CN105277717A - Magnetic particle separation and chemiluminescence immunoassay method of thyroglobulin - Google Patents
Magnetic particle separation and chemiluminescence immunoassay method of thyroglobulin Download PDFInfo
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Abstract
The invention discloses a quantitative detection kit of thyroglobulin. The quantitative detection kit of thyroglobulin comprises a fluorescein isothiocyanate labeled thyroglobulin monoclonal antibody solution, an alkaline phosphatase labeled thyroglobulin monoclonal antibody solution and a magnetic separation reagent. A detection method adopted in the invention is a combination of magnetic particle separation and chemiluminescence immunoassay, an enzyme labeling technology, a magnetic separation technology and a chemiluminescence technology, and has the advantages of convenient and simple operation, mild reaction conditions, stable luminescence value, and little affection by extraneous conditions. Compared with present detection methods, the method adopted in the invention has the advantages of simple sample processing process, low detection cost, fast detection, and accuracy and good repeatability of a test result.
Description
Technical field
The invention belongs to technical field of immune assay, relate to the immunoassay detection technique that food security is relevant, particularly relate to a kind of magnetic microparticle separating chemiluminescence immunologic detection method of thyroglobulin, be applicable to the quantitative detection of the thyroglobulin in human serum and blood plasma.
Background technology
Thyroglobulin (Thyroglobulin, Tg), be the 660ku glycoprotein of thyroid follicular epithelial cells secretion, each Tg about has 2 thyroxine (T4) and 0.5 triiodo thryonine (T3) molecule, is stored in follicular cavity.Lysosomal hydrolysis Tg surface T4, T3 also makes it to be released into blood, and a small amount of Tg is also released into blood simultaneously, and part Tg is secreted into blood through lymphatics of thyroid gland.Tg in blood circulation is removed by the macrophage of liver.The secretion factor of Tg is stimulated to comprise thyroid-stimulating hormone (TSH) and insulin-like growth factor-i (IGF-1); Inhibiting factor is gamma interferon, α-tumornecrosisfactor and vitamin A acid.
Thyroglobulin (TG) is the mark of differentiated thyroid carcinoma (DTC).Regular follow-up serum TG contributes to diagnosis to DTC recurrence and transfer, evaluation, and the result for the treatment of of monitoring patient prognosis.Other benign thyroid is sick, if hyperthyroidism, thyroid cyst, Hashimoto's disease etc. are due to the destruction of thyroid follicle, cause the TG in wherein colloid also to enter into blood circulation, serum TG levels is increased.In CH patient, detect that thyroglobulin can differentiate that thyroid gland lacks completely, thyreoaplasia or other pathological conditions.On the other hand, the damage of thyroid follicle wall can cause a large amount of thyroglobulins to enter blood, and therefore, thyroglobulin is also considered to the distinctive mark thing of thyroid body morphological integrity.Thyroglobulin measures and also can be used for differentiating subacute thyroiditis and false thyrotoxicosis.
The method of current measurement TG mainly contains radio immunoassay (RIA) and the large class of immune measurement analyze method (IMA) two.IMA comprises immunoradiometric assay (IRMA), fluoroimmunoassay (FIA), luminescent immunoassay (CLIA), Electrochemiluminescence assay (ECLIA) etc.
Summary of the invention
An object of the present invention is to provide a kind of thyroglobulin immue quantitative detection reagent box.
Kit provided by the invention is 1) or 2):
1) the thyroglobulin monoclonal antibody solution of marked by fluorescein isothiocyanate and the thyroglobulin monoclonal antibody solution of alkali phosphatase enzyme mark is comprised;
The thyroglobulin monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: solution A, cow's serum, horse serum, sheep blood serum, antiseptic mixing are obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, animal serum albumin and water composition;
2) thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate, the thyroglobulin monoclonal antibody of described alkali phosphatase enzyme mark and described anti-reagent buffer is comprised.
In mentioned reagent box,
Described animal serum albumin is bovine serum albumin(BSA), and described antiseptic is ProcLin-300;
The proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000g:23-28ml:1.5-2.5ml:4.0-5.0ml:0.5-2.0ml;
In described solution A, described Na
+concentration is 0.1-1mol/L, described Mg
2+concentration be 1mmol/L, described Zn
2+concentration be 0.1mmol/L, the mass percentage of described bovine serum albumin(BSA) is 0.2-1.0%, and described Trizmabase concentration is 0.01-0.5mol/L.
In mentioned reagent box,
In described anti-reagent buffer, the proportioning of described solution A, cow's serum, horse serum, sheep blood serum, ProcLin-300 and water is 1000g:25ml:2ml:4.5ml:1.0ml;
In described solution A, described Na
+come from NaCl, described NaCl concentration is 0.1mol/L;
Described Mg
2+come from MgCl
2, described MgCl
2concentration is 1.0mmol/L;
Described Zn
2+come from ZnCL
2, described ZnCL
2concentration is 0.1mmol/L;
The mass percentage of described bovine serum albumin(BSA) is 0.5%;
The concentration of described Trizmabase is 0.1mol/L.
In mentioned reagent box,
The thyroglobulin monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the thyroglobulin monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution is made up of alkali phosphatase enzyme mark thyroglobulin monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody is 0.5-1 μ g/mL;
Thyroglobulin monoclonal antibody in the thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate is different with thyroglobulin monoclonal antibody in described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody.
Mentioned reagent box also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
Mentioned reagent box also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of thyroglobulin antigen and calibration object damping fluid, and described thyroglobulin antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, protide protective agent, antiseptic, Trizmabase, animal blood serum and water composition, described Na
+concentration is 0.1-1mol/L; the protectant mass percentage of described protide is 0.2-1.0%; described antiseptic volumn concentration is 0.05-0.2%, and described Trizmabase concentration is 0.01-0.5mol/L, and the volumn concentration of described animal blood serum is 0.05-1.0%.
In mentioned reagent box,
Described Na
+come from NaCl, described NaCl concentration is 0.10mol/L;
Described protide protective agent is bovine serum albumin(BSA), and described protein amount percentage composition is 0.5%;
Described antiseptic is Proclin-300, and described Proclin-300 volumn concentration is 0.1%;
The concentration of described Trizmabase is 0.1mol/L;
Described animal blood serum is sheep blood serum, and described sheep blood serum volumn concentration is 0.2%.
In mentioned reagent box,
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6; To be concentration be described calibration object 0,10ng/mL, 50ng/mL, 150ng/mL, 300ng/mL, 750ng/mL thyroglobulin antigenic solution.
Described kit also comprises quality-control product, Sample dilution, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations; The calibration object of described quality-control product to be concentration be 10ng/mL and 300ng/mL.
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described acridan final concentration is 0.25mg/mL;
Described Sample dilution is made up of bovine serum albumin(BSA), trizmabase and water, and wherein, the mass percentage of described bovine serum albumin(BSA) is 0.2%-5%, and the concentration of described trizmabase is 0.1mol/L.
Another object of the present invention is to provide a kind of method preparing above-mentioned kit.
Method provided by the invention, comprises the steps: 8 kinds of equal independent packagings of component in mentioned reagent box; Entire package is kit again.
The application of above-mentioned kit in thyroglobulin quantitatively detects also is the scope of protection of the invention;
Or above-mentioned kit is also the scope of protection of the invention preparing the application in the quantitative testing product of thyroglobulin;
Or 8 kinds of components are also the scope of protection of the invention preparing the application in the quantitative testing product of thyroglobulin in above-mentioned kit;
Or thyroglobulin quantitative detecting method in a kind of sample to be tested, comprise the steps:
1) calibration object in above-mentioned kit, quality-control product and sample to be tested are mixed with the thyroglobulin monoclonal antibody solution of the marked by fluorescein isothiocyanate in above-mentioned kit and alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution respectively, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described immune reaction product and above-mentioned kit, reaction, obtain reaction product, by the precipitation in described reaction product and magnetic bead cleaning fluid suspendible, collecting precipitation, is Magneto separate product;
3) by the substrate solution mixing in described Magneto separate product and above-mentioned kit, obtain mix products, detect the luminous intensity of described mix products with Chemiluminescence Apparatus, calculate gamut thyroglobulin in sample to be tested by luminous intensity quantitative;
Described magnetic bead cleaning fluid is cleaning fluid and water in above-mentioned kit are mixed by 1:7, obtains solution;
Or the application of Magneto separate reagent in thyroglobulin quantitatively detects in above-mentioned kit.
The pH value of described anti-reagent buffer is 8.0 ± 0.05.
The pH value of described calibration object damping fluid is 8.0 ± 0.05.
The thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate is the product obtained with marked by fluorescein isothiocyanate thyroglobulin monoclonal antibody A;
Described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody is the product obtained with the monoclonal antibody B of alkali phosphatase enzyme mark thyroglobulin.
Detection method of the present invention is magnetic microparticle separating chemiluminescence immune detection, enzyme labeling technology, and magnetic separation technique and chemiluminescence combine, and it is convenient and simple for operation, and reaction conditions is gentle, and luminous value is stablized and affected by external condition less.Compared to existing detection method, it is simple that the present invention has sample process process, and testing cost is low, detects fast, the advantages such as test result is accurate, reproducible.
The present invention has the following advantages:
1, use magnetic microparticles to be solid phase, make immune response closer to liquid phase, reaction more fully with rapid, and makes the immune complex of combination more easily separated.；
2, the present invention utilizes magnetic microparticles to combine with chemiluminescence, provides the quantitative detecting method that a kind of sensing range is wide, highly sensitive, precision is good, measured value is stable, simple to operate, detects fast, can meet fast, detect in a large number the demand of sample.
Detection method of the present invention is magnetic microparticle separating chemiluminescence immune detection, enzyme labeling technology, and magnetic separation technique and chemiluminescence combine, and it is convenient and simple for operation, and reaction conditions is gentle, and luminous value is stablized and affected by external condition less.Compared to existing detection method, it is simple that the present invention has sample process process, and testing cost is low, detects fast, the advantages such as test result is accurate, reproducible.
Accompanying drawing explanation
Fig. 1 is that magnetic microparticle separating chemiluminescence immunization detects thyroglobulin schematic diagram.
Fig. 2 is that the detection kit of Beckman Ku Erte commerce and trade (China) company limited measures thyroglobulin result.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
All components in detection kit of the present invention all obtains from biological reagent or chemical reagents corporation's purchase by commercial sources.In the present invention, mark fluorescein isothiocynate monoclonal antibody used is that Fitzgerald company produces, and article No. is: 10-7834; Mark alkaline phosphatase antibodies is that Fitzgerald company produces, and article No. is: 10-7835; Antigen is that Fitzgerald company produces, and article No. is: 30-1219; Alkaline phosphatase is purchased from BBI company of Britain, and lot number is: 1693AA; Carboxyl magnetic bead colloidal solution is purchased from merk company; Goat-anti fluorescein isothiocynate antibody serum serum is purchased from animal used as test section of PLA General Hospital first affiliated hospital; Fluorescein isothiocynate (FITC) purchased from SIGMA company, CAT:3326-32-7, article No. is: F7250; SMCC is purchased from thermofisherscientific company, and article No. is: 22360; 2-IT purchased from thermofisherscientific company, CAS:4781-83-3; Article No. is: 26101; NHS purchased from SIMGA company, CAS:6066-82-6, article No.: 130672; EDC purchased from SIMGA company, CAS:25952-53-8, article No.: E7750.
Embodiment 1, thyroglobulin immue quantitative detection reagent box component and method thereof
One, the preparation of thyroglobulin immue quantitative detection reagent box
By after following 8 kinds of component independent packagings again entire package be kit, be thyroglobulin immue quantitative detection reagent box: 1, calibration object (containing the TG of a series of concentration, for Criterion curve); 2, quality-control product (containing certain density TG); 3, reagent A (the TG monoclonal antibody solution containing certain density marked by fluorescein isothiocyanate); 4, reagent B (the TG antibody containing certain density alkali phosphatase enzyme mark) 5, Sample dilution (trizmabase (tris) damping fluid containing 0.2-1.0% bovine serum albumin(BSA) (BSA)); 6, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody); 7, cleaning fluid (for preparing magnetic bead cleaning fluid); 8, substrate solution.In kit, 1,2,3,4,5,6,8 is indispensable reagent, and 7 replace using by buying other company's similar products.Mentioned reagent box is placed in the preservation of 4 degree, refrigerator.
Specific as follows:
1, calibration object
1) preparation of calibration object damping fluid
Calibration object damping fluid is by Na
+, bovine serum albumin(BSA), sheep blood serum, Proclin-300, Trizmabase and water composition, the final concentration of each component is as follows: Na
+come from NaCl, its concentration is 0.1mol/L; Bovine serum albumin(BSA), its concentration is 0.5% (mass percentage); Proclin-300, its concentration is 0.1% (volumn concentration); Trizmabase concentration is 0.1mol/L; Sheep blood serum, its concentration is 0.2% (volumn concentration); The pH value of calibration object damping fluid is 8.0 ± 0.05.
The collocation method of calibration object damping fluid is specific as follows:
A) Trizmabase12.06g is got, in NaCl5.82g and 1.0L reagent bottle;
B) get 600mL purified water in 1.0L reagent bottle, fully stir until dissolve completely, regulate PH to 10.0 ± 0.05;
C) adding sheep blood serum 2.0mL, bovine serum albumin(BSA) 5.0g, fully stirring and evenly mixing to dissolving completely;
D) add ProcLin-3001.0mL, purified water, quantitatively to 1000mL, fully mixes 4h;
E) PH most 8.0 ± 0.05 is regulated;
F) use 0.22 μm of membrane filtration and collect filtrate, posting label, 2 ~ 8 DEG C of preservations.
2) preparation of calibration object
By TG antigen, (antigen is that Fitzgerald company produces, article No. is: 30-1219) with above-mentioned 1) alignment savors damping fluid and is diluted to 750ng/mL, 300ng/mL, 150ng/mL, 50ng/mL, the calibration object of 10ng/mL, equivalent is distributed into that concentration is 0, the calibration object of 10ng/mL, 50ng/mL, 150ng/mL, 300ng/mL, 750ng/mL.
2, quality-control product (containing certain density TG)
Using the above-mentioned concentration calibration object that is 10ng/mL and 300ng/mL as quality-control product;
3, reagent A (the TG monoclonal antibody solution of marked by fluorescein isothiocyanate)
1), the preparation of anti-reagent buffer
Anti-reagent buffer is prepared as follows: solution A, cow's serum, horse serum, sheep blood serum and ProcLin-300 mixing is obtained anti-reagent buffer; Solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, bovine serum albumin(BSA) and water composition.The proportioning of solution A, cow's serum, horse serum, sheep blood serum, ProcLin-300 and water is 1000g:25ml:2ml:4.5ml:1.0ml;
In solution A, Na
+come from NaCl, its concentration is 0.1mol/L;
Mg
2+come from MgCl
2, its concentration is 1.0mmol/L;
Zn
2+come from ZnCL
2, its concentration is 0.1mmol/L;
The mass percentage of bovine serum albumin(BSA) is 0.5%;
The concentration of Trizmabase is 0.1mol/L.
The collocation method of anti-reagent buffer is specific as follows:
A) get Trizmabase12.06g, NaCl5.82g is in 1L beaker;
B) get 500g purified water in 1L beaker, fully stir until dissolve completely;
C) MgCl is added
295mg, ZnCL
213.6mg, fully dissolves mixing, regulates PH to 10.0 ± 0.05;
D) add bovine serum albumin(BSA) 5.0g, fully stir and evenly mix, place 1.0 hours;
E) add purified water surely to weigh to 1000g, after fully stirring and evenly mixing, regulate PH to 8.0 ± 0.05; Obtain solution A; Now liquor capacity is 1L;
F) cow's serum 25mL is drawn, horse serum 2.0mL, sheep blood serum 4.5mL, ProcLin-3001.0mL, fully mixing 4 hours;
G) adjust ph to 8.0 ± 0.05;
H) use 0.22 μm of membrane filtration and collect filtrate, posting label, 2 ~ 8 DEG C of preservations.
2), marked by fluorescein isothiocyanate TG monoclonal antibody
Marked by fluorescein isothiocyanate TG monoclonal antibody is the product obtained by marked by fluorescein isothiocyanate TG monoclonal antibody, and concrete grammar is as follows:
(antibody is that Fitzgerald company produces to get 1mgTG monoclonal antibody, article No. is: 10-7834), (slough original antibody with SephadexG25 pillar exchange buffering liquid and preserve damping fluid, be replaced by 0.2mol/L carbonate buffer solution PH9.5, 16.8g sodium bicarbonate is dissolved in 1000g purified water, PH9.5), and by centrifugal concentrating to 5mg/mL, (solvent is concentration is 0.2mol/L to add the fluorescein isothiocynate solution that concentration is 0.5mg/mL, pH value is 9.5 carbonate buffer solutions) 100 μ L (proportioning of TG monoclonal antibody and fluorescein isothiocynate is 1mg:0.05mg), mix.Room temperature lucifuge reacts 20 hours, obtains marked by fluorescein isothiocyanate TG monoclonal antibody.
Re-use SephadexG25 pillar and remove unreacted fluorescein isothiocynate, with 0.2mol/L carbonate buffer solution PH9.5 wash-out, collect yellow liquid, be stored in-20 DEG C, obtain marked by fluorescein isothiocyanate TG monoclonal antibody after purifying.
3), the preparation of reagent A
By above-mentioned 2) purifying after marked by fluorescein isothiocyanate TG monoclonal antibody and above-mentioned 1) anti-reagent buffer mix, obtain reagent A, wherein the concentration of the anti-TG of marked by fluorescein isothiocyanate is 0.3ug/ml.
4, reagent B (containing certain density alkali phosphatase enzyme mark TG monoclonal antibody)
1) alkali phosphatase enzyme mark TG monoclonal antibody
Alkali phosphatase enzyme mark TG monoclonal antibody is the product obtained by the another kind of monoclonal antibody of alkali phosphatase enzyme mark TG, and concrete grammar is as follows:
(Fitzgerald company produces to get the another kind of monoclonal antibody of 1mgTG, article No. is: 10-7835), with SephadexG25 pillar exchange buffering liquid (0.05mol/L Triethanolamine buffer, solvent is water, PH8.6), and by centrifugal concentrating to 2.5mg/mL, add activator 2-Iminothiolanehydrochloride (2-IT) solution (2-IT producer: thermofisherscientific, article No.: 26101 that concentration is 13.76mg/mL; Solvent is 0.05mol/L Triethanolamine buffer PH10.5) 5 μ L (being volume ratio 80:1), room temperature adds glycocoll and stops priming reaction after placing 20 minutes, ambient temperatare puts 10 minutes.Re-use SephadexG25 pillar desalination, collect the rear antibody of activation.
Get 1.5mg alkaline phosphatase (producer: BBI, lot number: 1693AA), upper SephadexG25 pillar exchange buffering liquid (0.05mol/L Triethanolamine buffer PH8.6), and by centrifugal concentrating to 5mg/mL, add Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) solution (the SMCC producer: thermofisherscientific that concentration is 6.69mg/mL, article No.: 22360, solvent is DMF, producer: sigma, article No.: 227056) 12 μ L, room temperature places 20 minutes, add glycocoll and stop priming reaction, room temperature places 10 minutes.Use SephadexG25 pillar desalination, obtain activating rear alkaline phosphatase.(illustrating: be comparatively universal method herein, need not describe in detail).
The ratio that TG antibody (adding as a solution) after activation and alkaline phosphatase (adding as a solution) after activation add 1mg alkaline phosphatase in 1mgTG antibody is mixed, add 5 μ L1M magnesium chloride brine mixings simultaneously, place 4 DEG C of reactions 20 hours.
Use Supperdex200 gel chromatography column separating purification, remove the TG antibody and alkaline phosphatase that do not connect, with 0.05mol/L Triethanolamine buffer PH7.0 wash-out, elution flow rate is 0.75ml/min, pillar diameter/length is 1.6/60cm, observe out peak position and connector collected by comparison standard molecule collection of illustrative plates, be stored in 4 DEG C, obtain alkali phosphatase enzyme mark TG monoclonal antibody after purifying.
2), the preparation of reagent B
By above-mentioned 1) purifying after in alkali phosphatase enzyme mark TG monoclonal antibody and above-mentioned 3 1) anti-reagent buffer mix, obtain reagent B, wherein the concentration of alkali phosphatase enzyme mark TG monoclonal antibody is 0.5ug/ml.
5, Sample dilution
Sample dilution is bovine serum albumin(BSA) (BSA), trizmabase and water are mixed, and obtains Sample dilution, and the mass percentage of bovine serum albumin(BSA) is the concentration of 0.5%, trizmabase is 0.1mol/L.
6, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody)
Magneto separate reagent is by covalently bound for goat-anti fluorescein isothiocynate antibody serum (polyclonal antibody, producer: animal used as test section of PLA General Hospital first affiliated hospital article No.: Y11005) surperficial at magnetic particle, obtains Magneto separate reagent; Wherein, the proportioning of anti-fluorescein isothiocynate antibody and magnetic particle is 150 μ g:1mg.
The diameter (producer: merck company, article No.: EM1-100/40) between 0.5-1.5 μm of magnetic particle, has superparamagnetism, and surface is containing carboxyl (COOH-) reactive group.
The concrete preparation method of Magneto separate reagent:
(1) by magnetic bead and activator EDC (purchased from SIMGA company, CAS:25952-53-8, article No.: E7750) and NHS (purchased from SIMGA company, CAS:6066-82-6, article No.: 130672) mix with certain proportion, room temperature reaction 120 minutes, obtains activated magnetic beads; Above-mentioned magnetic bead and activator mix ratio are: add 5mgEDC and 5mgNHS in 1000mg magnetic bead; (2) mixed with certain proportion with goat-anti fluorescein isothiocynate antibody by magnetic bead after activation, blending ratio is: add 150mg goat-anti fluorescein isothiocynate antibody in 1000mg magnetic bead, and 4 degree of oscillating reactionss 10 hours, obtain the magnetic bead of connection albumen; (3) processed by the magnetic bead confining liquid after connection albumen, room temperature closes 1 hour, obtains magnetic bead solution; Confining liquid is the MES damping fluid of pH7.0,0.01M containing 1g/100mLBSA; (4) the magnetic bead solution (3) obtained sedimentation under magnetic fields abandoned supernatant after 20 minutes; The stainless steel sift that 1500 order materials are 304 steel is crossed after magnetic bead being suspended in the MES damping fluid of 100mlpH7.0,0.01M, sieve in process, continuous vibration is also rinsed repeatedly with the MES damping fluid of pH7.0,0.01M, until compass screen surface remains the agglomerated particle that can not sieve, magnetic bead after sieving answers particle homogeneous, without aggegation after suspendible again; (5) the magnetic bead conserving liquid after sieving is diluted to 5mg/ml, room temperature suspendible is 4 DEG C of preservations after 2 hours, obtain the magnetic bead being combined with anti-fluorescein isothiocynate antibody; Conserving liquid is the trizmabase damping fluid containing 1% bovine serum albumin(BSA); Again the magnetic bead being combined with anti-fluorescein isothiocynate antibody is suspended in the trizmabase damping fluid containing 1% bovine serum albumin(BSA), obtains Magneto separate reagent;
Trizmabase damping fluid containing 1% bovine serum albumin(BSA) is bovine serum albumin(BSA), trizmabase and water are mixed, obtain the trizmabase damping fluid containing bovine serum albumin(BSA), wherein, the concentration of bovine serum albumin(BSA) is 1% (for mass percentage), the concentration of trizmabase is 0.1mol/L.
7, cleaning fluid
Cleaning fluid: the Tris-HCl damping fluid mixing of to be 8.0 ± 0.05 concentration by Tween-20, sodium chloride and pH be 0.15M, obtain cleaning fluid, wherein the concentration of Tween-20 is 0.04% (volumn concentration), and the concentration of sodium chloride is 0.25mol/L.
8, substrate solution
Substrate solution: to be 0.25M, pH by acridan and concentration be 8.0 ± 0.05 Tris-HCl damping fluid mix, dissolve, make acridan final concentration be 0.25mg/mL, the solution obtained.
Two, magnetic microparticles chemiluminescence immunoassay kit is utilized to carry out quantitative measurement to thyroglobulin content in sample
1, principle
Fig. 1 is that magnetic microparticle separating chemiluminescence immunization detects thyroglobulin schematic diagram.
The present invention is sandwich method.Magnetic microparticles chemiluminescence immune analysis method is utilized to carry out quantitative measurement to thyroglobulin content in sample.The know-why of reaction is: the thyroglobulin in the same sample of thyroglobulin antibody, calibration object or quality-control product that the thyroglobulin antibody that alkaline phosphatase (AP) marks and fluorescein isothiocynate (FITC) mark is combined, and forms the antigen antibody complex of " sandwich " structure.Add the magnetic particle being connected with anti-fluorescein antibody subsequently, antigen antibody complex is made to be connected on magnetic particle by the specific binding of anti-fluorescein antibody and fluorescein, Direct precipitation in externally-applied magnetic field, the compound that immune response is formed and other separating substances unconjugated.Abandon washing and precipitating compound after supernatant, add enzymatic chemical substrate solution.Substrate just catalytic pyrolysis under enzyme effect, forms unstable excited state intermediate, just sends photon when excited state intermediate gets back to ground state, forms luminescence-producing reaction.Use the luminous intensity of analyser detection reaction.In luminous intensity and sample, the content of thyroglobulin is proportional.
2, thyroglobulin magnetic microparticle separating chemiluminescence immunologic detection method
(1) immune response: respectively by the TG calibration object in the kit of 30 μ L above-mentioned, 30 μ L quality-control products, 30 μ L samples to be tested in differential responses pipe, again respectively to adding 30 μ L reagent A and 30 μ L reagent B in each reaction tube, be placed on oscillator and mix, 37 DEG C of incubation 15min;
(2) Magneto separate: add 30 μ L Magneto separate reagent again in each reaction tube processed through (1), be placed on oscillator and mix, 37 DEG C of incubation 15min, make magnetic particle sedimentation in magnetic field, remove supernatant, add 200 μ L magnetic bead cleaning fluids (cleaning fluid in the kit of above-mentioned one and purified water diluted by 1:7 and be mixed with magnetic bead cleaning fluid), remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then make magnetic particle sedimentation in magnetic field, remove supernatant; Repetition like this 2-4 time;
(3) read value: to each reaction tube processed through (2) often pipe add 200 μ L substrate solutions, detect luminous intensity with Chemiluminescence Apparatus.
Specific as follows:
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (CRP).
TG kit is in measurement range, and luminous intensity is directly proportional to TG concentration in sample to be tested, uses four parameter Logistic equation models of improvement to calculate TG concentration in sample.
y＝(A1-A0)/(1+(x/X0)^P)+A0
Y: luminous intensity
X: concentration value
A1: asymptotic line valuation on curve
A0: asymptotic line valuation under curve
X0: the concentration value that knee point is corresponding
P: the parameter that rate of curve is relevant
Embodiment 2, kit performance evaluation
According to the feature of external diagnosis reagent, detect the range of linearity of kit prepared by embodiment 1, sensitivity, accuracy, precision as usual.Concrete operation step is as follows:
One, reagent prepares:
Before experiment, first take out reagent A (concentration of marked by fluorescein isothiocyanate TG monoclonal antibody is 0.3ug/ml), reagent B (concentration of alkali phosphatase enzyme mark TG monoclonal antibody is 0.5ug/ml), calibration object, Magneto separate reagent, luminous substrate, sample diluting liquid, cleaning fluid concentrate, quality-control product, balance to room temperature.
Two, instrument prepares:
This kit adopts the ChemLite of Capitalbio Corporation Co., Ltd.
tM1200 Full-automatic chemiluminescence immunoassay analysis meters.
Specific as follows:
In sample cup, add calibration object, sample or quality-control product, sample single tube reaction application of sample amount is 30 μ L, and according to repeating addition in pipe number calculation sample cup, minimum application of sample amount is not less than 50 μ L, adds the reagent of sufficient quantity.
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (TG).
Four, performance index testing result:
1, the range of linearity:
Sample to be tested is exceed the sample of range of linearity upper concentration and exceed or equal the sample of range of linearity hypomere, be mixed at least 5 dilute concentrations (Xi), specifically as shown in table 1, the TG solution (solute: TG antigen, solvent: calibration object buffer concentration 750ng/mL) of wherein dilution ratio is the sample to be tested of 1 to be concentration be 750ng/mL.
With the kit of a preparation in embodiment 1 and the detection method of two, sample to be tested is detected, measure the range of linearity.Each dilute concentration tests 3 times.
Obtain the average (Yi) of measurement result respectively, with dilute concentration (Xi) for independent variable, with measurement result average (Yi) for dependent variable, obtain equation of linear regression, calculate linear regression coeffficient (r) by formula (1).
Use four parameter Logistic equation models of improvement, in 1.0-750ng/mL sensing range, dose-response curve correlation coefficient r=0.9996.This sensing range is far superior to the like product that patent report is crossed.
Table 1 range of linearity experimental result
2, accuracy:
Sample to be tested is dilution thyroglobulin (TG) international standard substance (BCR-457), make its ultimate density for (50 ± 10) ng/mL and (300 ± 60) ng/mL, sample step is to specifications it can be used as to detect, after duplicate measurements 3 times, its average results is designated as Cm, according to the relative deviation Cb of formula (1) computation and measurement concentration, deviation should in ± 10% scope.
Cb＝(Cm-Cr)/Cr×100％……………………………(1)
In formula:
Cb: the relative deviation of concentration
Cm: the average measuring concentration
Cr: demarcate concentration
Deviation is all in ± 10% scope, and concrete data are in table 2.
Table 2 accuracy experimental result
3, lowest detectable limit (sensitivity):
With 0 μ g/mL calibration object in kit as sample to be tested, replication 20 times, draw the RLU value (relative light unit) of 20 measurement results, calculate its mean value (M) and standard deviation (SD), draw RLU value corresponding to M+2SD, carry out 2 regression fits according to the concentration-RLU value result between zero-dose calibration object and adjacent calibration object and draw linear function, the RLU value of M+2SD is brought in above-mentioned equation, obtain corresponding concentration value.
By lowest detectable limit detection method in detection scheme, 3 repeated experiments, concrete data are in table 3.
Table 3 lowest detectable limit experimental result
| Sensitivity (ng/mL) | |
| Experiment 1 | 0.42 |
| Experiment 2 | 0.16 |
| Experiment 3 | 0.36 |
| Experiment 4 | 0.27 |
| Experiment 5 | 0.13 |
4, precision:
In batch: use TG (10 ± 5) ng/mL and the sample in (300 ± 60) ng/mL two concentration ranges as sample to be tested.The method of operating of by specification, carries out 10 replications to sample to be tested respectively with chemical luminescence detector device.Repeatability (CV) is calculated by formula (3).
measure the mean value of concentration for 10 times
X
i: measure concentration value at every turn
Between batch: use TG concentration to be that (300 ± 60) ng/mL is as sample to be tested.Use the kit of 3 different batches, the method for operating of by specification, with chemical luminescence detector device, 30 replications (every batch kit measurement 10 times) are carried out to sample to be tested.Difference between batch (CV) is calculated by formula (4).
In formula:
measure the mean value of concentration for 30 times
Measure the standard deviation of concentration for SD:30 time
Detect precision by precision detection method in detection scheme, concrete data are in table 4.
Table 4 Precision Experiment result
Embodiment 3, kit provided by the invention compare with home and abroad kit
1, kit provided by the invention and home and abroad kit performance index comparison
This kit of table 5 and the comparison of external kit performance index
2, kit provided by the invention and external kit carry out the comparison of clinical sample measured value
Detection kit chemoluminescence method with kit provided by the invention and Beckman Ku Erte commerce and trade (China) company limited) kit detects its testing result to 96 parts of human serum samples (patient knows the inside story) respectively simultaneously and sees accompanying drawing 2, the serum TG adopting kit provided by the invention to record is horizontal ordinate, with the TG detection kit of Beckman Ku Erte commerce and trade (China) company limited measure result be that ordinate does regretional analysis, dependent equation is: y=1.0598x+2.3965, and correlation coefficient r is: 0.992.Show through statistical procedures result, the clinical sample measured value correlativity that kit provided by the invention and external kit record is good.
Claims (10)
1. a thyroglobulin immue quantitative detection reagent box is 1) or 2):
1) the thyroglobulin monoclonal antibody solution of marked by fluorescein isothiocyanate and the thyroglobulin monoclonal antibody solution of alkali phosphatase enzyme mark is comprised;
The thyroglobulin monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: solution A, cow's serum, horse serum, sheep blood serum, antiseptic mixing are obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, animal serum albumin and water composition;
2) thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate, the thyroglobulin monoclonal antibody of described alkali phosphatase enzyme mark and described anti-reagent buffer is comprised.
2. kit according to claim 1, is characterized in that:
Described animal serum albumin is bovine serum albumin(BSA), and described antiseptic is ProcLin-300;
In described anti-reagent buffer, the proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000g:23-28ml:1.5-2.5ml:4.0-5.0ml:0.5-2.0ml;
In described solution A, described Na
+concentration is 0.1-1mol/L, described Mg
2+concentration be 1mmol/L, Zn
2+concentration be 0.1mmol/L, the mass percentage of described bovine serum albumin(BSA) is 0.2-1.0%, and described Trizmabase concentration is 0.01-0.5mol/L.
3. kit according to claim 1 or 2, is characterized in that:
In described anti-reagent buffer, the proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000g:25ml:2ml:4.5ml:1ml;
In described solution A, described Na
+come from NaCl, described NaCl concentration is 0.1mol/L;
Described Mg
2+come from MgCl
2, described MgCl
2concentration is 1.0mmol/L;
Described Zn
2+come from ZnCL
2, described ZnCL
2concentration is 0.1mmol/L;
The mass percentage of described bovine serum albumin(BSA) is 0.5%;
The concentration of described Trizmabase is 0.1mol/L.
4., according to described kit arbitrary in claim 1-3, it is characterized in that:
The thyroglobulin monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the thyroglobulin monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution is made up of alkali phosphatase enzyme mark thyroglobulin monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody is 0.5-1 μ g/mL;
Thyroglobulin monoclonal antibody in the thyroglobulin monoclonal antibody of described marked by fluorescein isothiocyanate is different with thyroglobulin monoclonal antibody in described alkali phosphatase enzyme mark thyroglobulin monoclonal antibody.
5., according to described kit arbitrary in claim 1-4, it is characterized in that:
Described kit also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
6., according to described kit arbitrary in claim 1-5, it is characterized in that: described kit also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of thyroglobulin antigen and calibration object damping fluid, and described thyroglobulin antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, protide protective agent, antiseptic, Trizmabase, animal blood serum and water composition, described Na
+concentration is 0.1-1mol/L; the protectant mass percentage of described protide is 0.2-1.0%; described antiseptic volumn concentration is 0.05-0.2%, and described Trizmabase concentration is 0.01-0.5mol/L, and the volumn concentration of described animal blood serum is 0.05-1.0%.
7. kit according to claim 6, is characterized in that:
Described Na
+come from NaCl, described NaCl concentration is 0.10mol/L;
Described protide protective agent is bovine serum albumin(BSA), and described protein amount percentage composition is 0.5%;
Described antiseptic is Proclin-300, and described Proclin-300 volumn concentration is 0.1%;
The concentration of described Trizmabase is 0.1mol/L;
Described animal blood serum is sheep blood serum, and described sheep blood serum volumn concentration is 0.2%.
8. the kit according to claim 6 or 7, is characterized in that:
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6;
Described kit also comprises quality-control product, Sample dilution, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations;
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described acridan final concentration is 0.25mg/mL;
Described Sample dilution is made up of bovine serum albumin(BSA), trizmabase and water, and wherein, the mass percentage of described bovine serum albumin(BSA) is 0.2%-5%, and the concentration of described trizmabase is 0.1mol/L.
9. prepare a method for arbitrary described kit in claim 1-8, comprise the steps: 8 kinds of equal independent packagings of component in described kit arbitrary in claim 1-8; Entire package is kit again.
10. the arbitrary described application of kit in thyroglobulin quantitatively detects in claim 1-8;
Or arbitrary described kit is preparing the application in the quantitative testing product of thyroglobulin in claim 1-8;
Or in claim 1-8 in arbitrary described kit 8 kinds of components preparing the application in the quantitative testing product of thyroglobulin;
Or thyroglobulin quantitative detecting method in a kind of sample to be tested, comprise the steps:
1) calibration object, quality-control product and the sample to be tested in described kit arbitrary in claim 1-8 is mixed with the thyroglobulin monoclonal antibody solution of the marked by fluorescein isothiocyanate in arbitrary described kit in claim 1-8 and alkali phosphatase enzyme mark thyroglobulin monoclonal antibody solution respectively, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described kit arbitrary in described immune reaction product and claim 1-8, reaction, obtain reaction product, by the precipitation in described reaction product and magnetic bead cleaning fluid suspendible, collecting precipitation, is Magneto separate product;
3) by the substrate solution mixing in described kit arbitrary in described Magneto separate product and claim 1-8, obtain mix products, detect the luminous intensity of described mix products with Chemiluminescence Apparatus, calculate gamut thyroglobulin in sample to be tested by luminous intensity quantitative;
Described magnetic bead cleaning fluid is cleaning fluid and water in described kit arbitrary in claim 1-8 are mixed by 1:7, obtains solution;
Or the application of Magneto separate reagent in thyroglobulin quantitatively detects in arbitrary described kit in claim 1-8.
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|---|---|---|---|
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| CN105353139A (en) * | 2015-09-28 | 2016-02-24 | 成都博奥新景医学科技有限公司 | Parathyroid hormone quantitative detection kit |
| CN106706927A (en) * | 2017-01-13 | 2017-05-24 | 广州华弘生物科技有限公司 | Thyroglobulin enzyme linked immunosorbent diagnostic kit |
| CN108226464A (en) * | 2018-02-05 | 2018-06-29 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring thyroglobulin content |
| CN112285353A (en) * | 2020-10-22 | 2021-01-29 | 武汉生之源生物科技股份有限公司 | Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system |
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| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
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| CN106706927A (en) * | 2017-01-13 | 2017-05-24 | 广州华弘生物科技有限公司 | Thyroglobulin enzyme linked immunosorbent diagnostic kit |
| CN108226464A (en) * | 2018-02-05 | 2018-06-29 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring thyroglobulin content |
| CN112285353A (en) * | 2020-10-22 | 2021-01-29 | 武汉生之源生物科技股份有限公司 | Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system |
| CN112557660A (en) * | 2020-12-09 | 2021-03-26 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence HER2 detection kit and preparation method thereof |
| CN113203863A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for interleukin-6 detection |
| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
| CN113203863B (en) * | 2021-04-28 | 2022-01-21 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for interleukin-6 detection |
| CN114878840A (en) * | 2022-07-12 | 2022-08-09 | 昆明思安生物科技有限公司 | Kit and method for measuring triiodothyronine by magnetic particle chemiluminescence |
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