CN105334316A - Reagent kit and method for detecting thyroglobulin antibody - Google Patents
Reagent kit and method for detecting thyroglobulin antibody Download PDFInfo
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- CN105334316A CN105334316A CN201510787584.6A CN201510787584A CN105334316A CN 105334316 A CN105334316 A CN 105334316A CN 201510787584 A CN201510787584 A CN 201510787584A CN 105334316 A CN105334316 A CN 105334316A
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- G—PHYSICS
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
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- G01N2800/046—Thyroid disorders
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Abstract
The invention discloses a reagent kit and method for detecting a thyroglobulin antibody. The reagent kit contains a first reagent, a second reagent, a magnetic separation reagent, a chemical light-emitting substrate, a calibration article, a quality control article and cleaning liquid. The first reagent is a biotin reagent marked by thyroglobulin. The second reagent is a thyroglobulin enzyme combination reagent. The magnetic separation reagent is a superparamagnetic nanometer magnetic particle reagent marked by streptavidine, and the reagent kit is adopted for detecting the thyroglobulin antibody. Due to the mode, the defects that radioimmunoassay is high in pollution, the period of validity is short, common microwell plate enzyme immunoassay precision is poor, and sensitivity is poor are overcome, an alkaline phosphatase enzyme catalysis chemiluminescence system, a magnetic particle separation system and an unique antigen and antibody coupling technology are used, and the sensitivity, the precision, the stability, the validity period, the safety and the environment friendliness of the method are greatly improved.
Description
Technical field
The present invention relates to vitro diagnostic techniques field, particularly relate to a kind of kit and the method that detect thyroglobulin antibody based on nanometer magnetic microparticle chemiluminescence and alkali phosphatase enzyme mark.
Background technology
Thyroglobulin (Tg) is a kind of large heterologous glycoprotein protein (MW660,000) be present in thyroid gland follicle cell.Thyroglobulin plays very important effect in the biosynthesizing of thyroid hormone, T3 and T4.In thyroid gland follicle cell, TPO (TPO) plays catalyzer in the iodination reaction of the tyrosyl group in thyroglobulin.Thyroglobulin through iodate will be kept in ovarian follicle colloid, serve as the conservator of T3 and T4.When thyroid gland is upset, thyroglobulin will be degraded, and now thyroid hormone, T3 and T4 will be discharged in blood.
The detection of thyroglobulin autoantibody is a kind of effective means whether qualification patient suffers from autoimmune thyroid disease.The patient that 80-100% suffers from the patient of struma lymphomatosa thyroiditis or chronic thyroiditis, 10-20% suffers from subacute thyroiditis and 60-70% suffer from hyperthyroid patient, and their anti-Tg antibody horizontal raises.Due to the heterogeneity of thyroglobulin, normal and euthyroid patient can be detected with it too the elderly patients and clinical manifestation that suffer from other illnesss to make antithyroglobulin antibodies.Also in the patient body suffering from Addison's disease (chronic adrenocortical hypofunction) and some type i diabetes, detect antithyroglobulin antibodies.
The detection method that thyroglobulin antibody is now commonly used has radiommunoassay and common microwell plate enzyme-linked immuno assay etc., but these methods exist some problems.There is high radioactivity, the term of validity healthy hidden danger that is short, that bring to operator and to shortcomings such as the pollutions of environment in radiommunoassay (RIA), there is precision difference, poor sensitivity, cannot realize the shortcomings such as quantitatively detection in common microwell plate enzyme-linked immuno assay (EIA), is also not easy to realize full-automation.
Summary of the invention
The technical matters that the present invention mainly solves is to provide a kind of kit and the method that detect thyroglobulin antibody, has and very easily realizes the advantages such as full-automation, highly sensitive, precision is high, the range of linearity is wide.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: provide a kind of kit detecting thyroglobulin antibody, comprise the first reagent, the second reagent, Magneto separate reagent, chemical luminous substrate, calibration object, quality-control product and cleaning fluid, described first reagent is thyroglobulin mark biotin reagent, described second reagent is thyroglobulin enzyme conjugates reagent, and described Magneto separate reagent is the superparamagnetic nano magnetic particulate reagent of marked by streptavidin.
In a preferred embodiment of the present invention, described first reagent or be thyroglobulin flag F ITC antigenic agents; Described enzyme in described second reagent is alkaline phosphatase or horseradish peroxidase; Thyroglobulin in described first reagent and described second reagent is natural extraction antigen or the gene recombinant antigens of humanized.
In a preferred embodiment of the present invention, described Magneto separate reagent is superparamagnetic nano magnetic particulate, and magnetic bead surfaces is tosyl activation, the diameter of described Magneto separate reagent is 1.05 μm, and isoelectric point is pH5.0, when pH7, electric charge is-10mV, and iron content is 26%; The direct or indirect labelled streptavidin of described superparamagnetic nano magnetic particulate.
In a preferred embodiment of the present invention, described chemical luminous substrate is the potpourri of AMPPD and reinforcing agent; Described cleaning fluid is concentrated cleaning solutions.
In a preferred embodiment of the present invention, the preparation method of described Magneto separate reagent is: by described magnetic particle 0.04 ~ 0.06mol/L, pH value be 4.5 ~ 5 MES damping fluid resuspended, the concentration of resuspended rear magnetic particle is 8 ~ 12mg/ml; Then add described Streptavidin, suspendible 30 ~ 60 minutes at 15 ~ 40 DEG C, the mass ratio of wherein said magnetic particle and described Streptavidin is 25 ~ 50:1; And then adding the carbodiimide aqueous solution of freshly prepared 8 ~ 12mg/ml, suspendible 2 ~ 12h at 15 ~ 40 DEG C, the volume ratio of wherein said MES damping fluid and described carbodiimide aqueous solution is 10 ~ 20:1; Magneto separate, remove supernatant, with containing mass ratio be 0.09 ~ 1.1% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome is that the TRIS buffer of 0.009 ~ 0.01mol/L is resuspended to described coupling and has the concentration of the magnetic particle of Streptavidin to be 0.2 ~ 1.0mg/ml, obtains described Magneto separate reagent;
The preparation method that described thyroglobulin (Tg) marks biotin reagent is: be the PBS damping fluid of 7 ~ 7.5 by 0.01 ~ 0.03mol/L, pH value by described thyroglobulin (Tg), dialysed overnight at 2 ~ 8 DEG C, is mixed with thyroglobulin (Tg) solution; Be dissolved in dimethyl sulfoxide (DMSO) by N-hydroxy-succinamide biotin ester and be mixed with N-hydroxy-succinamide biotin ester solution, the substance withdrawl syndrome of wherein said N-hydroxy-succinamide biotin ester is 8 ~ 12mM; Described N-hydroxy-succinamide biotin ester solution is joined in described thyroglobulin (Tg) solution, mixes, place 20 ~ 40 minutes at 15 ~ 40 DEG C; Add the TRIS buffer of 0.9 ~ 1.1M, cessation reaction, place 8 ~ 12 minutes at 15 ~ 40 DEG C, then with containing mass ratio be 2.5 ~ 3.2% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.04 ~ 0.06mol/L is diluted to containing the thyroglobulin (Tg) of N-hydroxy-succinamide biotin ester mark is 0.2 ~ 2.5 μ g/ml, obtain described thyroglobulin (Tg) and mark biotin reagent;
The preparation method of described thyroglobulin (Tg) enzyme conjugates reagent is: thyroglobulin (Tg) being joined concentration is in the 2-imido grpup sulfane hydrochloride coupling agent of 8 ~ 12mg/ml, 18 ~ 25 minutes are left standstill at 15 ~ 40 DEG C, add the glycine solution of 0.09 ~ 0.11mol/L, 4 ~ 5 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear thyroglobulin (Tg) of activation, 2 ~ 8 DEG C save backup; Alkaline phosphatase enzyme solutions is joined in 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution of 4 ~ 5mg/ml, 25 ~ 35 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, 2 ~ 8 DEG C save backup; By the alkaline phosphatase mixing after the thyroglobulin (Tg) after activation and activation, at 2-8 DEG C, leave standstill 12 ~ 24h, purify with Supperdex200 gel-purified post, obtain connector strong solution, save backup at 2 ~ 8 DEG C; By described connector strong solution with containing mass ratio be 0.4 ~ 0.6% bovine serum albumin(BSA), pH be 7.8 ~ 8.0, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.09 ~ 0.11mol/L is diluted to containing the thyroglobulin (Tg) of alkali phosphatase enzyme mark is 0.2 ~ 1 μ g/ml, obtains described thyroglobulin (Tg) enzyme conjugates reagent.
There is provided a kind of method detecting thyroglobulin antibody, comprising step is:
Step 1: add sample to be tested stoste in detector tube, then adds the first reagent and Magneto separate reagent successively, mixing, incubation 14 ~ 16 minutes at 36 ~ 38 DEG C;
Step 2: add magnetic field, make the system sedimentation in magnetic field after step 1 incubation, removes supernatant, and after cleaning fluid repeatedly cleans, remove magnetic field, concussion makes the abundant suspendible of magnetic particle;
Step 3: add magnetic field, make the magnetic particle sedimentation in magnetic field after step 2 suspendible, removes supernatant, then adds the second reagent, mixing, incubation 14 ~ 16 minutes at 36 ~ 38 DEG C;
Step 4: add magnetic field, make the system sedimentation in magnetic field after step 3 incubation, removes supernatant, and after cleaning fluid repeatedly cleans, remove magnetic field, concussion makes the abundant suspendible of magnetic particle;
Step 5: add magnetic field, make the magnetic particle sedimentation in magnetic field after step 3 suspendible, removes supernatant, then adds luminous substrate, removes magnetic field, relative luminous intensity value in detecting 1 second after abundant suspendible.
The invention has the beneficial effects as follows: the kit of detection thyroglobulin antibody of the present invention and method, overcome the high pollution of radiommunoassay, the term of validity be short and common microwell plate enzyme-linked immuno assay precision is poor, the shortcoming of poor sensitivity, use alkaline phosphatase enzyme-catalyzed chemical luminescence system, magnetic particle piece-rate system, unique antigen-antibody coupling technology, the sensitivity of the method, precision, stability, the term of validity, security, environmental-protecting performance are improved greatly.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:
Fig. 1 is thyroglobulin antibody detection method alignment product canonical plotting of the present invention;
Fig. 2 is serum sample testing result correlativity figure in thyroglobulin antibody detection method of the present invention.
Embodiment
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment one:
A kind of kit detecting thyroglobulin antibody is provided, comprise the first reagent, the second reagent, Magneto separate reagent, chemical luminous substrate, calibration object, quality-control product and cleaning fluid, described first reagent is that thyroglobulin (Tg) marks biotin reagent, described second reagent is thyroglobulin (Tg) enzyme conjugates reagent, and described Magneto separate reagent is the superparamagnetic nano magnetic particulate reagent of marked by streptavidin.
Described Magneto separate reagent comprises: the magnetic bead hydrophobicity magnetic bead surfaces that superparamagnetic nano magnetic particulate activates based on tosyl, and the diameter of described Magneto separate reagent is 1.05 μm, and isoelectric point is pH5.0, and when pH7, electric charge is-10mV, and iron content is 26%.The direct or indirect labelled streptavidin of described superparamagnetic nano magnetic particulate.Described superparamagnetic nano magnetic particulate is not limited to labelled streptavidin, also can mark anti-FITC antibody.
Thyroglobulin (Tg) in described first reagent and described second reagent is natural extraction antigen or the gene recombinant antigens of humanized.Described thyroglobulin (Tg) marks biotin reagent, but is not limited to thyroglobulin (Tg) and marks biotin, also can be thyroglobulin (Tg) flag F ITC antigen.Described in described thyroglobulin (Tg) enzyme conjugates reagent, enzyme can be alkaline phosphatase or horseradish peroxidase.
The key component of described chemical luminous substrate can be AMPPD and various effective reinforcing agent.Described cleaning fluid can be concentrated cleaning solutions.
The preparation method of described Magneto separate reagent is: by described magnetic particle 0.04 ~ 0.06mol/L, pH value be 4.5 ~ 5 MES damping fluid resuspended, the concentration of resuspended rear magnetic particle is 8 ~ 12mg/ml; Then add described Streptavidin, suspendible 30 ~ 60 minutes at 15 ~ 40 DEG C, the mass ratio of wherein said magnetic particle and described Streptavidin is 25 ~ 50:1; And then adding the carbodiimide aqueous solution of freshly prepared 8 ~ 12mg/ml, suspendible 2 ~ 12h at 15 ~ 40 DEG C, the volume ratio of wherein said MES damping fluid and described carbodiimide aqueous solution is 10 ~ 20:1; Magneto separate, remove supernatant, with containing mass ratio be 0.09 ~ 1.1% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome is that the TRIS buffer of 0.009 ~ 0.01mol/L is resuspended to described coupling and has the concentration of the magnetic particle of Streptavidin to be 0.2 ~ 1.0mg/ml, obtains described Magneto separate reagent.
The preparation method that described thyroglobulin (Tg) marks biotin reagent is: be the PBS damping fluid of 7 ~ 7.5 by 0.01 ~ 0.03mol/L, pH value by described thyroglobulin (Tg), dialysed overnight at 2 ~ 8 DEG C, is mixed with thyroglobulin (Tg) solution; Be dissolved in dimethyl sulfoxide (DMSO) by N-hydroxy-succinamide biotin ester and be mixed with N-hydroxy-succinamide biotin ester solution, the substance withdrawl syndrome of wherein said N-hydroxy-succinamide biotin ester is 8 ~ 12mM; Described N-hydroxy-succinamide biotin ester solution is joined in described thyroglobulin (Tg) solution, mixes, place 20 ~ 40 minutes at 15 ~ 40 DEG C; Add the TRIS buffer of 0.9 ~ 1.1M, cessation reaction, place 8 ~ 12 minutes at 15 ~ 40 DEG C, then with containing mass ratio be 2.5 ~ 3.2% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.04 ~ 0.06mol/L is diluted to containing the thyroglobulin (Tg) of N-hydroxy-succinamide biotin ester mark is 0.2 ~ 2.5 μ g/ml, obtain described thyroglobulin (Tg) and mark biotin reagent.
The preparation method of described thyroglobulin (Tg) enzyme conjugates reagent is: thyroglobulin (Tg) being joined concentration is in the 2-imido grpup sulfane hydrochloride coupling agent of 8 ~ 12mg/ml, 18 ~ 25 minutes are left standstill at 15 ~ 40 DEG C, add the glycine solution of 0.09 ~ 0.11mol/L, 4 ~ 5 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear thyroglobulin (Tg) of activation, 2 ~ 8 DEG C save backup; Alkaline phosphatase enzyme solutions is joined in 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution of 4 ~ 5mg/ml, 25 ~ 35 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, 2 ~ 8 DEG C save backup; By the alkaline phosphatase mixing after the thyroglobulin (Tg) after activation and activation, at 2-8 DEG C, leave standstill 12 ~ 24h, purify with Supperdex200 gel-purified post, obtain connector strong solution, save backup at 2 ~ 8 DEG C; By described connector strong solution with containing mass ratio be 0.4 ~ 0.6% bovine serum albumin(BSA), pH be 7.8 ~ 8.0, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.09 ~ 0.11mol/L is diluted to containing the thyroglobulin (Tg) of alkali phosphatase enzyme mark is 0.2 ~ 1 μ g/ml, obtains described thyroglobulin (Tg) enzyme conjugates reagent.
Embodiment two:
There is provided a kind of method detecting thyroglobulin antibody, comprising step is:
(1) in test tube, add sample 10 μ L to be checked successively, the superparamagnetic nano magnetic particulate reagent 50 μ L of marked by streptavidin and thyroglobulin (Tg) mark biotin reagent 100 μ L, mix rear 37 DEG C of reactions 15 minutes.
(2) add cleaning fluid 500 μ L, wash away unconjugated antigen-antibody and impurity, repeated washing 3 times.
(3) add thyroglobulin (Tg) enzyme conjugates reagent 100 μ L, mix rear 37 DEG C of reactions 15 minutes.
(4) add cleaning fluid 500 μ L, wash away unconjugated antigen-antibody and impurity, repeated washing 3 times.
(5) often pipe adds 150 μ L Chemoluminescent substrates, and 37 DEG C of reactions, after 5 minutes, are put into chemiluminescence analysis/analyzer and measured luminous intensity (RLU).
In above-mentioned steps after hatching, unconjugated component is removed in washing, and the complex catalysts substrate of formation produces photon, detects the concentration that photon intensity can calculate PCT.
The principle detected is: the TG antigen that biotin labeled TG antigen, alkaline phosphatase (ALP) mark, immune response is passed through with the anti-TG antibody in sample, form sandwich complex, add bag by the bead particulates of Streptavidin, pass through magnetic separation system, washing adds luminous substrate after removing unconjugated antibody, antigen and impurity, this complex catalysts luminous substrate sends photon, and luminous intensity is directly proportional to the content of anti-TG.
The present invention adopts biotinstreptatin signal amplifying system and alkali phosphatase enzyme mark, solve the problem that traditional ELISA sensitivity is lower, this kit adopts dual-antigen sandwich method to detect antibody, can IgG, IgM, IgA, IgE, IgD of detection specificity.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (6)
1. one kind is detected the kit of thyroglobulin antibody, it is characterized in that, comprise the first reagent, the second reagent, Magneto separate reagent, chemical luminous substrate, calibration object, quality-control product and cleaning fluid, described first reagent is thyroglobulin mark biotin reagent, described second reagent is thyroglobulin enzyme conjugates reagent, and described Magneto separate reagent is the superparamagnetic nano magnetic particulate reagent of marked by streptavidin.
2. the kit of detection thyroglobulin antibody according to claim 1, is characterized in that, described first reagent or be thyroglobulin flag F ITC antigenic agents; Described enzyme in described second reagent is alkaline phosphatase or horseradish peroxidase; Thyroglobulin in described first reagent and described second reagent is natural extraction antigen or the gene recombinant antigens of humanized.
3. the kit of detection thyroglobulin antibody according to claim 1, it is characterized in that, described Magneto separate reagent is superparamagnetic nano magnetic particulate, in described Magneto separate reagent, magnetic bead surfaces is tosyl activation, the diameter of described Magneto separate reagent is 1.05 μm, and isoelectric point is pH5.0, when pH7, electric charge is-10mV, and iron content is 26%; The direct or indirect labelled streptavidin of described superparamagnetic nano magnetic particulate.
4. the kit of detection thyroglobulin antibody according to claim 1, is characterized in that, described chemical luminous substrate is the potpourri of AMPPD and reinforcing agent; Described cleaning fluid is concentrated cleaning solutions.
5. the kit of detection thyroglobulin antibody according to claim 1, it is characterized in that, the preparation method of described Magneto separate reagent is: by described magnetic particle 0.04 ~ 0.06mol/L, pH value be 4.5 ~ 5 MES damping fluid resuspended, the concentration of resuspended rear magnetic particle is 8 ~ 12mg/ml; Then add described Streptavidin, suspendible 30 ~ 60 minutes at 15 ~ 40 DEG C, the mass ratio of wherein said magnetic particle and described Streptavidin is 25 ~ 50:1; And then adding the carbodiimide aqueous solution of freshly prepared 8 ~ 12mg/ml, suspendible 2 ~ 12h at 15 ~ 40 DEG C, the volume ratio of wherein said MES damping fluid and described carbodiimide aqueous solution is 10 ~ 20:1; Magneto separate, remove supernatant, with containing mass ratio be 0.09 ~ 1.1% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome is that the TRIS buffer of 0.009 ~ 0.01mol/L is resuspended to described coupling and has the concentration of the magnetic particle of Streptavidin to be 0.2 ~ 1.0mg/ml, obtains described Magneto separate reagent;
The preparation method that described thyroglobulin (Tg) marks biotin reagent is: be the PBS damping fluid of 7 ~ 7.5 by 0.01 ~ 0.03mol/L, pH value by described thyroglobulin (Tg), dialysed overnight at 2 ~ 8 DEG C, is mixed with thyroglobulin (Tg) solution; Be dissolved in dimethyl sulfoxide (DMSO) by N-hydroxy-succinamide biotin ester and be mixed with N-hydroxy-succinamide biotin ester solution, the substance withdrawl syndrome of wherein said N-hydroxy-succinamide biotin ester is 8 ~ 12mM; Described N-hydroxy-succinamide biotin ester solution is joined in described thyroglobulin (Tg) solution, mixes, place 20 ~ 40 minutes at 15 ~ 40 DEG C; Add the TRIS buffer of 0.9 ~ 1.1M, cessation reaction, place 8 ~ 12 minutes at 15 ~ 40 DEG C, then with containing mass ratio be 2.5 ~ 3.2% bovine serum albumin(BSA), pH value be 7 ~ 7.5, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.04 ~ 0.06mol/L is diluted to containing the thyroglobulin (Tg) of N-hydroxy-succinamide biotin ester mark is 0.2 ~ 2.5 μ g/ml, obtain described thyroglobulin (Tg) and mark biotin reagent;
The preparation method of described thyroglobulin (Tg) enzyme conjugates reagent is: thyroglobulin (Tg) being joined concentration is in the 2-imido grpup sulfane hydrochloride coupling agent of 8 ~ 12mg/ml, 18 ~ 25 minutes are left standstill at 15 ~ 40 DEG C, add the glycine solution of 0.09 ~ 0.11mol/L, 4 ~ 5 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear thyroglobulin (Tg) of activation, 2 ~ 8 DEG C save backup; Alkaline phosphatase enzyme solutions is joined in 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution of 4 ~ 5mg/ml, 25 ~ 35 minutes are left standstill at 15 ~ 40 DEG C, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, 2 ~ 8 DEG C save backup; By the alkaline phosphatase mixing after the thyroglobulin (Tg) after activation and activation, at 2-8 DEG C, leave standstill 12 ~ 24h, purify with Supperdex200 gel-purified post, obtain connector strong solution, save backup at 2 ~ 8 DEG C; By described connector strong solution with containing mass ratio be 0.4 ~ 0.6% bovine serum albumin(BSA), pH be 7.8 ~ 8.0, substance withdrawl syndrome be the concentration that the TRIS buffer of 0.09 ~ 0.11mol/L is diluted to containing the thyroglobulin (Tg) of alkali phosphatase enzyme mark is 0.2 ~ 1 μ g/ml, obtains described thyroglobulin (Tg) enzyme conjugates reagent.
6. detect a method for thyroglobulin antibody, it is characterized in that, comprising step is:
Step 1: add sample to be tested stoste in detector tube, then adds the first reagent and Magneto separate reagent successively, mixing, incubation 14 ~ 16 minutes at 36 ~ 38 DEG C;
Step 2: add magnetic field, make the system sedimentation in magnetic field after step 1 incubation, removes supernatant, and after cleaning fluid repeatedly cleans, remove magnetic field, concussion makes the abundant suspendible of magnetic particle;
Step 3: add magnetic field, make the magnetic particle sedimentation in magnetic field after step 2 suspendible, removes supernatant, then adds the second reagent, mixing, incubation 14 ~ 16 minutes at 36 ~ 38 DEG C;
Step 4: add magnetic field, make the system sedimentation in magnetic field after step 3 incubation, removes supernatant, and after cleaning fluid repeatedly cleans, remove magnetic field, concussion makes the abundant suspendible of magnetic particle;
Step 5: add magnetic field, make the magnetic particle sedimentation in magnetic field after step 3 suspendible, removes supernatant, then adds luminous substrate, removes magnetic field, relative luminous intensity value in detecting 1 second after abundant suspendible.
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