CN103336115A - Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof - Google Patents
Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof Download PDFInfo
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Abstract
The invention relates to a kit, in particular to a magnetic particle chemiluminescence immunodetection method for TPOAb (thyroid peroxidase antibody), and belongs to the technical field of immunodetection analysis. TPOAb antigen marked by FITC (fluorescein isothiocyanate) is combined with IgG (Intravenous Gamma Globulin) immune body marked by AP (alkaline phosphatase) to form an antigen-antibody-IgG-DON-HRP-conjugated sandwich immune complex which is like a sandwich structure. Later, magnetic particles connected with anti-Goat FITC antibody is added, through specific binding of the anti-Goat FITC antibody and the FITC, an antigen-antibody complex is connected to the magnetic particles, direct settling in an externally added magnetic field is performed, and compound formed by immune reaction can be separated from other uncombined substances without needing centrifugation. The kit provided by the invention combines chemiluminescence and the magnetic particles, provides a reaction system close to homogeneous phase, has the advantages of higher sensitivity, wide linearity range, high speed and the like, reduces product costs, and has a wide application prospect on aspect of clinical examination and the like.
Description
Technical field
The invention belongs to the immune detection analysis technical field, relate to the magnetic microparticle chemiluminescence immune assay kit and the method for testing thereof that detect human thyroid superoxide enzyme antibody (TPOAb) content in the serum.
Background technology
Thyroid peroxidase (thyroid peroxidase TPO) is the important enzyme of catalysis thyroid hormone, can the catalytic iodine ion and the addition reaction of thyroglobulin tyrosine residue, and with this synthetic thyroid element (T
4) or triiodo thryonine (T
3) wait thyroid hormone.TPO is synthetic by thyroid follicular cells, and it is made up of 933 amino acid residues, and molecular weight is 103kD, and is the abundantest at the microvillus punishment cloth of follicular cavity face.
TPOAb is as a kind of important autoantibody of autoimmune thyroid disease, has the important clinical meaning in the discriminating of autoimmune thyroid disease (if any Hashimoto thyroiditis, Graves disease) and diagnosis.The primary first is subtracted the patient, raise in conjunction with hTSH, can find that early stage first subtracts patient.The mensuration of TPOAb also can be used as the first-selected index of diagnosis and differential diagnosis such as Graves disease, Hashitoxicosis.
The chemiluminescence immunoassay detection technique is eighties of last century eighties, the emerging technology that grows up continue Enzyme-multiplied immune technique and after putting immune technology, have high sensitivity, high specific with respect to both chemiluminescence immunoassay technology of back, easy and simple to handle, quick, the mark bond is stable, therefore characteristics such as simultaneously "dead" isotope damage and pollution are extensively promoted the use of in the clinical detection analysis in recent years.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of human thyroid superoxide enzyme antibody (TPOAb) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out the TPOAb detection and have higher sensitivity and specificity, operate easylier, detection time is shorter.
The invention provides and detect the TPOAb chemical luminescence immune analysis reagent box, it is characterized in that this kit comprises: magnetic particle reagent; The TPOAb calibration object; The TPOAb quality-control product; The anti-reagent A of TPOAb; The anti-reagent B of TPOAb; Cleaning concentrate; Luminous substrate solution; The sample dilution.
Described magnetic particle reagent is for connecting the magnetic particle solution of goat-anti FITC antibody;
Described calibration object and quality-control product are the BSA damping fluid that contains a certain amount of TPOAb;
Described reagent A is for containing the TPO antigenic solution of a certain amount of fluorescein isothiocynate (FITC) mark;
Described reagent B i.e. two antienzymes mark reagent, is the human IgG antibody's of alkaline phosphatase (AP) mark potpourri;
Described cleaning concentrate is the damping fluid that contains Tris, NaCl and surfactant etc.;
Described luminous substrate solution is the damping fluid that contains the LumigenAPS-5 luminescent solution;
Described sample dilution is the solution that contains BSA.
The sample-pretreating method of detection TPOAb provided by the present invention may further comprise the steps:
1, sample pre-treatments
To clinical serum, centrifugal 5 minutes of 3000rpm gets upper strata liquid and can carry out assay determination.Must not deposit above 48 hours for testing sample 2-8 ℃, if do not detect in 48 hours, preserve below Ying Yu-20 ℃, but should be above 30 days.
2, prepare before the experiment
Need before the experiment all reagent are placed to room temperature; Be used for covering the plastic foil of magnetic separator when being ready to disposable flat based tubes and magnetic separator and incubation; Regulating the water bath temperature is 37 ℃; Prepare chemical luminescence detector, and read over the instrument operation instructions.
3, reagent is prepared
Before the experiment each reagent in the kit is being put abundant mixing on the mixing instrument; Should be even suspension behind the magnetic particle reagent mixing, do not have obvious aggegation.
4, utilize the chemical luminescence immune analysis reagent box test sample of above-mentioned detection TPOAb.
Detection method of the present invention is as follows:
(1) application of sample and immune response: in each flat based tubes, add 15 μ l TPOAb calibration objects, quality-control product or sample to be tested (carrying out the 1:20-100 dilution with the sample dilution in advance); 30 μ l reagent A, behind the mixing, 37 ℃ of incubations 30 minutes; Add 30 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 15 minutes.
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice.
(3) add two antienzymes mark reagent: in each flat based tubes, add 60 μ l reagent B, behind the mixing, 37 ℃ of incubations 30 minutes.
(4) washing: with (2) step.
(5) add luminous substrate solution: each test tube adds 200 μ l luminous substrate.
(6) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
The present invention has the following advantages:
1, uses magnetic bead to be solid phase, make immune response more near liquid phase, react more abundant and rapid, and make the immune complex of combination be more prone to separate, reduced non-specific adsorption.
2, the reagent B that uses is the monoclonal antibody potpourri, makes immunoreactive affinity higher, and the production differences between batches of monoclonal antibody are relatively little, easier assurance product batch between stable.
3, use chemical luminous substrate solution, the sensitivity of detection is improved, and the range of linearity is wideer.
4, the foundation of this method can provide the immunologic detection method of a kind of convenience, high sensitivity, accuracy and stability for the exploitation of other kits.
Main innovation part of the present invention is:
1, kit of the present invention combines chemiluminescence with immune magnetic particle, and a kind of reaction system near homogeneous phase is provided, and compared with prior art, kit of the present invention has higher detection sensitivity and specificity, and has reached preferable performance parameter.
2, the magnetic particle reagent in the kit, reagent A, reagent B, calibration object, quality-control product, cleaning concentrate, luminous substrate solution and sample dilution are the optimization formulas under this reaction system, and giving the use effect phase of kit and detecting performance provides powerful guarantee.
The indirect method that mainly adopts the magnetic microparticle chemiluminescence immune assay kit of detection of the present invention TPOAb quantitatively detects the content of TPOAb in the sample such as human serum; Pre-treatment requirement to sample is low, and the pre-treatment process is simple, and energy is quick, high flux detects gross sample; Adopted high special monoclonal antibody and super paramagnetic, high dispersive, magnetic particle that specific surface area is big, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Magnetic microparticle chemiluminescence immune assay kit of the present invention, simple in structure, easy to use, low price, carrying convenience are compared with enzyme linked immunological kit on the market etc.; the range of linearity is wide; effectively avoid hook effect, do not need the sample dilution, be applicable to the quantitative of batch samples screening.
Description of drawings
Fig. 1 is TPOAb chemiluminescence immunoassay examination criteria curve of the present invention, and wherein, horizontal ordinate is the concentration of TPOAb, and ordinate is relative luminous intensity (RLU).
Embodiment
Embodiment 1
One, magnetic bead buffer solution formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable vessel, add the pure water of 700ml;
2, take by weighing Tris6.02g, NaN
30.99g in container, mixing, stirring at room 2 hours;
3, detect pH value of solution, should be about 10;
4, modulation pH value is 8.0;
5, measure Tween-202.76ml; Take by weighing neomycinsulphate 0.99g; Tetracycline 0.03g; BSA4.97g; Casein 5.0g stirred adjust pH to 8.0 ± 0.05 1 hour in container;
6, be settled to 1L, adjust pH to 8.0 ± 0.05;
7, filter with the 0.22um filter, be stored in 4 ℃.
Two, the preparation process of magnetic particle reagent
Be that 0.1 micron tri-iron tetroxide microballoon activates with glutaraldehyde with diameter, the room temperature mixing is after 4 hours, with 0.01mol/L PBSpH7.4 buffer solution for cleaning three times, and suspends with this solution, and concentration is 50-100mg/ml; Then, add goat-anti FITC antibody 100 μ g in every milliliter of suspension, in 37 ℃ of mixing incubations 3-8 hour; Sealed 40 minutes in 37 ℃ with isopyknic 0.01mol/LPBS5%BSA pH7.4 damping fluid; At last, use 0.5%BSA0.02mol/L Tris-HCl pH8.0 buffer solution for cleaning three times, and prepare certain density working fluid with above-mentioned magnetic bead buffer solution.
Embodiment 2
One, alkaline phosphatase AP and human IgG antibody's coupling
Antibody is immunoglobulin (Ig), contains amino (NH
2), use the SMCC activator to activate, generate maleimide-immunoglobulin (Ig); Maleimide-immunoglobulin (Ig) contain can with the maleimide base group of-SH radical reaction, add and contain-alkaline phosphatase of SH, antibody and alkaline phosphatase can be linked together.
Two, the coupling of fluorescein isothiocynate FITC and TPO antigen
The conjugate of FITC molecule and TPO antigen is to carry out mark with common alkalies labelling method antagonist.When FITC reacted with antigen protein in alkaline solution, the amino sulphur carbon amine bond with fluorescein of the r of lysine was closed on the protein, forms the FITC-protein conjugate, i.e. fluorescence antibody or fluorescence bond.Generally multipotency is in conjunction with 15-20, and an IgG molecule can be in conjunction with the FITC of 2-8 molecule, and its reaction equation is as follows:
FITC-N=C=S+-NH
2-protein → FITC-NS-C-NH
2-protein
Embodiment 3
The preparation of calibration object/quality-control product:
1, peak: maximum concentration point is X, and impact point concentration is A, B, and C, D, E, F, during preparation V volume solution, the volume that needs to add raw material is respectively: table 1
| Concentration | Add calibration object dilution volume | Add the X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| F | V-F*V/X | F*V/X |
2, human thyroid superoxide enzyme antibody TPOAb measures kit TPOAb calibration object raw material (concentration is 5000IU/ml) to be mixed with concentration point with sample dilution (specifically fill a prescription and see embodiment 4) is 0,10,40,100,250,500IU/ml; Quality-control product concentration is respectively 10,250IU/ml.
3, fully the dissolving after, post label in 2-8 ℃ of preservation, the term of validity is 12 months.
Embodiment 4
One, cleaning concentrate formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable containers, add water outlet 0.7kg, take by weighing Tris12.11g; NaCl312.43g; Tween-2027.74g; Bronidox-L1g; Triton X-1001g;
2, placed 18 hours, transfer pH to 8.6 ± 0.05; Add pure water to 1L, filter.
Carry out 15 times of dilutions when 3, using.
Two, luminous substrate solution formulation operations rules: 1L is example with preparation:
1, according to amount of preparation, select suitable containers, take by weighing Tris36.36g; Na
2SO
310mg; SDS1.0g; Lucigenin 3.27mg; Lumigen APS-5140mg; Tween-200.31ml transfers pH to 9.35; Add pure water to 1L, filter.
2, test luminous value
Three, sample diluent preparing working specification: 1L is example with preparation:
1, adds the 500ml pure water in container, take by weighing trihydroxy aminomethane 6g; NaCl8.8g; BSA60g; Proclin3001ml transfers pH to 7.5 ± 0.05.
2, pure water is settled to 1L, filters.
Embodiment 5
The magnetic microparticle chemiluminescence immune detection of a kind of human thyroid superoxide enzyme antibody (TPOAb):
Operation steps:
(1) application of sample and immune response: in each flat based tubes, add 15 μ l TPOAb calibration objects, quality-control product or sample to be tested (carrying out the 1:20-100 dilution with the sample dilution in advance); 30 μ l reagent A, behind the mixing, 37 ℃ of incubations 30 minutes; Add 30 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 15 minutes.
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice.
(3) add two antienzymes mark reagent: in each flat based tubes, add 60 μ l reagent B, behind the mixing, 37 ℃ of incubations 30 minutes.
(4) washing: with (2) step.
(5) add luminous substrate solution: each test tube adds 200 μ l luminous substrate.
(6) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
Testing result:
Detection curve is seen Fig. 1.
Zero standard is carried out 20 repeated tests on schedule, get the mean value that zero standard measures on schedule and add 2 times standard deviation, be its sensitivity.The sensitivity of this method is≤1.0IU/ml.
Patients serum to 7 variable concentrations uses two batches of reagent to carry out 20 repeated tests respectively, and patients serum's concentration range is 1-400IU/ml, calculates its variation within batch.Variation within batch (CV) average out to 4.5% as a result.
Patients serum to 7 variable concentrations uses two batches of reagent to carry out 20 repeated tests respectively with different operating personnel, and patients serum's concentration range is 1-400IU/ml, calculates its batch variation.Batch variation (CV) average out to 9.6% as a result.
Claims (3)
1. the magnetic microparticle chemiluminescence immunity detection reagent of a human thyroid superoxide enzyme antibody TPOAb, it is characterized in that: be that the magnetic particle of 0.1-5 micron connects to form solid-phase reagent with goat-anti FITC antibody and diameter, form solid phase-Ag-Ab-ELIAS secondary antibody sandwich complex behind the FITC labelled antigen in the catching reaction system, human thyroid superoxide enzyme antibody and the two antienzyme labelled reagents; The enzyme that uses in the described two antienzyme labeling antibodies is alkaline phosphatase AP; The coupling method of described two antienzymes mark reagent is with N-succinamide 3-(2-pyridine dimercapto) antibody of propionic acid activation mixes with the ratio of 1:0.5-2 with the alkaline phosphatase of Traunt ' s reagent activation, and uses the gel chromatography column separating purification; Employed substrate solution is the damping fluid that contains Lumigen APS-5 luminescent solution.
2. TPOAb magnetic microparticle chemiluminescence immune assay kit as claimed in claim 1 is characterized in that this kit comprises: magnetic particle reagent; The TPOAb calibration object; The TPOAb quality-control product; The TPOAb reagent A; TPOAb reagent B; Cleaning concentrate; Luminous substrate solution; The sample dilution;
Described magnetic particle reagent is for connecting the magnetic particle solution of goat-anti FITC antibody;
Described calibration object and quality-control product are the BSA damping fluid that contains a certain amount of TPOAb;
Described reagent A is the TPO antigenic solution that contains a certain amount of FITC mark;
Described reagent B i.e. two antienzymes mark reagent, is the human IgG antibody's of alkaline phosphatase AP mark potpourri;
Described cleaning concentrate is the damping fluid that contains Tris, NaCl and surfactant etc.;
Described luminous substrate solution is the damping fluid that contains Lumigen APS-5 luminescent solution;
Described sample dilution is the solution that contains BSA.
3. kit as claimed in claim 1 or 2 is characterized in that, the using method of described kit is as follows:
(1) application of sample and immune response: add 15 μ l TPOAb calibration objects, quality-control product or sample to be tested in each flat based tubes, it all carries out the 1:20-100 dilution with the sample dilution in advance; 30 μ l reagent A, behind the mixing, 37 ℃ of incubations 30 minutes; Add 30 μ l magnetic particle reagent, behind the mixing, 37 ℃ of incubations 15 minutes;
(2) washing: flat based tubes was left standstill 2 minutes at magnetic separator, and camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Add 300 μ l cleaning fluids in every pipe, behind the mixing, flat based tubes was left standstill 2 minutes at magnetic separator, camber line is toppled over supernatant then, test tube and magnetic separator together is upside down on the thieving paper pat dry; Repeat twice;
(3) add two antienzymes mark reagent: in each flat based tubes, add 60 μ l reagent B, behind the mixing, 37 ℃ of incubations 30 minutes;
(4) washing: with (2) step;
(5) add luminous substrate solution: each test tube adds 200 μ l luminous substrate;
(6) read luminous value: the luminous value of measuring every pipe with Chemiluminescence Apparatus.
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Cited By (7)
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| CN104614535A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Thyroid microsomal antibody detection reagent kit as well as preparation method and application thereof |
| CN104698186A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hyaluronic acid and detection method and application of 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 |
| CN109387629A (en) * | 2017-08-04 | 2019-02-26 | 河北艾驰生物科技有限公司 | A kind of detection reagent detecting thyroid peroxidase antibody |
| CN111337673A (en) * | 2020-05-18 | 2020-06-26 | 博奥赛斯（天津）生物科技有限公司 | Synthetic polypeptide composition for novel coronavirus immunodetection and application |
| CN111337682A (en) * | 2020-05-18 | 2020-06-26 | 博奥赛斯（天津）生物科技有限公司 | Novel coronavirus IgM/IgG magnetic particle chemiluminescence immunoassay kit |
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Cited By (7)
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| 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 |
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| CN109387629A (en) * | 2017-08-04 | 2019-02-26 | 河北艾驰生物科技有限公司 | A kind of detection reagent detecting thyroid peroxidase antibody |
| CN111337673A (en) * | 2020-05-18 | 2020-06-26 | 博奥赛斯（天津）生物科技有限公司 | Synthetic polypeptide composition for novel coronavirus immunodetection and application |
| CN111337682A (en) * | 2020-05-18 | 2020-06-26 | 博奥赛斯（天津）生物科技有限公司 | Novel coronavirus IgM/IgG magnetic particle chemiluminescence immunoassay kit |
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Application publication date: 20131002 |