CN108205059A - A kind of kit and its test method for measuring calcitonin content - Google Patents
A kind of kit and its test method for measuring calcitonin content Download PDFInfo
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- CN108205059A CN108205059A CN201711215380.0A CN201711215380A CN108205059A CN 108205059 A CN108205059 A CN 108205059A CN 201711215380 A CN201711215380 A CN 201711215380A CN 108205059 A CN108205059 A CN 108205059A
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- calcitonin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
The invention discloses a kind of kits that calcitonin (CT) content in human serum is measured using Magnetism particulate immuno chemistry luminescence method.Kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate；The calcitonin coated antibody of its moderate resistance reagent marked by fluorescein isothiocyanate and the calcitonin labelled antibody of alkali phosphatase enzyme mark；Magnetic particle reagent is magnetic particle and goat-anti FITC attachments.Chemiluminescence is combined by the present invention with immune magnetic particle, it provides a kind of close to homogeneous reaction system, and employ one-step method reaction pattern, so that detection sensitivity, accuracy greatly improve, detection range expands, reaction time greatly shortens, from starting to be loaded onto testing result, the time is less than 35min, hence it is evident that is faster than similar kit；And multiple samples can be measured simultaneously on Full-automatic chemiluminescence apparatus, realize the rapid measure of high throughput of calcitonin, accuracy is high, and high specificity, accuracy and detection efficiency are enhanced.
Description
Technical field
The present invention relates to technological field of biochemistry, and in particular to a kind of to be measured in human body using Magnetism particulate immuno chemistry luminescence method
The kit of calcitonin (CT) content.
Background technology
Calcitonin (calcitonin, CT) is a kind of linear type peptide hormone containing 32 amino acid, in human body
It is to be manufactured by thyroid parafollicular cell (parafollicular cells, also known as C cells).In fish, reptile class, bird
This hormone is found with class, mammality.Major function is to reduce blood calcium.But calcitonin is generally for adjusting blood of human body
Middle calcium ion (Ca2+) it is constant there is no significant importance.It is to be secreted by parafollicular cells of thyroid gland.In other words, thyroid gland C
Cell is the endocrine cell for secreting calcitonin.Cell space is larger, rounded, oval or irregular shape, single to be embedded in thyroid gland
On folliculus wall, it is dispersed in close to basement membrane or in groups and filters in interfollicular tissue, also known as parafollicular cell.CT is applied to clinic, mainly sentences
The bone information state of disconnected metabolic bone disease, the effect of detecting and evaluate anti-bone information drug.Calcitonin affects calcium ion and phosphorus
The metabolic process of hydrochlorate, function are the effect of antagonism parathormone mostly.Calcitonin is mainly dropped through following four kinds of modes
Low blood calcium concentration：Inhibit absorption of the small intestine for calcium ion；Inhibit osteoclast (Osteoclast), reduce bone in calcium from
Son is lost in blood；Inhibit reabsorption of the renal tubule to phosphate radical, inhibit reabsorption of the renal tubule to calcium ion,
Increase calcium ion to be lost in from urine.
The calcitonin assay method being currently known has radioimmunology (RIA), enzyme linked immunosorbent assay (ELISA), latex
Enhance turbidimetry etc..Radioimmunology complex steps, reagent price is expensive, need to use mating instrument and there are radioactivity dirts
Dye.There are detection time is long, complicated for operation, poor repeatability, is unsuitable for emergency treatment and clinical patient and examines in time for enzyme linked immunosorbent assay
Disconnected needs.Latex enhancing immune turbidimetry is easy to operate, quick, but sensitivity is low, low value poor repeatability.
Invention content
The technical problem to be solved by the present invention is to overcome the defects of the prior art, providing, a kind of high use magnetic of accuracy is micro-
The kit of calcitonin (CT) content in grain chemiluminescence determination human serum.
In order to solve the above technical problem, the present invention provides following technical solutions：
A kind of kit for measuring calcitonin content, including calibration object, quality-control product, anti-reagent, magnetic particle reagent, shine bottom
Object；
The magnetic particle reagent is that anti-fluorescein isothiocynate antibody and the coupling of carboxyl magnetic bead are made, the luminous substrate
It is that ALPS is dissolved in luminous substrate buffer solution to be made；
Calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate are prepared respectively；By calibration object, quality-control product, anti-examination
Agent, magnetic particle reagent, luminous substrate, are separately applied in packing container, obtain the quantitative determination reagent kit of calcitonin；
(1), the preparation of the calibration object, quality-control product, includes the following steps：
Calcitonin antigen is dissolved with calibration object buffer solution, prepares the calibration object and quality-control product of anti-reagent；Wherein, the school
Quasi- product buffer solution is by adding in the tetracycline of 0.01g~0.05g and the sulphur of 0.1g~0.5g in the newborn bovine serum of 1L
Sour neomycin is prepared after being completely dissolved by 0.22 μm of filter membrane processing；
(2), the preparation of the anti-reagent：
1) preparation of anti-reagent buffer：
By the Na of 10g~20g2PO3H·12H2O, the NaPO of 1~2g3H2·12H2O, the sheep serum of 1g~5g, 3g~
The newborn bovine serum of 10g, the horse serum of 1g~5g are added in 1L purified waters, are stirred well to and are completely dissolved, and are adjusted with 4M HCl
The anti-reagent buffer is made in pH to 5~6；
2) fluorescein isothiocynate is coupled with calcitonin antibody, and the calcitonin coating for obtaining marked by fluorescein isothiocyanate is anti-
Body：
Fluorescein isothiocynate is configured to the fluorescein isothiocynate of a concentration of 1.0~5.0mg/mL with buffer solution first
Solution, then according to calcitonin antibody：Fluorescein isothiocynate solution=1:1.1~1:The two is transferred to by 1.5 mass ratio
In Brown Glass Brown glass bottles and jars only, abundant mixing；It is fully balanced after reaction using the carbonate buffer solution that pH is 8~9, then using gel layer
Analysis isolates and purifies to obtain the calcitonin coated antibody of marked by fluorescein isothiocyanate；
3) alkaline phosphatase is coupled with calcitonin antibody, obtains the calcitonin antibody of alkali phosphatase enzyme mark：
Alkaline phosphatase with buffer solution is configured to the alkaline phosphatase enzyme solutions of a concentration of 1.0~5.0mg/mL first, is dropped
Calcium element antibody and alkaline phosphatase reactive group is activated respectively respectively after according to molar ratio be alkaline phosphatase：Calcitonin=1:
1~1:3 reaction carries out coupling reaction than mixing abundant under the catalysis of catalyst, the use of pH is 8~9 fully after reaction
Tris buffer solutions balance, and gel column carries out isolating and purifying for different molecular big or small slice degree, obtains the drop calcium of alkali phosphatase enzyme mark
Plain labelled antibody；
The alkali that will be obtained in the calcitonin coated antibody of the marked by fluorescein isothiocyanate obtained in step 2) and step 3)
The calcitonin labelled antibody of acid phosphatase label is added in the phosphate buffer containing surfactant, is obtained after being sufficiently stirred
The anti-reagent；Preferably, the surfactant in the step 3) is Tween 20, in Triton X-100, Bronidox
It is one or more, the additive amount of surfactant is 0.01%~0.5%.
Further, which further includes cleaning solution, the configuration method of the cleaning solution will by 160gNaCl, 4gKCl,
24.2g trishydroxymethylaminomethanes, 1mL polysorbas20s are dissolved in 900ml distilled waters, are adjusted to PH7.4 with HCL, are determined with distilled water
Hold to 1000ml.With 15 times of dilutions of distilled water during use.
Further, the magnetic particle reagent is prepared according to following steps：
1) the carboxyl magnetic bead concentrate after abundant mixing is put into reaction bulb, which is placed in magnetic field to 15~
20min, supernatant is sucked after carboxyl magnetic bead all sedimentation, is added in into reaction bulb and is equivalent to carboxyl magnetic bead volume 2 in reaction bulb
~5 times of magnetic particle buffer solution, 20~30min of concussion cleaning；Reaction bulb is placed in magnetic field after 15~20min again and is sucked
Clearly；Repeated washing carboxyl magnetic bead 3 times；Finally by carboxyl magnetic bead solution constant volume to 10~50mg/mL, mixing is for use；
2) coupled reaction according to quality than carboxyl magnetic bead solution：Anti- fluorescein isothiocynate antibody=100:1 ratio exists
Anti- fluorescein isothiocynate antibody is added in carboxyl magnetic bead solution obtained by step 1), keeps mixing state anti-in 2~8 DEG C
It answers 18 hours；
3) reaction bulb places 15min in magnetic field, is washed 3 times with magnetic particle buffer solution after the sedimentation of carboxyl magnetic bead, then fixed
Hold to 10mg/mL, 2~8 DEG C of preservations, required for use magnetic particle reagent is made.Preferably, the configuration side of the magnetic particle buffer solution
Method is by the Tris of 12.12~15.26mg, the sodium chloride of 5.82~8.58mg, and it is pure that the Methyl cellulose ether of 50~60g is added to 1L
Change in water, be stirred well to and be completely dissolved to obtain the final product.
Further, the luminous substrate is fully molten with the luminous substrate buffer solution for being equivalent to 4~10 times of ALPS volumes
Solution ALPS is prepared；The configuration method of the luminous substrate buffer solution is by Tris, 5.82g of 12.12g~121.14g
Sodium chloride, 0.03g lucigenin be added in 1L purified waters, be stirred well to and be completely dissolved, be with salt acid for adjusting pH to 9.5
.
Using the method for the kit measurement calcitonin content, include the following steps：
1) three test tubes is taken to add 100 μ L calibration objects, 100 μ L quality-control products, 100 μ L samples to be tested respectively；
2) the 60 anti-reagents of μ L are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, is placed in 37
Water-bath 15min at DEG C；
3) 30 μ L magnetic particle reagents are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, puts
Water-bath 5min at 37 DEG C；
4) test tube on magnetic separator is precipitated to 3min, test tube and magnetic separator is slowly reversed, pours out supernatant；Falling
The test tube turned is placed on filter paper together with magnetic separator, flops magnetic separator bottom to remove all liquid being sticked on tube wall
Drop；
5) 300 μ L cleaning solutions are added in every test tube, with covered rearing with plastic film test tube, gently vibrate test tube 30s, after mixing
Test tube and magnetic separator are slowly reversed, pours out supernatant, the test tube of reversing together with magnetic separator, is placed on filter paper,
Separator bottom is firmly flopped to remove all drops being sticked on tube wall；
6) it is primary to repeat step 5)；
7) 200 μ L luminous substrates are added in every test tube, vibrate mixing 3s, luminous intensity is detected with Chemiluminescence Apparatus.
The magnetic particle reagent of the present invention is magnetic particle and goat-anti FITC attachments, the buffer solution containing BSA.
The calibration object of the present invention is the buffer solution containing BSA for being added to not same amount calcitonin antigen respectively.
The present invention technical principle be：The CT antibody of fluorescein isothiocynate (FITC) label is marked with alkaline phosphatase (AP)
CT in the CT pairing antibody and sample, calibration object or quality-control product of note, which is combined, forms " sandwich " compound.It then adds in and is connected with
The magnetic particle of anti-FITC antibody is incorporated in antigenantibody complex by the specific binding of anti-FITC antibody and FITC
On magnetic particle.Under the action of externally-applied magnetic field, the compound that immune response is formed with other unbonded substances is detached, is cleaned
After compound, enzyme-catalyzed chemical luminescence substrate is added in.Substrate, by catalytic pyrolysis, is formed among unstable excitation state under enzyme effect
Body sends out photon when excitation state intermediate returns to ground state, forms luminescence-producing reaction, you can uses Chemiluminescence Apparatus detection reaction
Luminous intensity.In detection range, luminous intensity is directly proportional to the content of the CT in sample, uses four parameters of improvement
Logistic equation models can calculate CT concentration in sample.
The advantageous effect that is reached of the present invention is：
1st, chemiluminescence is combined by the kit with immune magnetic particle, is provided a kind of close to homogeneous reactant
System, and employs one-step method reaction pattern so that detection sensitivity, accuracy greatly improve, detection range expands, during reaction
Between greatly shorten, from starting to be loaded onto testing result, the time is less than 35min, hence it is evident that is faster than similar kit；
2nd, a kind of new FITC antibody and magnetic particle coupling method have been invented, this method coupling efficiency is high, is firmly combined with, and
Process stabilizing while enhancing product performance, greatly reduces product cost.
3rd, kit moderate resistance reagent, magnetic particle reagent, calibration object, quality-control product, luminous substrate liquid and concentration washing lotion are these
Optimization formula under reaction system imitates the phase to the use of the kit and detection performance provides powerful guarantee.
4th, the present invention can measure multiple samples simultaneously on Full-automatic chemiluminescence apparatus, realize that the high throughput of calcitonin is fast
Speedization measures, and accuracy is high, and high specificity, accuracy and detection efficiency are enhanced.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Example is applied together for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the correlation of the kit and other commercial reagent box detection clinical serums of the present invention；Wherein abscissa is
The testing result of other commercial reagent boxes, ordinate are the testing result of kit of the present invention.
Specific embodiment
The preferred embodiment of the present invention is illustrated below, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
The operation sequence of kit test sample using the present invention is as follows：
1st, sample collection
Serum (sample of significant hemolysis or piarhemia cannot be used for measuring), the sample after collection are collected using correct medical technology
This may not exceed 8 hours being placed at room temperature for；Sample need to be positioned in 2-8 DEG C of refrigerator if detected not in 8 hours；If it needs
It preserves or transports within 72 hours or more, then should freeze in -20 DEG C hereinafter, avoiding multigelation.Room temperature is returned to before use, is gently shaken
Dynamic mixing.
2nd, prepare before experiment
1. one bottle of washing lotion is taken to carry out 15 times of dilutions with distilled water；
2. insulating box or water-bath pot temperature are adjusted to 37 DEG C, used after temperature stabilization；
3. by the abundant mixing of magnetic particle suspension to be visible by naked eyes precipitation.
3 experimental methods
1. taking out a certain amount of reaction vessel (flat based tubes), number.100ul calibration objects/matter is added according to requirement of experiment
Control product/clinical sample；
2. it is separately added into anti-reagent 60ul per hole；
3. solution in reaction vessel is uniformly mixed, 37 DEG C incubate 15 minutes；
4. shaking up magnetic particle suspension, 30ul is separately added into per hole；
5. solution in reaction vessel is uniformly mixed, 37 DEG C incubate 5 minutes；
6. reaction vessel is taken out, using Magneto separate and washing facility, by the wash liquid 3 times of magnetic particle in reaction vessel；
7. going washing lotion after the completion of washing, add luminous substrate liquid 200ul per hole, shake；
8. chemiluminescence detector device detects luminous intensity；
9. using four parameter fitting modes, using calibration object concentration value as X-axis, using calibration object luminous intensity values as Y-axis, establish
Calibration curve.Corresponding concentration value is back-calculated according to the luminous intensity values of sample to be tested.
Kit of the present invention according to methodology is identified, can reach following index：
Standard curve is linear：R ＞ 0.9900.
Minimum detection limit：≤0.5ng/ml.
Accuracy：TIANZHU XINGNAO Capsul 85%-115%.
Repeatability：Coefficient of variation CV≤8%.
Difference between batch：The coefficient of variation≤15%.
Linear dilution：R is more than 0.9900.
100 parts of serum sample measured values and commercially available calcitonin detection kit A are compared, kit more of the present invention with it is commercially available
The correlation of calcitonin kit testing result, the results are shown in Figure 1.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent replacement to which part technical characteristic.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Claims (7)
1. a kind of kit for measuring calcitonin content, which is characterized in that including calibration object, quality-control product, anti-reagent, magnetic particle examination
Agent, luminous substrate；
The magnetic particle reagent be by anti-fluorescein isothiocynate antibody and carboxyl magnetic bead coupling be made, the luminous substrate be by
ALPS is dissolved in luminous substrate buffer solution and is made；
Calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate are prepared respectively；By calibration object, quality-control product, anti-reagent,
Magnetic particle reagent, luminous substrate, are separately applied in packing container, obtain the quantitative determination reagent kit of calcitonin；
(1), the preparation of the calibration object, quality-control product, includes the following steps：
Calcitonin antigen is dissolved with calibration object buffer solution, prepares the calibration object and quality-control product of anti-reagent；Wherein, the calibration object
Buffer solution is new by tetracycline and the sulfuric acid of 0.1g~0.5g that 0.01g~0.05g is added in the newborn bovine serum of 1L
Mycin is prepared after being completely dissolved by 0.22 μm of filter membrane processing；
(2), the preparation of the anti-reagent：
1) preparation of anti-reagent buffer：
By the Na of 10g~20g2PO3H·12H2O, the NaPO of 1~2g3H2·12H2O, the sheep serum of 1g~5g, 3g~10g
In newborn bovine serum, the horse serum addition 1L purified waters of 1g~5g, it is stirred well to and is completely dissolved, pH to 5 is adjusted with 4M HCl
~6, the anti-reagent buffer is made；
2) fluorescein isothiocynate is coupled with calcitonin antibody, obtains the calcitonin coated antibody of marked by fluorescein isothiocyanate：
The fluorescein isothiocynate that fluorescein isothiocynate is configured to a concentration of 1.0~5.0mg/mL with buffer solution first is molten
Liquid, then according to calcitonin antibody：Fluorescein isothiocynate solution=1:1.1~1:The two is transferred to palm fibre by 1.5 mass ratio
In color vial, abundant mixing；It is fully balanced after reaction using the carbonate buffer solution that pH is 8~9, then using gel chromatography
It isolates and purifies to obtain the calcitonin coated antibody of marked by fluorescein isothiocyanate；
3) alkaline phosphatase is coupled with calcitonin antibody, obtains the calcitonin antibody of alkali phosphatase enzyme mark：
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions of a concentration of 1.0~5.0mg/mL, calcitonin with buffer solution first
Antibody and alkaline phosphatase reactive group is activated respectively respectively after according to molar ratio be alkaline phosphatase：Calcitonin=1:1~
1:3 reaction carries out coupling reaction than mixing abundant under the catalysis of catalyst, fully after reaction, uses the tris that pH is 8~9
Buffer solution balances, and gel column carries out isolating and purifying for different molecular big or small slice degree, obtains the calcitonin mark of alkali phosphatase enzyme mark
Remember antibody；
The alkaline phosphorus that will be obtained in the calcitonin coated antibody of the marked by fluorescein isothiocyanate obtained in step 2) and step 3)
The calcitonin labelled antibody of sour enzyme label is added in the phosphate buffer containing surfactant, after being sufficiently stirred described in acquisition
Anti- reagent.
2. the kit according to claim 1 for measuring calcitonin content, which is characterized in that the kit further includes cleaning
160gNaCl, 4gKCl, 24.2g trishydroxymethylaminomethane, 1mL polysorbas20s will be dissolved in by liquid, the configuration method of the cleaning solution
It in 900ml distilled waters, is adjusted with HCL to pH7.4,1000ml is settled to distilled water.
3. the kit of calcitonin content is measured according to claims 1 or 2 any one of them, which is characterized in that the step
3) surfactant in is one or more in Tween 20, Triton X-100, Bronidox, and surfactant adds
Dosage is 0.01%~0.5%.
4. the kit of calcitonin content is measured according to claims 1 or 2 any one of them, it is characterised in that the magnetic particle
Reagent is prepared according to following steps：
1) the carboxyl magnetic bead concentrate after abundant mixing is put into reaction bulb, which is placed in magnetic field to 15~
20min, supernatant is sucked after carboxyl magnetic bead all sedimentation, is added in into reaction bulb and is equivalent to carboxyl magnetic bead volume 2 in reaction bulb
~5 times of magnetic particle buffer solution, 20~30min of concussion cleaning；Reaction bulb is placed in magnetic field after 15~20min again and is sucked
Clearly；Repeated washing carboxyl magnetic bead 3 times；Finally by carboxyl magnetic bead solution constant volume to 10~50mg/mL, mixing is for use；
2) coupled reaction：According to quality than carboxyl magnetic bead solution：Anti- fluorescein isothiocynate antibody=100:1 ratio is in step
1) anti-fluorescein isothiocynate antibody is added in the carboxyl magnetic bead solution obtained by, mixing state response 18 is kept in 2~8 DEG C
Hour；
3) reaction bulb places 15min in magnetic field, is washed 3 times with magnetic particle buffer solution after the sedimentation of carboxyl magnetic bead, is then settled to
Required for use magnetic particle reagent is made in 10mg/mL, 2~8 DEG C of preservations.
5. the kit of calcitonin content is measured according to claim 4 any one of them, which is characterized in that the magnetic particle delays
The configuration method of fliud flushing is by the Tris of 12.12~15.26mg, the sodium chloride of 5.82~8.58mg, the Methyl cellulose of 50~60g
Ether is added in 1L purified waters, is stirred well to and is completely dissolved to obtain the final product.
6. the kit according to claim 1 for measuring calcitonin content, it is characterised in that：The luminous substrate is to use phase
When the luminous substrate buffer solution in 4~10 times of ALPS volumes fully dissolves what ALPS was prepared；The luminous substrate buffer solution
Configuration method be that the lucigenin of the sodium chloride of Tris, 5.82g of 12.12g~121.14g, 0.03g is added to 1L purified waters
In, it is stirred well to and is completely dissolved, with salt acid for adjusting pH to 9.5 to obtain the final product.
7. utilize the method for 2 any one of them kit measurement calcitonin content of claims 1 or 2, it is characterised in that this method
Include the following steps：
1) three test tubes is taken to add 100 μ L calibration objects, 100 μ L quality-control products, 100 μ L samples to be tested respectively；
2) the 60 anti-reagents of μ L are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, is placed at 37 DEG C
Water-bath 15min；
3) 30 μ L magnetic particle reagents are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, puts 37 DEG C
Lower water-bath 5min；
4) test tube on magnetic separator is precipitated to 3min, test tube and magnetic separator is slowly reversed, pours out supernatant；Reversing
Test tube is placed on filter paper together with magnetic separator, flops magnetic separator bottom to remove all drops being sticked on tube wall；
5) 300 μ L cleaning solutions are added in every test tube, with covered rearing with plastic film test tube, gently vibrate test tube 30s, after mixing slowly
Reversing test tube and magnetic separator, pour out supernatant, the test tube of reversing together with magnetic separator, be placed on filter paper, firmly
Separator bottom is flopped to remove all drops being sticked on tube wall；
6) it is primary to repeat step 5)；
7) 200 μ L luminous substrates are added in every test tube, vibrate mixing 3s, luminous intensity is detected with Chemiluminescence Apparatus.
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| CN104020295A (en) * | 2014-06-06 | 2014-09-03 | 北京玖佳宜科技有限公司 | Calcitonin detection kit and preparation method thereof |
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