CN103048477B - Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same - Google Patents
Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same Download PDFInfo
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Abstract
The invention relates to a nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as a preparation method and a detecting method of the nanometer magnetic particle chemiluminescence detection kit. The kit comprises a first reagent, a second reagent and a magnetic separating agent, wherein the first reagent is a solution containing a fluorescein labeled triiodothyronine antibody; the second agent is a solution containing an alkaline phosphatase labeled triiodothyronine antigen; and the magnetic separating agent is a suspension containing magnetic particles coated by a fluorescein antibody. According to the invention, the triiodothyronine can be quantitatively detected with lower cost, higher accuracy and higher precision.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind ofly combine nano magnetic particulate chemistry luminescent assay kit of the trilute (T3) of immune magnetic particle isolation technics and chemiluminescence immunoassay technology and preparation method thereof and detection method.
Background technology
Trilute (T3, T3) to be a kind of molecular weight be 651 iodotyrosine, the same with thyroxine (T3) is a kind of important thyroid hormone.T3, T3 all have in the circulating cycle in conjunction with state and free state two kinds of forms, remain mobile equilibrium under normal circumstances between two kinds of forms.
The mensuration of T3 is disorderly very meaningful with monitoring Hypothyroidism to diagnosis thyroid function.To hyperthyroid patient, total T3 of Most patients is consistent with the level of total T3.But in some case, hyperthyroidism is only because the generation of T3 increases (the T3 type hyperthyroidism disease) that cause, therefore at FT3(serum free thyroxine) normal hyperthyroid patient, suggestion detects T3, especially for FT3 and TSH(thyrotropic hormone) all normal patient's (general patient Chang Shouxian detects FT3 and TSH), more should detect T3 further.
Total T3 level of severe hypothyroidism patient is usually on the low side and the total T3 level of moderate patients with hypothyroidism may be normal.The serious patient of non-thyroid disorders or after surgery, the level of visible T3 level T3 on the low side and anti-raises.The T3 level of newborn and baby is general higher, but T3 level also can be on the low side in some gerontal case.
The detection method of current T3 all adopts immunological method at present.Clinical immunization detection technique is the technology utilizing antigen-antibody reaction principle to detect biological substance in vivo, the introducing of this technology makes clinical chemistry test there occurs revolutionary change: test item constantly increases, the sensitivity of method is higher, specificity is stronger, and automaticity is higher.After the eighties in last century, immunology detection technology develops rapidly along with the maturation of monoclonal antibody, artificial synthetic polypeptide, gene engineering expression antigen and various labelling technique, radiommunoassay (RIA) and enzyme immunoassay (EIA) (EIA) replace traditional immunoprecipitation and immunity cohesion gradually, make the susceptibility of detection, specificity improves all greatly, the application of chemiluminescence subsequently makes the susceptibility of immunology detection and the range of linearity be further improved.The quantitative detection that be applied as disease of these technology in clinical examination, observation of curative effect and CORD PARALYSIS provide more direct and objective data.
T3 is the routine immunization test item that Hospitals at Present is carried out, and has the important references that can not be substituted to be worth to the diagnosis of thyroid disease.The T3 immunologic function test reagent of current China approved listing has import and domestic two large classes, wherein import reagent has the product of the transnational quantitative detection companies such as Beckman, the methodology adopted mostly is chemiluminescence, and the methodology adopted in domestic reagent is most for putting the method for exempting from, nearly 2 years start to occur chemiluminescence series products.
Radioimmunoassay for detecting exist the range of linearity narrow, not easily realize the methodology limiting factors such as full-automatic.And chemiluminescence immunoassay have highly sensitive, detect linear wide ranges, easy and simple to handle, the advantages such as automaticity is high.Current chemiluminescence immunoassay technology has above-mentioned to be manyly widely used a little because of it.
But, in the immune detection of reality, because impurity component contained in testing sample is more, have impact on detection sensitivity and accuracy to a certain extent, so from the sample substrate of complexity quick separating, be purified into object determinand, be one of difficult problem of facing of clinical examination worker.
Magnetic particle immunoassay technology utilizes the Magnetic solid phases particulate of synthesis of polymer material certain particle size size to make carrier, with the method such as physisorption, chemical coupling bag, above be there is the various immunologic active materials such as the antibody of specificity affinity or antigen, have that velocity of separation is fast, efficiency is high, favorable repeatability, simple to operate, the feature such as biological character and function that do not affect separated cell or other biological material, orientable motion under additional magnetic fields, makes some special composition be separated, concentrates or purifying.
The open C N101949944A of Chinese invention patent has disclosed triiodothyronine quantitative detection kit and preparation method thereof a kind of; wherein, kit comprises the magnetic particle suspension, trilute series of calibration product, the trilute antibody of horseradish peroxidase-labeled, luminous substrate A liquid, luminous substrate B liquid and the concentrated washing lotion that are coated with T2-gelatin.Use kit disclosed in this patent successfully to make use of magnetic particle immunoassay technology and achieve accurate detection to trilute.But, preparation cost and the use cost of this kit are high, reason is, on the one hand, when carrying out the preparation of the magnetic particle suspension being coated with T2-gelatin, not only process is complicated, and the coating rate be directly coated on magnetic particle by T2-gelatin is lower, causes higher cost; On the other hand, it takes the trilute antibody of horseradish peroxidase-labeled, and the preparation of this antibody is also very loaded down with trivial details, and mark rate is low, limits its Detection results and causes cost to increase.In addition, the factor of not easily control and less stable that kit is existing in preparation technology, except the problem increased, also making the difference between batch of detection large, limiting the precision of detection method except causing foregoing cost.
Summary of the invention
Technical matters to be solved by this invention overcomes the deficiencies in the prior art; a kind of nano magnetic particulate chemistry luminescent assay kit of trilute is provided; it can prepare with lower cost, and can realize trilute accurately and the quantitative measurement of high precision ground.
The present invention also provides a kind of preparation method of nano magnetic particulate chemistry luminescent assay kit of trilute simultaneously, the method process stabilizing, and cost is low, and the precision of gained kit is high.
The preparation method of the easy and low cost of the kit that cancer antigen is used.
A nano magnetic particulate chemistry luminescent assay kit for trilute, it is characterized in that, this kit comprises:
First reagent: containing the solution of fluorescein-labeled trilute antibody;
Second reagent: containing the solution of the trilute antigen that alkaline phosphatase (ALP) marks;
Magneto separate agent: containing bag by the suspending liquid of the magnetic particle of anti-fluorescein antibody.
Preferably, the trilute antigen of this alkali phosphatase enzyme mark is connected and composed by crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and trilute antigen.
Further, in described magnetic particle reagent, the magnetic particle being coated with anti-fluorescein antibody by anti-fluorescein antibody and magnetic particle by the coupling of coupling agent phase chemistry.
Further, the concentration of the fluorescein-labeled trilute antibody in described first reagent is 0.5 ~ 1 μ g/mL, and the pH of described first reagent is 7-9; The concentration of the trilute antigen of the alkali phosphatase enzyme mark in described second reagent is 0.02 ~ 0.1 μ g/mL, and the pH of described second reagent is 7-9.
Those skilled in the art should know, kit of the present invention can further include other detect needed for reagent, such as substrate solution.But such as other reagent such as substrate solution can be bought separately or prepare, therefore, although can comprise these reagent in kit, they are for not essential 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 luminescent assay kit of above-mentioned trilute; it comprises the step preparing described first reagent, described second reagent and Magneto separate agent respectively, wherein: the preparation process of described second reagent is as follows:
1. make trilute antigen and crosslinking chemical disuccinimidyl suberate in dimethyl sulfoxide solvent, at room temperature react, make the connector of generation trilute antigen and disuccinimidyl suberate, save backup at 2 ~ 8 DEG C, wherein: described trilute antigen, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase exceedes
1000u/mg；
2. by the damping fluid containing alkaline phosphatase and step 1. gained solution mix according to the ratio that alkaline phosphatase and trilute antigen and the mol ratio of the connector of disuccinimidyl suberate are 1:1.1 ~ 1.3, at room temperature react, make the trilute antigen of the described alkali phosphatase enzyme mark of generation, reaction terminates, by reactant liquor by G-25 gel column desalination, select the damping fluid with proper pH value adjust concentration and pH value and get final product, the concentration of the damping fluid of its alkaline phosphatase is 0.5 ~ 1.5mg/ml.
Preferably, step 1. in, get trilute antigen, adding this antigen of dmso solution is 20 ~ 50mg/mL to concentration, adds crosslinking chemical disuccinimidyl suberate, room temperature reaction 2 ~ 3 hours, with dimethyl sulfoxide (DMSO), reactant liquor 1:10 is diluted, save backup at 2 ~ 8 DEG C.
Preferably, step 2. in, get the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/ml, be diluted to 0.5 ~ 1.5mg/ml with the sodium bicarbonate buffer liquid of pH9 ~ 10, add step 1. gained reactant liquor, room temperature leaves standstill reaction 20 ~ 60min.
Further, the preparation method of the first described reagent is as follows: the pH of preparation containing fluorescein is the damping fluid of 9 ~ 10, then be the ratio of 20 ~ 200:1 according to the molecular proportion of fluorescein and trilute antibody, the damping fluid be the damping fluid of 9 ~ 10 by the described pH containing fluorescein being 9 ~ 10 with the pH of trilute antibody mixes, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, the fluorescein that removing is free, obtain the solution containing fluorescein-labeled trilute antibody, then with damping fluid adjustment concentration and the pH with proper pH value, obtain the first described reagent.Wherein the damping fluid of proper pH value can be the TRIS damping fluid such as containing 0.5% bovine serum albumin(BSA), pH8.0.
Further, the preparation method of described Magneto separate agent is as follows: by the magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain described Magneto separate agent.Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μm, 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.
Preferably, the above-mentioned damping fluid with proper pH value is the TRIS damping fluid containing 0.5% bovine serum albumin(BSA), pH8.0.
Fluorescein of the present invention can be known various fluoresceins, and conventional has such as fluorescein isothiocynate, RB 200, TRITC etc.
The present invention additionally provides a kind of detection method adopting above-mentioned kit to be applied to triiodothyronine quantitative detection simultaneously, it is characterized in that, comprises the following steps:
(1) immune response: add sample to be tested stoste in detector tube, adds the first reagent and the second reagent successively, and mixing, carries out first time incubation, then add Magneto separate reagent at 25 ~ 40 DEG C, and mixing, carries out second time incubation at 25 ~ 40 DEG C;
(2) wash: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, then Magneto separate, removes supernatant;
(3) add substrate solution and detect luminous intensity: the chemical luminous substrate adding alkaline phosphatase enzymatic in detector tube, remove magnetic field, after abundant suspendible, detect luminous intensity values.
Further, the time of incubation first time described in step (1) can be 5 ~ 30mi n, is generally 15min; The time of second time incubation can be 2 ~ 10min, is generally 5min.
Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:
1. applicant finds, when taking disuccinimidyl suberate to carry out the coupling of alkaline phosphatase and trilute antigen as crosslinking chemical, have and compare higher coupling efficiency with other crosslinking chemical, while reduction preparation cost, be conducive to improving Detection results.Therefore, three kinds of reagent in kit of the present invention all can be prepared by stable preparation technology, and production cost is low, and due to the stability of preparation technology, kit assay difference between batch is little, and between the analysis of detection, precision improves.
2. the preparation method of the solution of the trilute antigen containing alkali phosphatase enzyme mark in kit of the present invention, can effectively by trilute antigen and alkaline phosphatase coupling, coupling efficiency is high, reduces the low Detection results with guaranteeing kit of cost of kit further.
3, take kit of the present invention to detect, accuracy is good, and precision is high, highly sensitive, and sensing range is wide, and sample, without the need to pre-dilution, simple to operately to save time.Compared with the method adopting import reagent box to carry out detecting, 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 sensitivity evaluation matched curve;
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 ng/mL, and ordinate y is Abbott's kit sample measured value, and concentration unit is ng/mL).
Embodiment
The preparation of embodiment 1 first reagent
(1) material and instrument: trilute (T3) monoclonal antibody (more than 95wt%, concentration is 2mg/mL to purity) of preserving with phosphate buffer; Fluorescein isothiocynate (FITC), the reagent such as sodium carbonate should reach chemical pure; The buying of G-25 gel-purified post is from GE company.
(2) preparation process:
1. the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1 ~ 0.2mol/L pH9.0 ~ 10.0 is used;
2. the ratio being 1:20 according to trilute monoclonal antibody and FITC molecular proportion adds step and is 1. joined FITC solution in antibody-solutions, and mix, room temperature leaves standstill 12h hour, and reaction generates T3 antibody-FITC connector;
3. be separated through step reactant liquor 2. by G-25 gel column, remove unreacted FITC, obtain the solution containing T3 antibody-FITC connector (i.e. the T3 antibody of FITC mark);
4. the step solution that 3. gained contains T3 antibody-FITC connector being diluted to T3 antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0 is 0.5 ~ 1 μ g/mL, is the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: T3 antigen (pressed powder, purity is more than 95wt%); With the alkaline phosphatase (ALP solution, ALP purity is about 99%, and specific activity is about 1500U/mg, and concentration is 10mg/mL) that phosphate buffer is preserved; Cross liner DS S is purchased from THERMO company, and the chemical reagent such as TRIS should reach chemical pure; G-25 gel-purified post is GE Products.
(2) preparation process:
1. get 1mg T3 antigen, adding DMSO, to dissolve this antigen to concentration be 20 ~ 50mg/mL, and add DSS0.5mg, room temperature reaction 2 hours, diluted by reactant liquor 1:10 with DMSO, 2-8 DEG C saves backup;
2. the ALP solution of 1mg is got, with the NaHCO of 0.1M pH9.5
3damping fluid by ALP solution dilution to 1mg/ml, add the T3-DMSO solution that 1. step prepared in ALP damping fluid after dilution and carry out coupled reaction, add that T3-DMSO liquor capacity is ALP damping fluid volume 1/20, room temperature leaves standstill reaction 30min, with G-25 gel column desalination, 2-8 DEG C saves backup;
3. by step solution 2. with containing 0.5% bovine serum albumin(BSA) BSA) the TRIS damping fluid of the 0.1mol/L of pH8.0 is diluted to 0.02 ~ 0.1 μ g/ml and pH9 ~ 10, is the second reagent.
The preparation of embodiment 3 Magneto separate reagent
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle is containing carboxyl (COOH) active group, and every gram (g) magnetic particle (dry weight) carboxyl-content is not less than 0.4 mM (mmol), has superparamagnetism, and diameter is between 0.5-2 μm.
Anti-FITC antibody: can be polyclonal antibody also can be monoclonal antibody, and purity is more than 90wt%, and dilution is tired more than 1:100 ten thousand;
MES (MES), carbodiimide (EDC), TRI S and other reagent should reach chemical pure.
(2) preparation process:
1. get the suspending liquid of 100mg magnetic particle, Magneto separate removes supernatant, and with 0.05mol/L, pH4.5 ~ 5MES damping fluid, 10mL is resuspended;
2. the anti-FITC antibody of 2 ~ 4mg is added, room temperature suspendible 30 ~ 60min;
3. the EDC aqueous solution of the freshly prepared 10mg/mL of 0.5 ~ 1mL is added, room temperature suspendible 2 ~ 12h;
4. Magneto separate, removes supernatant, is resuspended to 1mg/mL, pH8.0, is Magneto separate reagent with the TRIS damping fluid of the 0.1mol/L containing 0.5% bovine serum albumin(BSA) (BSA) pH8.0.
The nano magnetic particulate chemistry luminescent assay kit of embodiment 4 trilute
This kit comprises:
The first reagent (concentration is 0.75 μ g/mL) prepared according to embodiment 1 method, 5mL;
The second reagent (concentration is 0.06 μ g/mL) prepared according to embodiment 2 method, 5mL;
The Magneto separate reagent 5mL prepared according to embodiment 3 method.
The nano magnetic particulate chemistry luminescent assay kit of embodiment 5 trilute
This kit comprises:
The first reagent (concentration is 0.5 μ g/mL) prepared according to embodiment 1 method, 5mL;
The second reagent (concentration is 0.02 μ g/mL) prepared according to embodiment 2 method, 5mL;
The Magneto separate reagent 5mL prepared according to embodiment 3 method.
The nano magnetic particulate chemistry luminescent assay kit of embodiment 6 trilute
This kit comprises:
The first reagent (concentration is 1 μ g/mL) prepared according to embodiment 1 method, 5mL;
The second reagent (concentration is 0.1 μ g/mL) prepared according to embodiment 2 method, 5mL;
The Magneto separate reagent 5mL prepared according to embodiment 3 method.
Embodiment 7 takes the kit of embodiment 4 to carry out the quantitative detection of trilute
(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 first reagent, 50 μ L second reagent, mixing, incubation 15min under 37 ± 1 DEG C of conditions; Add 50 μ L Magneto separate reagent, mixing, incubation 5min under 37 ± 1 DEG C of conditions;
2. wash: 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, then Magneto separate, 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 substrates solution (Beijing Apis Biotechnology Co., Ltd. APCL-I), concussion makes the abundant suspendible of magnetic particle, in 5min, detect luminous intensity.
(2) calibration object typical curve is drawn
Calibration object typical curve is see 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, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and draw linear function, M-2SD value is brought in above-mentioned equation, obtains corresponding concentration value, be lowest detectable limit.The sensitivity of this method is not more than 5ng/mL.Wherein: A point luminous value is respectively see table 1:
Table 1
The luminous average X=114657 of A point
SD=5273
X-2SD=104111
B point luminous value is respectively see table 2.
The luminous average X=64179 of B point
Table 2
| T3-STD-B(RLU) |
| 68001 |
| 60357 |
A, B point connects some matched curve see Fig. 2.Sensitivity=0.104ng/mL.
(4) precision evaluation
1. precision in analyzing
By a collection of for the kit of embodiment 4, measure the serum of basic, normal, high three kinds of variable concentrations respectively, 10 hole replicate determinations, result, see table 3, show that variation within batch coefficient is 3.88% ~ 6.24%.
The test of interior precision analyzed by table 3
| Measure serum-concentration (ng/mL) | Measure number of times | CV (%) in analyzing |
| 0.875 | 10 | 6.24 |
| 3.432 | 10 | 4.50 |
| 6.546 | 10 | 3.88 |
2. precision between analyzing
The kit of embodiment 4 is got three batches, often criticizes the serum that kit all measures basic, normal, high three kinds of variable concentrations, 10 hole replicate determinations.Every part of serum obtains 30 concentration measured values, and see table 4, the coefficient of variation between statistical study is 6.04% ~ 7.22%.
Precision test between table 4 analysis
| Measure serum-concentration (ng/mL) | Measure number of times | CV (%) between analysis |
| 0.875 | 30 | 7.22 |
| 3.432 | 30 | 6.65 |
| 6.546 | 30 | 6.04 |
(5) accuracy estimating
In 2 routine pooled serum samples, add different amount T3 standard items, the serum forming 3 concentration levels adds sample, and additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by the following formulae discovery recovery.This method serum matrix recovery is between 90-110%.Data are see table 5.
R: the recovery;
V: the volume adding standard solution;
V
0: the volume of people source sample;
C: people source sample adds the detectable concentration after standard solution;
C
0: 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
Choose the test variable concentrations thyroid hormone (T4), reverse triiodothyronine (rT3) and diiodothyronine (T2) sample that there are similar structures with trilute to kit specific assay, be mixed with the sample being greater than physiological concentration, measure with this method.The results are shown in Table 6, the equal no cross reaction of cross reacting rate of this law and T4, rT3, T2.
Table 6 specificity experiments
(7) relativity evaluation
With the chemical luminescence reagent kit of kit and Abbott, 100 parts of human serum samples are detected simultaneously.Its testing result is see accompanying drawing 3, and serum T 3 concentration of survey is in the process of the present invention horizontal ordinate, and with the result of Abbott's kit measurement for ordinate does regretional analysis, dependent equation is: y=-0.02689+1.0434x, and related coefficient is: 0.9689.Show through statistical procedures result, this method is good with external kit clinical sample measured value correlativity.
(8) Evaluation of Thermal Stability
4 DEG C of 12 months and 37 DEG C of stability experiments of 7 days are carried out respectively to kit, result show kit standard product luminous intensity change, batch in and the index such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalences done according to Spirit Essence of the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (2)
1. a nano magnetic particulate chemistry luminescent assay kit for trilute, described kit comprises:
First reagent: containing the solution of fluorescein-labeled trilute antibody;
Second reagent: containing the solution of the trilute antigen of alkali phosphatase enzyme mark;
Magneto separate agent: containing bag by the suspending liquid of the magnetic particle of anti-fluorescein antibody;
The trilute antigen of described alkali phosphatase enzyme mark is connected and composed by crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and trilute antigen;
The concentration of the fluorescein-labeled trilute antibody in described first reagent is 0.5 ~ 1 μ g/mL, and the pH of described first reagent is 7-9; The concentration of the trilute antigen of the alkali phosphatase enzyme mark in described second reagent is 0.02 ~ 0.1 μ g/mL, and the pH of described second reagent is 7-9;
The nano magnetic particulate chemistry luminescent assay kit of described trilute is prepared by following preparation method; described preparation method comprise the described solution containing fluorescein-labeled trilute antibody of preparation respectively, the described trilute antigen containing alkali phosphatase enzyme mark solution and described in be coated with the step of the suspending liquid of the magnetic particle of anti-fluorescein antibody, it is characterized in that: the preparation process of the solution of the described trilute antigen containing alkali phosphatase enzyme mark is as follows:
1. trilute antigen is got, 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, save backup at 2 ~ 8 DEG C, wherein: described trilute antigen, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase is more than 1000u/mg;
2. the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/ml is got, 0.5 ~ 1.5mg/ml is diluted to the sodium bicarbonate buffer liquid of pH 9 ~ 10, add step 1. gained reactant liquor, room temperature leaves standstill reaction 20 ~ 60min, make the trilute antigen of the described alkali phosphatase enzyme mark of generation, reaction terminates, by reactant liquor by G-25 gel column desalination, select the damping fluid with proper pH value adjust concentration and pH value and get final product, the concentration of the damping fluid of its alkaline phosphatase is 0.5 ~ 1.5mg/ml;
The preparation method of described first reagent is as follows: the pH of preparation containing fluorescein is the damping fluid of 7 ~ 9, then be the ratio of 20 ~ 200:1 according to the molecular proportion of fluorescein and trilute antibody, the damping fluid be the damping fluid of 7 ~ 9 by the described pH containing fluorescein being 7 ~ 9 with the pH of trilute antibody mixes, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, the fluorescein of removing, obtain the solution containing fluorescein-labeled trilute antibody, then with damping fluid adjustment concentration and the pH with proper pH value, obtain the first reagent,
The preparation method of described Magneto separate agent is as follows: make magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain described Magneto separate agent; Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μm, on every gram of magnetic particle with the content of carboxyl-reactive group be more than or equal to 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 containing 0.5% bovine serum albumin(BSA), pH8.0.
2. the preparation method of the nano magnetic particulate chemistry luminescent assay kit of a trilute as claimed in claim 1; it comprise the described solution containing fluorescein-labeled trilute antibody of preparation respectively, the described trilute antigen containing alkali phosphatase enzyme mark solution and described in be coated with the step of the suspending liquid of the magnetic particle of anti-fluorescein antibody, it is characterized in that: the preparation process of the solution of the described trilute antigen containing alkali phosphatase enzyme mark is as follows:
1. trilute antigen is got, 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, save backup at 2 ~ 8 DEG C, wherein: described trilute antigen, the purity of described alkaline phosphatase are all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase is more than 1000u/mg;
2. the phosphate buffer that concentration is more than or equal to the alkaline phosphatase of 5mg/ml is got, 0.5 ~ 1.5mg/ml is diluted to the sodium bicarbonate buffer liquid of pH 9 ~ 10, add step 1. gained reactant liquor, room temperature leaves standstill reaction 20 ~ 60min, make the trilute antigen of the described alkali phosphatase enzyme mark of generation, reaction terminates, by reactant liquor by G-25 gel column desalination, select the damping fluid with proper pH value adjust concentration and pH value and get final product, the concentration of the damping fluid of its alkaline phosphatase is 0.5 ~ 1.5mg/ml;
The preparation method of described first reagent is as follows: the pH of preparation containing fluorescein is the damping fluid of 7 ~ 9, then be the ratio of 20 ~ 200:1 according to the molecular proportion of fluorescein and trilute antibody, the damping fluid be the damping fluid of 7 ~ 9 by the described pH containing fluorescein being 7 ~ 9 with the pH of trilute antibody mixes, after mixing, room temperature leaves standstill reaction, then reactant liquor is separated by G-25 gel column, the fluorescein of removing, obtain the solution containing fluorescein-labeled trilute antibody, then with damping fluid adjustment concentration and the pH with proper pH value, obtain the first reagent,
The preparation method of described Magneto separate agent is as follows: make magnetic particle containing carboxyl-reactive group and anti-fluorescein antibody under the existence of coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction terminates, Magneto separate, remove supernatant, by damping fluid adjustment pH and the concentration with proper pH value, obtain described Magneto separate agent; Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μm, on every gram of magnetic particle with the content of carboxyl-reactive group be more than or equal to 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 containing 0.5% bovine serum albumin(BSA), pH8.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210550222.1A CN103048477B (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210550222.1A CN103048477B (en) | 2012-12-18 | 2012-12-18 | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103048477A CN103048477A (en) | 2013-04-17 |
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| CN103217414B (en) * | 2013-04-27 | 2015-02-11 | 江苏福隆生物技术有限公司 | Chemiluminescent immunoassay method for 3,5,3'-triiodothyronine in blood serum |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
| CN104730257A (en) * | 2015-02-10 | 2015-06-24 | 深圳市新产业生物医学工程股份有限公司 | RT3 chemiluminiscence immunodetection kit as well as detection method and application thereof |
| CN108776218A (en) * | 2018-05-31 | 2018-11-09 | 湖南远璟生物技术有限公司 | A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| 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 |
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| CN101545913A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles |
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| EP2600973B1 (en) * | 2010-08-05 | 2016-05-25 | Abbott Point Of Care, Inc. | Immunoassay method and device with magnetically susceptible bead capture |
| US9329175B2 (en) * | 2010-08-05 | 2016-05-03 | Abbott Point Of Care Inc. | Oscillating immunoassay method and device |
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| CN1580281A (en) * | 2003-08-06 | 2005-02-16 | 宋克 | Gene chip enzyme-method microsphere amplifying label |
| CN101545913A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles |
| CN102426255A (en) * | 2011-08-31 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | Quantitative detection kit and detection method of free tetraiodothyronine (FT4) |
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