CN106248970A - A kind of test kit measuring type i collagen C-terminal peptide content and method of testing thereof - Google Patents
A kind of test kit measuring type i collagen C-terminal peptide content and method of testing thereof Download PDFInfo
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- CN106248970A CN106248970A CN201610672100.8A CN201610672100A CN106248970A CN 106248970 A CN106248970 A CN 106248970A CN 201610672100 A CN201610672100 A CN 201610672100A CN 106248970 A CN106248970 A CN 106248970A
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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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- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of employing Magnetism particulate immuno chemistry luminescence method and measure the test kit of type i collagen C-terminal peptide (CTX) content in human serum.Test kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate；The type i collagen C-terminal peptide coated antibody of the most anti-reagent marked by fluorescein isothiocyanate and the peptide-labeled antibody of type i collagen C-terminal of alkali phosphatase enzyme mark；Magnetic particle reagent is magnetic particle and goat-anti FITC junctional complex.Chemiluminescence is combined by the present invention with immunity magnetic particle, provide a kind of close to homogeneous reaction system, and have employed one-step method reaction pattern, detection sensitivity, elaboration are greatly improved, detection range expands, response time is greatly shortened, from starting to be loaded onto testing result, the time is less than 35min, hence it is evident that be faster than similar test kit；And can measure multiple sample on Full-automatic chemiluminescence apparatus, it is achieved the rapid mensuration of high flux of type i collagen C-terminal peptide, accuracy is high, and high specificity, degree of accuracy and detection efficiency are enhanced simultaneously.
Description
Technical field
The present invention relates to technological field of biochemistry, be specifically related to a kind of employing in Magnetism particulate immuno chemistry luminescence method mensuration human body
The test kit of type i collagen C-terminal peptide (CTX) content.
Background technology
Type i collagen is the collagen protein form that human body is the abundantest, is unique collagen component in bone, accounts for the 90% of bone matrix
Above.In the constantly reconstruction of whole skeletal bones substrate, type i collagen is degraded, and lower fragment is released into blood, and part occurs in urine
In.The content and the change that measure these small fragments in blood, urine can evaluate bone resorption state, both contributed to examining of metabolic osteopathy
Disconnected, also can monitor and evaluate the curative effect of anti-bone resorption medicine.Type i collagen C-terminal peptide (CTX), is in these little fragments of peptides
Kind.Complete type i collagen C-terminal peptide contains 26 aminoacid, its 15th～22 aminoacid be followed successively by paddy-rely-the third=group-
My god-Dong-Gan-sweet-arginine (EKAHDGGR), this part has specificity for type i collagen α 1 (I) chain C end, referred to as 8 ammonia
Base acid sequence (8AA).CTX is only derived from ripe collagen fiber, does not derive from new rubber alloy former；The most it is not degraded and is not the most weighed
New utilization, can directly reflect the degraded situation of collagen fiber；Collagen diet is on its nothing impact；Specificity is higher, glue in reflection bone
The minor variations of former conversion is sensitiveer.CTX is applied to clinic, mainly judges the bone resorption state of metabolic osteopathy, detect with
Evaluate the curative effect of anti-bone resorption medicine.
The type i collagen C-terminal peptide assay method being currently known has radioimmunology (RIA), enzyme linked immunosorbent assay
(ELISA), latex-enhanced turbidimetry etc..Radioimmunology complex steps, reagent is expensive, need to use supporting instrument and deposit
At radioactive pollution.There is detection time length, operation complexity, poor repeatability, be unsuitable for emergency treatment and clinic in enzyme linked immunosorbent assay
The needs that patient diagnoses in time.Latex enhancing immune turbidimetry is simple to operate, quick, but sensitivity is low, low value poor repeatability.
Summary of the invention
The technical problem to be solved in the present invention is the defect overcoming prior art, it is provided that the employing magnetic that a kind of accuracy is high is micro-
The test kit of type i collagen C-terminal peptide (CTX) content in grain chemiluminescence determination human serum.
In order to solve above-mentioned technical problem, the invention provides following technical scheme:
A kind of test kit measuring type i collagen C-terminal peptide content, including calibration object, quality-control product, anti-reagent, magnetic particle examination
Agent, luminous substrate；
Described magnetic particle reagent is to be made with carboxyl magnetic bead coupling by anti-Fluorescein isothiocyanate antibody, described luminous substrate
It is to be dissolved in ALPS in luminous substrate buffer to make；
Prepare calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate respectively；Type i collagen C-terminal peptide is calibrated
Product, type i collagen C-terminal peptide quality-control product, anti-reagent, magnetic particle reagent, luminous substrate, be separately applied in packing container, obtain I
The quantitative determination reagent kit of Collagen Type VI C-terminal peptide；
(1), the preparation of described calibration object, quality-control product, comprise the following steps:
Dissolve type i collagen C-terminal peptide antigen with calibration object buffer solution, prepare calibration object and the quality-control product of anti-reagent；Its
In, described calibration object buffer solution be by add in the new-born calf serum of 1L the tetracycline of 0.01g～0.05g and 0.1g～
The polygynax of 0.5g, processes through 0.22 μm filter membrane after being completely dissolved and is prepared from；
(2), the preparation of described anti-reagent:
1) preparation of anti-reagent buffer:
By the Na of 10g～20g2PO3H·12H2The NaPO of O, 1～2g3H2·12H2The sheep serum of O, 1g～5g, 3g～
The new-born calf serum of 10g, the horse serum of 1g～5g add in 1L purified water, are stirred well to be completely dissolved, regulate with 4M HCl
PH to 5～6, prepares described anti-reagent buffer；
2) Fluorescein isothiocyanate and type i collagen C-terminal peptide antibody coupling, it is thus achieved that the I type of marked by fluorescein isothiocyanate
Collagen C-terminal peptide coated antibody:
First with buffer, Fluorescein isothiocyanate is configured to the Fluorescein isothiocyanate that concentration is 1.0～5.0mg/mL
Solution, then according to type i collagen C-terminal peptide antibody: the mass ratio of Fluorescein isothiocyanate solution=1:1.1～1:1.5 is by two
Person transfers to, in Brown Glass Brown glass bottles and jars only, fully mix；The carbonate buffer solution balance fully using pH to be 8～9 after reaction, then makes
With the isolated and purified type i collagen C-terminal peptide coated antibody obtaining marked by fluorescein isothiocyanate of gel chromatography；
3) alkali phosphatase and type i collagen C-terminal peptide antibody coupling, it is thus achieved that the type i collagen C-terminal of alkali phosphatase enzyme mark
Peptide antibody:
First with buffer, alkali phosphatase is configured to the alkaline phosphatase enzymatic solution that concentration is 1.0～5.0mg/mL, I type
Collagen C-terminal peptide antibody and alkali phosphatase are alkali phosphatase according to mol ratio after being activated respectively by reactive group respectively: I type
The reaction of collagen C-terminal peptide=1:1～1:3 carries out coupling reaction than fully mixing under the catalysis of catalyst, fully after reaction,
The tris buffer balance using pH to be 8～9, gel column carries out the isolated and purified of different molecular big or small slice degree, obtains alkalescence phosphorus
The peptide-labeled antibody of type i collagen C-terminal of acid enzyme labelling；
By step 2) in the type i collagen C-terminal peptide coated antibody of marked by fluorescein isothiocyanate that obtains and step 3) in
The peptide-labeled antibody of type i collagen C-terminal of the alkali phosphatase enzyme mark obtained adds the phosphate buffer containing surfactant
In, obtain described anti-reagent after being sufficiently stirred for；Preferably, described step 3) in surfactant be Tween 20, Triton
One or more in X-100, Bronidox, the addition of surfactant is 0.01%～0.5%.
Further, this test kit also includes cleanout fluid, the collocation method of described cleanout fluid just 160gNaCl, 4gKCl,
24.2g trishydroxymethylaminomethane, 1mL polysorbas20 are dissolved in 900ml distilled water, adjust to PH7.4 with HCL, fixed with distilled water
Hold to 1000ml.With distilled water 15 times dilution during use.
Further, described magnetic particle reagent is prepared according to following steps:
1) will fully mixing after carboxyl magnetic bead concentrated solution put in reaction bulb, this reaction bulb is placed in magnetic field 15～
20min, sucks supernatant after carboxyl magnetic bead all settles, and adds and be equivalent to carboxyl magnetic bead volume 2 in reaction bulb in reaction bulb
～the magnetic particle buffer of 5 times, concussion cleans 20～30min；Suck after reaction bulb is placed in magnetic field 15～20min again
Clearly；Repeated washing carboxyl magnetic bead 3 times；Finally by carboxyl magnetic bead solution constant volume to 10～50mg/mL, mix stand-by；
2) coupled reaction according to quality than carboxyl magnetic bead solution: the ratio of anti-Fluorescein isothiocyanate antibody=100:1 exists
Step 1) obtained by carboxyl magnetic bead solution in add anti-Fluorescein isothiocyanate antibody, in 2～8 DEG C keep mixing state anti-
Answer 18 hours；
3) 15min placed in magnetic field by reaction bulb, washes 3 times with magnetic particle buffer after carboxyl magnetic bead settles, fixed subsequently
Hold to 10mg/mL, 2～8 DEG C of preservations, prepare required stand-by magnetic particle reagent.Preferably, the configuration side of described magnetic particle buffer
Method is that it is pure that the Methyl cellulose ether of 50～60g joins 1L by the Tris of 12.12～15.26mg, the sodium chloride of 5.82～8.58mg
Change in water, be stirred well to be completely dissolved and get final product.
Further, described luminous substrate is the most molten with the luminous substrate buffer being equivalent to ALPS volume 4～10 times
Solve what ALPS was prepared from；The collocation method of described luminous substrate buffer is by Tris, 5.82g of 12.12g～121.14g
Sodium chloride, the lucigenin of 0.03g join in 1L purified water, be stirred well to be completely dissolved, with salt acid for adjusting pH to 9.5 i.e.
?.
The method using this kit measurement type i collagen C-terminal peptide content, comprises the following steps:
1) take three test tubes and add 100 μ L calibration objects, 100 μ L quality-control products, 100 μ L samples to be tested respectively；
2) every test tube adds the 60 anti-reagent of μ L, uses covered rearing with plastic film test tube, gently tube shaken 30s, be placed in 37
Water-bath 15min at DEG C；
3) every test tube adds 30 μ L magnetic particle reagent, uses covered rearing with plastic film test tube, gently tube shaken 30s, put
Water-bath 5min at 37 DEG C；
4) test tube precipitating on magnetic separator 3min, reversing test tube and magnetic separator, pour out supernatant lentamente；Falling
The test tube turned, together with magnetic separator, is placed on filter paper, bounces all liquid being bonded on tube wall bottom magnetic separator with removing
Drip；
5) every test tube adds 300 μ L cleanout fluid, uses covered rearing with plastic film test tube, gently tube shaken 30s, after mixing
Reversing test tube and magnetic separator, pour out supernatant slowly, the test tube of reversing together with magnetic separator, is placed on filter paper,
Firmly bounce all drops that separator bottom is bonded on tube wall with removing；
6) step 5 is repeated) once；
7) every test tube adds 200 μ L luminous substrate, vibration mixing 3s, detects luminous intensity with Chemiluminescence Apparatus.
The magnetic particle reagent of the present invention is magnetic particle and goat-anti FITC junctional complex, the buffer containing BSA.
The calibration object of the present invention is the buffer containing BSA that with the addition of the most commensurability type i collagen C-terminal peptide antigen respectively.
The know-why of the present invention is: the CTX antibody of Fluorescein isothiocyanate (FITC) labelling and alkali phosphatase (AP)
The CTX combination that the CTX of labelling matches in antibody and sample, calibration object or quality-control product forms " sandwich " complex.It is subsequently added
It is connected with the magnetic particle of anti-FITC antibody, makes antigenantibody complex tie by anti-FITC antibody and the specific binding of FITC
It is combined on magnetic particle.Under the effect of externally-applied magnetic field, the complex that immunoreation is formed is separated with other materials unconjugated,
After cleaning complex, add enzyme-catalyzed chemical luminescence substrate.Substrate by catalytic pyrolysis, forms unstable excited state under enzyme effect
Intermediate, sends photon when excited state intermediate returns to ground state, forms luminescence-producing reaction, and Chemiluminescence Apparatus detection can be used anti-
The luminous intensity answered.In detection range, luminous intensity is directly proportional to the content of the CTX in sample, uses four parameters of improvement
Logistic equation model can calculate CTX concentration in sample.
The present invention is reached to provide the benefit that:
1, chemiluminescence is combined by this test kit with immunity magnetic particle, it is provided that a kind of close to homogeneous reactant
System, and have employed one-step method reaction pattern so that detection sensitivity, elaboration are greatly improved, detection range expands, during reaction
Between be greatly shortened, from starting to be loaded onto testing result, the time be less than 35min, hence it is evident that be faster than similar test kit；
2, having invented a kind of new FITC antibody and magnetic particle coupling method, the method coupling efficiency is high, is firmly combined with, and
Process stabilizing, while enhancing product performance, greatly reduces product cost.
3, in test kit, anti-reagent, magnetic particle reagent, calibration object, quality-control product, luminous substrate liquid and concentration washing liquid are all these
Optimization formula under reaction system, imitates the phase to the use of this test kit and detection performance provides powerful guarantee.
4, the present invention can measure multiple sample on Full-automatic chemiluminescence apparatus simultaneously, it is achieved type i collagen C-terminal peptide
The rapid mensuration of high flux, accuracy is high, and high specificity, degree of accuracy and detection efficiency are enhanced.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, with the reality of the present invention
Execute example together for explaining the present invention, be not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is the test kit dependency with other commercial reagent box detection clinical serum of the present invention；Wherein abscissa is
The testing result of other commercial reagent boxes, vertical coordinate is the testing result of test kit of the present invention.
Detailed description of the invention
Hereinafter the preferred embodiments of the present invention are illustrated, it will be appreciated 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 using the test kit test sample of the present invention is as follows:
1, sample collection
Correct medical technology is used to collect serum (sample of significant hemolysis or lipidemia cannot be used for measuring), the sample after collection
This is placed in room temperature and may not exceed 8 hours；Sample need to be positioned in the refrigerator of 2-8 DEG C if do not detected in 8 hours；If needing
Within more than 72 hours, preserve or transport, then should be frozen in less than-20 DEG C, it is to avoid multigelation.It is returned to room temperature before using, shakes gently
Dynamic mixing.
2, prepare before experiment
1. take one bottle of washing liquid distilled water and carry out 15 times of dilutions；
2. calorstat or water-bath temperature are adjusted to 37 DEG C, use after temperature stabilization；
3. magnetic particle suspension is fully mixed to being visible by naked eyes precipitation.
3 experimental techniques
1. a certain amount of reaction vessel (flat based tubes) is taken out, numbering.100ul calibration object/matter is added according to requirement of experiment
Control product/clinical sample；
2. every hole is separately added into anti-reagent 60ul；
3. by solution mix homogeneously in reaction vessel, 37 DEG C of incubations 15 minutes；
4. shaking up magnetic particle suspension, every hole is separately added into 30ul；
5. by solution mix homogeneously in reaction vessel, 37 DEG C of incubations 5 minutes；
6. take out reaction vessel, use Magneto separate and washing facility, by the wash liquid 3 times of magnetic particle in reaction vessel；
7. washing liquid, every hole is gone to add luminous substrate liquid 200ul after having washed, concussion；
8. chemiluminescence detector device detection luminous intensity；
9. use four parameter fitting modes, with calibration object concentration value as X-axis, with calibration object luminous intensity values as Y-axis, set up
Calibration curve.Luminous intensity values according to sample to be tested is back-calculated corresponding concentration value.
Test kit of the present invention is identified according to methodology, can reach following index:
Standard curve is linear: R ＞ 0.9900.
Lowest detectable 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 are contrasted with commercially available type i collagen C-terminal peptide detection kit A, compares reagent of the present invention
Box and the dependency of commercially available type i collagen C-terminal peptide reagent box testing result, result is as shown in Figure 1.
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention,
Although being described in detail the present invention with reference to previous embodiment, for a person skilled in the art, it still may be used
So that the technical scheme described in foregoing embodiments to be modified, or wherein portion of techniques feature is carried out equivalent.
All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included in the present invention's
Within protection domain.
Claims (7)
1. the test kit measuring type i collagen C-terminal peptide content, it is characterised in that include calibration object, quality-control product, anti-reagent,
Magnetic particle reagent, luminous substrate；
Described magnetic particle reagent is to be made with carboxyl magnetic bead coupling by anti-Fluorescein isothiocyanate antibody, described luminous substrate be by
ALPS is dissolved in luminous substrate buffer and makes；
Prepare calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate respectively；By calibration object, quality-control product, anti-reagent,
Magnetic particle reagent, luminous substrate, be separately applied in packing container, obtains the quantitative determination reagent kit of type i collagen C-terminal peptide；
(1), the preparation of described calibration object, quality-control product, comprise the following steps:
Dissolve type i collagen C-terminal peptide antigen with calibration object buffer solution, prepare calibration object and the quality-control product of anti-reagent；Wherein, institute
Stating calibration object buffer solution is tetracycline and 0.1g～0.5g by adding 0.01g～0.05g in the new-born calf serum of 1L
Polygynax, after being completely dissolved through 0.22 μm filter membrane process be prepared from；
(2), the preparation of described anti-reagent:
1) preparation of anti-reagent buffer:
By the Na of 10g～20g2PO3H·12H2The NaPO of O, 1～2g3H2·12H2The sheep serum of O, 1g～5g, 3g～10g
New-born calf serum, the horse serum of 1g～5g add in 1L purified water, are stirred well to be completely dissolved, regulate pH to 5 with 4M HCl
～6, prepare described anti-reagent buffer；
2) Fluorescein isothiocyanate and type i collagen C-terminal peptide antibody coupling, it is thus achieved that the type i collagen C of marked by fluorescein isothiocyanate
Terminal peptide coated antibody:
First with buffer, Fluorescein isothiocyanate is configured to the Fluorescein isothiocyanate that concentration is 1.0～5.0mg/mL molten
Liquid, then according to type i collagen C-terminal peptide antibody: the mass ratio of Fluorescein isothiocyanate solution=1:1.1～1:1.5 incite somebody to action the two
Transfer to, in Brown Glass Brown glass bottles and jars only, fully mix；The carbonate buffer solution balance fully using pH to be 8～9 after reaction, then uses
The isolated and purified type i collagen C-terminal peptide coated antibody obtaining marked by fluorescein isothiocyanate of gel chromatography；
3) alkali phosphatase and type i collagen C-terminal peptide antibody coupling, it is thus achieved that the type i collagen C-terminal peptide of alkali phosphatase enzyme mark resists
Body:
First with buffer, alkali phosphatase is configured to the alkaline phosphatase enzymatic solution that concentration is 1.0～5.0mg/mL, type i collagen
C-terminal peptide antibody and alkali phosphatase are alkali phosphatase according to mol ratio after being activated respectively by reactive group respectively: type i collagen
The reaction of C-terminal peptide=1:1～1:3 carries out coupling reaction than fully mixing under the catalysis of catalyst, fully after reaction, uses
PH is the tris buffer balance of 8～9, and gel column carries out the isolated and purified of different molecular big or small slice degree, obtains alkali phosphatase
The peptide-labeled antibody of type i collagen C-terminal of labelling；
By step 2) in the type i collagen C-terminal peptide coated antibody of marked by fluorescein isothiocyanate that obtains and step 3) in obtain
Alkali phosphatase enzyme mark the peptide-labeled antibody of type i collagen C-terminal add containing surfactant phosphate buffer in, fill
Described anti-reagent is obtained after dividing stirring.
The test kit of mensuration type i collagen C-terminal peptide content the most according to claim 1, it is characterised in that this test kit is also
Including cleanout fluid, the collocation method of described cleanout fluid just 160gNaCl, 4gKCl, 24.2g trishydroxymethylaminomethane, 1mL tell
Temperature 20 is dissolved in 900ml distilled water, adjusts to PH7.4 with HCL, is settled to 1000ml with distilled water.
3. according to the test kit measuring type i collagen C-terminal peptide content described in any one of claim 1 or 2, it is characterised in that
Described step 3) in surfactant be Tween 20, one or more in Triton X-100, Bronidox, live in surface
The addition of property agent is 0.01%～0.5%.
4. according to the test kit measuring type i collagen C-terminal peptide content described in any one of claim 1 or 2, it is characterised in that institute
State magnetic particle reagent to prepare according to following steps:
1) will fully mixing after carboxyl magnetic bead concentrated solution put in reaction bulb, this reaction bulb is placed in magnetic field 15～
20min, sucks supernatant after carboxyl magnetic bead all settles, and adds and be equivalent to carboxyl magnetic bead volume 2 in reaction bulb in reaction bulb
～the magnetic particle buffer of 5 times, concussion cleans 20～30min；Suck after reaction bulb is placed in magnetic field 15～20min again
Clearly；Repeated washing carboxyl magnetic bead 3 times；Finally by carboxyl magnetic bead solution constant volume to 10～50mg/mL, mix stand-by；
2) coupled reaction: according to quality than carboxyl magnetic bead solution: the ratio of anti-Fluorescein isothiocyanate antibody=100:1 is in step
1) the carboxyl magnetic bead solution obtained by adds anti-Fluorescein isothiocyanate antibody, in 2～8 DEG C, keeps mixing state response 18
Hour；
3) 15min placed in magnetic field by reaction bulb, washes 3 times with magnetic particle buffer, be settled to subsequently after carboxyl magnetic bead settles
10mg/mL, 2～8 DEG C of preservations, prepare required stand-by magnetic particle reagent.
5. according to the test kit measuring type i collagen C-terminal peptide content described in any one of claim 4, it is characterised in that described
The collocation method of magnetic particle buffer is by the Tris of 12.12～15.26mg, the sodium chloride of 5.82～8.58mg, 50～60g
Methyl cellulose ether joins in 1L purified water, is stirred well to be completely dissolved and get final product.
The test kit of mensuration type i collagen C-terminal peptide content the most according to claim 1, it is characterised in that: the described luminous end
Thing is fully to dissolve ALPS with the luminous substrate buffer being equivalent to ALPS volume 4～10 times to be prepared from；The described luminous end
The collocation method of thing buffer is the sodium chloride of Tris, 5.82g of 12.12g～121.14g, the lucigenin of 0.03g to be joined
In 1L purified water, it is stirred well to be completely dissolved, with salt acid for adjusting pH to 9.5 and get final product.
7. the method utilizing kit measurement type i collagen C-terminal peptide content described in any one of claim 1 or 2, its feature exists
Comprise the following steps in the method:
1) take three test tubes and add 100 μ L calibration objects, 100 μ L quality-control products, 100 μ L samples to be tested respectively；
2) every test tube adds the 60 anti-reagent of μ L, uses covered rearing with plastic film test tube, gently tube shaken 30s, be placed at 37 DEG C
Water-bath 15min；
3) every test tube adds 30 μ L magnetic particle reagent, uses covered rearing with plastic film test tube, gently tube shaken 30s, put 37 DEG C
Lower water-bath 5min；
4) test tube precipitating on magnetic separator 3min, reversing test tube and magnetic separator, pour out supernatant lentamente；Reversing
Test tube, together with magnetic separator, is placed on filter paper, bounces all drops being bonded on tube wall bottom magnetic separator with removing；
5) every test tube adds 300 μ L cleanout fluid, uses covered rearing with plastic film test tube, gently tube shaken 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
Bounce all drops that separator bottom is bonded on tube wall with removing；
6) step 5 is repeated) once；
7) every test tube adds 200 μ L luminous substrate, vibration mixing 3s, detects luminous intensity with Chemiluminescence Apparatus.
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| CN108169486A (en) * | 2017-12-07 | 2018-06-15 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
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| CN108152273A (en) * | 2017-12-07 | 2018-06-12 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring intact PTH content |
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| CN110361543A (en) * | 2019-07-29 | 2019-10-22 | 清华大学深圳研究生院 | NTx Test paper, NTx detection kit and detection method |
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