CN208476939U - A kind of chemical luminescence reagent kit detecting EGFR - Google Patents
A kind of chemical luminescence reagent kit detecting EGFR Download PDFInfo
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- CN208476939U CN208476939U CN201821240347.3U CN201821240347U CN208476939U CN 208476939 U CN208476939 U CN 208476939U CN 201821240347 U CN201821240347 U CN 201821240347U CN 208476939 U CN208476939 U CN 208476939U
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Abstract
The utility model discloses a kind of chemical luminescence reagent kits for detecting EGFR, and including the reagent bottle in kit body, kit body, kit body includes box body, box cover, and box cover the cover is on box body；Kit body is PMMA material, it is sturdy and durable, it will not damage easily during transportation, box cover upper surface is equipped with the first notch, box body lower surface is equipped with non-slip mat, non-slip mat matches with the first notch, when several kits are stacked, save space, it is readily transported, equipped with resilient cushion and fixed pad, it can play the role of fixed and protect reagent bottle, coated EGFR antibody and sample to be tested form the sandwich complex structure of " solid-phase coating antibody-determined antigen-enzyme labelled antibody " on the EGFR antibody and microwell plate of the utility model horseradish peroxidase-labeled, with horseradish peroxidase enzyme catalytic luminous substrate luminol, sensitivity greatly improves, it can be provided for clinical diagnosis more special, quickly, reliable foundation.
Description
Technical field
The utility model relates to medical immunology external diagnosis reagent fields, and in particular to a kind of chemiluminescence for detecting EGFR
Kit.
Background technique
EGF-R ELISA (Epidermal Growth Factor Receptor；It EGFR) is epidermal growth factor
(EGF) receptor of cell Proliferation and signal transduction.EGFR is a kind of glycoprotein, belongs to tyrosine kinase receptor, and cell membrane passes through
It is logical, molecular weight 170kDa.During research and production, needs to measure EGFR protein content in solution and change, carry out monitoring experiment
Condition and monitoring industrial processes.It is thin that EGFR is distributed widely in mammalian epithelial cell, fibroblast, spongiocyte, cutin
The cell surfaces such as born of the same parents, EGFR signal path play an important role to physiology courses such as the growth of cell, proliferation and differentiation.EGFR
The activity or cellular localization of key factor are abnormal in equal protein tyrosine kinases afunction or its associated signal paths, can draw
Play the generation of tumour, diabetes, immune deficiency and cardiovascular disease.
EGFR measuring method has colloidal gold immunochromatographimethod (GICA) at present；Western blotting (WB)；Enzyme-linked Immunosorbent Assay examination
It tests (ELISA), chemical luminescence method immune analysis (CLIA)；The advantages of GICA, ELISA, WB is less expensive, the disadvantage is that clever
Sensitivity is low, examines and does not measure in the case that EGFR protein content is few, and immunoassay of the CLIA as on-radiation has sensitivity
High, the features such as required time is short, pollution-free, detection range is wide, as various automatic luminous analysis instruments emerge and inhomogeneity
The continuous release of the chemical luminescence immune analysis reagent box of type, becomes the substitution of time resolution, radio-immunity, ELISA
Person, but it is also seldom for the chemical luminescence reagent kit of EGFR currently on the market.
Utility model content
The purpose of this utility model is to provide a kind of chemical luminescence reagent kits for detecting EGFR, have filled up EGFR in the market
Chemical luminescence reagent kit blank, have many advantages, such as that testing result high sensitivity, detection range are wide.
To achieve the above object, the utility model provides the following technical solutions:
A kind of chemical luminescence reagent kit detecting EGFR, including the reagent bottle in kit body, kit body and admittedly
Phase carrier, the reagent bottle include 2 large size reagent bottles, 3 medium size reagent bottles, 8 small size reagent bottles, 2 large size reagent bottles
In be respectively provided with concentrated cleaning solution, dilution, 3 medium size reagent bottles are respectively provided with enzyme labelled antibody, Chemoluminescent substrate A, change
Luminous substrate liquid B is learned, 8 small size reagent bottles are respectively provided with positive control, negative control, 6 standard solutions, the kit
Box body includes box body, box cover, and for the box cover the cover on box body, lid inner surface is equipped with a circle gasket, box cover appearance
Four vertex in face are equipped with the first notch, and the section of the first notch is square structure, and the height of the first notch is d1, box sheet
Internal portion is equipped with fixed pad, resilient cushion, and 13 reagent bottle holes, fixed pad, resilient cushion examination are equipped on the fixed pad, resilient cushion
The position in agent bottle hole is corresponding, and resilient cushion is located at fixed pad lower section, and fixed pad is connected with resilient cushion by the first connection strap, box
The vertex of ontology lower surface four is equipped with non-slip mat, and the section of non-slip mat is square structure, the size of non-slip mat, shape and the
One notch matches, and the height of non-slip mat is d2, d2=d1.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the kit body is PMMA material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the gasket is rubber material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the resilient cushion, fixed pad, the first connection strap are
Foam.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the non-slip mat is PVC material, PU material, silica gel
One of material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the solid phase carrier is the 96 of polystyrene material
Hole microtiter plate.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the solid phase carrier is coated with EGFR antibody, the enzyme
Labeling antibody be horseradish peroxidase-labeled EGFR antibody, Chemoluminescent substrate A be containing 5ug/mL to iodophenol,
The Tris-HCl buffer (0.1M, pH=8.5) of 5.0mg/mL luminol, Chemoluminescent substrate B are to contain 100mg/mL mistake
The citrate buffer solution (0.1M, pH=4.5) of hydrogen oxide, 15mg/mL horseradish peroxidase.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the dilution is by 100ml bovine serum albumin
Thimerosal that white, 20ml weight percent concentration is 1%, 10ml concentration are the 0.1M potassium ferricyanide, the Tween-20 of 0.5ml, 10ml
Gentamicin that concentration is 10mg/ml, surplus are that PBS buffer solution (0.01M, pH=7.4) is settled to what 1000ml was mixed to get
Solution.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein it is 2.96g that the concentrated cleaning solution, which is by weight,
NaH2PO4·2H2O, weight is the Na of 29g2HPO4·12H2O, weight is NaCl the and 20ml Tween-20 of 234g, adds distilled water
It is settled to the solution that 1000ml is mixed to get.
A kind of above-mentioned chemical luminescence reagent kit for detecting EGFR, wherein the positive control is prepared by EGFR positive serum,
The negative control is prepared by Healthy Human Serum, the standard solution be 6 concentration gradients EGFR antigen, the concentration according to
It is secondary are as follows: 0ng/ml, 50ng/ml, 100ng/ml, 250ng/ml, 500ng/ml and 1000ng/ml.
This kit using when also need to use coating buffer, confining liquid, the coating buffer is carbonate buffer solution
(0.03mol/L, pH=9.6), the confining liquid are to be by 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration
The solution that 0.03mol/L phosphate solution (pH=7.4) is mixed to get.
The utility model has EGFR antibody and microwell plate the utility model has the advantages that the utility model horseradish peroxidase-labeled
Upper coated EGFR antibody and sample to be tested form the sandwich complex knot of " solid-phase coating antibody-determined antigen-enzyme labelled antibody "
Structure, therefore the double antibody sandwich method that the utility model uses not only had efficiently utilized chemiluminescence principle, but also have applied horseradish
Catalyzed Synthesis By Peroxidase luminous substrate luminol, the optical signal that detection luminol generates, sensitivity greatly improves, than enzyme now
Join immuning adsorpting analysis sensitivity and improve about 10 times, more special, quick, reliable foundation can be provided for clinical diagnosis.
Detailed description of the invention
FIG. 1 is a schematic structural view of the utility model；
Fig. 2 is the top view of Fig. 1；
Fig. 3 is box cover upper surface schematic diagram；
Fig. 4 is the bottom view of Fig. 3；
Fig. 5 is fixed pad, resilient cushion, the first connection strap schematic diagram；
Fig. 6 is reagent bottle schematic diagram；
In figure: 1- box body, 2- large size reagent bottle, 3- medium size reagent bottle, 4- trumpet reagent bottle, 5- box cover, 6- gasket,
The first notch of 7-, the fixed pad of 8-, 9- resilient cushion, 10- reagent bottle hole, the first connection strap of 11-, 12- non-slip mat.
Specific embodiment
The following will be combined with the drawings in the embodiments of the present invention, carries out the technical scheme in the embodiment of the utility model
Clearly and completely describe.
Experimental method used in embodiment is conventional method unless otherwise specified；Material used in embodiment,
Reagent etc., is commercially available unless otherwise specified.
Embodiment 1, the structure of chemical luminescence reagent kit
As shown in figures 1 to 6, a kind of chemical luminescence reagent kit detecting EGFR, including in kit body, kit body
Reagent bottle and solid phase carrier, the reagent bottle includes 2 large size reagent bottles, 3 medium size reagent bottles, 8 small size reagent bottles, 2
Concentrated cleaning solution, dilution are respectively provided in a large size reagent bottle, 3 medium size reagent bottles are respectively provided with enzyme labelled antibody, chemistry hair
Light substrate solution A, Chemoluminescent substrate B, it is molten that 8 small size reagent bottles are respectively provided with positive control, negative control, 6 standard items
Liquid, the kit body include box body 1, box cover 5, and for the box cover the cover on box body, lid inner surface is equipped with a circle
Gasket 6, four vertex of lid outer surface are equipped with the first notch 7, and the section of the first notch is square structure, and first lacks
The height of mouth is d1, and fixed pad 8, resilient cushion 9 are equipped with inside box body, is equipped with 13 reagents on the fixation pad, resilient cushion
Bottle hole 10, fixed pad, the position in resilient cushion reagent bottle hole are corresponding, and resilient cushion is located at fixed pad lower section, fixed pad and resilient cushion
It is connected by the first connection strap 11, the vertex of box body lower surface four is equipped with non-slip mat 12, and the section of non-slip mat is pros
Shape structure, size, the shape of non-slip mat match with the first notch, and the height of non-slip mat is d2, d2=d1.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the kit body is PMMA material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the gasket is rubber material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the resilient cushion, fixed pad, the first connection strap are
Foam.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the non-slip mat is PVC material, PU material, silica gel
One of material.
Above-mentioned a kind of chemical luminescence reagent kit for detecting EGFR, wherein the solid phase carrier is the 96 of polystyrene material
Hole microtiter plate.
This kit body is PMMA material, more sturdy and durable, will not be damaged easily during transportation, table on box cover
Face is equipped with the first notch, and box body lower surface is equipped with non-slip mat, and non-slip mat matches with the first notch, and several kits are stacked on
When together, space is on the one hand saved, on the other hand will not collapse easily, be readily transported, lid inner surface is equipped with gasket, reagent
It is equipped with resilient cushion and fixed pad in box box body, can play the role of fixed and protects reagent bottle.
The composition of embodiment 2, chemical luminescence reagent kit
This kit includes stating concentrated cleaning solution, dilution, enzyme labelled antibody, Chemoluminescent substrate A, chemiluminescent substrate
Liquid B, positive control, negative control, standard solution, coating buffer, confining liquid.
The solid phase carrier is coated with EGFR antibody, and the enzyme labelled antibody is the EGFR antibody of horseradish peroxidase-labeled,
Chemoluminescent substrate A is containing 5ug/mL to Tris-HCl buffer (0.1M, pH=of iodophenol, 5.0mg/mL luminol
8.5), Chemoluminescent substrate B is the lemon acid buffering containing 100mg/mL hydrogen peroxide, 15mg/mL horseradish peroxidase
Liquid (0.1M, pH=4.5).
The dilution is by 100ml bovine serum albumin(BSA), the thimerosal that 20ml weight percent concentration is 1%, 10ml
Concentration is the 0.1M potassium ferricyanide, the Tween-20 of 0.5ml, 10ml concentration are 10mg/ml gentamicin, surplus are PBS buffer solution
(0.01M, pH=7.4) is settled to the solution that 1000ml is mixed to get.
The concentrated cleaning solution is the NaH for being 2.96g by weight2PO4·2H2O, weight is the Na of 29g2HPO4·12H2O、
Weight is NaCl the and 20ml Tween-20 of 234g, and distilled water is added to be settled to the solution that 1000ml is mixed to get.
The positive control is prepared by EGFR positive serum, and the negative control is prepared by Healthy Human Serum, the standard
Product solution is the EGFR antigen of 6 concentration gradients, and the concentration is successively are as follows: 0ng/ml, 50ng/ml, 100ng/ml, 250ng/
Ml, 500ng/ml and 1000ng/ml.
This kit using when also need to use coating buffer, confining liquid, the coating buffer is carbonate buffer solution
(0.03mol/L, pH=9.6), the confining liquid are to be by 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration
The solution that 0.03mol/L phosphate solution (pH=7.4) is mixed to get.
Embodiment 3, the preparation method of kit
Positive control is prepared with EGFR positive serum, negative control is prepared with Healthy Human Serum, is matched with EGFR positive serum
Standard items processed, microtiter plate are coated with EGFR antibody, and horseradish peroxidase-labeled EGFR antibody prepares Chemoluminescent substrate
A and B prepares concentrated cleaning solution and dilution, dispenses above-mentioned component, and be assembled into finished product.
(1) be coated with: it is anti-to be configured to the EGFR that peridium concentration is 2ug/ml with the carbonate buffer solution that 30mM pH value is 9.6
Body be coated with buffer, and by coating buffer be carried in microtiter plate (hole 100ul/), 2-8 DEG C coating 18-24 hours.
(2) it closes: being that 0.03mol/L phosphate is molten with 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration
The confining liquid that liquid (pH=7.4) is mixed to get closes the microtiter plate after above-mentioned washing.
(3) enzyme labelled antibody: taking HRP5mg to be dissolved in 0.2mol/L, and in pH=5.6 acetate buffer 1ml, 1%DNFB is added
Ethanol solution 0.1ml, gentle agitation 1h, is added the 0.1mol/LNaIO of fresh configuration at room temperature40.5ml, at this time solution
Become blackish green from former brown, 4 DEG C of placement 30min, solution becomes blackish green from former brown at this time, and 2.5% ethylene glycol is added
1ml, gentle agitation 1h, terminates reaction, antibody 5-10mg to be marked is added at room temperature, and with 1mol/L, pH=9.5 carbonate is slow
Fliud flushing adjusts pH value to 9.0, mixes, set 4 DEG C overnight；Sodium borohydride solution 0.1ml is added, mixes, 4 DEG C of placement 3h are right
0.01mol/L, pH=7.4 phosphate buffer, 4 DEG C of dialysed overnights are changed liquid 3 times, and 3000r/min is centrifuged 30min, removal precipitating
Object, collecting supernatant is enzyme labelled antibody.
(4) chemiluminescent substrate: Chemoluminescent substrate A is containing 5ug/mL to iodophenol, 5.0mg/mL luminol
Tris-HCl buffer (0.1M, pH=8.5), Chemoluminescent substrate B are peppery containing 100mg/mL hydrogen peroxide, 15mg/mL
The citrate buffer solution (0.1M, pH=4.5) of root catalase.
Embodiment 4, the application method of this kit
(1) microtiter plate after taking out coating in 2-8 DEG C of refrigerator, is placed at room temperature for 15 minutes.
(2) each 2 hole of positive control, negative control, standard items every hole 50ul, sample to be tested 50ul are set, 37 after oscillation mixing
DEG C incubate 30 minutes.
(3) dereaction liquid is got rid of, every hole is filled it up with the cleaning solution after dilution, board-washing 5 times, finally buckled on clean blotting paper
It is dry.
(4) every hole is separately added into 100uL enzyme labelled antibody, and 37 DEG C incubate 20 minutes.
(5) dereaction liquid is got rid of, every hole is filled it up with the cleaning solution after dilution, board-washing 5 times, finally buckled on clean blotting paper
It is dry.
(6) every hole adds each 50 μ L of Chemoluminescent substrate A, B, is sufficiently vibrated and is uniformly mixed with micro oscillator, room temperature (20
~25 DEG C) it is protected from light 5 minutes.
(7) it must measure in adding in the after Chemoluminescent substrate the 5th~20 minute, be measured on chemiluminescence measuring instrument
The luminous intensity (RLU) in each hole, 1 second/hole of time of measuring.
While there has been shown and described that the embodiments of the present invention, for the ordinary skill in the art,
It is understood that these embodiments can be carried out with a variety of variations in the case where not departing from the principles of the present invention and spirit, repaired
Change, replacement and variant, the scope of the utility model is defined by the appended claims and the equivalents thereof.
Claims (6)
1. a kind of chemical luminescence reagent kit for detecting EGFR, which is characterized in that including the examination in kit body, kit body
Agent bottle and solid phase carrier, the reagent bottle include 2 large size reagent bottles, 3 medium size reagent bottles, 8 small size reagent bottles, and 2 greatly
It is respectively provided with concentrated cleaning solution, dilution in number reagent bottle, 3 medium size reagent bottles are respectively provided with enzyme labelled antibody, chemiluminescence bottom
Thing liquid A, Chemoluminescent substrate B, 8 small size reagent bottles are respectively provided with positive control, negative control, 6 standard solutions, institute
Stating kit body includes box body, box cover, and for the box cover the cover on box body, lid inner surface is equipped with a circle gasket,
Four vertex of lid outer surface are equipped with the first notch, and the section of the first notch is square structure, the height of the first notch
For d1, box body inside is equipped with fixed pad, resilient cushion, and the fixation pads, is equipped with 13 reagent bottle holes on resilient cushion, fixed
Pad, the position in resilient cushion reagent bottle hole are corresponding, and resilient cushion is located at fixed pad lower section, and fixed pad is connect with resilient cushion by first
Item is connected, and the vertex of box body lower surface four is equipped with non-slip mat, and the section of non-slip mat is square structure, non-slip mat it is big
Small, shape matches with the first notch, and the height of non-slip mat is d2, d2=d1.
2. a kind of chemical luminescence reagent kit for detecting EGFR as described in claim 1, which is characterized in that the kit body
For PMMA material.
3. a kind of chemical luminescence reagent kit for detecting EGFR as described in claim 1, which is characterized in that the gasket is rubber
Glue material matter.
4. a kind of chemical luminescence reagent kit for detecting EGFR as described in claim 1, which is characterized in that the resilient cushion is consolidated
Fixed pad, the first connection strap are foam.
5. a kind of chemical luminescence reagent kit for detecting EGFR as described in claim 1, which is characterized in that the non-slip mat is
One of PVC material, PU material, silica gel material.
6. a kind of chemical luminescence reagent kit for detecting EGFR as described in claim 1, which is characterized in that the solid phase carrier is
96 hole microtiter plates of polystyrene material.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201821240347.3U CN208476939U (en) | 2018-08-02 | 2018-08-02 | A kind of chemical luminescence reagent kit detecting EGFR |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201821240347.3U CN208476939U (en) | 2018-08-02 | 2018-08-02 | A kind of chemical luminescence reagent kit detecting EGFR |
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| Publication Number | Publication Date |
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| CN208476939U true CN208476939U (en) | 2019-02-05 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109100512A (en) * | 2018-08-02 | 2018-12-28 | 宁波奥丞生物科技有限公司 | A kind of chemical luminescence reagent kit detecting EGFR |
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2018
- 2018-08-02 CN CN201821240347.3U patent/CN208476939U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109100512A (en) * | 2018-08-02 | 2018-12-28 | 宁波奥丞生物科技有限公司 | A kind of chemical luminescence reagent kit detecting EGFR |
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