CN106596968A - Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit - Google Patents
Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit Download PDFInfo
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- 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
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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention provides a dot gold infiltration kit for detecting urine microalbumin, and application of the dot gold infiltration kit. The dot gold infiltration kit comprises a sample diluent, a washing liquid, a colloidal gold labeled human serum albumin antibody, a urine microalbumin chromatographic device and a calibration card, wherein the sample diluents comprises Triton-100, a phosphate buffer solution and a Tris buffer solution; the washing liquid comprises bovine serum albumin, a phosphate buffer solution and a Tris buffer solution. Compared with the prior art, the dot gold infiltration kit for detecting the urine microalbumin has the advantages that colloidal gold particles are provided with negative charges on the surfaces and can form firm combination with the positive charge groups of urine microalbumin antibody protein molecules due to electrostatic interaction; meanwhile, the gold particles have the characteristic of high electron density, and when the antibody labeled by the gold particles is combined with a corresponding antigen ligand and a large number of the god particles are gathered at the site of the antibody ligand, macroscopic red dots are formed. The dot gold infiltration kit for detecting the urine microalbumin, provided by the invention, has the advantages of short detection time, high precision degree, low requirements on instruments and environment, and convenience in clinical popularization and application.
Description
Technical field
The present invention relates to technical field of medical examination, more particularly to a kind of detection microdose urine protein spot gold diafiltration reagent
Box and its application.
Background technology
Albumin is the normal protein in a kind of blood, but only occurs the white egg of very small amount in urine in physiological conditions
In vain.Microalbuminuria reflects renal abnormality proteinaceous effluent.Under normal circumstances, the albumin in urine is few, tool for body metabolism
Body to every liter of urinary albumin less than 20mg (<20mg/L), so being microalbumin.If found after health check-up micro- in urine
Amount albumin belongs to microalbuminuria in the range of 20mg/L-200mg/L, just.In clinic, commonly used urine micro protein refers to
Mark to monitor the generation of kidney trouble.
The detection of urine micro protein is that early detection ephrosis is most sensitive, one of most reliable diagnosis index.It is micro by urinating
Albuminous numerical value, just can accurately diagnose the state of an illness and judge the course of disease with reference to the statement of incidence, symptom and medical history.
After Canonical management kidney, the nephron is repaired, and albuminous amount recovers normal level in urine, so urine Microalbunin
In vain can be used as kidney trouble prognosis index.
Microdose urine protein detection technique mainly has immunoturbidimetry, colloidal gold method, ELISA etc..Immunoturbidimetry
Relative operation is relatively complicated and easily occurs hook effect in the presence of high concentration antigen, so as to the result for affecting to measure；
Simultaneously it is not suitable for small lot packaging, limits its popularization in basic hospital.The colloidal gold method testing time is longer, precision compared with
Difference, and it is easily influenced by environmental conditions.The operation of enzyme linked immunological rule is extremely loaded down with trivial details and higher to operator's requirement, and detection is time-consuming
Long, its popularization and application in clinic is limited.
The content of the invention
Present invention solves the technical problem that be provide a kind of detection microdose urine protein spot gold diafiltration kit and its
Using, with detection time it is short, precision is good the characteristics of.
In view of this, the invention provides a kind of spot gold of detection microdose urine protein is percolated kit, including：PH value
For cleaning solution that 6.5～6.7 Sample dilution, pH value are 6.2～6.4, colloid gold label human serum albumins antibody, urinate micro-
Amount albumin chromatography device and calibration card, the Sample dilution is slow including Triton-100, phosphate buffer and Tris
Rush liquor；The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
Preferably, the concentration of Triton-100 is 0.1～10% in the Sample dilution, phosphate buffer
Concentration is 0.005～1mol/L, and the concentration of Tris buffer solns is 0.01～1mol/L；Bovine serum albumin in the cleaning solution
White concentration is 0.1～10%, and the concentration of phosphate buffer is 0.01mol/L, and the concentration of Tris buffer solns is
0.01～1mol/L.
Preferably, the phosphate buffer is prepared by following raw material：Na2HPO40.263-0.376 weight portions,
KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, ox blood are pure
The weight portion of albumen 7；The Tris buffer solns are by trishydroxymethylaminomethane 4.6-5.5 weight portions, KH2PO43.0-2.1 weight
Amount part, the weight portions of NaCl 8, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
Preferably, the colloid gold label human serum albumins antibody is prepared as follows：It is 1% by mass fraction
Gold chloride and distilled water by volume 1：79 are made into A liquid；It is 1% by trisodium citrate that mass fraction is 1%, mass fraction
Tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～0.080：15.84～15.93 are made into B
Liquid；By the A liquid and B liquid while being heated to 59～61 DEG C, stirring A liquid in rapidly joining B liquid, continues to stir 1 point in water-bath
Clock, was heated to boiling in 10～13 minutes, obtained collaurum liquid；Using 0.25M K2CO3Adjusting the collaurum liquid pH value is
6.8～7.1；By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, in absorption
Clear liquid；The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing obtains human serum albumins
Antibody；The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the human serum albumins resists
Body is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, sequentially adds 1～30%
Sodium azide and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) are with the volume ratio of the collaurum liquid
500μl：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min, removes supernatant, sinks
Starch is colloid gold label human serum albumins antibody.
Preferably, the trisodium citrate, tannic acid, K2CO3It is 4 with the volume ratio of distilled water：0.08：0.08：15.84, institute
State the colloid gold particle that a diameter of 10 ＜ D ＜ 11nm are formed in collaurum liquid.
Preferably, the reaction box of a diameter of 2.8cm, the reaction box bag are provided with the microdose urine protein chromatography device
Include cassette bottom and lid, the lid top surface central authorities are provided with upper surface a diameter of 0.56cm, a diameter of 0.27cm of lower surface
Small sircle hole；The full absorbent material of pad in the reaction box；Place between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of one 1.18 × 1.18cm.
Preferably, the nitrocellulose filter of the coating human serum albumins antibody is prepared according to following steps：By human blood
Pure albumen coated antibody stoste is at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；Will be described
Supernatant loads bag filter, with distilled water dialysis desalination, bottling freezing after ultrafiltration concentration, obtains human serum albumins and resists
Body；The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, obtains 0.8mg/ml's
Coating buffer；The coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking oven by every diaphragm 0.02ml
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after middle drying.
Preferably, the calibration card detects the corresponding detection of color by human serum albumins gradient mass concentration solution
Signal value.According to the signal value that Test paper shows, compare with calibration card signal value, you can quantitatively judge its mass concentration.
Preferably, the gradient mass concentration is respectively 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 120mg/
L, 200mg/L, and the signal of the gradient mass concentration detection is corresponding with calibration card.
Accordingly, the present invention also provides a kind of application of the spot gold diafiltration kit of detection microdose urine protein, including
Following steps：Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant is extremely
Mix in Sample dilution, obtain sample to be tested；Sample to be detected is accurately drawn using pipettor, the nitre in reaction box circular hole is put
On acid cellulose face；After sample to be detected penetrates into, colloid gold label human serum albumins antibody, institute is added dropwise to reaction box face
The volume ratio that colloid gold label human serum albumins antibody is stated with the detection sample is 1：1；Treat colloid gold label human seralbumin
After protein antibodies penetrate into, cleaning solution is added dropwise on reaction box face, the cleaning solution is 1 with the volume ratio of the detection sample：
1；Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value, by instrument
Detection sample microdose urine protein concentration is calculated with reference to calibration card information, its mass concentration is quantitatively judged.
The invention provides a kind of spot gold diafiltration kit of detection microdose urine protein and its application, including：PH value
For cleaning solution that 6.5～6.7 Sample dilution, pH value are 6.2～6.4, colloid gold label human serum albumins antibody, urinate micro-
Amount albumin chromatography device and calibration card, the Sample dilution is slow including Triton-100, phosphate buffer and Tris
Rush liquor and；The cleaning solution includes bovine serum albumin(BSA) and phosphate buffer and Tris buffer solns.With it is existing
Have technology to compare, due to the surface of colloid gold particle it is negatively charged, can be with the positive electricity of microdose urine protein antibody protein molecule
Lotus group firm combination because electrostatic interaction is formed, while gold grain has the characteristic of high electron density again, when being marked by gold grain
Antibody combine with corresponding antigen ligand, gold grain just forms macroscopic red when antibody ligand site is assembled in a large number
Color spot point.The microdose urine protein spot gold diafiltration kit of the present invention has detection time short, and precision is good, to instrument and ring
The requirement in border is relatively low, the advantages of be easy to clinical application.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described with reference to embodiment, but
It should be appreciated that these descriptions are simply to further illustrate the features and advantages of the present invention, rather than to the claims in the present invention
Limit.
The embodiment of the invention discloses a kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is
6.5～6.7 Sample dilution, the cleaning solution that pH value is 6.2～6.4, colloid gold label human serum albumins antibody, urine are micro
Albumin chromatographs device and calibration card, and the Sample dilution includes Triton-100, phosphate buffer and Tris buffering
Liquor；The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
Dot immunogold filtration assay is to allow sample to be tested vertical penetration to cross microporous barrier (for example：Nitrocellulose filter), envelope
Upper coated capture probe capture, then allow collaurum mark probe to be percolated microporous barrier in the same fashion, tie with captured part
Aggregation after conjunction forms macroscopic punctation.Dot Immunogold Filtration Assay of the present invention based on affinity chromatography principle,
Nitrocellulose filter is adopted for solid phase carrier, to adsorb antibody on celluloid as solid phase, with antigen in human blood and
Antibody labeling collaurum is liquid phase, and while using antibody labeling collaurum as probe and indicator, and set up a kind of new
The immunologic detection method of type.The negatively charged positive electricity with microdose urine protein antibody protein molecule in surface of wherein colloid gold particle
Lotus group firm combination because electrostatic interaction is formed, while gold grain has the characteristic of high electron density again, when being marked by gold grain
Antibody combine with corresponding antigen ligand, gold grain just forms macroscopic red when antibody ligand site is assembled in a large number
Color spot point.
Preferably, the concentration of Triton-100 is 0.1～10% in the Sample dilution, phosphate buffer
The concentration of solution is 0.005～1mol/L, and the concentration of Tris buffer solns is 0.01～1mol/L；Ox blood in the cleaning solution
Clear albuminous concentration is 0.1～10%, and the concentration of phosphate buffer is 0.005～1mol/L, and Tris buffer solutions are molten
The concentration of liquid is 0.01～1mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO4 0.263-
0.376 weight portion, KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight of distilled water 1000
Part, the weight portion of bovine serum albumin(BSA) 7；The Tris buffer solns by trishydroxymethylaminomethane 4.6-5.5 weight portions,
KH2PO43.0-2.1 weight portions, the weight portions of NaCl 8, the weight portions of KCl 0.3, the weight portion of distilled water 1200, bovine serum albumin(BSA) 9
Weight portion is formulated.
Preferably, the colloid gold label human serum albumins antibody is prepared as follows：By quality point
Number for 1% gold chloride and distilled water by volume 1：79 are made into A liquid；By trisodium citrate, quality point that mass fraction is 1%
Number is 1% tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～0.080：15.84～
15.93 it is made into B liquid；The A liquid and B liquid are heated in water-bath 59～61 DEG C simultaneously, A liquid is stirred, in rapidly joining B liquid,
Continue to stir 1 minute, boiling was heated in 10～13 minutes, obtain collaurum liquid；Using 0.25M K2CO3Adjust the glue
Body gold liquid pH value is 6.8～7.1；By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations
60 minutes, Aspirate supernatant；The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is obtained
Human serum albumins antibody；The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the people
Seralbumin antibody is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, according to
1～30% sodium azide of secondary addition and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) and the colloid
The volume ratio of golden liquid is 500 μ l：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min,
Supernatant is removed, sediment is colloid gold label human serum albumins antibody.
Preferably, by trisodium citrate, the tannic acid that mass fraction is 1%, 25mM that mass fraction is 1%
K2CO3With distilled water by volume 4：0.08：0.08：15.84 are made into B liquid, and a diameter of 10 ＜ D ＜ are formed in the collaurum liquid
The colloid gold particle of 11nm.
Preferably, the reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, it is described anti-
Box is answered to include cassette bottom and lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, lower surface is a diameter of
The small sircle hole of 0.27cm；The full absorbent material of pad in the reaction box；Below the small sircle hole and above the absorbent material it
Between place 1.18 × 1.18cm coating human serum albumins antibody nitrocellulose filter, it is anabolic reaction to close tight lid
Box.The non-relevant areas of most of sample to be tested and label probe from beyond spot can be avoided to be lost in, affect detection sensitivity, can
So that the technology is widely used in clinical examination.
Preferably, the nitrocellulose filter of the coating human serum albumins antibody is prepared according to the following steps：Will
Human serum albumins coated antibody stoste is at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；Will
The supernatant loads bag filter, and with distilled water dialysis desalination, bottling freezing, obtains human serum albumins after ultrafiltration concentration
Antibody；The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, 0.8mg/ml is obtained
Coating buffer；The coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.02ml
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after being dried in case.
Preferably, the calibration card detects that color is corresponding by human serum albumins gradient mass concentration solution
Detected signal value.The gradient mass concentration be respectively preferably 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L,
120mg/L、200mg/L。
Accordingly, the present invention also provides a kind of application of detection microdose urine protein spot gold diafiltration kit, including with
Lower step：Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant is to sample
Mix in this dilution, obtain sample to be tested；Sample to be detected is accurately drawn using pipettor, the nitric acid in reaction box circular hole is put
On cellulose face；After sample to be detected penetrates into, colloid gold label human serum albumins antibody is added dropwise to reaction box face, it is described
Colloid gold label human serum albumins antibody is 1 with the volume ratio of the detection sample：1；Treat colloid gold label human seralbumin egg
After Bai Kangti penetrates into, cleaning solution is added dropwise on reaction box face, the cleaning solution is 1 with the volume ratio of the detection sample：1；
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value is tied by instrument
Close calibration card information and calculate detection sample microdose urine protein concentration, quantitatively judge its mass concentration.
From above scheme as can be seen that the present invention is simple and convenient, the color that device shows is chromatographed according to microdose urine protein, with
Correspondence batch targeted message, you can the mass concentration of quantitative material to be detected.
For a further understanding of the present invention, the technical scheme that the present invention is provided is carried out specifically with reference to embodiment
Bright, protection scope of the present invention is not limited by the following examples.
The raw material and chemical reagent that the embodiment of the present invention is adopted is commercial.
Embodiment 1
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.6 Sample dilution, pH
It is worth the cleaning solution for 6.3, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
5.6%, the concentration of phosphate buffer is 0.58mol/L, and the concentration of Tris buffer solns is 0.55mol/L；It is described
The concentration of bovine serum albumin(BSA) is 0.47% in cleaning solution, and the concentration of phosphate buffer is 0.49mol/L, and Tris is buffered
The concentration of liquor is 0.52mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.313 weight
Amount part, KH2PO41.05 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, bovine serum albumin
White 7 weight portion；The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 4.9, KH2PO42.45 weight portions, NaCl
8 weight portions, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
9500r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.055：0.051：15.88 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 60.5 DEG C, stir A liquid, it is quick plus
Enter B liquid, continue to stir 1 minute, boiling was heated in 30 seconds 11 minutes, make the collaurum of particle diameter 10.2nm, the glue
Body gold liquid color by it is black → blue → purple → red when；(2) step (1) collaurum liquid is used in the adjustment of collaurum pH value
0.25M K2CO3Adjustment pH value be 6.98 after it is standby；(3) preparation of human serum albumins antibody and dialysis, by antibody at 4 DEG C
9500r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing is standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum
Albumin antibody 0.288mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes,
Sequentially add the μ l of 10% sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 3.3 DEG C after stirring evenly, under the conditions of 6000r/min from
The heart 30 minutes, remove supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 50min, 3000r/min centrifugation 30min,
The μ L of Aspirate supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：Accurately inhaled with pipettor
25 μ L detection samples are taken, is put on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins is added dropwise
Antibody：After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) treat
After colloid gold label human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record
As a result：Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value passes through
Instrument combines calibration card information and calculates detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Embodiment 2
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.5 Sample dilution, pH
It is worth the cleaning solution for 6.2, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
0.%, the concentration of phosphate buffer is 0.05mol/L, and the concentration of Tris buffer solns is 0.01mol/L；It is described to wash
The concentration for washing bovine serum albumin(BSA) in liquid is 0.1%, and the concentration of phosphate buffer is 0.005mol/L, Tris buffer solutions
The concentration of solution is 0.01mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.263 weight
Part, KH2PO41.0 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, bovine serum albumin(BSA) 7
Weight portion；The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 4.6, KH2PO42.1 weight portions, the weights of NaCl 8
Amount part, the weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
6000r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.035：0.035：15.84 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 59 DEG C, stir A liquid, rapidly join
B liquid, continues to stir 1 minute, and boiling was heated in 10 minutes, makes the collaurum of particle diameter 8nm, the collaurum liquid color
By it is black → blue → purple → red when；(2) adjustment of collaurum pH value is by step (1) collaurum liquid 0.25M K2CO3Adjustment
PH value be 6.8 after it is standby；(3) preparation of human serum albumins antibody and dialysis, the 6000r/min centrifugations 60 at 4 DEG C by antibody
Minute, Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with distilled water dialysis desalination
Afterwards, bottling freezing, standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum albumins antibody
0.27mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes, sequentially adds 10%
The μ l of sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 2.5 DEG C after stirring evenly, are centrifuged 45 minutes under the conditions of 4600r/min, remove
Supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 15min, 2000r/min centrifugation 45min,
The μ L of Aspirate supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：Accurately inhaled with pipettor
25 μ L detection samples are taken, is put on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins is added dropwise
Antibody：After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) treat
After colloid gold label human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record
As a result：Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value passes through
Instrument combines calibration card information and calculates detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Embodiment 3
A kind of spot gold diafiltration kit of detection microdose urine protein, including：PH value is 6.7 Sample dilution, pH
It is worth the cleaning solution for 6.4, colloid gold label human serum albumins antibody, microdose urine protein chromatography device and calibration to block, the sample
This dilution includes Triton-100, phosphate buffer and Tris buffer solns；The cleaning solution includes cow's serum
Albumin, phosphate buffer and Tris buffer solns.The concentration of Triton-100 is in the Sample dilution
10%, the concentration of phosphate buffer is 1mol/L, and the concentration of Tris buffer solns is 1mol/L；In the cleaning solution
The concentration of bovine serum albumin(BSA) is 10%, and the concentration of phosphate buffer is 1mol/L, the concentration of Tris buffer solns
For 1mol/L.The phosphate buffer is preferably prepared by following raw material：Na2HPO40.376 weight portion, KH2PO4
1.109 weight portions, the weight portions of NaCl 6, the weight portions of KCl 0.2, the weight portion of distilled water 1000, the weight portion of bovine serum albumin(BSA) 7；
The Tris buffer solns are by the weight portion of trishydroxymethylaminomethane 5.5, KH2PO43.0 weight portions, the weight portions of NaCl 8,
The weight portions of KCl 0.3, the weight portion of distilled water 1200, the weight portion of bovine serum albumin(BSA) 9 are formulated.
The reaction box of a diameter of 2.8cm is provided with the microdose urine protein chromatography device, the reaction box includes cassette bottom
And lid, the lid top surface central authorities are provided with a diameter of 0.56cm in upper surface, the roundlet of a diameter of 0.27cm of lower surface
Hole；The full absorbent material of pad in the reaction box；One is placed between below the small sircle hole and above the absorbent material
The nitrocellulose filter of the coating human serum albumins antibody of 1.18 × 1.18cm, it is anabolic reaction box to close tight lid.
The nitrocellulose filter for being coated with human serum albumins antibody is prepared from according to the following steps：
(1) purifying of antibody, purifying and the dialysis of human serum albumins coated antibody, by antibody stoste at 4 DEG C
13000r/min is centrifuged 60 minutes, and Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with double steamings
After water dialysis desalination, bottling freezing, standby after ultrafiltration concentration；(2) it is molten with phosphate buffer and Tris buffer solutions
It is coating buffer that liquid dilutes above-mentioned antibody to 0.8mg/ml；(3) coating buffer is dropped on the nitrocellulose filter for cutting, per
Diaphragm 0.02ml, diaphragm is placed in after being dried in baking oven and makes the nitrocellulose filter for being coated with human serum albumins antibody.
The colloid gold label human serum albumins antibody is prepared from according to the following steps：(1) preparation of collaurum is with matter
Amount fraction is respectively 1% gold chloride, 1% trisodium citrate, 1% tannic acid, 25mM K2CO3And distilled water is raw material, first by 1% chlorine
Auric acid presses volume 1 with distilled water：79 are made into A liquid；By 1% trisodium citrate, 1% tannic acid, 25mMK2CO3Volume is pressed with distilled water
4：0.08：0.08：15.93 it is made into B liquid；Again by A liquid, B liquid in water-bath while heated to 61 DEG C, stir A liquid, rapidly join B
Liquid, continues to stir 1 minute, and boiling was heated in 13 minutes, makes the collaurum of particle diameter 12nm, the collaurum liquid color
By it is black → blue → purple → red when；(2) adjustment of collaurum pH value is by step (1) collaurum liquid 0.25M K2CO3Adjustment
PH value be 7.1 after it is standby；(3) preparation of human serum albumins antibody and dialysis, the 13000r/min centrifugations 60 at 4 DEG C by antibody
Minute, Aspirate supernatant is human serum albumins antibody；The antibody is loaded into bag filter, with distilled water dialysis desalination
Afterwards, bottling freezing, standby；(4) colloid gold label human serum albumins antibody, by step (3) human serum albumins antibody
0.30mg, is slowly added to be coated with the 100ml collaurum liquid of step (2) preparation, after stirring 15 minutes, sequentially adds 10%
The μ l of sodium azide 500, the μ l of 1% bovine serum albumin(BSA) 100, at 4.0 DEG C after stirring evenly, are centrifuged 19 minutes under the conditions of 8000r/min, remove
Supernatant, colloid gold label human serum albumins antibody.
One kind detection microdose urine protein spot gold diafiltration kit detection method, operates according to the following steps：(1) treat
The collection of inspection human urine：Middle segment urine sample is collected with centrifuge tube, is mixed, put room temperature and place 2h, 4000r/min centrifugation 20min, drawn
The μ L of supernatant 25 mix as detection sample in Sample dilution；(2) sample application is detected：25 μ are accurately drawn with pipettor
L detects sample, puts on celluloid face in reaction box circular hole；(3) colloid gold label human serum albumins antibody is added dropwise：
After sample to be detected penetrates into, then 25 μ L colloid gold label human serum albumins antibody are added dropwise toward reaction box face；(4) collaurum is treated
After mark human serum albumins antibody penetrates into, then 25 μ L cleaning solutions are added dropwise toward on reaction box face；(5) detect and record result：
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value is tied by instrument
Close calibration card information and calculate detection sample microdose urine protein concentration, you can quantitatively judge its mass concentration.
Upper embodiment and embodiment are both needed to the usage amount that appropriateness as the case may be adjusts temperature and distilled water, this explanation
It is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that for the ordinary skill people of the art
Member for, under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention, these improve and
Modification is also fallen in the protection domain of the claims in the present invention.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or using the present invention.
Various modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The most wide scope for causing.
Claims (10)
1. a kind of spot gold of detection microdose urine protein is percolated kit, it is characterised in that include：
Cleaning solution, colloid gold label human serum albumins that Sample dilution that pH value is 6.5～6.7, pH value are 6.2～6.4
Antibody, microdose urine protein chromatography device and calibration card,
The Sample dilution includes Triton-100, phosphate buffer and Tris buffer solns；
The cleaning solution includes bovine serum albumin(BSA), phosphate buffer and Tris buffer solns.
2. spot gold according to claim 1 is percolated kit, it is characterised in that
The concentration of Triton-100 is 0.1～10% in the Sample dilution, and the concentration of phosphate buffer is 0.005
The concentration of～1mol/L, Tris buffer soln is 0.01～1mol/L；
The concentration of bovine serum albumin(BSA) is 0.1～10% in the cleaning solution, the concentration of phosphate buffer is 0.005～
The concentration of 1mol/L, Tris buffer soln is 0.01～1mol/L.
3. spot according to claim 1 gold diafiltration kit, it is characterised in that the phosphate buffer by with
Lower raw material is prepared：Na2HPO40.263-0.376 weight portions, KH2PO41.109-1.000 weight portions, the weight portions of NaCl 6,
KCl0.2 weight portions, the weight portion of distilled water 1000, the weight portion of bovine serum albumin(BSA) 7；The Tris buffer solns are by three hydroxyl first
Base aminomethane 4.6-5.5 weight portions, KH2PO43.0-2.1 weight portions, the weight portions of NaCl 8, the weight portions of KCl 0.3, distilled water
1200 weight portions, the weight portion of bovine serum albumin(BSA) 9 are formulated.
4. spot gold according to claim 1 is percolated kit, it is characterised in that the colloid gold label human seralbumin egg
Bai Kangti is prepared as follows：
By gold chloride that mass fraction is 1% and distilled water by volume 1：79 are made into A liquid；By the lemon that mass fraction is 1%
Sour trisodium, mass fraction are 1% tannic acid, 25mM K2CO3With distilled water by volume 4：0.035～0.080：0.035～
0.080：15.84～15.93 are made into B liquid；The A liquid and B liquid are heated in water-bath 59～61 DEG C simultaneously, A liquid is stirred, soon
Speed is added in B liquid, continues to stir 1 minute, and boiling was heated in 10～13 minutes, obtains collaurum liquid；
Using 0.25M K2CO3The collaurum liquid pH value is adjusted for 6.8～7.1；
By human serum albumins antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, Aspirate supernatant；
The supernatant is loaded into bag filter, after distilled water dialysis desalination, bottling freezing obtains human serum albumins antibody；
The human serum albumins antibody is slowly added to be marked in the collaurum liquid, the human serum albumins antibody
It is 0.27～0.30mg with the mass volume ratio of the collaurum liquid：100ml, after stirring 15 minutes, sequentially adds 1～30% and changes
Nitrogen sodium and 0.1～10% bovine serum albumin(BSA), the sodium azide, bovine serum albumin(BSA) are with the volume ratio of the collaurum liquid
500μl：100μl：100ml, at 2.5～4.0 DEG C after stirring evenly, is centrifuged 30 minutes under the conditions of 6000r/min, removes supernatant, sinks
Starch is colloid gold label human serum albumins antibody.
5. spot gold according to claim 4 is percolated kit, it is characterised in that the trisodium citrate, tannic acid, K2CO3
It is 4 with the volume ratio of distilled water：0.08：0.08：15.84, the colloid of a diameter of 10 ＜ D ＜ 11nm is formed in the collaurum liquid
Gold grain.
6. spot gold according to claim 1 is percolated kit, it is characterised in that in the microdose urine protein chromatography device
The reaction box of a diameter of 2.8cm is provided with, the reaction box includes cassette bottom and lid, and the lid top surface central authorities are provided with one
The a diameter of 0.56cm in upper surface, the small sircle hole of a diameter of 0.27cm of lower surface；The full absorbent material of pad in the reaction box；Described
The coating human serum albumins antibody of a 1.18 × 1.18cm is placed between small sircle hole lower section and absorbent material top
Nitrocellulose filter.
7. spot gold according to claim 6 is percolated kit, it is characterised in that the coating human serum albumins antibody
Nitrocellulose filter according to following steps prepare：
By human serum albumins coated antibody stoste at 4 DEG C with 6000～13000r/min centrifugations 60 minutes, in absorption
Clear liquid；
The supernatant is loaded into bag filter, with distilled water dialysis desalination, bottling freezing, obtains human serum after ultrafiltration concentration
Albumin antibody；
The human serum albumins antibody is diluted with phosphate buffer and Tris buffer solns, obtains 0.8mg/ml's
Coating buffer；
The coating buffer is dropped on the nitrocellulose filter for cutting, every diaphragm 0.02ml is placed in diaphragm in baking oven
Obtain being coated with the nitrocellulose filter of human serum albumins antibody after drying.
8. spot gold according to claim 1 is percolated kit, it is characterised in that the calibration card is human serum albumins
Gradient mass concentration solution detects the corresponding detected signal value of color.
9. spot gold according to claim 8 is percolated kit, it is characterised in that the gradient mass concentration is respectively
5mg/L、10mg/L、20mg/L、40mg/L、80mg/L、120mg/L、200mg/L。
10. the application of the spot gold diafiltration kit of a kind of detection microdose urine protein, it is characterised in that comprise the following steps：
Collection mud-stream urine, room temperature places 15min-2h, and with 3000r/min centrifugation 30min, Aspirate supernatant to sample is dilute
Release in liquid and mix, obtain sample to be tested；
Sample to be detected is accurately drawn using pipettor, is put on celluloid face in reaction box circular hole；
After sample to be detected penetrates into, colloid gold label human serum albumins antibody, the collaurum mark is added dropwise to reaction box face
Note human serum albumins antibody is 1 with the volume ratio of the detection sample：1；
After colloid gold label human serum albumins antibody penetrates into, be added dropwise cleaning solution on reaction box face, the cleaning solution with
The volume ratio of the detection sample is 1：1；
Observing response box face, after cleaning solution fully penetrates into face, detection microdose urine protein chromatography device signal value, by instrument
Device combines calibration card information and calculates detection sample microdose urine protein concentration, quantitatively judges its mass concentration.
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| CN201611094803.3A Pending CN106855573A (en) | 2016-08-23 | 2016-12-02 | Detection PSA spot gold diafiltration kit and its detection method |
| CN201611094793.3A Pending CN106771144A (en) | 2016-08-23 | 2016-12-02 | One kind detection C reactive proteins dot immunogold diafiltration kit and quantitative detecting method |
| CN201611095552.0A Pending CN106596968A (en) | 2016-08-23 | 2016-12-02 | Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit |
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Cited By (2)
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|---|---|---|---|---|
| CN108303550A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of the spot gold diafiltration kit and its quantitative detecting method of detection adiponectin |
| CN113009150A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting urine microalbumin in sweat, detection kit comprising same and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112485449A (en) * | 2020-11-23 | 2021-03-12 | 爱若维生物科技(苏州)有限公司 | Spot immunogold filtration kit for detecting cat SAA and semi-quantitative detection method |
| CN113588610B (en) * | 2021-07-21 | 2024-02-02 | 重庆创芯生物科技有限公司 | Filtering membrane pretreatment liquid for improving immunofluorescence detection sensitivity, and preparation method and application thereof |
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| CN106771144A (en) | 2017-05-31 |
| CN106855573A (en) | 2017-06-16 |
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