CN106290911A - A kind of retinol binding protein measures test kit and method of testing thereof - Google Patents
A kind of retinol binding protein measures test kit and method of testing thereof Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/02—Nutritional disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
The invention discloses the test kit of retinol binding protein (RBP) content during a kind of employing Magnetism particulate immuno chemistry luminescence method measures.Test kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate；The retinol binding protein coated antibody of the most anti-reagent marked by fluorescein isothiocyanate and the retinol binding protein traget antibody 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 25min, 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 retinol binding protein, 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 retinol binding protein (RBP) content and method of testing thereof.
Background technology
Retinol binding protein (RBP) is mainly synthesized by hepatocyte, be distributed widely in human serum, cerebrospinal fluid, urine and
In other body fluid.In blood, RBP exists, in transporter with the composite form of 1:1:1 (mol) with retinol, prealbumin
The retinol of 90% is to body tissue, and when RBP is combined with the RBP receptor of cell surface, retinol enters intracellular, complex
Disintegrating, free RBP leaches from glomerule, and wherein the overwhelming majority is heavily absorbed by proximal renal tubular epithelial cells, and is decomposed, and supplies
Tissue utilizes, and only has and discharges from urine on a small quantity.When the diseases such as internal zinc, iron deficiency and severe infections can reduce the biological conjunction of RBP
Become.
Retinol binding protein (RBP) is the lipophilic carrier albumen of a kind of low-molecular-weight, belongs to Lipocalin superfamily protein
Member.Its function is to epithelial tissue from hepatic transport vitamin A, and can specifically be combined with retinal epithelial cells, for
Retina provides vitamin A.RBP is widely present in blood of human body, urine and other body fluid, due to its have molecular weight little and
The feature that half-life is short, measures the functional lesion of retinol binding protein energy early discovery renal tubules, and the sensitive reflection kidney of energy is near
The extent of damage of convoluted tubule, is alternatively arranged as the index of liver function Random early Detection and monitoring and therapeutic.Machine can be reflected specifically again because of RBP
The nutritional status of body, is the most also the sensitive indicator of a diagnosis early malnutrition.
The retinol binding protein 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 retinol binding protein (RBP) content in grain chemiluminescence determination human serum.
In order to solve above-mentioned technical problem, the invention provides following technical scheme:
A kind of retinol binding protein measures test kit, including calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminescence
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；Retinol binding protein is calibrated
Product, retinol binding protein quality-control product, anti-reagent, magnetic particle reagent, luminous substrate, be separately applied in packing container, obtain
The quantitative determination reagent kit of retinol binding protein；
(1), the preparation of described calibration object, quality-control product, comprise the following steps:
Dissolve retinol binding protein 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 retinol binding protein antibody coupling, it is thus achieved that regarding of marked by fluorescein isothiocyanate is yellow
Alcohol associated proteins 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 retinol binding protein 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 retinol binding protein coated antibody obtaining marked by fluorescein isothiocyanate of gel chromatography；
3) alkali phosphatase and retinol binding protein antibody coupling, it is thus achieved that the retinol of alkali phosphatase enzyme mark combines egg
Bai Kangti:
First with buffer, alkali phosphatase is configured to the alkaline phosphatase enzymatic solution that concentration is 1.0～5.0mg/mL, depending on
Flavol associated proteins antibody and alkali phosphatase are alkali phosphatase according to mol ratio after being activated respectively by reactive group respectively: regard
The reaction of flavol associated proteins=1:1～1:3 carries out coupling reaction than fully mixing under the catalysis of catalyst, fully reacts
After, 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
The retinol binding protein traget antibody of phosphatase enzyme mark；
By step 2) in the retinol binding protein coated antibody of marked by fluorescein isothiocyanate that obtains and step 3) in
The retinol binding protein traget antibody 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.
?.
Use this test kit retinol binding protein assay method, comprise the following steps:
1) take three test tubes and add 20 μ L calibration objects, 20 μ L quality-control products, 20 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 5min 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 retinol binding protein antigen respectively.
The know-why of the present invention is: the RBP antibody of Fluorescein isothiocyanate (FITC) labelling and alkali phosphatase (AP)
The RBP combination that the RBP 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 RBP in sample, uses four parameters of improvement
Logistic equation model can calculate RBP 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 25min, 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 retinol binding protein
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.20uL calibration object/Quality Control is added according to requirement of experiment
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 5 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.
106 parts of serum sample measured values are contrasted with commercially available Retinal-binding protein detection test kit A, compares reagent of the present invention
Box and the dependency of commercially available retinol binding protein test kit 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. a retinol binding protein measures test kit, 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 retinol binding protein；
(1), the preparation of described calibration object, quality-control product, comprise the following steps:
Dissolve retinol binding protein 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 retinol binding protein antibody coupling, it is thus achieved that the retinol knot of marked by fluorescein isothiocyanate
Hop protein 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 retinol binding protein 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 retinol binding protein coated antibody obtaining marked by fluorescein isothiocyanate of gel chromatography；
3) alkali phosphatase and retinol binding protein antibody coupling, it is thus achieved that the retinol binding protein 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, retinol
Associated proteins antibody and alkali phosphatase are alkali phosphatase according to mol ratio after being activated respectively by reactive group respectively: retinol
The reaction of associated proteins=1:1～1:3 carries out coupling reaction than fully mixing under the catalysis of catalyst, fully after reaction, makes
By the tris buffer balance that pH is 8～9, gel column carries out the isolated and purified of different molecular big or small slice degree, obtains alkaline phosphatase
The retinol binding protein traget antibody of enzyme labelling；
By step 2) in the retinol binding protein coated antibody of marked by fluorescein isothiocyanate that obtains and step 3) in obtain
Alkali phosphatase enzyme mark retinol binding protein traget antibody add containing surfactant phosphate buffer in, fill
Described anti-reagent is obtained after dividing stirring.
Retinol binding protein the most according to claim 1 measures test kit, it is characterised in that this test kit also includes clearly
Washing liquid, the collocation method of described cleanout fluid just 160gNaCl, 4gKCl, 24.2g trishydroxymethylaminomethane, 1mL polysorbas20 are molten
In 900ml distilled water, adjust to PH7.4 with HCL, be settled to 1000ml with distilled water.
3. measure test kit according to the retinol binding protein described in any one of claim 1 or 2, it is characterised in that described step
Rapid 3) surfactant in is Tween 20, one or more in Triton X-100, Bronidox, surfactant
Addition is 0.01%～0.5%.
4. measure test kit according to the retinol binding protein described in any one of claim 1 or 2, it is characterised in that described magnetic is micro-
Grain 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 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. measure test kit according to the retinol binding protein described in any one of claim 4, it is characterised in that described magnetic particle
The collocation method of buffer is that the methyl of 50～60g is fine by the Tris of 12.12～15.26mg, the sodium chloride of 5.82～8.58mg
Dimension ether joins in 1L purified water, is stirred well to be completely dissolved and get final product.
Retinol binding protein the most according to claim 1 measures test kit, it is characterised in that: described luminous substrate is to use
The luminous substrate buffer being equivalent to ALPS volume 4～10 times fully dissolves what ALPS was prepared from；Described luminous substrate buffers
The collocation method of liquid is that the sodium chloride of Tris, 5.82g of 12.12g～121.14g, the lucigenin of 0.03g are joined 1L purification
In water, it is stirred well to be completely dissolved, with salt acid for adjusting pH to 9.5 and get final product.
7. utilizing the retinol binding protein described in any one of claim 1 or 2 to measure the method for testing of test kit, its feature exists
Comprise the following steps in the method:
1) take three test tubes and add 20 μ L calibration objects, 20 μ L quality-control products, 20 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 5min；
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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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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 |
| CN108226464A (en) * | 2018-02-05 | 2018-06-29 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring thyroglobulin content |
| CN108318695A (en) * | 2018-01-30 | 2018-07-24 | 江苏晶红生物医药科技股份有限公司 | People RBP colloidal gold immunochromatographimethod quantitative testing test paper cards and its clinical application |
| CN109142715A (en) * | 2018-07-03 | 2019-01-04 | 江南大学 | A kind of novel nano magnetic particle suspension system and its preparation method |
| CN111719003A (en) * | 2020-07-14 | 2020-09-29 | 安徽省天长市周氏羊业有限公司 | RBP1 gene and application thereof, sheep ovary in-vitro development quality evaluation method and amplification primer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| 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 |
| CN108318695A (en) * | 2018-01-30 | 2018-07-24 | 江苏晶红生物医药科技股份有限公司 | People RBP colloidal gold immunochromatographimethod quantitative testing test paper cards and its clinical application |
| CN108318695B (en) * | 2018-01-30 | 2020-08-11 | 江苏晶红生物医药科技股份有限公司 | Human RBP colloidal gold immunochromatographic assay quantitative detection test paper card and clinical application thereof |
| CN108226464A (en) * | 2018-02-05 | 2018-06-29 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring thyroglobulin content |
| CN109142715A (en) * | 2018-07-03 | 2019-01-04 | 江南大学 | A kind of novel nano magnetic particle suspension system and its preparation method |
| CN111719003A (en) * | 2020-07-14 | 2020-09-29 | 安徽省天长市周氏羊业有限公司 | RBP1 gene and application thereof, sheep ovary in-vitro development quality evaluation method and amplification primer |
| CN112159733A (en) * | 2020-09-25 | 2021-01-01 | 芯朗道(天津)医疗科技有限责任公司 | Cleaning solution for magnetic particle chemiluminescence immunoassay and preparation method thereof |
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