CN107966563A - A kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits and preparation method thereof - Google Patents
A kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits and preparation method thereof Download PDFInfo
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- CN107966563A CN107966563A CN201611018772.3A CN201611018772A CN107966563A CN 107966563 A CN107966563 A CN 107966563A CN 201611018772 A CN201611018772 A CN 201611018772A CN 107966563 A CN107966563 A CN 107966563A
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- acridinium ester
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- G—PHYSICS
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- 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
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/901—Antibodies with enzymatic activity; e.g. abzymes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/328—Vasculitis, i.e. inflammation of blood vessels
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Abstract
The invention discloses a kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits, the kit includes：The magnetic particle of myeloperoxidase antigen coat, the mouse anti-human IgG antibodies of acridinium ester label, antimyeloperoxidase antibody IgG calibrations product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits.The advantages that kit of the present invention is easy to operate compared with available reagent box, high sensitivity, detection range is wide.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, and specifically, the present invention provides a kind of inspection of chemiluminescence immunoassay
Survey antimyeloperoxidase antibody IgG kits and preparation method thereof.
Background technology
Myeloperoxidase（Myeloperoxidase, MPO）It is glycosylated hemoprotein, is primarily present in medullary system
Cell（Mainly neutrophil cell and monocyte）Aniline blue particles in, environmental stimuli can cause neutrophil leucocyte to gather
Collect, release MPO, 95% MPO is to be released into blood by the neutrophil degranulation activated in blood.MPO is by 2
The dimer that subunit polymerization forms, each subunit is again by a heavy chain（α chains）With a light chain（β chains）Form, 2 sub- single
Position is connected at α chains by 1 disulfide bond.
MPO levels are the activation of neutrophil leucocyte with there is extremely significant correlation between neutrophil activation degree
Diagnosis on marker Present clinical to acute coronary syndrome is with assessment mainly using indexs such as myocardial enzymes.Such index
Level would generally occur significantly raised after substantial damage occurs for cardiac muscle, and therefore, such index cannot be to the hair of angiocardiopathy
It is raw that early warning is provided.The measure of MPO antibody can be used for the diseases such as diagnosis of autoimmune vasculitis, be treated simultaneously for monitoring
It is significant with state of an illness tracking and judging prognosis etc..
The main method of clinical detection antimyeloperoxidase antibody IgG is enzyme linked immunosorbent assay, but this method exists
Following shortcomings：
（1）Antigen coat apparatus and reaction are used as using 12 × 8 types, 6 × 8 types, 8 × 12 types or 96 hole Special micro porous plate of complete plate
Container, can only be divided into 12 batches, 6 batches, 8 batches or whole plate first use when in use, can not carry out independent, single part
Detection；
（2）Quantitative determination reagent type used is more, each detection reagent will be contained with reagent bottle, and often be used
It is required for replacing imbibition nozzle during a kind of reagent to be filled into the micropore of microwell plate respectively, not only reagent bottle species is more, filling examination
The operation of agent is also extremely cumbersome；
（3）Lack the corresponding mark to detection information, just can only will appreciate that or know by checking the mark of kit external packing box
The product batch number and term of validity information of detection reagent are known, and the information known is uncontrolled in detection process, has very big
Randomness；
（4）Detection reagent in open space, easily causes the cross contamination between various reagents and shadow in detection process
Ring the accuracy of testing result；
（5）For detection process more using manual operations, the dosage of reagent or sample is not bery accurate, and operating process is extremely cumbersome and multiple
It is miscellaneous, bust easily occurs, the accuracy of testing result and precision are poor；
（6）In the quantity configuration of detection project reagent set and using being above the person-portion of item number × 48/96, if necessary to detect
10 projects, then the configuration of reagent and use number must be 10 × 48/96 person-portions, if only a sample needs detection 10 not
Same project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, there is the shortcomings that inadequate economical rationality.
The content of the invention
Antimyeloperoxidase antibody IgG detection techniques have the following disadvantages at present：Testing cost is high, detection sensitivity
Low, the linear narrow range of detection, reappearance is low, cannot quantify, is complicated etc..
It is linear to disclose a kind of low testing cost, high sensitivity, detection precisely in order to overcome shortcoming described above by the present invention
Scope is wide, reappearance is high, can quantify, antimyeloperoxidase antibody IgG kits easy to operate and preparation method thereof.This
Invention prepares chemical luminescence immune analysis reagent box first, mainly includes：Antimyeloperoxidase antibody IgG monoclonal antibody bag
Magnetic particle, the coated acridinium ester of antimyeloperoxidase antibody IgG monoclonal antibody and the antimyeloperoxidase antibody of quilt
IgG calibrates product；Then calibration product are detected using Full-automatic chemiluminescence immunoassay analysis meter, draw standard curve, it is built-in
In computer software, actual sample is tested, concentration of specimens is calculated according to sample luminous value；Finally to antimyeloperoxidase antibody
IgG automatic chemiluminescence immunoassays system carries out performance（Sensitivity, linear, precision, interference）Evaluation.
It is of the invention compared with current technology, has the following advantages：
1st, present invention selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, which is
Direct chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, which is not required the participation of enzyme, more cost-effective；
2nd, the acridinium ester chemiluminescent immunoassay system detection sensitivity that the present invention selects is high, can reach 1.5 AU/mL；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention selects is wide, can reach 1.5-300 AU/mL；
4th, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention selects is high, and in batch and difference between batch is within 5%,
This is that other chemiluminescence immunoassay systems are unapproachable；
5th, chemiluminescence immunoassay system of the invention has realized quantifying for sample, soft to testing by built-in standard curve
Part, only needs test sample to directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized full-automation, and the addition of reagent and sample has the instrument complete entirely
Into operation is easier, reduces artificial error.
Brief description of the drawings
Fig. 1 is the antimyeloperoxidase antibody IgG canonical plottings that embodiment 3 obtains.
Claims (10)
1. a kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits, the kit include：Marrow peroxidating
The nanometer magnetic microsphere of thing enzyme antigen coat, the mouse anti-human IgG antibodies of acridinium ester label, Chemoluminescent substrate, anti-marrow peroxidating
Thing enzyme antibody IgG calibrates product.
2. kit according to claim 1, it is characterised in that the solid phase carrier of the myeloperoxidase antigen coat
For magnetic particle.
3. kit according to claim 1, it is characterised in that the solid phase carrier of the myeloperoxidase antigen coat
Particle diameter for carboxylated is 0.05-1um magnetic particles.
4. kit according to claim 1, it is characterised in that the chemiluminescent labels are acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
5. kit according to claim 1, it is characterised in that the preferred acridinium ester of chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid.
7. kit according to claim 1, it is characterised in that the chemiluminescence preexciting liquid is mass fraction
0.005% ~ 0.5% hydrogen peroxide (H2O2) solution, exciting liquid is the sodium hydroxide of 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
8. kit according to claim 1, it is characterised in that the antimyeloperoxidase antibody IgG calibrates product and is
Antimyeloperoxidase antibody IgG is configured to concentration as 0 AU/mL, 20AU/mL, 40 AU/mL, 80 with standard items buffer solution
AU/mL, 160 AU/mL, 320 AU/mL, 4 DEG C save backup.
9. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include the preparation of the magnetic particle of myeloperoxidase antigen coat, acridinium ester label mouse anti-human IgG antibodies preparation, chemiluminescence
Preparation, the preparation of antimyeloperoxidase antibody IgG calibration product of substrate solution.
10. according to the preparation method of kit described in claim 1 and claim 9, it is characterised in that comprise the following steps：
1）The preparation of the magnetic particle of myeloperoxidase antigen coat：
The nanometer magnetic bead suspension of carboxylated is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, and add EDC aqueous solutions, activate magnetic
Bead surface carboxyl, adds myeloperoxidase antigen, at room temperature suspension 2-10 h, Magneto separate, removes supernatant, Tris buffer solutions
It is resuspended, obtains the magnetic particle of myeloperoxidase antigen coat；Optionally, a diameter of 0.1 μm ~ 2.0 μ of carboxylated nanometer magnetic bead
m；MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the mouse anti-human IgG antibodies of acridinium ester label：
Mouse anti-human IgG antibodies are taken, add carbonate buffer solution, are mixed, acridinium ester is then added and mixes, lucifuge is reacted at room temperature,
Taken out after 1-2 h, centrifuge desalting column desalting processing, handled respectively with pure water and TBS buffer solutions first in desalination processes,
The mouse anti-human IgG antibodies that are eventually adding derive the acridine ester solution of substance markers, collect liquid in centrifuge tube to preserving pipe
Obtain the acridinium ester that mouse anti-human IgG antibodies derive substance markers；
3）Antimyeloperoxidase antibody IgG calibrates the preparation of product：
Antimyeloperoxidase antibody IgG is configured to concentration as 0 AU/mL, 20AU/mL, 40 AU/ with standard items buffer solution
ML, 80 AU/mL, 160 AU/mL, 320 AU/mL, packing is lyophilized, and 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, sequentially add the hydrogen peroxide (H that 0.5 ~ 100uL mass fractions are 20%2O2), 0.5 ~ 5 gram it is anti-
Rotten agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge storage；Optionally, preservative for commercialization sodium azide, PC300,
Surfactant is polysorbas20, Tween 80, Triton X-100, Triton X-405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, sequentially add 0.2 ~ 1 gram of sodium hydroxide, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surface work
Property agent, shake up the storage of rear lucifuge；Optionally, preservative is commercialization sodium azide, PC300, and surfactant is polysorbas20, spits
Temperature 80, Triton X-100, Triton X-405.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620680259X | 2016-06-30 | ||
| CN201620680259 | 2016-06-30 |
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| Publication Number | Publication Date |
|---|---|
| CN107966563A true CN107966563A (en) | 2018-04-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201611018772.3A Pending CN107966563A (en) | 2016-06-30 | 2016-11-21 | A kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269317A (en) * | 2020-02-12 | 2020-06-12 | 珠海丽珠试剂股份有限公司 | Method for coating MPO on solid support, product and application thereof |
| CN111879758A (en) * | 2020-07-28 | 2020-11-03 | 江苏扬新生物医药有限公司 | Acridinium ester antibody labeling method and application thereof |
| CN113945711A (en) * | 2021-10-18 | 2022-01-18 | 北京和杰创新生物医学科技有限公司 | Processing method for reducing non-specific adsorption of magnetic beads in autoimmune antibody detection |
Citations (4)
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|---|---|---|---|---|
| CN103033624A (en) * | 2012-12-16 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human myeloperoxidase chemiluminescent immunodetection kit |
| CN103185797A (en) * | 2011-12-30 | 2013-07-03 | 深圳市亚辉龙生物科技有限公司 | Reagent device and method for detecting anti-myeloperoxidase antibody |
| CN104459107A (en) * | 2014-04-05 | 2015-03-25 | 北京中航赛维生物科技有限公司 | Quantitative measurement kit for myeloperoxidase antibody (MPO-Ab) and detection method thereof |
| CN104897901A (en) * | 2015-05-12 | 2015-09-09 | 西安金磁纳米生物技术有限公司 | Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 |
-
2016
- 2016-11-21 CN CN201611018772.3A patent/CN107966563A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103185797A (en) * | 2011-12-30 | 2013-07-03 | 深圳市亚辉龙生物科技有限公司 | Reagent device and method for detecting anti-myeloperoxidase antibody |
| CN103033624A (en) * | 2012-12-16 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human myeloperoxidase chemiluminescent immunodetection kit |
| CN104459107A (en) * | 2014-04-05 | 2015-03-25 | 北京中航赛维生物科技有限公司 | Quantitative measurement kit for myeloperoxidase antibody (MPO-Ab) and detection method thereof |
| CN104897901A (en) * | 2015-05-12 | 2015-09-09 | 西安金磁纳米生物技术有限公司 | Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 |
Non-Patent Citations (1)
| Title |
|---|
| M. MAHLER ET AL.: "Development and performance evaluation of novel chemiluminescence assays for detection of anti-PR3 and anti-MPO antibodies", 《CLINICA CHIMICA ACTA》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269317A (en) * | 2020-02-12 | 2020-06-12 | 珠海丽珠试剂股份有限公司 | Method for coating MPO on solid support, product and application thereof |
| CN111269317B (en) * | 2020-02-12 | 2022-04-08 | 珠海丽珠试剂股份有限公司 | Method for coating MPO on solid support, product and application thereof |
| CN111879758A (en) * | 2020-07-28 | 2020-11-03 | 江苏扬新生物医药有限公司 | Acridinium ester antibody labeling method and application thereof |
| CN113945711A (en) * | 2021-10-18 | 2022-01-18 | 北京和杰创新生物医学科技有限公司 | Processing method for reducing non-specific adsorption of magnetic beads in autoimmune antibody detection |
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