CN109387629A - A kind of detection reagent detecting thyroid peroxidase antibody - Google Patents
A kind of detection reagent detecting thyroid peroxidase antibody Download PDFInfo
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- CN109387629A CN109387629A CN201710658714.5A CN201710658714A CN109387629A CN 109387629 A CN109387629 A CN 109387629A CN 201710658714 A CN201710658714 A CN 201710658714A CN 109387629 A CN109387629 A CN 109387629A
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- thyroid peroxidase
- reagent
- preservative
- peroxidase antibody
- containing protein
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- 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
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
Abstract
The invention discloses a kind of detection reagents for detecting thyroid peroxidase antibody, comprising: reagent one: the magnetic particle of thyroid peroxidase antigen coat, preservative 0.1%, the phosphate buffer containing protein stabiliser；Reagent two: preservative 0.1%, the phosphate dilution containing protein stabiliser；Reagent three: the acridinium ester of mouse anti-human igg label, preservative 0.1%, the phosphate buffer containing protein stabiliser；Calibration object: 1 32.14IU/mL containing thyroid peroxidase antibody of level, preservative 0.1%, the phosphate buffer containing protein stabiliser；2 159.24IU/mL containing thyroid peroxidase antibody of level, preservative 0.1%, the phosphate buffer containing protein stabiliser.Quality-control product: 21.18-36.38IU/mL containing thyroid peroxidase antibody, preservative 0.1%, the phosphate buffer containing protein stabiliser；Detection reagent sensitivity with higher and accuracy, the Full-automatic chemiluminescence immunoassay analysis meter produced with our company, which matches, greatly reduces reagent cost.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of detection examination for detecting thyroid peroxidase antibody
Agent.
Background technique
Thyroid peroxidase antibody is main parathyroid tissue autoantibody, is thyroid hormone synthesis process
Key enzyme, it is closely related with parathyroid tissue immune injury.In recent years, it is ground by immunology, biochemistry and genetic recombination
Study carefully, have proven to the main component that thyroid peroxidase antibody is thyroid microsomal, thyroid peroxidase antibody is
The active constituent of thyroid microsomal antibodies, therefore the thyroid peroxidase antibody for being present in patient's body is exactly thyroid gland
Microsome.Thyroid peroxidase antibody is also the important autoantigen of autoimmune thyroid disease, can stimulate machine
Body immune system generates anti-thyroid peroxidase antibody.It measures the clinical meaning of anti-thyroid peroxidase antibody and resists
Thyroglobulin antibody is also roughly the same, mainly for chronic lymphocytic thyroiditis, hyperthyroidism, original
Hair property hypothyroidism has auxiliary diagnosis, efficacy assessment value.Certain patient's anti-thyroglobulin antibody are negative,
But anti-thyroid peroxidase antibody is positive, thus two kinds of antibody detect the positive that antithyroid autoantibody can be improved simultaneously
Recall rate, and can be used as the important evidence of clinical diagnosis and antidiastole autoimmune thyroiditis.
Detection thyroid peroxidase antibody helps to solve the problems, such as that clinical diagnosis occurs, such as abnormal high rush first
Shape hormone levels are considered as Asia if thyroid peroxidase antibody increases with the free thyroxine of normal level simultaneously
Clinical thyroid hypofunction and early stage chronic lymphocytic thyroiditis.Low-level thyroid peroxidase antibody exists
10% is accounted in asymptomatic patient, indication is susceptible to suffer from Autoimmune thyroid diseases；85% hyperthyroidism and first subtract patient and show high level
Thyroid peroxidase antibody, therefore in the diagnosis of most of Autoimmune thyroid diseases, thyroid gland peroxidating
Object enzyme antibody and thyroglobulin antibody joint-detection have higher clinical value.In addition, postpartum thyroiditis, atrophic
Thyroid gland, part nodular goiter patient, thyroid peroxidase antibody can be the positive；Certain autoimmune diseases
As rheumatism, the visible thyroid peroxidase antibody of systemic lupus hung classes increase.
At present clinically detect thyroid peroxidase antibody method have radioimmunoassay, EIA enzyme immunoassay,
Immunofluorescence analysis, Time-resolved Fluoroimmunoassay and chemiluminescence immune assay etc..Radioimmunoassay high sensitivity,
Still this method has radioactive pollution to high specificity, and cumbersome, market is gradually dispirited；EIA enzyme immunoassay is easy to operate, examines
It is short and pollution-free to survey the time, but sensitivity is not good enough；It is special that immunofluorescence analysis and Time-resolved Fluoroimmunoassay need
Instrument and equipment, testing cost is more expensive；Chemiluminescence immune assay has high sensitivity, and high specificity, detection range are wide, special
It is the advantages such as "dead" harm, is gradually replacing radioimmunoassay and EIA enzyme immunoassay.It is real in medical institutions and clinic
It tests in room, the reagent of better performances has Roche, Abbott Laboratories, Beckman, Siemens, Hitachi etc., and these types of detection method is resulting to face
The detected value of bed sample can more actually react the morbid state of patient, and high specificity, high sensitivity, rate of missed diagnosis and misdiagnosis rate are all
Lower, often as the ideal method of clinical samples detection, but mentioned reagent must be matched with expensive necessary instrument,
Reagent cost is considerably increased, large hospital and scientific research institution are primarily adapted for use in.It is directed to for national second level and following hospital
It is very high using import reagent cost, it is not suitable for promoting.The detection reagent of detection thyroid peroxidase antibody domestic at present
Using Beijing Yuan De Bioisystech Co., Ltd as representative, domestic reagent does not need the instrument of special expensive, and testing cost is lower,
Suitable for small-middle hospital and some township hospital, but there are certain stability is bad, phenomena such as repeatability is slightly poor.Therefore,
It is necessary to develop to locate domestic high performance chemiluminescence immune assay detection reagent.
Summary of the invention
The purpose of the present invention is to provide a kind of detection reagent for detecting thyroid peroxidase antibody, the detection reagents
Sensitivity with higher and accuracy, the Full-automatic chemiluminescence immunoassay analysis meter produced with our company matches to drop significantly
Low reagent cost.
Technical solution of the present invention includes the following contents: 1, a kind of detection examination for detecting thyroid peroxidase antibody
Agent, comprising: reagent one: the magnetic particle suspension of thyroid peroxidase antigen coat；Reagent two: reaction dilution；Reagent
Three: the acridinium ester marked with mouse anti-human igg；Calibration object and quality-control product.It is characterized in that by following proportion: one group of reagent becomes
The magnetic particle suspension 2.5mg of thyroid peroxidase antigen coat, 7.5 μ L of preservative, dilution are containing protein stabilized
The phosphate buffer of agent.Two groups of reagent become the phosphate buffer containing protein stabiliser, 7.5 μ L of preservative.Reagent three
Group becomes the acridinium ester 2.5mg marked with mouse anti-human igg, 7.5 μ L of preservative, and dilution is the phosphate containing protein stabiliser
Buffer.Calibration object group becomes level 1: thyroid peroxidase antibody 32.14IU/mL, 1 μ L of preservative, containing protein stabilized
The phosphate buffer of agent；Level 2: thyroid peroxidase antibody 159.24IU/mL, 1 μ L of preservative contain protein stabiliser
Phosphate buffer.Quality-control product group becomes 1 21.18-36.38IU/mL containing thyroid peroxidase antibody of quality-control product, anti-corrosion
1 μ L of agent, the phosphate buffer containing protein stabiliser；2 103.07- containing anti-thyroid peroxidase antibody of quality-control product
147.27IU/m, 1 μ L of preservative, the phosphate buffer containing protein stabiliser；
In the magnetic particle suspension of the thyroid peroxidase antigen coat, used magnetic particle has superparamagnetic
Property, partial size is 1.0-1.2 μm.
The sample of detection reagent of the present invention is human serum or blood plasma, is also applied for containing ethylenediamine tetra-acetic acid, sodium citrate
Or the sample of anticoagulant heparin agent.
Reagent of the invention uses chemoluminescence method magnetic particle detection method, and stable processing technique is feasible, and production cost is low, right
The system evaluation of the reagent shows that it, with accuracy height, the sensitivity and specificity of height and is not easily susceptible to various chaff interferents
The influence of matter.Reagent is reproducible, and validity period up to 12 months, is suitable for clinical application.
Specific embodiment
A kind of detection reagent detecting thyroid peroxidase antibody, comprising:
Reagent one: the magnetic particle suspension of thyroid peroxidase antigen coat；
Reagent two: reaction dilution；
Reagent three: the acridinium ester of mouse anti-human igg label；
Calibration object accuses product.
The preparation of 1 reagent one of embodiment:
Step 1: magnetic particle solution is mixed well, and 1mL magnetic particle homogeneous solution (content 1%) is taken to be placed in micro-pipe
In.Micro-pipe is stood 1 minute on the magnetic base, and removes supernatant.1mL reaction solution is taken to be added in above-mentioned micro-pipe, sufficiently
100 μ g thyroid peroxidase antigens are added in reaction solution after mixing concussion 30 minutes, continues room temperature concussion and mixes 30
Minute.Micro-pipe is set 1 minute on the magnetic base, supernatant is removed.After being rinsed with 1mL cleaning buffer solution to magnetic particle,
It stands and removes supernatant, repeated washing obtains the magnetic particle suspension of thyroid peroxidase antigen coat afterwards three times.Addition refers to
The quantitative phosphate buffer containing protein stabiliser mixes to obtain reagent one.
Step 2: the determination of one concentration of reagent
By the magnetic particle diluted of the magnetic particle assay approval of thyroid peroxidase antigen coat, with sample-adding
It measures volume and carries out orthogonal experiment, dilution ratio is respectively 1: 6.25,1: 12.5,1: 25 and 1: 50,25 μ L of sample-adding amount volume, 50 μ
L.Reaction system is evaluated using TPO antigen gradient dilution sample, the results are shown in Table 1.
The dilution ratio and sample-adding amount orthogonal experiment of 1 magnetic particle of table
As can be known from the results of Table 1: the dilution of magnetic particle 1: 12.5,50 μ L are in use, linearly dependent coefficient r is best
0.9982, slope also preferably 0.9341 at this time, and background is lower.Comprehensively consider linearly dependent coefficient r, slope, variation and sheet
Bottom, it is 50 μ L that the magnetic particle dilution ratio of last preferably thyroid peroxidase antigen coat, which is 1: 12.5, sample-adding amount,.
The preparation of 2 reagent two of embodiment:
Step 1: (1) taking mouse anti-human IgG antibodies 1mg, is dialysed 24 hours with combination buffer, and matched with combination buffer
Concentration 1mg/mL is made.(2) it takes acridinium ester 1mg to be dissolved in 1mL dimethyl sulfoxide and obtains concentration as 1mg/mL.(3) it takes above-mentioned dense
The 80 μ L of acridine ester solution of degree is added in antibody-solutions, and is sufficiently stirred 2 hours.(4) with storage buffer in step (3)
Solution carry out dialysis 24 hours, mixed well after adding 50% glycerol.The phosphoric acid containing protein stabiliser of specified amount is added
Salt buffer obtains reagent two after mixing.
Step 2: the determination of two concentration of reagent
Mouse anti-human igg label acridinium ester is carried out according to 1: 100,1: 500,1: 1000 and 1: 2000 ratio respectively dilute
It releases, the sample-adding amount volume with 50 μ L, 100 μ L carries out orthogonal experiment.Use thyroid peroxidase antigen gradient dilution sample
Reaction system is evaluated, testing result is shown in Table 2.
2 mouse anti-human igg of table marks the concentration gradient and sample-adding amount orthogonal experiment of acridinium ester
As can be seen from Table 2, the acridinium ester of mouse anti-human igg label is linearly related in 1: 100 100 μ L sample-adding amount of dilution
Coefficient r is best, is 0.9963, slope is preferably also 0.9385 at this time.It is therefore preferable that the acridinium ester dilution of mouse anti-human igg label
Ratio is 1/100, sample-adding amount is 100 μ L.
3 reagent of embodiment, three diluted concentration determines
Reaction dilution raw material is diluted respectively according to 1: 100,1: 200,1: 400,1: 800, by detecting first shape
Gland Peroxidase Antibody gradient dilution sample, evaluates reaction system.Testing result is shown in Table 3.
The dilution ratio of the reaction dilution of table 3
As can be seen from Table 3, for reaction dilution in 1: 200 dilution, linearly dependent coefficient r is best, is 0.9972, at this time
Linear reference product slope is preferably also 0.9580.Therefore final preferred reaction diluted ratio is 1: 200.
The preparation of 4 calibration object of embodiment
It takes thyroid peroxidase antibody to be added to after the phosphate buffer containing protein stabiliser mixes well, uses state
Family's standard items carry out assignment.
The preparation of the charge product of embodiment 5
It takes thyroid peroxidase antibody to be added to after the phosphate buffer containing protein stabiliser mixes well, uses state
Family's standard items carry out assignment.
6 detection method of embodiment:
The first step mixes the magnetic particle of sample, reaction dilution and Anti-thyroid Peroxidase antigen coat simultaneously
It is incubated for, the thyroid peroxidase antibody in sample is combined with the magnetic particle of Anti-thyroid Peroxidase antigen coat.
Enter second step after washing, the acridinium ester that anti-human igg label is added is incubated for form " thyroid peroxidase antigen magnetic particle-
Thyroid peroxidase antibody-anti-human igg acridinium ester " compound, after washing, in the reactive mixture be added preexciting agent and
Exciting agent.The phase that thyroid peroxidase IgG antibody content and Full-automatic chemiluminescence immune system in sample detect
It is directly proportional to light value, thyroid peroxidase antibody concentration is determined by calibration curve.This reagent uses in methodology
The chemoluminescence method of high sensitivity, easy to operate, sensitivity for analysis is high, can substantially reduce testing cost, and in methodology
On can keep away enzyme-linked immunosorbent assay due to missing inspection caused by thyroid peroxidase antibody low titre in serum or blood plasma.
The verifying of 7 range of linearity of embodiment:
With the buffer system of detection reagent by anti-thyroid peroxidase antibody national standard, according to the line of this reagent
Property range (2.00IU/mL~1000.00IU/mL), dilute at least five concentration point in proportion, replication 3 times, it is flat to calculate it
Result average value and dilution ratio are carried out straight line fitting with least square method by mean value, and calculating linearly dependent coefficient r should not be low
In 0.9900.
The test of 4 range of linearity of table
8 sensitivity test of embodiment:
It is operated in strict accordance with specification, detects zero calibration object, replication 20 times, obtain the luminous value of 20 measurement results
(relative light unit) calculates its average and standard deviation, obtains the sum of average value and twice of standard deviation, according to zero-dose calibration object
Concentration and luminous value (relative light unit) between calibration object level one carry out two o'clock recurrence, obtain linear function, will be averaged
The sum of value and twice of standard deviation are brought into this linear function, obtain corresponding concentration value, as minimum detection limit, as a result Ying Bugao
In 1.00IU/mL.
5 sensitivity test of table
9 stability experiment of embodiment:
Under 2-8 DEG C of condition of storage, quality-control product is measured 0 month, 12 months respectively, each sample measures 10
It is secondary, take mean value.The result shows that (see the table below), the coefficient of variation can satisfy requirement, illustrate the detection reagent in embodiment 1,2 in 2-
It can at least stablize 12 months under 8 DEG C of conditions of storage.
The test of 6 stability experiment of table
It is provided for the embodiments of the invention a kind of anti-thyroid peroxidase antibody detection reagent above, has carried out in detail
Thin to introduce, used herein a specific example illustrates the principle and implementation of the invention, and above embodiments are said
It is bright to be merely used to help understand method and its core concept of the invention；At the same time, for those skilled in the art, foundation
Thought of the invention, there will be changes in the specific implementation manner and application range, in conclusion the content of the present specification is not
It is interpreted as limitation of the present invention.
Claims (2)
1. a kind of detection reagent for detecting thyroid peroxidase antibody, comprising: reagent one: thyroid peroxidase antigen
Coated magnetic particle suspension；Reagent two: reaction dilution；Reagent three: the acridinium ester marked with mouse anti-human igg；Calibration object and
Quality-control product, it is characterised in that be by following proportion: one group of the reagent magnetic particle for becoming thyroid peroxidase antigen coat is outstanding
Supernatant liquid 2.5mg, 7.5 μ L of preservative, dilution are the TRIS buffer containing protein stabiliser.Two groups of reagent
Dilution, 7.5 μ L of preservative are reacted as the trishydroxymethylaminomethane containing protein stabiliser.Three groups of reagent become anti-with mouse
The acridinium ester 2.5mg of human IgG label, 7.5 μ L of preservative, dilution are slow for the trishydroxymethylaminomethane containing protein stabiliser
Fliud flushing.Calibration object group becomes 1 32.14IU/mL containing anti-thyroid peroxidase antibody of level, 1 μ L of preservative, containing protein stabilized
The phosphate buffer of agent, 2 159.24IU/mL containing anti-thyroid peroxidase antibody of level, 1 μ L of preservative are steady containing albumen
Determine the phosphate buffer of agent.Quality-control product group becomes 1 21.18-36.38IU/ containing anti-thyroid peroxidase antibody of quality-control product
ML, 1 μ L of preservative, the phosphate buffer containing protein stabiliser, quality-control product 2 contain anti-thyroid peroxidase antibody
103.07-147.27IU/m 1 μ L of preservative, the phosphate buffer containing protein stabiliser.
2. a kind of detection reagent for detecting thyroid peroxidase antibody according to claim 1, it is characterised in that anti-
The coated magnetic particle of thyroid peroxidase antibody has superparamagnetism, and partial size is 1.0-1.2 μm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710658714.5A CN109387629A (en) | 2017-08-04 | 2017-08-04 | A kind of detection reagent detecting thyroid peroxidase antibody |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710658714.5A CN109387629A (en) | 2017-08-04 | 2017-08-04 | A kind of detection reagent detecting thyroid peroxidase antibody |
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| CN109387629A true CN109387629A (en) | 2019-02-26 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336115A (en) * | 2013-07-17 | 2013-10-02 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof |
| CN104237513A (en) * | 2014-09-30 | 2014-12-24 | 博奥赛斯（天津）生物科技有限公司 | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit |
| CN105467122A (en) * | 2015-11-17 | 2016-04-06 | 苏州浩欧博生物医药有限公司 | Kit and method for detection of thyroid peroxidase antibody |
| CN106053440A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Mycoplasma pneumoniae IgG chemiluminescence immunoassay kit and preparation method thereof |
-
2017
- 2017-08-04 CN CN201710658714.5A patent/CN109387629A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336115A (en) * | 2013-07-17 | 2013-10-02 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof |
| CN104237513A (en) * | 2014-09-30 | 2014-12-24 | 博奥赛斯（天津）生物科技有限公司 | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit |
| CN105467122A (en) * | 2015-11-17 | 2016-04-06 | 苏州浩欧博生物医药有限公司 | Kit and method for detection of thyroid peroxidase antibody |
| CN106053440A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Mycoplasma pneumoniae IgG chemiluminescence immunoassay kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王晓娟，徐军发，徐霞主编: "《临床免疫学检验实验》", 28 February 2014, 武汉：华中科技大学出版社 * |
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