg/mL to ensure that the concentration is 0.8 mu g/mL;
(3) and SA-PE: diluting the stock solution with protein stabilizing solution 50 times under the condition that the concentration of the stock solution is 1mg/mL to ensure that the concentration is 2 mug/mL;
(4) working fluid: diluting the stock solution with protein stabilizing solution 6.7 times under the condition that the concentration of the stock solution is 40.6mg/mL to ensure that the concentration is 0.6 mg/mL;
(5) experiment buffer solution: adding 50g BSA, 1mL Tween-20, 3mL Proclin300 and 0.5mL RPE2520 into 1000mL of phosphate buffer, stirring for more than 30min, and performing sterile filtration with the diameter of at least 0.45 μm after the solution is clear and free of impurities;
(6) washing buffer solution: 5mL Tween-20, 3mL Proclin300 and 0.3mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
The production process of the kit mainly comprises the following steps:
preparing an activation buffer solution, wherein the main components of the activation buffer solution comprise: namely 8.0g/L NaCl reagent, 2.9g/L Na2HPO4 & 12H2O reagent, 2.4g/L KCl reagent, 2.4g/L KH2PO4 reagent, 0.5g/L Tris reagent, 0.5mL/L Tween20 reagent and 0.5g/L SodiumAzide reagent;
② activation and coating of microspheres, adding 1mL of microspheres with specific numbers into a 1.5mL centrifuge tube, adding 5-50uLEDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 5-50uLSuLfo-NHS (N-hydroxy thiosuccinimide), adding 10-100ug of specific antibody, vortex mixing for 30 seconds, ultrasonic mixing for 30 seconds, and shaking for overnight at 2-8 ℃.
When the detection kit is produced, a table concentrator for placing 4 antibody bottles simultaneously needs to be arranged to coat microspheres with different numbers respectively, and the steps are as follows:
1) washing: performing suction filtration washing, and transferring the washed microbeads to an activation buffer solution;
2) quality control: verifying quality by using a quality control panel;
3) mixing multiple coated microbeads, washing residual microbeads on the wall of the bottle with a small amount of activating buffer solution, mixing, diluting to constant volume with the activating buffer solution, and packaging after inspection is qualified.
Preparing a detection antibody: when the concentration of the stock solution was 0.5mg/mL, the stock solution was diluted 62.5-fold with the protein stabilizing solution to a concentration of 0.8. mu.g/mL.
Preparing SA-PE: when the concentration of the stock solution was 1mg/mL, the stock solution was diluted 50-fold with the protein stabilizing solution to a concentration of 2. mu.g/mL.
Preparing working solution: when the concentration of the stock solution was 40.6mg/mL, the stock solution was diluted 6.7-fold with the protein stabilizing solution to a concentration of 0.6 mg/mL.
Preparing an experimental buffer solution: 50g BSA, 1mL Tween-20, 3mL Proclin300 and 0.5mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
Preparing a washing buffer solution: 5mL Tween-20, 3mL Proclin300 and 0.3mL RPE2520 were added to 1000mL of phosphate buffer, stirred for more than 30min, and sterile filtered to at least 0.45 μm after the solution was clear and free of impurities.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A mycoplasma pneumoniae and chlamydia antibody detection kit based on a magnetic particle luminescence method is characterized by comprising: (1) a plurality of sealed reagent bottles which are respectively filled with capture microsphere mixed liquor, detection antibody, SA-PE, experiment buffer solution, washing buffer solution and working solution, and (2) a kit which separates and integrally packages the reagent bottles; the microbeads are polystyrene microspheres with uniform size, and the polystyrene microspheres are dyed and numbered by different fluorescent dyes, so that microbeads with different codes are obtained; the capture microsphere mixed solution contains protein detection microbeads, and the method specifically comprises the following steps:
the detection micro-beads are coated with anti-human MP-IgG antibody, anti-human MP-IgM antibody, anti-human CP-IgG antibody and anti-human CP-IgM antibody on the surfaces of the micro-beads with different codes respectively.
2. The kit for detecting antibodies to mycoplasma pneumoniae and chlamydia based on the magnetic particle luminescence method according to claim 1, wherein the diameter of the micro-beads is 5-7 microns.
3. The kit for detecting antibodies against mycoplasma pneumoniae and chlamydia based on the magnetic particle luminescence method as claimed in claim 1, wherein the fluorescent marker is phycoerythrin-labeled streptavidin.
4. The kit for detecting antibodies against mycoplasma pneumoniae and chlamydia based on the magnetic particle luminescence method according to claim 1, wherein the preparation method of the capture microsphere mixture comprises the following steps: and respectively coating different mycoplasma pneumoniae and chlamydia antibody antibodies by using different coded microbeads, and mixing to prepare the detection microbeads to obtain the capture microsphere mixed solution.
5. The kit for detecting antibodies against mycoplasma pneumoniae and chlamydia based on the magnetic particle luminescence method according to claim 1, wherein the detection beads are prepared by the following method: preparing a microbead activating solution, adding any one antibody according to the proportion that each milliliter of microbead contains 10-100ug of antibody, fully mixing, coating different antibodies, and using microbeads with different codes; the microbead activating solution is prepared by adding microbeads of 1000 unit volumes into a reaction tube, and adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride of 5-50 unit volumes and N-hydroxy thiosuccinimide of 5-50 unit volumes for activation.
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| CN202111240285.2A CN114113590A (en) | 2021-10-25 | 2021-10-25 | Mycoplasma pneumoniae and chlamydia antibody detection kit based on magnetic particle luminescence method |
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| CN202111240285.2A CN114113590A (en) | 2021-10-25 | 2021-10-25 | Mycoplasma pneumoniae and chlamydia antibody detection kit based on magnetic particle luminescence method |
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2021
- 2021-10-25 CN CN202111240285.2A patent/CN114113590A/en active Pending
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| CN102928587A (en) * | 2012-11-16 | 2013-02-13 | 南京凯基生物科技发展有限公司 | Colloidal gold method detection test strip and reagent kit for IgM and IgG antibodies of mycoplasma pneumoniae and preparation method of reagent kit |
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