CN103063852B - Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof - Google Patents
Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof Download PDFInfo
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Abstract
The invention relates to a free thyroxine nanometer magnetic particle chemiluminescence assay kit and a preparation method thereof and a detection method thereof. The free thyroxine nanometer magnetic particle chemiluminescence assay kit comprises solutions containing fluorescein-labeled free thyroid antibodies, suspension liquid of magnetic particles covered with fluorescein-resistance antibodies and solutions containing alkaline-phosphatase-labeled free thyroxine antigens, wherein the alkaline-phosphatase-labeled free thyroxine antigens are formed by the connection of alkaline phosphatase and the free thyroxine antigens through crosslinking agent suberic acid succinimide ester. The free thyroxine nanometer magnetic particle chemiluminescence assay kit and the preparation method thereof and the detection method thereof have the advantage of enabling the free thyroxine to be quantificationally detected on the condition of lower cost, higher accuracy and higher precision.
Description
Technical field
The invention belongs to biological technical field, be specifically related to nano magnetic particulate chemistry luminescence assays kit of a kind of free thyroxine that combines immune magnetic particle isolation technics and nano magnetic particulate chemistry luminescence assays technology and preparation method thereof.The present invention is also particularly related to the preparation method of this kit.
Background technology
Thyroxine, 3,5,3', 5'-tetraiodothyronine (T4) is that a kind of molecular weight is 777 thyroid hormone.T4 has in conjunction with state and two kinds of forms of free state in circulation, is keeping mobile equilibrium under normal circumstances between two kinds of forms.In circulation T4 have 75% with thyroid binding globulin (TBG) combination, 15% and prealbumin, 10% is combined with albumin.Free T4 (FT4) only accounts for 0.03%, is but bringing into play real physiological action.The gestational period, hepatitis, congenital TBG increase, take estrogen (oral contraceptive) etc. while causing TBG Enrichment, and the concentration of total T3, T4 also increases.Ephrosis, liver failure, congenital TBG lack, take androgen or high dose glucocorticoid causes that, when TBG concentration reduces, the concentration of total T3, T4 also decreases.In these situations, measure total T3, T4 and can not accurately reflect thyroid functional status.The level of free T3, the free T4 degree that is affected is less, measures the thyroid hormone of free state and can better understand thyroid function in therefore a lot of situations.
FT4 is the routine immunization test item that Hospitals at Present is carried out, and to the diagnosis of thyroid disease, has the important references can not be substituted to be worth.The FT4 immunologic function test reagent of China's approved listing at present has import and domestic two large classes, wherein import reagent has the product of the transnational quantitative detection company such as Abbott Laboratories, the methodology adopting mostly is chemiluminescence, and the methodology adopting in domestic reagent is most for putting the method for exempting from, within nearly 2 years, start to occur chemiluminescence series products.
There is narrow, the difficult methodology limiting factors such as full-automation that realize of the range of linearity in radioimmunoassay for detecting.And that nano magnetic particulate chemistry luminescent assays has is highly sensitive, detect the advantages such as linear wide ranges, easy and simple to handle, automaticity height.Nano magnetic particulate chemistry luminescence assays technology is widely used because it has above-mentioned plurality of advantages at present.
Yet, in actual immune detection, because impurity component contained in testing sample is more, detection sensitivity and accuracy have been affected to a certain extent, so from complicated sample substrate fast separated, be purified into object determinand, be one of difficult problem of facing of clinical examination worker.Magnetic particle immunoassay technology is to utilize the magnetic solid phase particle of synthesis of polymer material certain particle size size to make carrier, on coated with methods such as physisorption, chemical couplings, there are the various immunologic active materials such as the antibody of specificity affinity or antigen, have that velocity of separation is fast, efficiency is high, the feature such as favorable repeatability, simple to operate, the biological character that do not affect separated cell or other biological material and function, orientable motion under additional magnetic fields, makes some special composition be able to separation, concentrate or purifying.
The open CN101639481A of Chinese invention patent discloses the magnetic particle nano magnetic particulate chemistry luminescence assays kit of free thyroxine a kind of; this kit comprises free thyroxine series calibration object; the magnetic particle solution that anti-fluorescein isothiocynate monoclonal antibody is coated; the free thyroxine antibody-solutions of marked by fluorescein isothiocyanate; the free thyroxine of horseradish peroxidase-labeled; Chemoluminescent substrate, concentrated cleaning solution.In this kit, coated magnetic particle solution, the free thyroxine antibody-solutions of marked by fluorescein isothiocyanate and the free thyroxine of horseradish peroxidase-labeled of anti-fluorescein isothiocynate monoclonal antibody forms the immune response reagent that immune response step is used.Although this kit can be realized quickly and accurately and detecting, but wherein, the preparation process of the free thyroxine of horseradish peroxidase-labeled is very loaded down with trivial details, and technology stability is bad, and mark rate is low, limits its detection effect and particularly analyze a precision and cause cost to increase.
Summary of the invention
Technical matters to be solved by this invention is to overcome the deficiencies in the prior art; a kind of nano magnetic particulate chemistry luminescence assays kit of free thyroxine is provided; it can prepare with lower cost, and can realize free thyroxine accurately and high precision quantitative measurement.
The present invention also provides a kind of preparation method of nano magnetic particulate chemistry luminescence assays kit of free thyroxine simultaneously, the method process stabilizing, cost is low, and the precision of gained kit particularly to analyze a precision high.
For solving above technical matters, a kind of technical scheme that the present invention takes is:
The nano magnetic particulate chemistry luminescence assays kit of a kind of free thyroxine (FT4), it comprises the immune response reagent for immune response step, this immune response reagent comprises the solution that contains fluorescein-labeled free thyroid antibody and is coated with the suspending liquid of the magnetic particle of anti-fluorescein (FITC) antibody, it is characterized in that: described immune response reagent also comprises the solution of the free thyroxine antigen that contains alkaline phosphatase (ALP) mark, the free thyroxine antigen of this alkali phosphatase enzyme mark is connected and composed by crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and free thyroxine antigen.
Further, the pH of the described solution that contains fluorescein-labeled free thyroid antibody is 7 ~ 9, and the concentration of fluorescein-labeled free thyroid antibody is wherein 0.5 ~ 1 μ g/mL.The pH of the solution of the described free thyroxine antigen that contains alkali phosphatase enzyme mark is 7 ~ 9, and wherein the concentration of the free thyroxine antigen of alkali phosphatase enzyme mark is 0.02 ~ 0.1 μ g/mL.
According to the present invention, described contain fluorescein-labeled anti-free thyroxine antibody and can easily prepare by ripe and stable technique.
According to the present invention, described in be coated with the magnetic particle of anti-fluorescein antibody preparation be to be also comparatively easy to, and the existing ripe preparation technology of prior art can reference.For example can prepare by conventional physisorption or the coated mode of chemical coupling, be not particularly limited.As a kind of preferred embodiment of the present invention: this is coated with in the magnetic particle of anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and magnetic particle.In the described magnetic particle that is coated with anti-fluorescein antibody, phase chemistry coupling between anti-fluorescein antibody and magnetic particle.
Those skilled in the art should know, and kit of the present invention can further include other and detects required reagent, for example substrate solution.But such as other reagent such as substrate solutions, can buy separately or prepare, therefore, although can comprise these reagent in kit, they be not essential for kit of the present invention.
The another technical scheme that the present invention takes is: a kind of preparation method of nano magnetic particulate chemistry luminescence assays kit of above-mentioned free thyroxine; it comprise respectively the solution that contains fluorescein-labeled free thyroid antibody described in preparation, described in contain alkali phosphatase enzyme mark the solution of free thyroxine antigen and the step of suspending liquid that is coated with the magnetic particle of anti-fluorescein antibody, wherein: described in contain alkali phosphatase enzyme mark the preparation process of solution of free thyroxine antigen as follows:
1. make free thyroxine antigen and crosslinking chemical disuccinimidyl suberate in dimethyl sulfoxide solvent, at room temperature reaction, make to generate the connector of thyroxine antigen and disuccinimidyl suberate, at 2 ~ 8 ℃, save backup, wherein: the purity of described free thyroxine antigen, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpass 1000u/mg;
2. the damping fluid that contains alkaline phosphatase and the step ratio that 1. gained solution is 1:1.1 ~ 1.3 according to the mol ratio of alkaline phosphatase and thyroxine antigen and the connector of disuccinimidyl suberate is mixed, at room temperature reaction, make to generate the free thyroxine antigen of described alkali phosphatase enzyme mark, reaction finishes, reactant liquor is passed through to G-25 gel column desalination, selection has the damping fluid of proper pH value to be adjusted concentration and pH value and get final product, and the concentration of the damping fluid of its alkaline phosphatase is 0.5 ~ 1.5mg/mL.
According to a concrete aspect, step 1. in, get free thyroxine antigen, adding this antigen of dmso solution to concentration is 20 ~ 50mg/mL, add crosslinking chemical disuccinimidyl suberate, room temperature reaction 2 ~ 3 hours,, saves backup at 2 ~ 8 ℃ reactant liquor 1:10 dilution with dimethyl sulfoxide (DMSO).
According to another concrete aspect, step 2. in, get the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/mL, with the sodium bicarbonate buffer liquid of pH9 ~ 10, be diluted to 0.5 ~ 1.5mg/mL, add 1. gained reactant liquor of step, the standing reaction 20 ~ 60min of room temperature.
In a concrete embodiment, step 2. in, the damping fluid that contains alkaline phosphatase and the step ratio that 1. gained solution is 1:1.2 according to the mol ratio of alkaline phosphatase and thyroxine antigen and the connector of disuccinimidyl suberate is mixed.
Further, the preparation method of the described solution that contains fluorescein-labeled free thyroid antibody is as follows: the damping fluid that the pH that preparation contains fluorescein is 9 ~ 10, then the ratio that is 20 ~ 200:1 according to the molecular proportion of fluorescein and anti-free thyroxine antibody, the damping fluid that is 9 ~ 10 by the described pH that contains fluorescein mixes with the damping fluid that the pH of anti-free thyroxine antibody is 9 ~ 10, after mixing, the standing reaction of room temperature, then reactant liquor is carried out to separation by G-25 gel column, remove free fluorescein, obtain the solution that contains fluorescein-labeled anti-free thyroxine antibody, then with the damping fluid with proper pH value, adjust concentration and pH, obtain.Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of the suspending liquid of the described magnetic particle that is coated with anti-fluorescein antibody is as follows: the magnetic particle that makes to contain carboxyl reactive group and anti-fluorescein antibody are under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic is separated, remove supernatant, with the damping fluid with proper pH value, adjust pH and concentration, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5 ~ 2 μ m, on every gram of magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described anti-fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired and is greater than 1:100 ten thousand.Wherein the damping fluid of proper pH value can be for for example containing the TRIS damping fluid of 0.5% bovine serum albumin(BSA), pH8.0.
Preferably, the above-mentioned damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
Fluorescein of the present invention can be known various fluoresceins, and conventional have for example fluorescein isothiocynate, RB 200, a TRITC etc.
Step when kit of the present invention is applied to chemiluminescence immunoassay and detects is the same with traditional detection method, comprises the immune response step of carrying out successively, uses step that magnetic separation and washing facility wash immunoreactive reactant liquor and to adding substrate solution in the reactant liquor after washing and utilizing the step of chemiluminescence detector detection luminous intensity.
Preferably, described immune response step is specifically implemented as follows: in test sample tube, add sample to be tested stoste or dilution, the solution of the free thyroxine antigen that then add successively the solution that contains fluorescein-labeled free thyroid antibody, contains alkali phosphatase enzyme mark, mix, at 25 ~ 40 ℃, carry out incubation for the first time, then the suspending liquid that adds the magnetic particle that is coated with anti-fluorescein antibody, mixes, and carries out incubation for the second time at 25 ~ 40 ℃; Described substrate solution is alkaline phosphatase chemical luminous substrate solution.
Further, the time of described incubation for the first time can be 10 ~ 20min, is generally 15min; The time of incubation can be 3 ~ 15min for the second time, is generally 5min.
The step that above-mentioned use magnetic separation and washing facility wash immunoreactive reactant liquor and all can implementing with reference to conventional method to the step that adds substrate solution and utilize chemiluminescence detector to detect luminous intensity in the reactant liquor after washing, equipment used and device are also conventional.Wherein, alkaline phosphatase chemical luminous substrate solution is also known for those skilled in the art, commercially available acquisition.
Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:
1. applicant finds, while taking disuccinimidyl suberate to carry out the coupling of alkaline phosphatase and free thyroxine antigen as crosslinking chemical, have and compare higher coupling efficiency with other crosslinking chemical, when reducing preparation cost, be conducive to improve detection effect.Therefore, three kinds of reagent in kit of the present invention all can prepare by stable preparation technology, and production cost is low, and due to preparation technology's stability, it is little that kit is analyzed difference between batch, and between the analysis of detection, precision improves;
2. the preparation method of the solution of the free thyroxine antigen that contains alkali phosphatase enzyme mark in kit of the present invention, can be effectively by free thyroxine antigen and alkaline phosphatase coupling, coupling efficiency is high, further reduces the low detection effect with guaranteeing kit of cost of kit.
3, take kit of the present invention to detect, accuracy is good, and precision is high, highly sensitive, and sensing range is wide, the unordered pre-dilution of sample, simple to operate saving time.Compare with the method that adopts import reagent box to detect, detection method of the present invention has significant advantage on cost.
Accompanying drawing explanation
Fig. 1 is detection calibration product typical curves;
Fig. 2 is that matched curve is evaluated in sensitivity;
Fig. 3 is serum sample testing result correlativity (wherein horizontal ordinate x is the kit sample measured value that embodiment 4 is prepared into, and concentration unit is pg/mL, and ordinate y is Abbott's kit sample measured value, and concentration unit is pg/mL).
Embodiment
For convenience of description, hereinafter, with the first reagent, the second reagent and magnetic separation agent represent respectively the solution that contains fluorescein-labeled free thyroid antibody in the present invention, the solution of the free thyroxine antigen that contains alkaline phosphatase (ALP) mark and be coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody.
The preparation of embodiment 1 first reagent
(1) material and instrument: the anti-FT4 monoclonal antibody of preserving with phosphate buffer (purity surpasses 95wt%, and concentration is 2mg/mL); Fluorescein isothiocynate (FITC), the reagent such as sodium bicarbonate should reach chemical pure; G-25 gel-purified post buying ZiGE company.
(2) preparation process:
1. use the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1 ~ 0.2mol/L pH9.0 ~ 10.0;
2. the ratio that is 1:20 according to FT4 antibody and FITC molecular proportion adds step 1. to be joined FITC solution in antibody-solutions, mixes, and the standing 12h hour of room temperature, reaction generates FT4 antibody-FITC connector;
3. by carrying out separation through step reactant liquor 2. by G-25 gel column, remove unreacted FITC, obtain the solution that contains FT4 antibody-FITC connector (being the FT4 antibody of FITC mark);
4. the step solution that 3. gained contains FT4 antibody-FITC connector being diluted to FT4 antibody-FITC connector concentration with the TRIS damping fluid that contains the 0.1mol/L of 0.5% bovine serum albumin(BSA) (BSA) pH8.0 is 0.5 ~ 1 μ g/mL, pH is 7 ~ 9, is the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: FT4 antigen (pressed powder, purity surpasses 95%); The alkaline phosphatase of preserving with phosphate buffer (ALP solution, ALP purity is approximately 99%, specific activity is about 1500U/mg, concentration is 10mg/mL); Coupling agent DSS is purchased from THERMO company, and the chemical reagent such as TRIS should reach chemical pure; G-25 gel-purified Zhu Wei GE company product.
(2) preparation process:
1. get 1mg FT4 antigen, adding DMSO to dissolve this antigen to concentration is 20 ~ 50mg/mL, adds DSS0.5mg, room temperature reaction 2 hours, and with DMSO, by reactant liquor 1:10 dilution, 2-8 ℃ saves backup;
2. get the ALP solution of 1mg, with the NaHCO of 0.1M pH9.5
3damping fluid by ALP solution dilution to 1mg/mL, in ALP damping fluid after dilution, add the FT4-DMSO solution that 1. step is prepared to carry out coupled reaction, adding FT4-DMSO liquor capacity is 1/20 of ALP damping fluid volume, the standing reaction of room temperature 30min, with G-25 gel column desalination, 2-8 ℃ saves backup;
3. step solution is 2. diluted to 0.02 ~ 0.1 μ g/mL and pH9 ~ 10 with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0, is the second reagent.
The preparation of embodiment 3 magnetic separation agents
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle is containing the active group of carboxyl (COOH), and every gram of (g) magnetic particle (dry weight) carboxyl-content is not less than 0.4 mM (mmol), has superparamagnetism, and diameter is between 0.5-2 μ m
Anti-FITC mAb: can be polyclonal antibody, can be also monoclonal antibody, and purity is more than 90wt%, dilution is tired over 1:100 ten thousand;
MES (MES), carbodiimide (EDC), TRIS and other reagent should reach chemical pure.
(2) preparation process:
1. the suspending liquid of getting 100mg magnetic particle, magnetic divides the supernatant of leaving away, and with 0.05mol/L, 10mL is resuspended for pH4.5 ~ 5MES damping fluid;
2. the anti-FITC mAb that adds 2 ~ 4mg, room temperature suspendible 30 ~ 60min;
3. the EDC aqueous solution that adds the freshly prepared 10mg/mL of 0.5 ~ 1mL, room temperature suspendible 2 ~ 12h;
4. magnetic is separated, removes supernatant, with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0, is resuspended to 1mg/mL, and pH8.0, is magnetic separation agent.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 4 free thyroxines
This kit comprises:
The first reagent of preparing according to embodiment 1 method (concentration is 0.75 μ g/mL), 50mL;
The second reagent of preparing according to embodiment 2 methods (concentration is 0.05 μ g/mL), 50mL;
The magnetic separation reagent 50mL preparing according to embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 5 free thyroxines
This kit comprises:
The first reagent of preparing according to embodiment 1 method (concentration is 0.5 μ g/mL), 5mL;
The second reagent of preparing according to embodiment 2 methods (concentration is 0.02 μ g/mL), 5mL;
The magnetic separation reagent 5mL preparing according to embodiment 3 methods.
Embodiment 6 takes the kit of embodiment 4 to carry out the quantitative detection of free thyroxine
(1) detecting step:
1. immune response: add 30 μ l sample to be tested (serum or blood plasma) stostes in detector tube, then add 50 μ l the first reagent, 50 μ l the second reagent, mix, incubation 15min under 37 ± 1 ℃ of conditions; Add 50 μ l magnetic separation agents, mix incubation 5min under 37 ± 1 ℃ of conditions;
2. washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add the cleaning fluid of 600 μ l, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic is separated, removes supernatant.This step repeats 3 times;
3. add substrate solution and detect luminous intensity: in detector tube, add 150 μ l alkaline phosphatase chemical luminous substrate solution (Beijing APCL-of A Pisi Bioisystech Co., Ltd I), concussion makes the abundant suspendible of magnetic particle, detects unit interval inner glow intensity.
(2) draw calibration object typical curve
Calibration object typical curve is referring to Fig. 1.
(3) sensitivity evaluation
Detect " 0 " concentration samples, duplicate detection 20 times, calculate mean value (M) and the standard deviation (SD) of relative luminous intensity (RLU), and calculate M-2SD value, according to the concentration-RLU between zero-dose calibration object and adjacent calibration object, carry out 2 regression fits and draw linear function, M-2SD value is brought in above-mentioned equation, obtain corresponding concentration value, be lowest detectable limit.The sensitivity of this method is not more than 2pg/mL.Wherein: A point luminous value is respectively referring to table 1:
Table 1
The luminous average X=654270 of A point
SD=13021
X-2SD=628228
B point luminous value is respectively referring to table 2.
The luminous average X=367576 of B point
Table 2
| FT4-STD-B(RLU) |
| 367598 |
| 367554 |
A, B point connects some matched curve referring to Fig. 2.Sensitivity=0.454pg/mL.
(4) precision evaluation
1. precision in analyzing
The kit of embodiment 4 is a collection of, measure respectively the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations, result, referring to table 3, show that variation within batch coefficient is 4.01%～6.36%.
Table 3 is analyzed interior precision test
| Measure serum-concentration (pg/mL) | Measure number of times | CV (%) in analyzing |
| 8.335 | 10 | 6.36 |
| 20.158 | 10 | 5.25 |
| 60.335 | 10 | 4.01 |
2. precision between analyzing
The kit of embodiment 4 is got to three batches, and every batch of kit is all measured the serum of basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations.Every part of serum obtains 30 concentration measured values, and referring to table 4, the coefficient of variation between statistical study, is 5.39%～8.24%.
Precision test between table 4 analysis
| Measure serum-concentration (pg/mL) | Measure number of times | CV between analysis (%) |
| 8.335 | 30 | 7.65 |
| 20.158 | 30 | 5.44 |
| 60.335 | 30 | 5.69 |
(5) accuracy estimating
In 2 routine pooled serum samples, add different amount FT4 standard items, the serum that forms 3 concentration levels adds sample, and additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by following formula calculate recovery rate.This method serum matrix recovery is between 90-110%.Data are referring to table 5.
R: the recovery;
V: the volume that adds standard solution;
V0: the volume of people source sample;
C: people source sample adds the detectable concentration after standard solution;
C0: the detectable concentration of people source sample;
Cs: the concentration of standard solution.
Table 5 accuracy estimating-interpolation recovery experiment data
(6) kit Evaluation on specificity
To kit specificity, check is to choose test variable concentrations trilute (T3), reverse triiodothyronine (rT3) and diiodothyronine (T2) sample that has similar structures with FT4, be mixed with the sample that is greater than physiological concentration, with this method, measure, and calculate cross reacting rate.The results are shown in Table 6, the equal no cross reaction of cross reacting rate of this law and T3, rT3, T2.
The experiment of table 6 specificity
| Cross reaction thing | Actual concentrations | FT4 measures concentration (pg/mL) |
| T3 | 500ng/mL | ＜2 |
| 3,3 ', 5 '-reverse triiodothyronine | 50ng/mL | ＜2 |
| 3,3 '-T2 | 50ng/mL | ＜2 |
(7) correlativity evaluation
With the chemical luminescence reagent kit of kit and Abbott, 100 parts of human serum samples are detected simultaneously.Its testing result is referring to accompanying drawing 3, and serum FT 4 concentration of survey of the inventive method of take are horizontal ordinate, and the result of Abbott's kit measurement of take is done regretional analysis as ordinate, and dependent equation is: y=-1.1875+1.0528x, related coefficient is: 0.9874.Learn by statistics result and show, this method is good with external kit clinical sample measured value correlativity.
(8) Evaluation of Thermal Stability
Kit is carried out respectively to 4 ℃ of 12 months and 37 ℃ of stability experiments of 7 days, result show kit standard items luminous intensity variation, batch in and the index such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Embodiment 7 takes the kit of embodiment 5 to carry out the quantitative detection of free thyroxine
(1) detecting step: with embodiment 6.
(2) sensitivity evaluation: the kit sensitivity in this embodiment is 0.454pg/mL, reaches domestic and international similar reagent top standard, meets preferably clinical practice.
(3) precision evaluation: see that with analysis precision is all less than 10% in the kit analysis in this embodiment, difference between batch is little.
(4) evaluation of the accuracy: the kit serum in this embodiment adds the recovery between 90-110%, and clinical practice measuring value accuracy is reliable.
Above-described embodiment is only explanation technical conceive of the present invention and feature; its object is to allow person skilled in the art can understand content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (2)
1. the chemical luminescence immune analysis reagent box of a free thyroxine, described kit comprises the immune response reagent for immune response step, described immune response reagent comprises the solution that contains fluorescein-labeled free thyroid antibody and is coated with the suspending liquid of the magnetic particle of anti-fluorescein antibody, it is characterized in that: described immune response reagent also comprises the solution of the free thyroxine antigen that contains alkali phosphatase enzyme mark, the free thyroxine antigen of this alkali phosphatase enzyme mark is connected and composed by crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and free thyroxine antigen,
The pH of the described solution that contains fluorescein-labeled free thyroid antibody is 7～9, the concentration of fluorescein-labeled free thyroid antibody is wherein 0.5～1 μ g/mL, the preparation method of the solution that this contains fluorescein-labeled free thyroid antibody is as follows: the damping fluid that the pH that preparation contains fluorescein is 9～10, then the ratio that is 20～200:1 according to the molecular proportion of fluorescein and anti-free thyroxine antibody, the damping fluid that is 9～10 by the described pH that contains fluorescein mixes with the damping fluid that the pH of anti-free thyroxine antibody is 9～10, after mixing, the standing reaction of room temperature, then reactant liquor is carried out to separation by G-25 gel column, remove free fluorescein, obtain the solution that contains fluorescein-labeled anti-free thyroxine antibody, then with the damping fluid with proper pH value, adjust concentration and pH, obtain,
The pH of the solution of the described free thyroxine antigen that contains alkali phosphatase enzyme mark is 7～9, wherein the concentration of the free thyroxine antigen of alkali phosphatase enzyme mark is 0.02～0.1 μ g/mL, described in contain alkali phosphatase enzyme mark the preparation process of solution of free thyroxine antigen as follows:
1. get free thyroxine antigen, adding this antigen of dmso solution to concentration is 20～50mg/mL, add crosslinking chemical disuccinimidyl suberate, room temperature reaction 2～3 hours, with dimethyl sulfoxide (DMSO), reactant liquor 1:10 is diluted, at 2～8 ℃, save backup, wherein: the purity of described free thyroxine antigen, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg;
2. the damping fluid that contains alkaline phosphatase and the step ratio that 1. gained solution is 1:1.1～1.3 according to the mol ratio of alkaline phosphatase and thyroxine antigen and the connector of disuccinimidyl suberate is mixed, at room temperature reaction, make to generate the free thyroxine antigen of described alkali phosphatase enzyme mark, reaction finishes, reactant liquor is passed through to G-25 gel column desalination, selection has the damping fluid of proper pH value and adjusts concentration and pH value and get final product, the concentration of the damping fluid of its alkaline phosphatase is 0.5～1.5mg/ml, get the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/ml, with the sodium bicarbonate buffer liquid of pH 9～10, be diluted to 0.5～1.5mg/ml, add 1. gained reactant liquor of step, the standing reaction 20～60min of room temperature,
The preparation method of the suspending liquid of the described magnetic particle that is coated with anti-fluorescein antibody is as follows: the magnetic particle that makes to contain carboxyl reactive group and anti-fluorescein antibody are under the existence of coupling agent carbodiimide, room temperature reaction 2～18 hours, reaction finishes, magnetic is separated, remove supernatant, with the damping fluid with proper pH value, adjust pH and concentration, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5～2 μ m, on every gram of magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described anti-fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired and is greater than 1:100 ten thousand;
The described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
2. the preparation method of the chemical luminescence immune analysis reagent box of a free thyroxine as claimed in claim 1, it comprise respectively the solution that contains fluorescein-labeled free thyroid antibody described in preparation, described in contain alkali phosphatase enzyme mark free thyroxine antigen solution and described in be coated with the step of suspending liquid of the magnetic particle of anti-fluorescein antibody, it is characterized in that:
The preparation method of the solution that contains fluorescein-labeled free thyroid antibody is as follows: the damping fluid that the pH that preparation contains fluorescein is 9～10, then the ratio that is 20～200:1 according to the molecular proportion of fluorescein and anti-free thyroxine antibody, the damping fluid that is 9～10 by the described pH that contains fluorescein mixes with the damping fluid that the pH of anti-free thyroxine antibody is 9～10, after mixing, the standing reaction of room temperature, then reactant liquor is carried out to separation by G-25 gel column, remove free fluorescein, obtain the solution that contains fluorescein-labeled anti-free thyroxine antibody, then with the damping fluid with proper pH value, adjust concentration and pH, obtain,
The preparation process of the solution of the described free thyroxine antigen that contains alkali phosphatase enzyme mark is as follows:
1. get free thyroxine antigen, adding this antigen of dmso solution to concentration is 20～50mg/mL, add crosslinking chemical disuccinimidyl suberate, room temperature reaction 2～3 hours, with dimethyl sulfoxide (DMSO), reactant liquor 1:10 is diluted, at 2～8 ℃, save backup, wherein: the purity of described free thyroxine antigen, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg;
2. the damping fluid that contains alkaline phosphatase and the step ratio that 1. gained solution is 1:1.1～1.3 according to the mol ratio of alkaline phosphatase and thyroxine antigen and the connector of disuccinimidyl suberate is mixed, at room temperature reaction, make to generate the free thyroxine antigen of described alkali phosphatase enzyme mark, reaction finishes, reactant liquor is passed through to G-25 gel column desalination, selection has the damping fluid of proper pH value and adjusts concentration and pH value and get final product, the concentration of the damping fluid of its alkaline phosphatase is 0.5～1.5mg/ml, get the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/ml, with the sodium bicarbonate buffer liquid of pH 9～10, be diluted to 0.5～1.5mg/ml, add 1. gained reactant liquor of step, the standing reaction 20～60min of room temperature,
The preparation method of the suspending liquid of the described magnetic particle that is coated with anti-fluorescein antibody is as follows: the magnetic particle that makes to contain carboxyl reactive group and anti-fluorescein antibody are under the existence of coupling agent carbodiimide, room temperature reaction 2～18 hours, reaction finishes, magnetic is separated, remove supernatant, with the damping fluid with proper pH value, adjust pH and concentration, obtain, wherein said magnetic particle has superparamagnetism, its diameter is 0.5～2 μ m, on every gram of magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described anti-fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired and is greater than 1:100 ten thousand;
The described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
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| CN108562752A (en) * | 2018-05-31 | 2018-09-21 | 湖南远璟生物技术有限公司 | A kind of free thyroxine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN113214380B (en) * | 2021-05-06 | 2023-05-09 | 三诺生物传感股份有限公司 | Preparation method of alkaline phosphatase labeled thyroxine |
| CN113686841B (en) * | 2021-09-01 | 2023-06-30 | 北京利德曼生化股份有限公司 | Kit for quantitatively detecting thyroxine binding force, preparation method and detection method thereof |
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