CN106918698A - A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme - Google Patents
A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
The present invention relates to a kind of phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK).The phospho-AB chip agent box of detection people RTK of the present invention includes：Solid phase carrier, to be coated with the standard diaphragm or slide of specific antibody；Cleaning solution, including 20X concentrated cleaning solutions and its dilution containing 0.1% polysorbas20；Sample dilution；Dilution for diluting antibody and HRP streptavidins；Biotinylated detection antibody mixture；300X concentrates fluorescein streptavidin solution；Sample treatment solution, is 2X cell pyrolysis liquids；The specific antibody is directed to 71 kinds of antibody of protein.RTK phospho-ABs chip agent box of the present invention can by once test it is rapid, simple and direct and exactly determine RTK paths in 71 Phosphorylation status of protein, changed by monitoring the protein phosphorylation in experimental model system, just the pathway activation effect in cell can be quickly detected, without by cumbersome immuno-precipitation and blotting.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of phospho-AB chip agent box, more particularly, to a kind of people
The phospho-AB chip agent box of receptor tyrosine kinase.
Background technology
Protein phosphorylation refers to intracellular protein under outer signals stimulation, on specific amino acid sites by egg
It is white it is kinase catalytic occur phosphorylation occur and produce phosphorylation albumen will successively activate downstream stages signaling molecule, by level
Connection reaction finally realizes biological effect.One protein can be produced on one or more site by multiple protein kinases
Raw phosphorylation.Cell-membrane receptor EGFR families include EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3 and ErbB4/
The different albumen of tetra- kinds of HER4.Under normal physiological status, ErbB receptor is for regulation cell propagation, differentiation, motion and apoptosis
Signal transduction play vital effect.EGF receptor family shows notable difference in different acceptors, displays that big
Intercrossing.Wherein, ErbB1 most gametophytes in family member, and the multiple gametophyte of the combination with ceiling rate receives
Body tyrosine residue；ErbB3 is characterized as multiple phosphatidylinositol-3-kinases (PI3K) binding site；ErbB2 has little
Association gametophyte, Shc is most normal one；ErbB1 and ErbB4 has with reference to many kinds of phosphate acceptors junket of Grb2 or Grb2 and Shc
Histidine residue, and show than ErbB2 and the larger range of gametophytes of ErbB3；The ErbB1 and ErbB2 generally mistakes in cancer cell
Expression is amplified so that they turn into important target protein in medicine is used or is developed.Phosphorylation it is whether normal, also often
Often imply that whether cellular signal transduction exception occurs, and the conduction path of exception often causes some diseases.Therefore, to phosphorus
The research of acidifying shows critically important biology and clinical value.Due to the diversity of phosphorylation site, how to come quick effective
Ground understands intracellular phosphorylation state, is always a problem for perplexing vast researcher.
Before without anti-tyrosine phosphorylation antibody, the tyrosine phosphorylation of protein and enzyme can only be by abnormally dangerous
And very time-consuming radioactivity experiment detect.And utilize anti-tyrosine phosphorylation antibody, then can be by Western
Blot or other immunological methods easily detect that phosphorylation signal.Conventional detection method includes：Use anti-tyrosine phosphatase
Change antibody and endogenous or heterogenous expression phosphorylated protein is detected on Western Blot.If the content of target protein is relatively low,
The method that immunoprecipitation can also be used first is enriched with the tyrosine protein that phosphorylation occurs, then the level for detecting target protein.It can be seen that,
Detect that the phosphorylation of receptor tyrosine kinase is cumbersome, high cost；Further, since being typically with for scientist's research is new
Phosphorylation site, and the phospho-AB for preparing albumen specific site is often more wasted time and energy than the preparation of general antibody, effect
Fruit is also bad.
The content of the invention
It is an object of the invention to provide a kind of phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK),
The RTK phospho-ABs chip agent box can by once test it is rapid, simple and direct and exactly determine RTK paths in 71 eggs
The Phosphorylation status of white matter, are changed by monitoring the protein phosphorylation in experimental model system, just can be quickly detected in cell
Pathway activation effect, without by cumbersome immuno-precipitation and blotting (Western Blotting).
The phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme of the present invention includes：Solid phase carrier,
To be coated with the standard diaphragm or slide of specific antibody；Cleaning solution, including the 20X concentrated cleaning solutions containing 0.1% polysorbas20 and
Its dilution；Sample dilution；Dilution for diluting antibody and HRP- streptavidins；Biotinylated detection antibody mixing
Thing；300X concentrates fluorescein-streptavidin solution；Sample treatment solution, is 2X cell pyrolysis liquids；Wherein, the specific antibody
It is directed to the antibody selected from following 71 kinds of protein：ABL1、ACK1、ALK、Ax1、Blk、BMX、Btk、Csk、Dtk、EGFR、
EphA1、EphA1、EphA2、EphA3、EphA4、EphA5、EphA6、EphA7、EphA8、EphB1、EphB2、EphB3、
EphB4, EphB6, ErbB2, ErbB3, ErbB4, FAK, FER, FGFR1, FGFR2, FGFR2 (α types), Fgr, FRK, FYN, Hck,
HGFR、IGF-1R、insulin R、Itk、JAK1、JAK2、JAK3、LCK、LTK、Lyn、MATK、M-CSFR、MUSK、NGFR、
PDGFR-a、PDGFR-b、PYK2、RET、ROR1、ROR2、ROS、RYK、SCFR、SRMS、SYK、Tec、Tie-1、Tie-2、
TNK1、TRKB、TXK、Tyk2、TYRO10、VEGFR2、VEGFR3、ZAP70。
The further spy of the phospho-AB chip agent box according to detection human receptor tyrosine kinase enzyme of the present invention
Levy, the solid phase carrier is porous fiber film, is selected from：Nitrocellulose filter, fiber filter film, polyvinylidene fluoride film (PVDF) or
Person's nylon membrane.
Preferably, the perforated membrane is 0.05 to 0.1% surfactant or is formed under severe oxidative conditions hydrophilic
At 20 to 26 DEG C after group, combined closely with specific antibody under 40% damp condition.
It is highly preferred that the perforated membrane is the pvdf membrane processed by nonionic surfactant.
The further spy of the phospho-AB chip agent box according to detection human receptor tyrosine kinase enzyme of the present invention
Levy, the solid phase carrier is slide.
Preferably, the slide is after glutaraldehyde or poly-D-lysine, APG or APES adhesives are processed
Combined closely with specific antibody under the conditions of 20 to 26 DEG C.
It is highly preferred that the slide is the slide processed by hydrophilic reagent APG.
It is highly preferred that the hydrophilic reagent is APG, 0.01~0.1% that mass fraction is 0.01~0.2%
Glycerine, the ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method of hydrophilic reagent treatment slide is that slide exists
Soaked 3~5 minutes in hydrophilic reagent, dried.
Most preferably, the hydrophilic reagent is APG, 0.05% glycerine, 0.01% that mass fraction is 0.1%
Macrogol 4000 ultra-pure water solution；The method of hydrophilic reagent treatment slide is that slide is soaked into 3 points in hydrophilic reagent
Clock, dries.
It is highly preferred that the detection antibody mixture under 24 DEG C, 40% damp condition point sample on solid phase carrier.
The further spy of the phospho-AB chip agent box according to detection human receptor tyrosine kinase enzyme of the present invention
Levy, the sample treatment solution is the RIPA buffer solutions comprising protease and phosphatase inhibitor cocktail.
Preferably, the inhibitors of phosphatases is to be selected from：Na3VO4、NaF。
Antibody chip kit of the present invention can determine RTK by the detection method similar to sandwich method ELISA
Phosphorylated protein.Its operating procedure includes：
1) sample for diluting respective concentration with Sample dilution is incubated in reactive tank；
2) wash, add the biotinylated detection antibody of dilution to be incubated；
3) wash, add the fluorescein-labeled streptavidin solution of dilution to be incubated；
4) wash, slide be imaged and judged result in laser scanner.
Imaging is scanned to the slide that reaction terminates with the laser scanner containing Cy3 passages, is swept from suitable laser
Retouching parameter makes highest signal on chip, close to saturation, gained image is stored as tiff files.Then will be every with chip reading software
The fluorescence signal of individual point is converted into digital signal.Each is drawn by the digital signal of the RTK phosphorylated protein objects of reference for diluting
The signal of sample, then judges RTK phosphorylated proteins expression in the sample by corresponding standard reference point.
Preferably, result judgement is according to following calculation：
X (Ny)=X (y) * P1/P (y), after subtracting background letter throat value, wherein P1 represents letter of the positive control in reference to group
Number intensity, P (y) represents signal intensity of the positive control in reaction group, and X (y) represents signal intensity of the sample in reaction group,
Represent detected value of the sample in reaction group.When detected value is more than 1.5 times of reference value or less than 0.65 times of reference value, can be respectively
It is judged to positive or negative.
Because the present invention is using the good characteristic of sampling liquid, i.e. specific antibody and contains 0.5 to 1.5% casein
PH7.4 phosphate buffers are mixed to form mixtures of antibodies, make antibody chip kit of the present invention by specific antibody
The step of being fixed on slide is greatly simplified, without general in prior art (immuno-precipitation or common ELISA method)
The operating procedure of the active ingredient on the point sample rear enclosed slide for using.
In one embodiment of the invention, in the printing operation of step (1), using U.S. Bole (Bio-Rad) company
Or the full automatic point sampling instrument of platinum Ai Ermo (Perkin Elmer) company production.Each specific antibody chip dot matrix is arranged in
Diaphragm, and in specific operating process, the arrangement of each specific antibody can need to be adjusted according to experimental design, according to
Different antibody chip arrangement arrays, controls full-automatic point sample instrument, prepares required intermediate products.
The phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK) of the present invention, with advantages below：
(1) can simultaneously detect 71 protein phosphorylation levels, and the parallel detection of multisample multi objective can be realized, overcome
Prior art operation is cumbersome, Testing index is single, need to have the low defect of expense instrument, sensitivity, with cheap, convenient, sensitive, standard
Really, high flux, sample consumption it is few, can common lab promote and scale；(2) phospho-AB core of the present invention
Piece is suitable for using full-automatic point sample instrument come point sample, thus with high flux, many sites it is specific；(3) present invention preferably employs
Porous fiber film is optimized treatment to diaphragm as solid phase carrier during point sample and coating, so as to improve this
Performance of the antibody chip kit at aspects such as sensitivity, accuracy, high fluxs.
In sum, the phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK) of the present invention is special
Suitable for the detection of biology, medical science, medicament research and development, disease treatment and its association area.
Brief description of the drawings
Fig. 1 is the membrane DNA chip of the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme of the present invention
The sample application array figure of (using diaphragm as solid phase carrier).
Fig. 2 is the glass core of the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme of the present invention
The sample application array figure of piece (using slide as solid phase carrier).
Fig. 3 is testing result of the A431 cells in chip；Detection of the A431 cells that left figure display EGF is not induced in chip
As a result；Testing result of the A431 cells that right figure display EGF has been induced in chip.This embodiment uses membrane DNA chip.
Fig. 4 is people RTK phospho-ABs chip agent box inspection Cont-ODN and gal-1-ODN treatment of the present invention
The result figure of trophocyte.This embodiment uses glass-chip.
Specific embodiment
Embodiment 1：The screening of optimum antibody chip carrier.
Conventional antibody chip is more with slide as carrier, because slide is frangible and testing equipment is expensive, is not suitable for small-scale
Using also for the reagent conveyer belt of diagnostic field carrys out problem.And the frangible background of traditional nitrocellulose filter is high, as a result fluctuate
Greatly.We have screened the diaphragm and slide for being processed without activity methods on the market, and either diaphragm is drawn by substantial amounts of examination
Or the active agent formulation on slide, temperature and surface is only the key for determining point sample effect.
(1) screening of diaphragm
In order that antibody is fixed on membrane surface, we have screened distinct methods treatment diaphragm, such as following table.Again with automatically
Point sample instrument luminescent solution point on the surface of diaphragm, then with UV scanners come reading.
The processing method of each group diaphragm is as follows：
1st group：Nitrocellulose diaphragm, is purchased from FISHER companies, and article No. is LC2009.
2nd group：Kynoar (PVDF) film, is purchased from WHATMAN companies, and article No. is 10485289.
3rd group：Pvdf membrane in 2nd group is soaked in 0.02% to 0.075% nonionic surfactant Tween80
The aqueous solution in.
4th group：Pvdf membrane hydrogen peroxide of the addition containing catalyst in 2nd group is carried out into Strong oxdiative reaction, H is added2SO4
Form hydrophilic radical.
Specific antibody is pressed into gradient dilution point on above-mentioned 4 groups of diaphragms respectively, secondary antibody is subsequently adding and substrate is examined
Survey, according to the pore homogeneity of point sample, test limit and background selection select most suitable point sample film, as a result see the table below 1.
Table 1：The comparing of different disposal method diaphragm performance
| The type of film | Homogeneity | Background | LDL |
| 1 | Loading wells homogeneity is poor | It is more visible | 0.4ng/ml |
| 2 | Point sample aperture is homogeneous without diffusion | There is miscellaneous point | 0.2ng/ml |
| 3 | Point sample aperture is homogeneous without diffusion | Clearly | 0.2ng/ml |
| 4 | Point sample aperture is homogeneous | Clearly | 0.4ng/ml |
As seen from the above table, the pvdf membrane for being processed by surfactant has homogeneity and minimum test limit higher,
The test limit of the pvdf membrane processed by strong oxidizer is more untreated low, but background is more visible.And the albumen of common NC films is inhaled
Attached property is poor, and sensitivity is relatively low.In summary index, the pvdf membrane that prioritizing selection of the present invention is processed by nonionic surfactant
As solid phase carrier.
(2) screening of slide
The processing method of each group slide is as follows：
1st group of amination slide：Purchased from Corning Incorporated, article No. is UltraGAPS40019.
2nd group of aldehyde radical slide：Add glutaraldehyde to soak 40 minutes the 1st group of slide and make aldehyde radical slide.
3rd group of APES slides：Will common slide add the APES of acetone dilution in soak 0.5 to 1 minute, then with pure third
Ketone cleaning is made APES slides.
4th group of poly-D-lysine slide：Add the poly-D-lysine immersion 0.5 to 1 that PBS dilutes small common slide
When, then cleaned with pure water and be made poly-D-lysine slide.
5th group of the slide processed through hydrophilic reagent：By common slide with hydrophilic reagent process, hydrophilic reagent be 0.01 to
0.1% APG.
The point sample effect of various slides is not quite similar, and adds the slide point sample effect of hydrophilic coating more apparent, point sample effect
More untreated height.The more other type effects of slide wherein by amination and hydrophilic treated are higher.In 20 to 26 degree
Under the conditions of, film glass/piece point sample effect is more visible, and background value is low；And 27 to 30 spend under the conditions of, no matter the point of which kind of film/slide
All there are diffusion phenomena in sample effect, and temperature humidity is higher, spreads more obvious.
Secondary antibody and substrate, the sensitivity at different temperature and damp condition are added after the slide binding antibody of each group
(ng/ml) the selection result such as table 2 below.
Table 2：
In summary index, selects the slide by the treatment of hydrophilic reagent APG as solid phase carrier.
Embodiment 2：The preparation of kit.
Antibody chip kit of the present invention includes following component：
Solid phase carrier：The standard diaphragm or slide of coated antibody.
Cleaning solution：20X concentrated cleaning solutions containing polysorbas20.1X cleaning solutions are pH 7.2, containing 0.1% polysorbas20,0.1mol/
The phosphate buffer of L.
Dilution：2 bottles of 15ml 5X concentration and dilution liquid D for being used for diluted sample, 1 bottle is used to dilute antibody and HRP- chains parent
With the 15ml 5X concentration and dilution liquid B of element.1X dilutions B is 15mM, the PBS of p H7.4, solute and its in the dilution
Mass concentration or molar concentration or volumetric concentration in liquid B is as follows：0.5% casein, 2-4% sucrose, 150mM NaCl.1X is dilute
Liquid D is released for 15mM, the PBS of pH6.5, solute and its mass concentration or molar concentration in the sample diluent or
Volumetric concentration is as follows：2-4% sucrose, 150mM NaCl.
Detection antibody：Biotinylated detection antibody mixture.
200 μ l 300X concentrate fluorescein-streptavidin solution.
Sample treatment solution：2X cell pyrolysis liquids 10ml, 1X cell pyrolysis liquid include 10mM pH 7.5, Tris.HCl, 25mM
NaCl, 1% NaTDC, 1%Triton X-100, comprising phosphatase and protease inhibitors.
Coated specific antibody is for the antibody selected from following 71 kinds of protein on solid phase carrier (referring to table 3).
These antibody are commercially available, such as purchased from companies such as R＆D Systems, PeproTech, Raybiotech.
Table 3：The targeted antigen protein title of specific antibody
These specific antibodies complete printing operation by full-automatic point sample instrument, and specific antibody is fixed on into point sample film
Method is comprised the following steps：
1) specific antibody is mixed to form antibody and mixes with the pH7.4 phosphate buffers containing 0.5 to 1.5% casein
Thing；
2) each antibody content is fixed on the loading wells with 0.01 to 2ng in mixtures of antibodies, each antibody sets
Put 2 to 4 repeating holes；
3) antibody chip dot matrix is arranged in diaphragm, diaphragm 10 to 100 specific antibodies of point every square centimeter.Each specificity
The arrangement of antibody can need to be adjusted according to experimental design, complete by control according to different antibody chip arrangement arrays
Auto sample applicator, prepares required intermediate products.
A kind of dot matrix order of specific antibody can be found in Fig. 2.
The diaphragm also includes positive control and negative control.Positive control wells are correspondence specific antibody with corresponding
The biotinylation IgG antibody of concentration, the concentration of the biotinylated IgG antibody of each reaction is consistent, in order to standardize detection.
Negative control hole is the biotinylated irrelevant antibody with respective concentration, each reaction biotinylated irrelevant antibody it is dense
Degree is consistent, in order to standardize detection.
According to the selection result of embodiment 1, solid phase carrier uses porous fiber film, including：Nitrocellulose filter, fiber mistake
Filter membrane, fiberglass substrate, polyvinylidene fluoride film (PVDF) or nylon membrane.
Tunica fibrosa facile hydrolysis under high temperature, high pressure and acid-base condition, is also easily decomposed by the microorganisms, and preserves also volatile for a long time
Water and dry, electrically charged and become fragile, durability is poor.Therefore, the present invention is by 0.05 to 0.1% surfactant, 0.1 to
0.5% sodium azide treatment perforated membrane, combines closely under the conditions of 18 to 22 DEG C with specific antibody, the point sample for ultimately forming
Hole complete display.
In the present embodiment, full-automatic point sample instrument is the product that Bio Rad Laboratories or platinum Ai Ermo companies produce；Slide
It is Corning Incorporated's product.Certainly, in the above-mentioned steps of inventive technique scheme, the use of instrument and material is not limited to
The present embodiment is enumerated, but can solve the problem that technical problem of the invention, and realize that corresponding technique effect is foundation.
Embodiment 3：RTK phosphorylated proteins are detected with the kit of embodiment 2
Counterdie is put in supporting square box, due to multiple chip dot matrix being distributed with the counterdie of the present embodiment, so at this
Square box is provided with 8 grids in embodiment, is divided into each chip dot matrix by the grid between square box separate anti-
Area is answered, what is used in the present embodiment is provided with 8 grid square boxes, room temperature is positioned in each grid plus after 2 milliliters of confining liquids
Lower culture 30 minutes, then carries out the rapid operation of following steps successively：
1st, it is loaded
The confining liquid in each grid is suctioned out, 100 microlitres~5 milliliters samples diluted through confining liquid are placed with film
In grid, then it is placed on shaking table and shakes at room temperature 1 to 2 hour, or can be also reacted 12-18 hours at 4 DEG C.
2nd, film is washed
Cleaning solution I is cleaned：Sample is suctioned out from grid, is cleaned with 1~5 milliliter 1 times of cleaning solution I, shaking table is placed on afterwards
Upper room temperature is shaken 5 minutes, repeats this cleaning step twice.
Cleaning solution II is cleaned：Raffinate is suctioned out from grid, is cleaned with 1~5 milliliter 1 times of cleaning solution II, be placed on shake afterwards
Room temperature is shaken 5 minutes on bed, repeats this cleaning step once.
3rd, the antibody mixed liquor for being marked with biotin is added
Biotin labeling is added to each grid, the specific antibody for listed antigen protein in table 1 is mixed with
500 microlitres~2 milliliters of mixed liquor, be then placed on shaking table room temperature and shake 1 to 2 hour.Also 12-18 can be reacted at 4 DEG C small
When.Then suctioned out from grid and be marked with the antibody mixed liquor of biotin, repeat step 2 washes film step.
4th, HRP-Streptavidin is added
500 microlitres~2 milliliters avidins of the horseradish peroxidase-labeled for diluting are added to each grid
Streptavidin (HRP-Streptavidin), is then placed on shaking table under the conditions of room temperature and shakes 1 to 2 hour, can also be reacted at 4 DEG C
12-18 hours.HRP-Streptavidin is then suctioned out from grid, repeat step 2 washes film step.
HRP-Streptavidin is commercially available from BD companies (production number 554066), and experiment is preceding, it is necessary to be carried out with confining liquid
20,000 times of dilutions.
It should be noted that the step of this implementation 1 in step 4, use it is following easily, its composition can compound method such as
Under：
2M Tris buffer solutions (pH7.5)：Trizma Base 484g, purified water 1.3L, regulation acid-base value to 7.5, purifying
Water adds to 2L.
The compound method of confining liquid is as follows：First by 20 × PBS (KCl 16g, NaCl 640g, KH2PO416g,
Na2HPO492g, after being dissolved in 2.6L purified waters, then adds purified water to 4 liters) it is diluted to 1 × PBS (20 × PBS 200ml, purifying
Water 3800ml), then prepare 10%BSA (BSA400g, 1 × PBS add to 4 liters), finally prepare confining liquid (4 liters of 10%BSA,
4 liters of Casein, mixes).
20 × cleaning solution II (20 × TBS) partition is as follows：2M Tris buffer solution (pH7.5) 800ml, 5M NaCl
4800ml (purified water adds to 5 liters after dissolving for NaCl 1461g, 3.3 liters of purified water), after mixing, purified water adds to 8 liters.Use
When, by 20 × cleaning solution II doubling dilution.
20 × cleaning solution I (2%Tween/20 × TBS) partition is as follows：20 × cleaning solution II 1L, Tween 20ml, mixes
It is even.When using, by 20 × cleaning solution II doubling dilution.
5th, detect
Pressing from both sides out counterdie with tweezers and being placed in plumbness makes unnecessary liquid drip-dry；Film is then placed on the plastic sheet of cleaning
On, it is ensured that what counterdie was fixed with antibody one faces up；Add 500 microlitres of (A for preparing:B=1:1) luminescent solution (commercially available from
BIOFX companies, production number LUMB-0500-01) on every counterdie and placing 2 minutes at room temperature, this process must assure that hair
Light liquid is completely covered whole film；Counterdie surface is covered in another clean plastic sheet again, carefully by plastic sheet
Bubble is extruded away, should be avoided during the bubble in plastic sheet is extruded away on film firmly.After above-mentioned steps,
Counterdie is placed on and is imaged within 5 to 10 seconds at room temperature, low-temperature CCD low is used during shooting, and using receive data in the UV scanners for matching
According to.
Embodiment 4：The application of kit
1) 37 DEG C, the EGF10 of 100ng/ml are exposed to after squama epidermal cell cancer cell A431 is processed through serum starvation overnight
Minute, compared with control cells equally through Nature enemy but is not exposed in EGF.Two groups of cells are incubated during antibody chip is added after cracking
Educate, add the anti-phosphate acceptor phosphotyrosine antibody of biotin labeling to detect the phosphate acceptor junket ammonia on activated receptor
Acid.By after fluorescent dye effect, using laser scanner detection signal.
Testing result of the A431 cells that the left figure display EGF of Fig. 3 is not induced in chip；The right figure display EGF of Fig. 3 has been lured
Testing result of the A431 cells led in chip.
Result shows：The EGFR of the A431 cells induced by EGF, the expression quantity ratio of ErbB2, ErbB3 albumen is not induced
Height.
2) people RTK phospho-ABs chip agent box detection Cont-ODN and gal-1-ODN of the present invention is processed
Trophocyte.
GAL-1 plays crucial by adjusting the expression of HLA-G on embryonic feeder cells in trimester maternal immunological regulation
Effect.GAL-1 levels can be as the factor of prediction miscarriage.
Human receptor tyrosine kinase enzyme phospho-AB chip can monitor effects of the GAL-1 to cell protein phosphotidic level.
The trophocyte (gal-1-ODN) of embryonic feeder cells cell (Cont-ODN) and its suppression GAL-1 expression is by cracking simultaneously
Human receptor tyrosine kinase enzyme phospho-AB chip is separately added into, fully after washing, the anti-phosphoric acid junket of biotin labeling is added
Propylhomoserin antibody, HRP labels streptomysin and luminous substrate.
Result shows：The expression of FGFR2, SRMS, TXK albumen is suppressed by different degrees of.
Claims (10)
1. it is a kind of detect human receptor tyrosine kinase enzyme phospho-AB chip agent box, it is characterised in that including：
Solid phase carrier, to be coated with the standard diaphragm or slide of specific antibody；
Cleaning solution, including 20X concentrated cleaning solutions and its dilution containing 0.1% polysorbas20；
Sample dilution；
Dilution for diluting antibody and HRP- streptavidins；
Biotinylated detection antibody mixture；
300X concentrates fluorescein-streptavidin solution；
Sample treatment solution, is 2X cell pyrolysis liquids；
Wherein, the specific antibody is directed to the antibody selected from following 71 kinds of protein：ABL1、ACK1、ALK、Ax1、Blk、
BMX、Btk、Csk、Dtk、EGFR、EphA1、EphA1、EphA2、EphA3、EphA4、EphA5、EphA6、EphA7、EphA8、
EphB1、EphB2、EphB3、EphB4、EphB6、ErbB2、ErbB3、ErbB4、FAK、FER、FGFR1、FGFR2、FGFR2(α
Type), Fgr, FRK, FYN, Hck, HGFR, IGF-1R, insulin R, Itk, JAK1, JAK2, JAK3, LCK, LTK, Lyn,
MATK、M-CSFR、MUSK、NGFR、PDGFR-a、PDGFR-b、PYK2、RET、ROR1、ROR2、ROS、RYK、SCFR、SRMS、
SYK、Tec、Tie-1、Tie-2、TNK1、TRKB、TXK、Tyk2、TYRO10、VEGFR2、VEGFR3、ZAP70。
2. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 1, its feature exists
In：The solid phase carrier is porous fiber film, is selected from：Nitrocellulose filter, fiber filter film, polyvinylidene fluoride film (PVDF) or
Person's nylon membrane.
3. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 2, its feature exists
In：The perforated membrane be 0.05 to 0.1% surfactant or under severe oxidative conditions formed hydrophilic radical after 20 to
26 DEG C, combined closely with specific antibody under 40% damp condition.
4. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 3, its feature exists
In：The perforated membrane is the pvdf membrane processed by nonionic surfactant.
5. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 1, its feature exists
In：The solid phase carrier is slide.
6. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 5, its feature exists
In：The slide is combined closely under the conditions of 20 to 26 DEG C by after hydrophilic reagent treatment with specific antibody.
7. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 6, its feature exists
In the hydrophilic reagent is to be selected from：Glutaraldehyde or poly-D-lysine, APG or APES adhesives.
8. it is according to claim 6 detection human receptor tyrosine kinase enzyme phospho-AB chip agent box, it is described hydrophilic
Reagent is APG, 0.01~0.1% glycerine that mass fraction is 0.01~0.2%, 0.01~0.05% poly- second two
The ultra-pure water solution of alcohol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3~5 minutes, is dried in the air
It is dry.
9. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 1, its feature exists
In：The sample treatment solution is the RIPA buffer solutions comprising protease and phosphatase inhibitor cocktail.
10. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 8, its feature exists
In the inhibitors of phosphatases is to be selected from：Na3VO4、NaF。
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| CN201511005672.2A CN106918698B (en) | 2015-12-25 | 2015-12-25 | A kind of phospho-AB chip agent box detecting human receptor tyrosine kinase enzyme |
| PCT/CN2016/097197 WO2017107541A1 (en) | 2015-12-25 | 2016-08-29 | Phosphorylation antibody-array test kit for detecting receptor tyrosine kinase |
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| WO2017107541A1 (en) | 2017-06-29 |
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