CN103048477A - 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 of nano magnetic particulate chemistry luminescence assays kit and preparation method thereof and detection method of the trilute (T3) that combines immune magnetic particle isolation technics and chemiluminescence immunoassay technology.
Background technology
(T3 is that a kind of molecular weight is 651 iodotyrosine T3) to trilute, and the same with thyroxine (T3) is a kind of important thyroid hormone.T3, T3 all have in circulation in conjunction with attitude and two kinds of forms of free state, are keeping mobile equilibrium between two kinds of forms under normal circumstances.
The mensuration of T3 is disorderly very meaningful with the monitoring Hypothyroidism to the diagnosis thyroid function.To hyperthyroid patient, total T3 of Most patients is consistent with the level of total T3.But in some case, hyperthyroidism only is because the generation of T3 increases (the T3 type hyperthyroidism disease) that causes, therefore at the FT3(serum free thyroxine) normal hyperthyroid patient, suggestion detects T3, especially for FT3 and TSH(thyrotropic hormone) equal normal patients (general patient Chang Shouxian detects FT3 and TSH), more should further detect T3.
Total T3 level of the serious hypothyroidism patient usually total T3 level of on the low side and moderate hypothyroidism patient may be normal.The serious patient of Non-thyrogenous disease 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 generally higher, but the T3 level also can be on the low side in some gerontal case.
The detection method of T3 all adopts immunological method at present at present.Clinical immunoassay technology is the technology of utilizing antigen-antibody reaction principle detection of biological substance in vivo, the introducing of this technology makes clinical chemistry test that revolutionary variation occur: test item constantly increases, the sensitivity of method is higher, specificity is stronger, and automaticity is higher.After the eighties in last century, the immunology detection technology develops rapidly along with the maturation of monoclonal antibody, artificial synthetic polypeptide, gene engineering expression antigen and various labelling techniques, radiommunoassay (RIA) and enzyme immunoassay (EIA) (EIA) replace traditional immunoprecipitation and immunity cohesion gradually, susceptibility, the specificity of detection are all improved greatly, and the subsequently application of chemiluminescence is further improved the susceptibility of immunology detection and the range of linearity.The quantitative detection, observation of curative effect and the CORD PARALYSIS that are applied as disease of these technology in clinical examination provides 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 at present China's approved listing has import and domestic two large classes, wherein import reagent has the product of the transnational quantitative detection such as Beckman company, the methodology that adopts mostly is chemiluminescence, and the methodology that adopts in the domestic reagent is most for putting the method for exempting from, and begins to occur the chemiluminescence series products in nearly 2 years.
There are the methodology limiting factors such as narrow, the difficult realization full-automation of the range of linearity in radioimmunoassay for detecting.And that chemiluminescence immunoassay has is highly sensitive, detect the advantages such as linear wide ranges, easy and simple to handle, automaticity height.At present chemiluminescence immunoassay technology has above-mentioned manyly be widely used a little because of it.
Yet, in the immune detection of reality, owing to impurity component contained in the testing sample is more, detection sensitivity and accuracy have been affected to a certain extent, so from the sample substrate of complexity, separate fast, be purified into the purpose determinand, it is one of difficult problem of facing of clinical examination worker.
The magnetic particle immunoassay technology is to utilize the magnetic solid phase particle of synthesis of polymer material certain particle size size to make carrier, have the various immunologic active materials such as the antibody of specificity affinity or antigen on coated with methods such as physisorption, chemical couplings, have that velocity of separation is fast, efficient is high, the characteristics such as favorable repeatability, simple to operate, the biological character that do not affect separated cell or other biological material and function, adding orientable motion under the magnetic fields, so that some special composition is separated, concentrated or purifying.
The open C N101949944A of Chinese invention patent discloses triiodothyronine quantitative detection kit and preparation method thereof a kind of; wherein, kit comprises that the magnetic particle suspension, trilute series calibration object, the trilute antibody of horseradish peroxidase-labeled, luminous substrate A liquid, the luminous substrate B liquid that are coated with T2-gelatin reach concentrated washing lotion.Use the kit of this patent disclosure successfully to utilize the magnetic particle immunoassay technology to realize accurate detection to trilute.Yet, the preparation cost of this kit and use cost are high, reason is, on the one hand, when the preparation of the magnetic particle suspension that is coated with T2-gelatin, not only process is complicated, and the coating rate that directly T2-gelatin is coated on the magnetic particle is lower, causes higher cost; On the other hand, it takes the trilute antibody of horseradish peroxidase-labeled, and the preparation of this antibody also is very loaded down with trivial details, and mark rate is low, limits it and detects effect and cause cost to increase.In addition, the factor of kit existing difficult control and less stable on preparation technology except causing problem that foregoing cost increases, also so that the difference between batch that detects is large, has limited the precision of detection method.
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 trilute is provided; it can prepare with lower cost, and can realize the accurate and high precision ground quantitative measurement of trilute.
The present invention also provides a kind of preparation method of nano magnetic particulate chemistry luminescence assays kit of trilute simultaneously, the method process stabilizing, and cost is low, and the precision of gained kit is high.
The kit that cancer antigen is used easy and preparation method cheaply.
A kind of nano magnetic particulate chemistry luminescence assays kit of trilute is characterized in that this kit comprises:
The first reagent: the solution that contains fluorescein-labeled trilute antibody;
The second reagent: the solution that contains the trilute antigen of alkaline phosphatase (ALP) mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle of coated fluorescein antibody.
Preferably, the trilute antigen of this alkali phosphatase enzyme mark is connected and composed by the crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and trilute antigen.
Further, in the described magnetic particle reagent, the magnetic particle that is coated with fluorescein antibody passes through the coupling of coupling agent phase chemistry by fluorescein antibody and magnetic particle.
Further, the concentration of the fluorescein-labeled trilute antibody in described the first reagent is 0.5 ~ 1 μ g/mL, and the pH of described the first reagent is 7-9; The concentration of the trilute antigen of the alkali phosphatase enzyme mark in described the second reagent is 0.02 ~ 0.1 μ g/mL, and the pH of described the second reagent is 7-9.
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 can buy separately or prepare such as other reagent such as substrate solutions, therefore, although can comprise these reagent in the 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 trilute; it comprises the step for preparing respectively described the first reagent, described the second reagent and magnetic separating agent, and wherein: the preparation process of described the second reagent is as follows:
1. make trilute antigen and crosslinking chemical disuccinimidyl suberate in dimethyl sulfoxide solvent, at room temperature reaction, make the connector that generates trilute antigen and disuccinimidyl suberate, under 2 ~ 8 ℃, save backup, wherein: the purity of described trilute antigen, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses
1000u/mg；
The damping fluid that 2. will contain alkaline phosphatase and step 1. gained solution are that the ratio of 1:1.1～1.3 is mixed according to alkaline phosphatase and trilute antigen and the mol ratio of the connector of disuccinimidyl suberate, at room temperature reaction, make the trilute antigen that generates described alkali phosphatase enzyme mark, reaction finishes, reactant liquor is passed through G-25 gel column desalination, selection has damping fluid adjustment concentration and the pH value of proper pH value and get final product, and 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 to concentration is 20 ~ 50mg/mL, adds the crosslinking chemical disuccinimidyl suberate, room temperature reaction 2 ~ 3 hours,, save backup under 2 ~ 8 ℃ reactant liquor 1:10 dilution with dimethyl sulfoxide (DMSO).
Preferably, step 2. in, get concentration more than or equal to the phosphate buffer of the alkaline phosphatase of 5mg/ml, be diluted to 0.5 ~ 1.5mg/ml with the sodium bicarbonate buffer liquid of pH9 ~ 10, add 1. gained reactant liquor of step, room temperature leaves standstill reaction 20 ~ 60min.
Further, the preparation method of described the first reagent is as follows: the pH that preparation contains fluorescein is 9 ~ 10 damping fluid, then the molecular proportion according to fluorescein and trilute antibody is the ratio of 20 ~ 200:1, be 9 ~ 10 damping fluid with the described pH that contains fluorescein with the pH of trilute antibody be that 9 ~ 10 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, remove free fluorescein, obtain containing the solution of fluorescein-labeled trilute antibody, then adjust concentration and pH with the damping fluid with proper pH value, namely get described the first reagent.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 described magnetic separating agent is as follows: will contain the magnetic particle of carboxyl reactive group and fluorescein antibody in the presence of the coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, namely get described magnetic separating agent.Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram magnetic particle with the content of carboxyl reactive group be not less than 0.4mmol; Described fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired greater than 1:100 ten thousand.
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 commonly used have for example fluorescein isothiocynate, RB 200, a TRITC etc.
The present invention also provides simultaneously a kind of and has adopted above-mentioned kit to be applied to the detection method that triiodothyronine quantitative detects, and it is characterized in that, may further comprise the steps:
(1) immune response: add sample to be tested stoste in detector tube, add successively the first reagent and the second reagent, mixing carries out the incubation first time under 25 ~ 40 ℃, then add the magnetic separation agent, and mixing carries out the incubation second time under 25 ~ 40 ℃;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant;
(3) add substrate solution and detect luminous intensity: in detector tube, add the chemical luminous substrate of alkaline phosphatase enzymatic, remove magnetic field, fully detect luminous intensity values behind the suspendible.
Further, the time of incubation can for 5～30mi n, be generally 15min for the first time described in the step (1); The time of incubation can be 2 ~ 10min for the second time, is generally 5min.
Because the enforcement of above technical scheme, the present invention compared with prior art has following advantage:
1. the applicant finds, when taking disuccinimidyl suberate to carry out the coupling of alkaline phosphatase and trilute antigen as crosslinking chemical, have with other crosslinking chemical and compare higher coupling efficiency, when reducing preparation cost, be conducive to improve the detection effect.Therefore, three kinds of reagent in the kit of the present invention all can prepare by stable preparation technology, and production cost is low, and because preparation technology's stability, it is little that kit is analyzed difference between batch, and precision improves between the analysis of detection.
2. the preparation method of the solution of the trilute antigen that contains alkali phosphatase enzyme mark in the kit of the present invention, can be effectively with trilute antigen and alkaline phosphatase coupling, coupling efficiency is high, and it is low and guarantee the detection effect of kit further to reduce the cost of kit.
3, take kit of the present invention to detect, accuracy is good, and precision is high, and highly sensitive, sensing range is wide, and sample need not pre-dilution, simple to operate saving time.Compare with the method that adopts the import reagent box to detect, detection method of the present invention has significant advantage at cost.
Description of drawings
Fig. 1 is detection calibration product typical curves;
Fig. 2 is that matched curve is estimated 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 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: with trilute (T3) monoclonal antibody (purity surpasses 95wt%, and concentration is 2mg/mL) of phosphate buffer preservation; 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. use the FITC solution of the carbonate buffer solution preparation 0.5mg/mL of 0.1～0.2mol/L pH9.0 ~ 10.0;
2. add step according to trilute monoclonal antibody and FITC molecular proportion by the ratio of 1:20 in antibody-solutions and 1. joined FITC solution, mix, room temperature left standstill 12h hour, and reaction generates T3 antibody-FITC connector;
3. will separate by the G-25 gel column through step reactant liquor 2., remove unreacted FITC, obtain containing the solution of T3 antibody-FITC connector (being the T3 antibody of FITC mark);
4. with step 3. the gained solution that contains T3 antibody-FITC connector to be diluted to T3 antibody-FITC connector concentration with the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 be 0.5～1 μ g/mL, be the first reagent.
The preparation of embodiment 2 second reagent
(1) material and instrument: T3 antigen (pressed powder, purity surpasses 95wt%); The alkaline phosphatase of preserving with phosphate buffer (ALP solution, ALP purity are about 99%, and specific activity is about 1500U/mg, and concentration is 10mg/mL); Cross liner DS S is available from THERMO company, and the chemical reagent such as TRIS should reach chemical pure; G-25 gel-purified post is GE company product.
(2) preparation process:
1. get 1mg T3 antigen, it is 20 ~ 50mg/mL that adding DMSO dissolves this antigen to concentration, adds DSS0.5mg, room temperature reaction 2 hours, and with reactant liquor 1:10 dilution, 2-8 ℃ saves backup with DMSO;
2. get the ALP solution of 1mg, with the NaHCO of 0.1M pH9.5
3Damping fluid with the ALP solution dilution to 1mg/ml, the T3-DMSO solution that 1. the adding step prepares in the ALP damping fluid after the dilution carries out coupled reaction, and adding the T3-DMSO liquor capacity is 1/20 of ALP damping fluid volume, and room temperature leaves standstill reaction 30min, with G-25 gel column desalination, 2-8 ℃ saves backup;
3. with 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 magnetic separation agents
(1) material and instrument:
The suspending liquid of magnetic particle: magnetic particle content 5wt%, magnetic particle contain the active group of carboxyl (COOH), 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 μ m.
Anti-FITC mAb: can be polyclonal antibody, also can be monoclonal antibody, and purity is more than the 90wt%, and dilution is tired above 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, magnetic divides the supernatant of leaving away, and uses 0.05mol/L, and 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 separates, and removes supernatant, uses the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH8.0 to be resuspended to 1mg/mL, and pH8.0 is the magnetic separation agent.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 4 trilutes
This kit comprises:
According to first reagent (concentration is 0.75 μ g/mL) of embodiment 1 method preparation, 5mL;
According to second reagent (concentration is 0.06 μ g/mL) of embodiment 2 methods preparation, 5mL;
Magnetic separation agent 5mL according to the preparation of embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 5 trilutes
This kit comprises:
According to first reagent (concentration is 0.5 μ g/mL) of embodiment 1 method preparation, 5mL;
According to second reagent (concentration is 0.02 μ g/mL) of embodiment 2 methods preparation, 5mL;
Magnetic separation agent 5mL according to the preparation of embodiment 3 methods.
The nano magnetic particulate chemistry luminescence assays kit of embodiment 6 trilutes
This kit comprises:
According to first reagent (concentration is 1 μ g/mL) of embodiment 1 method preparation, 5mL;
According to second reagent (concentration is 0.1 μ g/mL) of embodiment 2 methods preparation, 5mL;
Magnetic separation agent 5mL according to the preparation of embodiment 3 methods.
(1) detecting step:
1. immune response: in detector tube, add 30 μ L sample to be tested (serum or blood plasma) stostes, then add 50 μ L the first reagent, 50 μ L the second reagent, mixing, incubation 15min under 37 ± 1 ℃ of conditions; Add 50 μ L magnetic separation agents, mixing, 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 separates, and removes supernatant; This step repeats 3 times;
3. add substrate solution and detect luminous intensity: add 150 μ L alkaline phosphatase chemical luminous substrate solution (Beijing Ah APCL-of this Bioisystech Co., Ltd I) in detector tube, concussion makes the abundant suspendible of magnetic particle, detects luminous intensity in 5min.
(2) draw the calibration object typical curve
The 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 calculating M-2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and the adjacent calibration object and draw linear function, the M-2SD value is brought in the above-mentioned equation, obtain 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 referring to table 1:
Table 1
The luminous average X=114657 of A point
SD=5273
X-2SD=104111
B point luminous value is respectively referring to table 2.
The luminous average X=64179 of B point
Table 2
| T3-STD-B(RLU) |
| 68001 |
| 60357 |
A, B point connect the some matched curve referring to Fig. 2.Sensitivity=0.104ng/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, the result is referring to table 3, and drawing the variation within batch coefficient is 3.88%～6.24%.
Precision test in table 3 is analyzed
| 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, 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 6.04%～7.22%.
Precision test between table 4 is analyzed
| 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
Add different amount T3 standard items in 2 routine pooled serum samples, the serum that forms 3 concentration levels adds sample, and the 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;
V
0: the volume of people source sample;
C: people source sample adds the detectable concentration behind the 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
Check is to choose test variable concentrations thyroid hormone (T4), reverse triiodothyronine (rT3) and diiodothyronine (T2) sample that similar structures is arranged with trilute to the kit specificity, be mixed with the sample greater than physiological concentration, measure with this method.The results are shown in Table 6, the equal no cross reaction of the cross reacting rate of this law and T4, rT3, T2.
The experiment of table 6 specificity
(7) correlativity evaluation
Chemical luminescence reagent kit with kit and Abbott detects simultaneously to 100 parts of human serum samples.Its testing result is referring to accompanying drawing 3, take serum T 3 concentration of the survey of the inventive method as horizontal ordinate, does regretional analysis take the result of Abbott's kit measurement as ordinate, and dependent equation is: y=-0.02689+1.0434x, related coefficient is: 0.9689.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 4 ℃ of 12 months and 37 ℃ of stability experiments of 7 days, the result show kit standard items luminous intensity variation, batch in and the indexs such as betweenrun precision, accuracy all within normal range, the kit term of validity can reach 12 months.
Above-described embodiment only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the personage who is familiar with technique can understand content of the present invention and according to this enforcement; can not limit protection scope of the present invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (10)
1. the nano magnetic particulate chemistry luminescence assays kit of a trilute, described kit comprises:
The first reagent: the solution that contains fluorescein-labeled trilute antibody;
The second reagent: the solution that contains the trilute antigen of alkali phosphatase enzyme mark;
Magnetic separating agent: the suspending liquid that contains the magnetic particle of coated fluorescein antibody.
2. the nano magnetic particulate chemistry luminescence assays kit of trilute according to claim 1, it is characterized in that: the trilute antigen of described alkali phosphatase enzyme mark is connected and composed by the crosslinking chemical disuccinimidyl suberate by alkaline phosphatase and trilute antigen.
3. the nano magnetic particulate chemistry luminescence assays kit of trilute according to claim 1 and 2, it is characterized in that: the concentration of the fluorescein-labeled trilute antibody in described the first reagent is 0.5 ~ 1 μ g/mL, and the pH of described the first reagent is 7-9; The concentration of the trilute antigen of the alkali phosphatase enzyme mark in described the second reagent is 0.02 ~ 0.1 μ g/mL, and the pH of described the second reagent is 7-9.
4. the preparation method of the nano magnetic particulate chemistry luminescence assays kit of a trilute as claimed in claim 3; the step that it comprises the suspending liquid of the solution for preparing respectively the described solution that contains fluorescein-labeled trilute antibody, the described trilute antigen that contains alkali phosphatase enzyme mark and the described magnetic particle that is coated with fluorescein antibody is characterized in that: the preparation process of the solution of the described trilute antigen that contains alkali phosphatase enzyme mark is as follows:
1. make trilute antigen and crosslinking chemical disuccinimidyl suberate in dimethyl sulfoxide solvent, at room temperature reaction, make the connector that generates trilute antigen and disuccinimidyl suberate, under 2 ~ 8 ℃, save backup, wherein: the purity of described trilute antigen, described alkaline phosphatase is all more than or equal to 95wt%, and the specific activity of described alkaline phosphatase surpasses 1000u/mg;
The damping fluid that 2. will contain alkaline phosphatase and step 1. gained solution are that the ratio of 1:1.1 ~ 1.3 is mixed according to alkaline phosphatase and trilute antigen and the mol ratio of the connector of disuccinimidyl suberate, at room temperature reaction, make the trilute antigen that generates described alkali phosphatase enzyme mark, reaction finishes, reactant liquor is passed through G-25 gel column desalination, selection has damping fluid adjustment concentration and the pH value of proper pH value and get final product, and the concentration of the damping fluid of its alkaline phosphatase is 0.5 ~ 1.5mg/ml.
5. preparation method according to claim 4, it is characterized in that: step 1. in, get trilute antigen, adding this antigen of dmso solution to concentration is 20 ~ 50mg/mL, add the crosslinking chemical disuccinimidyl suberate, room temperature reaction 2 ~ 3 hours, saves backup under 2 ~ 8 ℃ reactant liquor 1:10 dilution with dimethyl sulfoxide (DMSO).
6. according to claim 4 or 5 described preparation methods, it is characterized in that: step 2. in, get concentration more than or equal to the phosphate buffer of the alkaline phosphatase of 5mg/ml, sodium bicarbonate buffer liquid with pH 9 ~ 10 is diluted to 0.5 ~ 1.5mg/ml, add 1. gained reactant liquor of step, room temperature leaves standstill reaction 20 ~ 60min.
7. preparation method according to claim 4, it is characterized in that: the preparation method of described the first reagent is as follows: the pH that preparation contains fluorescein is 7 ~ 9 damping fluid, then the molecular proportion according to fluorescein and trilute antibody is the ratio of 20 ~ 200:1, be 7 ~ 9 damping fluid with the described pH that contains fluorescein with the pH of trilute antibody be that 7 ~ 9 damping fluid mixes, behind the mixing, room temperature leaves standstill reaction, then reactant liquor is separated by the G-25 gel column, the fluorescein of removing, obtain containing the solution of fluorescein-labeled trilute antibody, then adjust concentration and pH with the damping fluid with proper pH value, namely get the first reagent.
8. preparation method according to claim 4, it is characterized in that: the preparation method of described magnetic separating agent is as follows: make the magnetic particle that contains the carboxyl reactive group and fluorescein antibody in the presence of the coupling agent carbodiimide, room temperature reaction 2 ~ 18 hours, reaction finishes, magnetic separates, remove supernatant, adjust pH and concentration with the damping fluid with proper pH value, namely get described magnetic separating agent; Wherein said magnetic particle has superparamagnetism, and its diameter is 0.5 ~ 2 μ m, on every gram magnetic particle with the content of carboxyl reactive group more than or equal to 0.4mmol; Described fluorescein antibody is monoclonal antibody or polyclonal antibody, and purity is more than or equal to 90wt%, and dilution is tired greater than 1:100 ten thousand.
9. according to claim 4 or 7 or 8 described preparation methods, it is characterized in that: described damping fluid with proper pH value is the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0.
10. the described kit of each claim is characterized in that for the detection method that triiodothyronine quantitative is detected in the employing claim 1 ~ 3, may further comprise the steps:
(1) immune response: add sample to be tested stoste in detector tube, add successively the first reagent and the second reagent, mixing carries out the incubation first time under 25 ~ 40 ℃, then add the magnetic separation agent, and mixing carries out the incubation second time under 25 ~ 40 ℃;
(2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant;
(3) add substrate solution and detect luminous intensity: in detector tube, add the chemical luminous substrate of alkaline phosphatase enzymatic, remove magnetic field, fully detect luminous intensity values behind the suspendible.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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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 |
| 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 |
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