CN109085335A - The immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 - Google Patents
The immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 Download PDFInfo
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- CN109085335A CN109085335A CN201810969563.XA CN201810969563A CN109085335A CN 109085335 A CN109085335 A CN 109085335A CN 201810969563 A CN201810969563 A CN 201810969563A CN 109085335 A CN109085335 A CN 109085335A
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- kit
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/54346—Nanoparticles
Abstract
The invention discloses the immunofluorescence technique kits of quantitative detection blood vessel endothelium marker CD146, including kit body and the test strips being set in kit body, the kit body includes box body, lid, test strips include bottom plate, water absorption pad, nitrocellulose coated film, bonding pad and sample pad, quantum dot-labeled CD146 monoclonal antibody I is coated on bonding pad, it is coated with CD146 monoclonal antibody II in detection line, rabbit-anti mouse lgG is coated on nature controlling line；Immunofluorescent reagent box of the present invention uses quantum dot-labeled technology, quantum dot is modified with ethyl thioglycollic acid, it ensure that the stability of quantum dot-labeled antibody, have many advantages, such as that testing result high sensitivity, detection time are short, specific good, kit exquisiteness of the present invention is light, easy to carry, there is no rigid requirement to operationlocation and environment, simple and quick, detecting step is simple, has quantitative detection function, is suitable for market further genralrlization and uses.
Description
Technical field
The present invention relates to external diagnosis reagent fields, and in particular to quantitative detection blood vessel endothelium marker CD146's is immune
Fluorescence method kit.
Background technique
Angiogenesis suffers from extremely important meaning to the growth, transfer or even prognosis of malignant entity tumor.State at present
Inherence research tumour in new vessels when, common vascular endothelial cell marker include CD34, CD31, CD146, CD146 and
VWF etc., angiogenesis are that malignant entity tumor is broken through after basement membrane of epithelium necessary to further growth, are newly formed in tumour
Blood vessel has its own unique design feature, and it is imperfect to show as tube wall, no smooth muscle elements, only by porose endothelial cell
Constituted with the basement membrane of sheet, most scholars think, the design feature of new vessels make malignant tumor tissue occur DISTANT METASTASES IN at
It is possible.Therefore, quantifying for new vessels is considered as a kind of important independent prognostic marker in malignant entity tumor.
CD146 is a kind of membrane glycoprotein of calcium independence cell adhesion molecule, contactin at
Member, the interaction between mediated cell and cell or between cell and extracellular matrix, CD146 selectivity in new vessels
Height expression, participates in the generation of new vessels, provides necessary nutrition for the growth and transfer of tumour, CD146 can promote tumour raw
Long, hematogenous metastasis, it is closely related with invasion and metastasis of tumor, it can be used as the Research of predicting markers of invasion and metastasis of tumor, CD146 exists
It plays an important role in Tumor Angiongesis, generation, development and transfer, and is likely to become the reference index of tumor prognosis judgement.
Currently, the method for detection vascular endothelial cell marker CD146 is presently mainly Immunohistochemical Method, Immunohistochemical Method
It is mainly used for qualitative analysis, sensitivity is lower, and disturbing factor is more, and repeatability is bad, and detection time is long.Therefore Immunohistochemical Method
Be not suitable for clinic quick diagnosis, how to produce quick quantitative detection equipment and urgently solved the problems, such as needs.
Summary of the invention
The purpose of the present invention is to provide the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146, tools
Have emission spectrum narrow range and it is symmetrical, Stocks displacement is big, ambient noise is small, high sensitivity, detection time are short, specific good
The advantages that.
To achieve the above object, the invention provides the following technical scheme: quantitative detection blood vessel endothelium marker CD146's exempts from
Epidemic disease fluorescence method kit, including kit body and the test strips being set in kit body, the kit body packet
Box body, lid are included, box body, lid are connected by buckle, and the lid inner surface is equipped with the bolt for being used to fix lid and box body,
Bolt is symmetrical set, and for bolt between the upper and lower every 3 pairs of setting, cartridge inner surface is equipped with the jack being adapted with bolt, the lid
Body is equipped with well, observation window, and well is circular through hole structure, and observation window is rectangular through holes structure, lid upper surface
Equipped with project label, detection mark, lid inner surface is equipped with the detent protrusion for fixing test strips, and the cartridge inner surface is set
There are a baffle, strip projected parts, baffle includes the lagging of upper and lower ends and the perpendicular baffle that connect vertically with lagging, the strut rail
Plate and perpendicular baffle form semi-closed structure, and the test strips are located in the semi-closed structure of upper and lower ends, and the strip projected parts are left
The right side is symmetrical arranged, and the distance between strip projected parts are equal to the width of test strips；
The test strips include bottom plate, water absorption pad, nitrocellulose coated film, bonding pad and sample pad, the nitric acid
Cellulose coated film is covered in the intermediate position of the bottom plate, and detection line separately is provided on nitrocellulose coated film
And nature controlling line, the detection line is close to the bonding pad, nature controlling line package amount on the water absorption pad, the bonding pad
Son puts the CD146 monoclonal antibody I of label, is coated with CD146 monoclonal antibody II in the detection line, is coated on nature controlling line
Rabbit-anti mouse lgG.
Preferably, the sample-adding nose end two sides of the kit body are equipped with waveform sawtooth.
Preferably, the quantum dot is CdSe quantum dots or cadmiumsulfide quantum dot.
Preferably, the bonding pad is polyurethane material.
Preferably, the sample pad is glass fibre element film.
Preferably, the difference of the CD146 monoclonal antibody I and CD146 monoclonal antibody II is to combine CD146 antigen
Site it is different.
Preferably, the water absorption pad, nitrocellulose coated film, bonding pad, between sample pad, successively with and only with it is adjacent
Position is in contact and partly overlaps.
The immunofluorescence technique kit of above-mentioned quantitative detection blood vessel endothelium marker CD146 includes following preparation step:
(1) it prepares quantum dot-labeled CD146 monoclonal antibody I: taking latex particle that PBS buffer solution (0.02M, pH is added
=7.2) in, centrifuge centrifugation；Remove supernatant, precipitating particulate matter is resuspended with PBS buffer solution (0.02M, pH=7.2), obtains
Latex particle solution, 4 DEG C save for use；Latex particle solution is dilute by latex particle with MES buffer (0.01M, pH=6.1)
It releases to 10mg/ml, ethyl thioglycollic acid then is added in the latex particle solution after dilution, after mixing, centrifuge centrifugation is discarded
Clearly, and with PBS buffer solution (0.02M, pH=7.2) precipitating particulate matter is resuspended, centrifuge is centrifuged again later, is discarded supernatant, and is sunk
Shallow lake particulate matter redissolves in 0.02M, the PBS buffer solution of pH7.4, obtains ethyl thioglycollic acid-latex compound suspension；By sulfenyl second
Acid-latex compound suspension is diluted to 10mg/ml with MES buffer (0.01M, pH=6.1), then by the sulphur after dilution
CdSe quantum dots are added in the compound suspension of guanidine-acetic acid-latex, and ultrasound mixes, and centrifuge centrifugation discards supernatant, and slow with PBS
Precipitating particulate matter is resuspended in fliud flushing (0.02M, pH=7.2), and centrifuge is centrifuged again later, discards supernatant, and precipitating particulate matter redissolves
PBS buffer solution (0.02M, pH=7.2) in, obtain quantum dot-ethyl thioglycollic acid-latex compound suspension；By quantum dot-sulfenyl
Acetic acid-latex compound suspension is diluted to 10mg/ml with MES buffer (0.01M, pH=6.1), then in quantum dot-sulphur
Carbodiimide is added in guanidine-acetic acid-latex compound, stirs and evenly mixs, and centrifuge centrifugation discards supernatant, and use PBS buffer solution
Precipitate particles are resuspended in (0.02M, pH=7.2), later according to CD146 monoclonal antibody I and quantum dot-ethyl thioglycollic acid-latex
CD146 monoclonal antibody I is added in the ratio of the mass ratio 1:10 of compound, stirs and evenly mixs, and centrifuge centrifugation removes supernatant, will
Sediment is redissolved in the PBS buffer solution (0.02M, pH=7.2) containing 5wt%BSA to get quantum dot-labeled CD146 Dan Ke
Grand antibody I；
(2) bonding pad is prepared: will be quantum dot-labeled using the PBS buffer solution (0.02M, pH=7.2) containing 1wt% sucrose
CD146 monoclonal antibody I be diluted to the concentration of 2mg/ml, reusing quantitative spray film instrument will be quantum dot-labeled with the amount of 2ul/cm
CD146 monoclonal antibody I be sprayed on bonding pad, 37 DEG C of drying 1h under the conditions of being protected from light, be made monoclonal antibody bonding pad；
(3) nitrocellulose coated film is prepared: using the PBS buffer solution (0.02M, pH=7.2) containing 1wt% sucrose point
CD146 monoclonal antibody II, rabbit anti-mouse igg antibody are not diluted to the concentration of 2mg/ml, reuse quantitative spray film instrument with 1ul/
The two interval 0.8cm is sprayed on nitrocellulose filter by the amount of cm, 37 DEG C of drying 1h, be added desiccant be placed in 4 DEG C seal up for safekeeping it is standby
With；
(4) sample pad is prepared: with PBS buffer solution (0.01M, pH=containing 1wt%BSA, 0.1wt%tritonx-100
7.2) sample pad 1.5h is impregnated, 37 DEG C are dried for standby；
(5) it assembles kit: the nitrocellulose coated film for being marked with antibody being pasted on to the middle position of bottom plate, in nitre
One end of acid cellulose coated film overlaps water absorption pad, and the other end overlaps bonding pad, overlaps sample pad on bonding pad, test strips are set
In the designated position of box body, lid is covered, is placed in sealing space of the humidity lower than 15% and saves.
The present invention has the utility model has the advantages that immunofluorescent reagent box of the present invention uses ethyl thioglycollic acid using quantum dot-labeled technology
Quantum dot is modified, ensure that the stability of quantum dot-labeled antibody, with testing result high sensitivity, ambient noise is small, detects
The advantages that time is short, specific good, and box body is easily assembled to lid, is equipped with waveform sawtooth, is slided convenient for grasping and being not easy
It falling, guarantees the stability of kit detection, kit exquisiteness of the present invention is light, and it is easy to carry, do not have to operationlocation and environment
Rigid requirement, simple and quick, detecting step is simple, testing result is stable, has quantitative detection function, is suitable for market into one
Step is promoted the use of.
Detailed description of the invention
Fig. 1 is schematic structural view of the invention；
Fig. 2 is lid inner surface schematic diagram；
Fig. 3 is cartridge inner surface schematic diagram；
Fig. 4 is test strips structure schematic diagram；
In figure: 1- test strips, 2- box body, 3- lid, 4- bolt, 5- jack, 6- well, 7- observation window, 8- targets
Knowing, 9- detection mark, 10- detent protrusion, 11- lagging, 12- erects baffle, 13- strip projected parts, 14- bottom plate, 15- water absorption pad,
16- nitrocellulose coated film, 17- bonding pad, 18- sample pad, 19- waveform sawtooth, 20- detection line, 21- nature controlling line.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description.
Embodiment 1
As shown in Figs 1-4, the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146, including kit
Box body and the test strips 1 being set in kit body, the kit body include box body 2, lid 3, box body 2, lid 3
It is connected by buckling, 3 inner surface of lid is equipped with the bolt 4 for being used to fix lid 3 Yu box body 2, and 4 bilateral symmetry of bolt is set
It sets, for bolt 4 between the upper and lower every 3 pairs of setting, 2 inner surface of box body is equipped with the jack 5 being adapted with bolt 4, and the lid 3, which is equipped with, to be added
Sample hole 6, observation window 7, well 6 are circular through hole structure, and observation window 7 is rectangular through holes structure, and lid upper surface is equipped with item
Target knows 8, detection mark 9, and 3 inner surface of lid is equipped with the detent protrusion 10 for fixing test strips 1, the width of detent protrusion 10
With the equivalent width of test strips 1,2 inner surface of box body is equipped with baffle, strip projected parts 13, and baffle includes the strut rail of upper and lower ends
Plate 11 and the perpendicular baffle 12 connecting vertically with lagging 11, the lagging 11 form semi-closed structure, institute with perpendicular baffle 12
It states test strips 1 to be located in the semi-closed structure of upper and lower ends, the strip projected parts 13 are symmetrical set, between strip projected parts 13
Distance be equal to test strips 1 width；
The test strips include bottom plate 14, water absorption pad 15, nitrocellulose coated film 16, bonding pad 17 and sample pad
18, the nitrocellulose coated film 16 is covered in the intermediate position of the bottom plate 14, and is set on nitrocellulose coated film 16
It is equipped with detection line 20 separately and nature controlling line 21, for the detection line 20 close to the bonding pad 17, the nature controlling line 21 is close
The water absorption pad 15 is coated with quantum dot-labeled CD146 monoclonal antibody I on the bonding pad 17, wraps in the detection line 20
There is CD146 monoclonal antibody II, rabbit-anti mouse lgG is coated on nature controlling line 21.
Preferably, the sample-adding nose end two sides of the kit body are equipped with waveform sawtooth 19；Convenient for grasping, avoid sliding
It falls.
Preferably, the quantum dot is CdSe quantum dots or cadmiumsulfide quantum dot.
Preferably, the bonding pad 17 is polyurethane material.
Preferably, the sample pad 18 is glass fibre element film.
Preferably, the difference of the CD146 monoclonal antibody I and CD146 monoclonal antibody II is to combine CD146 antigen
Site it is different.
Preferably, the water absorption pad 15, nitrocellulose coated film 16, bonding pad 17, between sample pad 18, successively with and
Only it is in contact and partly overlaps with adjacent regions.
Mentioned reagent box box body structure is reasonable, exquisite light, easy to carry, wants to operationlocation and environment without hardness
It asks, simple and quick, detecting step is simple, testing result is stable, has quantitative detection function.
Embodiment 2
The preparation step of the immunofluorescence technique kit of above-mentioned quantitative detection blood vessel endothelium marker CD146 is as follows:
(1) it prepares quantum dot-labeled CD146 monoclonal antibody I: taking 1ml latex particle that the PBS buffer solution of 10ml is added
In (0.02M, pH=7.2), centrifuge 8000rpm is centrifuged 5min；Remove supernatant, with PBS buffer solution (0.02M, pH of 5ml
=7.2) precipitating particulate matter is resuspended, obtains latex particle solution, 4 DEG C save for use；By latex particle solution MES buffer
Latex particle is diluted to 10mg/ml by (0.01M, pH=6.1), and 3ml sulphur is added in the latex particle solution after then taking 5ml to dilute
Guanidine-acetic acid, after mixing 24 hours, centrifuge 8000rpm centrifugation is discarded supernatant, and heavy with PBS buffer solution (0.02M, pH=7.2)
Outstanding precipitating particulate matter, centrifuge 5000rpm is centrifuged again later, is discarded supernatant, and precipitating particulate matter is redissolved in 0.02M, pH7.4's
In PBS buffer solution, ethyl thioglycollic acid-latex compound suspension is obtained；By ethyl thioglycollic acid-latex compound suspension, buffered with MES
Liquid (0.01M, pH=6.1) is diluted to 10mg/ml, and the compound suspension of ethyl thioglycollic acid-latex after then taking 20ml to dilute is added
20mg CdSe quantum dots, ultrasound mix 8h, and centrifuge 5000rpm centrifugation discards supernatant, and with PBS buffer solution (0.02M, pH
=7.2) be resuspended precipitating particulate matter, later again centrifuge 5000rpm be centrifuged, discard supernatant, precipitating particulate matter redissolve in
In the PBS buffer solution of 0.02M, pH7.4, quantum dot-ethyl thioglycollic acid-latex compound suspension is obtained；By quantum dot-sulfenyl second
Acid-latex compound suspension is diluted to 10mg/ml with MES buffer (0.01M, pH=6.1), then according to quantum dot-sulphur
The mass ratio of guanidine-acetic acid-latex compound and carbodiimide is 1:8, and carbodiimide is added, stirs and evenly mixs 10min, 5000rpm from
The heart discards supernatant, and precipitate particles are resuspended with PBS buffer solution (0.02M, pH=7.2), anti-according to CD146 monoclonal later
CD146 monoclonal antibody I is added in body I and quantum dot-ethyl thioglycollic acid-latex compound mass ratio 1:10 ratio, and stirring is mixed
After even 3 hours, 5000rpm is centrifuged 5min, removes supernatant, and sediment is redissolved in the PBS buffer solution containing 5wt%BSA
(0.02M, pH=7.2) is to get quantum dot-labeled CD146 monoclonal antibody I；
(2) bonding pad is prepared: will be quantum dot-labeled using the PBS buffer solution (0.02M, pH=7.2) containing 1wt% sucrose
CD146 monoclonal antibody I be diluted to the concentration of 2mg/ml, reusing quantitative spray film instrument will be quantum dot-labeled with the amount of 2ul/cm
CD146 monoclonal antibody I be sprayed on bonding pad, 37 DEG C of drying 1h under the conditions of being protected from light, be made monoclonal antibody bonding pad；
(3) nitrocellulose coated film is prepared: using the PBS buffer solution (0.02M, pH=7.2) containing 1wt% sucrose point
CD146 monoclonal antibody II, rabbit anti-mouse igg antibody are not diluted to the concentration of 2mg/ml, reuse quantitative spray film instrument with 1ul/
The two interval 0.8cm is sprayed on nitrocellulose filter by the amount of cm, 37 DEG C of drying 1h, be added desiccant be placed in 4 DEG C seal up for safekeeping it is standby
With；
(4) sample pad is prepared: with PBS buffer solution (0.01M, pH=containing 1wt%BSA, 0.1wt%tritonx-100
7.2) sample pad 1.5h is impregnated, 37 DEG C are dried for standby；
(5) it assembles kit: the nitrocellulose coated film for being marked with antibody being pasted on to the middle position of bottom plate, in nitre
One end of acid cellulose coated film overlaps water absorption pad, and the other end overlaps bonding pad, overlaps sample pad on bonding pad, test strips are set
In the designated position of box body, lid is covered, is placed in sealing space of the humidity lower than 15% and saves.
Embodiment 3
The present embodiment is substantially the same manner as Example 2, and different places is, the quantum dot used for cadmiumsulfide quantum dot,
It is 5 hours that antibody ultrasound mixing time is added in quantum dot-ethyl thioglycollic acid-latex suspension of activation.
Embodiment 4
Clinical sample detection method: to be measured after 10min in sample pad prepared by 100ul sample to be tested addition embodiment 2
The test strips of sample are put into fluorescence immune chromatography detector, read the luminous value of T/C line, and are recorded, by sample to be tested T/C value
It brings into standard curve, obtains the concentration of CD146；The present invention can detect the concentration of CD146, easy to operate, knot with direct quantitative
Fruit is reliable.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146, which is characterized in that including kit body
And the test strips in kit body are set to, the kit body includes box body, lid, and box body, lid pass through buckle
Be connected, the lid inner surface, which is equipped with, to be used to fix the bolt of lid and box body, and bolt is symmetrical set, bolt between the upper and lower every
3 pairs of setting, cartridge inner surface are equipped with the jack being adapted with bolt, and the lid is equipped with well, observation window, and well is
Circular through hole structure, observation window are rectangular through holes structure, and lid upper surface is equipped with project label, detection mark, table in lid
Face is equipped with the detent protrusion for fixing test strips, and the cartridge inner surface is equipped with baffle, strip projected parts, and baffle includes upper and lower two
The lagging at end and the perpendicular baffle connecting vertically with lagging, the lagging and perpendicular baffle form semi-closed structure, described
Test strips are located in the semi-closed structure of upper and lower ends, and the strip projected parts are symmetrical set, the distance between strip projected parts
Equal to the width of test strips；
The test strips include bottom plate, water absorption pad, nitrocellulose coated film, bonding pad and sample pad, the cellulose nitrate
Plain coated film is covered in the intermediate position of the bottom plate, and detection line and matter separately is provided on nitrocellulose coated film
Line is controlled, the detection line is coated with quantum dot on the water absorption pad, the bonding pad close to the bonding pad, the nature controlling line
The CD146 monoclonal antibody I of label is coated with CD146 monoclonal antibody II in the detection line, is coated with rabbit-anti on nature controlling line
Mouse lgG.
2. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 as described in claim 1, feature exist
In the sample-adding nose end two sides of the kit body are equipped with waveform sawtooth.
3. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 as described in claim 1, feature exist
In the quantum dot is CdSe quantum dots or cadmiumsulfide quantum dot.
4. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 as described in claim 1, feature exist
In the bonding pad is polyurethane material.
5. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 as described in claim 1, feature exist
In the sample pad is glass fibre element film.
6. the immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 as described in claim 1, feature exist
Between, the water absorption pad, nitrocellulose coated film, bonding pad, sample pad, successively with and only be in contact with adjacent regions and
It partly overlaps.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810969563.XA CN109085335A (en) | 2018-08-23 | 2018-08-23 | The immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810969563.XA CN109085335A (en) | 2018-08-23 | 2018-08-23 | The immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 |
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| CN109085335A true CN109085335A (en) | 2018-12-25 |
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ID=64794498
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| CN201810969563.XA Pending CN109085335A (en) | 2018-08-23 | 2018-08-23 | The immunofluorescence technique kit of quantitative detection blood vessel endothelium marker CD146 |
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