CN106918698B - A kind of phospho-AB chip agent box detecting human receptor tyrosine kinase enzyme - Google Patents
A kind of phospho-AB chip agent box detecting human receptor tyrosine kinase enzyme Download PDFInfo
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- 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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Abstract
The present invention relates to a kind of phospho-AB chip agent boxes 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, for the standard diaphragm or slide for being coated with specific antibody；Cleaning solution, 20X concentrated cleaning solution and its dilution including containing 0.1% polysorbas20；Sample dilution；For diluting the dilution of antibody and HRP- streptavidin；Biotinylated detection antibody mixture；Fluorescein-streptavidin solution is concentrated in 300X；Sample treatment solution is 2X cell pyrolysis liquid；The specific antibody is the antibody for 71 kinds of protein.RTK phospho-AB chip agent box of the present invention can by once test it is rapid, simple and direct and accurately measure RTK access in 71 protein Phosphorylation status, pass through the protein phosphorylation variation in monitoring experimental model system, just the pathway activation effect that can be quickly detected in cell, without passing through cumbersome immuno-precipitation and blotting.
Description
Technical field
The invention belongs to field of biotechnology, are 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 technique
Protein phosphorylation refer to intracellular protein outer signals stimulation under, on specific amino acid sites by egg
The white kinase catalytic albumen that phosphorylation generation occurs and generates phosphorylation will successively activate downstream stages signaling molecule, pass through grade
Connection reaction is final to realize biological effect.One protein can be produced on one or more site by multiple protein kinases
Raw phosphorylation.Cell-membrane receptor EGFR family includes EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3 and ErbB4/
Tetra- kinds of HER4 different albumen.Under normal physiological status, ErbB receptor is for adjusting cell Proliferation, differentiation, movement and apoptosis
Signal transduction play a crucial role.EGF receptor family shows notable difference in different receptors, also shows big
Intercrossing.Wherein, ErbB1 most gametophytes in family member, and with ceiling rate the multiple gametophytes of combination by
Body tyrosine residue；ErbB3 is characterized as multiple phosphatidylinositol-3-kinases (PI3K) binding site；ErbB2 has seldom
It is associated with gametophyte, Shc is most normal one；ErbB1 and ErbB4, which has, combines more kinds of phosphate acceptors junket of Grb2 or Grb2 and Shc
Histidine residue, and show gametophyte more larger range of than ErbB2 and ErbB3；ErbB1 and ErbB2 mistake usually in cancer cell
Expression or amplification, so that they become important target protein in drug is used or developed.Phosphorylation it is normal whether, also often
Often imply whether cellular signal transduction exception occurs, and abnormal conduction path often leads to some diseases.Therefore, to phosphorus
The research of acidification shows critically important biology and clinical value.Due to the diversity of phosphorylation site, how to come fast and effective
Ground understands intracellular phosphorylation state, is always a problem for perplexing vast researcher.
Before no anti-tyrosine phosphorylation antibody, the tyrosine phosphorylation of protein and enzyme can only be by abnormally dangerous
And very time-consuming radioactivity test to detect.And utilize anti-tyrosine phosphorylation antibody, then it can pass through Western
Blot or other immunological methods easily detect that phosphorylation signal.Conventional detection method includes: with anti-tyrosine phosphatase
Change antibody and detects endogenous or heterogenous expression phosphorylated protein on Western Blot.If the content of target protein is lower,
The method that immunoprecipitation can also be used first is enriched with the tyrosine protein that phosphorylation occurs, then detects the level of target protein.As it can be seen that
The phosphorylation for detecting receptor tyrosine kinase is cumbersome, at high cost；In addition, usually using newly due to scientist's research
Phosphorylation site, and it is more time-consuming and laborious to prepare preparation of the phospho-AB of albumen specific site often than general antibody, effect
Fruit is also bad.
Summary of the invention
The purpose of the present invention is to provide a kind of phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK),
The RTK phospho-AB chip agent box can by once test it is rapid, simple and direct and accurately measure RTK access in 71 eggs
The Phosphorylation status of white matter is changed by the protein phosphorylation in monitoring 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,
For the standard diaphragm or slide for being coated with specific antibody；Cleaning solution, including containing 0.1% polysorbas20 20X concentrated cleaning solution and
Its dilution；Sample dilution；For diluting the dilution of antibody and HRP- streptavidin；Biotinylated detection antibody mixing
Object；Fluorescein-streptavidin solution is concentrated in 300X；Sample treatment solution is 2X cell pyrolysis liquid；Wherein, the specific antibody
Be for selected from following 71 kinds of protein antibody: 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。
The further spy of the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to the present invention
Sign, the solid phase carrier be porous fiber film, be selected from: nitrocellulose filter, fiber filter film, polyvinylidene fluoride film (PVDF) or
Person's nylon membrane.
Preferably, the perforated membrane be 0.05 to 0.1% surfactant or formed under severe oxidative conditions hydrophilic
It combines closely under 20 to 26 DEG C, 40% damp condition with specific antibody after group.
It is highly preferred that the perforated membrane is the pvdf membrane handled by nonionic surfactant.
The further spy of the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to the present invention
Sign, the solid phase carrier are slide.
Preferably, the slide is by after glutaraldehyde or poly-D-lysine, alkyl glycosides or the processing of APES adhesive
It combines closely under the conditions of 20 to 26 DEG C with specific antibody.
It is highly preferred that the slide is the slide handled by hydrophilic reagent alkyl glycosides.
It is highly preferred that the hydrophilic reagent be mass fraction be 0.01~0.2% alkyl glycosides, 0.01~0.1%
Glycerol, the ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method of hydrophilic reagent processing slide is that slide exists
It impregnates 3~5 minutes, dries in hydrophilic reagent.
Most preferably, the hydrophilic reagent be mass fraction be 0.1% alkyl glycosides, 0.05% glycerol, 0.01%
Macrogol 4000 ultra-pure water solution；The method of hydrophilic reagent processing slide is that slide is impregnated 3 points in hydrophilic reagent
Clock dries.
It is highly preferred that the detection antibody mixture is under 24 DEG C, 40% damp condition on point sample to solid phase carrier.
The further spy of the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to the present invention
Sign, the sample treatment solution are the RIPA buffer 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 measure RTK by being similar to the detection method of sandwich method ELISA
Phosphorylated protein.Its operating procedure includes:
1) it is incubated in reactive tank with the sample of Sample dilution dilution respective concentration；
2) it washs, diluted biotinylated detection antibody incubation is added；
3) it washs, diluted fluorescein-labeled streptavidin solution is added and is incubated for；
4) it washs, be imaged simultaneously judging result to slide in laser scanner.
It is scanned imaging with the slide that the laser scanner in the channel containing Cy3 terminates reaction, suitable laser is selected to sweep
Retouching parameter makes the close saturation of highest signal, gained image on chip be stored as tiff file.It then will be every with chip reading software
The fluorescence signal of a point is converted into digital signal.It is drawn by the digital signal of diluted RTK phosphorylated protein object of reference each
Then the signal of sample judges the expression of RTK phosphorylated protein 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 indicates letter of the positive control in reference group
Number intensity, P (y) indicate signal strength of the positive control in reaction group, and X (y) indicates signal strength of the sample in reaction group,
Indicate detected value of the sample in reaction group.When detected value is greater than 1.5 times of reference value or less than 0.65 times of reference value, can distinguish
It is determined as positive or negative.
Since the present invention uses the good characteristic of sampling liquid, i.e., specific antibody and contain 0.5 to 1.5% casein
PH7.4 phosphate buffer is mixed to form antibody mixture, makes antibody chip kit of the present invention by specific antibody
The step of being fixed on slide is greatly simplified, without general in the prior art (immuno-precipitation or common ELISA method)
The operating procedure of effective component on the point sample rear enclosed slide used.
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, has the advantage that
(1) 71 protein phosphorylation levels can be detected simultaneously, and can be realized the parallel detection of multisample multi objective, overcome
Prior art operation is cumbersome, Testing index is single, need to have the defects such as expense instrument, sensitivity is low, has cheap, convenient, sensitive, quasi-
Really, high-throughput, sample dosage is few, can promote in common lab and scale；(2) phospho-AB core of the present invention
Piece is suitable for using full-automatic point sample instrument come point sample, therefore has high-throughput, multidigit point specific；(3) present invention preferably employs
Processing is optimized to diaphragm as solid phase carrier, and during point sample and coating in porous fiber film, to improve this
Antibody chip kit sensitivity, accuracy, in terms of performance.
In conclusion the phospho-AB chip agent box of human receptor tyrosine kinase enzyme (RTK) of the present invention is special
It is detected suitable for biology, medicine, medicament research and development, disease treatment and its related fields.
Detailed description of the invention
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 cell in chip；Detection of the A431 cell that left figure display EGF is not induced in chip
As a result；Testing result of the A431 cell that right figure display EGF has been induced in chip.This embodiment is using membrane DNA chip.
Fig. 4 is that people RTK phospho-AB chip agent box of the present invention examines Cont-ODN and gal-1-ODN processing
The result figure of trophocyte.This embodiment is using glass-chip.
Specific embodiment
Embodiment 1: the screening of optimum antibody chip carrier.
Conventional antibody chip is mostly using slide as carrier, since slide is frangible and detection device is expensive, is not suitable for small-scale
Using also carrying out problem for the reagent conveyer belt of diagnostic field.And the frangible background of traditional nitrocellulose filter is high, as a result fluctuates
Greatly.We have screened the diaphragm and slide for not having to activity methods processing on the market, obtain either diaphragm by a large amount of screening
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 to make antibody be fixed on membrane surface, we have screened distinct methods processing diaphragm, such as following table.Again with full-automatic
Point sample instrument on the surface in diaphragm, then reads luminescent solution point with UV scanner.
The processing method of each group diaphragm is as follows:
1st group: nitrocellulose diaphragm is purchased from FISHER company, article No. LC2009.
2nd group: Kynoar (PVDF) film is purchased from WHATMAN company, article No. 10485289.
3rd group: the pvdf membrane in the 2nd group is soaked in 0.02% to 0.075% nonionic surfactant Tween80
Aqueous solution in.
4th group: the hydrogen peroxide containing catalyst is added in the pvdf membrane in the 2nd group and carries out Strong oxdiative reaction, adds H2SO4
Form hydrophilic radical.
Specific antibody is pressed into gradient dilution point on above-mentioned 4 groups of diaphragms respectively, secondary antibody then is added and substrate is examined
It surveys, according to the pore homogeneity of point sample, detection limit and background selection select most suitable point sample film, as a result see the table below 1.
Table 1: the comparison of different disposal method diaphragm performance
| The type of film | Homogeneity | Background | Minimum detection limit |
| 1 | Loading wells homogeneity is poor | It is more visible | 0.4ng/ml |
| 2 | The uniform no diffusion in point sample aperture | There is miscellaneous point | 0.2ng/ml |
| 3 | The uniform no diffusion in point sample aperture | Clearly | 0.2ng/ml |
| 4 | Point sample aperture is uniform | Clearly | 0.4ng/ml |
As seen from the above table, the pvdf membrane homogeneity with higher and minimum detection handled by surfactant limits,
The detection limit of pvdf membrane by strong oxidizer processing is more untreated low, but background is more visible.And the albumen of common NC film is inhaled
Attached property is poor, and sensitivity is lower.In summary index, the present invention preferentially select the pvdf membrane by nonionic surfactant processing
As solid phase carrier.
(2) screening of slide
The processing method of each group slide is as follows:
1st group of amination slide: Corning Incorporated, article No. UltraGAPS40019 are purchased from.
2nd group of aldehyde radical slide: glutaraldehyde is added in the 1st group of slide and impregnates 40 minutes production aldehyde radical slides.
3rd group of APES slide: common slide is added in the diluted APES of acetone and is impregnated 0.5 to 1 minute, then uses pure C
APES slide is made in ketone cleaning.
4th group of poly-D-lysine slide: it is 0.5 to 1 small that the diluted poly-D-lysine immersion of PBS is added in common slide
When, then cleaned with pure water and poly-D-lysine slide is made.
5th group through hydrophilic reagent handle slide: common slide is handled with hydrophilic reagent, hydrophilic reagent be 0.01 to
0.1% alkyl glycosides.
The point sample effect of various slides is not quite similar, and the slide point sample effect that hydrophilic coating is added is more apparent, point sample effect
More untreated height.The more other type effects of slide wherein Jing Guo 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 degree under the conditions of, no matter which kind of film/slide point
All there are diffusion phenomena in sample effect, and temperature humidity is higher, and diffusion is 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 the following table 2.
Table 2:
In summary index is selected by the slide of hydrophilic reagent alkyl glycosides processing 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: the 20X concentrated cleaning solution containing polysorbas20.1X cleaning solution is pH 7.2, contains 0.1% polysorbas20,0.1mol/
The phosphate buffer of L.
Dilution: 2 bottles of 15ml 5X concentration and dilution liquid D for diluted sample, 1 bottle for diluting antibody and HRP- chain parent
With the 15ml 5X concentration and dilution liquid B of element.1X dilution B is 15mM, the PBS buffer solution 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
Release liquid D be 15mM, the PBS buffer solution 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.
Detect antibody: biotinylated detection antibody mixture.
Fluorescein-streptavidin solution is concentrated in 200 μ l 300X.
Sample treatment solution: 2X cell pyrolysis liquid 10ml, 1X cell pyrolysis liquid includes 10mM pH 7.5, Tris.HCl, 25mM
NaCl, 1% NaTDC, 1%Triton X-100 include phosphatase and protease inhibitors.
Coated specific antibody is for the antibody for being selected from following 71 kinds of protein on solid phase carrier (referring to table 3).
These antibody are commercially available, such as purchased from the 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 point sample film
Method the following steps are included:
1) specific antibody is mixed to form antibody with the pH7.4 phosphate buffer containing 0.5 to 1.5% casein and mixes
Object；
2) each antibody content is fixed on the loading wells with 0.01 to 2ng in antibody mixture, each antibody is set
Set 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 controlling according to different antibody chip arrangement arrays
Auto sample applicator prepares required intermediate products.
A kind of dot matrix sequence of specific antibody can be found in Fig. 2.
The diaphragm further includes positive control and negative control.Positive control wells are that having for corresponding specific antibody is corresponding
The concentration of the biotinylation IgG antibody of concentration, the biotinylated IgG antibody of each reaction is consistent, in order to standardize detection.
Negative control hole be the biotinylated irrelevant antibody with respective concentration, the biotinylated irrelevant antibody of each reaction 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, comprising: 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 saves for a long time also volatile
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 handles perforated membrane, combines closely under the conditions of 18 to 22 DEG C with specific antibody, finally formed point sample
Hole complete display.
In the present embodiment, full-automatic point sample instrument is the product that Bio Rad Laboratories or platinum Ai Ermo company produce；Slide
For 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 to be able to solve technical problem of the invention, and realize that corresponding technical effect is foundation.
Embodiment 3: RTK phosphorylated protein is detected with the kit of embodiment 2
Counterdie is put in mating square box, since multiple chip dot matrix being distributed on 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 mutually independent anti-
Area is answered, what is used in the present embodiment is provided with 8 grid square boxes, is placed in room temperature into each grid plus after 2 milliliters of confining liquids
Lower culture 30 minutes then successively carries out the rapid operation of following steps:
1, it is loaded
The confining liquid in each grid is sucked out, is put into film through the sample that confining liquid diluted for 100 microlitres~5 milliliters
It in grid, is then placed on shaking table and shakes at room temperature 1 to 2 hour, or can also be reacted 12-18 hours at 4 DEG C.
2, film is washed
Cleaning solution I cleaning: sample is sucked out out of grid, is cleaned with 1~5 milliliter 1 times of cleaning solution I, is placed on shaking table later
Upper room temperature is shaken 5 minutes, repeats this cleaning step twice.
Cleaning solution II cleaning: raffinate is sucked out out of grid, is cleaned with 1~5 milliliter 1 times of cleaning solution II, is placed on shakes later
Room temperature is shaken 5 minutes on bed, and it is primary to repeat this cleaning step.
3, the antibody mixed liquor for being marked with biotin is added
Biotin labeling is added to each grid, is mixed with the specific antibody for antigen protein listed in table 1
500 microlitres~2 milliliters of mixed liquor, be then placed on shaking table room temperature and shake 1 to 2 hour.Also it is small 12-18 can be reacted at 4 DEG C
When.Then go out to be marked with the antibody mixed liquor of biotin from grid interior suction, repeat step 2 washes film step.
4, HRP-Streptavidin is added
The avidin of 500 microlitres~2 milliliters horseradish peroxidase-labeleds diluted is added to each grid
Streptavidin (HRP-Streptavidin), is then placed on shaking table and shakes under room temperature 1 to 2 hour, can also react at 4 DEG C
12-18 hours.HRP-Streptavidin is then sucked out out of grid, repeat step 2 washes film step.
HRP-Streptavidin is commercially available from BD company (production number 554066), before experiment, needs to be carried out with confining liquid
20,000 times of dilutions.
It should be noted that in step 1 to the step 4 of this implementation, use it is following be easy, ingredient can preparation method such as
Under:
2M Tris buffer (pH7.5): Trizma Base 484g, purified water 1.3L adjust pH value to 7.5, purify
Water adds to 2L.
The preparation method of confining liquid is as follows: first by 20 × PBS (KCl 16g, NaCl 640g, KH2PO416g,
Na2HPO492g, after being dissolved in 2.6L purified water, then plus purified water to 4 liters) be 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,
It 4 liters of Casein, mixes).
20 × cleaning solution II (20 × TBS) partition is as follows: 2M Tris buffer (pH7.5) 800ml, 5M NaCl
4800ml (NaCl 1461g, 3.3 liters of purified water, purified water adds to 5 liters after dissolution), after mixing, purified water adds to 8 liters.It uses
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.In use, by 20 × cleaning solution II doubling dilution.
5, it detects
Pressing from both sides out counterdie with tweezers and being placed in plumbness makes extra liquid drip-dry；Film is then placed on clean plastic sheet
On, guarantee that counterdie is fixed with the one side of antibody upward；500 microlitres of prepared (A:B=1:1) luminescent solutions are added (to be commercially available from
BIOFX company, production number LUMB-0500-01) to placing 2 minutes on every counterdie and at room temperature, this process must assure that hair
Whole film is completely covered in light liquid；It is covered in counterdie surface with another clean plastic sheet again, it carefully will be in plastic sheet
Bubble squeezes away, should be avoided on film during squeezing away the bubble in plastic sheet firmly.After above-mentioned steps,
Counterdie is placed on and is imaged within 5 to 10 seconds at room temperature, uses low-temperature CCD low when shooting, and using reading number in the UV scanner to match
According to.
Embodiment 4: the application of kit
1) squama epidermal cell cancer cell A431 is exposed to 37 DEG C after serum starvation overnight is handled, the EGF10 of 100ng/ml
Minute, control cell equally through Nature enemy but is not exposed in EGF.Two groups of cells are added in antibody chip after cracking incubates
It educates, adds the anti-phosphate acceptor phosphotyrosine antibody of biotin labeling to detect the phosphate acceptor junket ammonia on activated receptor
Acid.After fluorescent dye acts on, signal is detected using laser scanner.
Testing result of the A431 cell that the left figure display EGF of Fig. 3 is not induced in chip；The right figure of Fig. 3 shows that EGF has been lured
Testing result of the A431 cell led in chip.
As the result is shown: the EGFR of the A431 cell by EGF induction, the expression quantity ratio of ErbB2, ErbB3 albumen do not induce
Height.
2) people RTK phospho-AB chip agent box detection Cont-ODN and gal-1-ODN of the present invention is handled
Trophocyte.
GAL-1 is played crucial by the expression of HLA-G on adjusting embryonic feeder cells in trimester maternal immunological regulation
Effect.GAL-1 level can be used as the factor of prediction miscarriage.
Human receptor tyrosine kinase enzyme phospho-AB chip can monitor effect of the GAL-1 to cell protein phosphotidic level.
Embryonic feeder cells cell (Cont-ODN) and its trophocyte (gal-1-ODN) for inhibiting GAL-1 to express are by cracking simultaneously
It is separately added into human receptor tyrosine kinase enzyme phospho-AB chip, sufficiently after washing, adds the anti-phosphoric acid junket of biotin labeling
Propylhomoserin antibody, HRP marker streptomysin and luminous substrate.
As the result is shown: the expression of FGFR2, SRMS, TXK albumen is inhibited by different degrees of.
Claims (4)
1. a kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme characterized by comprising
Solid phase carrier is porous fiber film, is selected from: nitrocellulose filter, fiber filter film, polyvinylidene fluoride film or nylon membrane；
The porous fiber film is under the surface modification of 0.05 to 0.1% surfactant or under severe oxidative conditions shape
At what is combined closely under 20 to 26 DEG C, 40% damp condition with specific antibody after hydrophilic radical；
Cleaning solution, 20X concentrated cleaning solution and its dilution including containing 0.1% polysorbas20；
Sample dilution；
For diluting the dilution of antibody and HRP- streptavidin；
Biotinylated detection antibody mixture；
Fluorescein-streptavidin solution is concentrated in 300X；
Sample treatment solution, for the RIPA buffer comprising protease and phosphatase inhibitor cocktail, the inhibitors of phosphatases
It is to be selected from: Na3VO4,NaF；
Wherein, the specific antibody for the antibody of following 55 kinds of protein by forming: ABL1, ACK1, Ax1, Blk, BMX,
Btk、Csk、Dtk、EphA1、EphA1、EphA3、EphA4、EphA5、EphA6、EphA7、EphA8、EphB6、FAK、FER、
FGFR2α、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、ROR2、ROS、RYK、SRMS、Tec、Tie-1、
Tie-2、TNK1、TRKB、TXK、Tyk2、TYRO10、VEGFR3。
2. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 1, feature exist
In: the perforated membrane is the pvdf membrane handled by nonionic surfactant.
3. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 2, feature exist
In: the nonionic surfactant is Tween80.
4. the phospho-AB chip agent box of detection human receptor tyrosine kinase enzyme according to claim 1, feature exist
In: the perforated membrane is pvdf membrane, and Xiang Suoshu pvdf membrane, which is added in the hydrogen peroxide containing catalyst, carries out Strong oxdiative reaction, is added
H2SO4Form hydrophilic radical.
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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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| WO2017127664A1 (en) | 2016-01-20 | 2017-07-27 | The Scripps Research Institute | Ror1 antibody compositions and related methods |
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| CN109957139A (en) * | 2019-03-14 | 2019-07-02 | 天韧膜科技(苏州)有限公司 | A kind of post-processing approach for nitrocellulose membrane |
| CN114076823B (en) * | 2020-08-13 | 2024-05-17 | 深圳迈瑞生物医疗电子股份有限公司 | Method for preparing solid phase component and prepared solid phase component |
| CN115097129B (en) * | 2022-08-24 | 2023-03-10 | 山东子峰生物技术有限公司 | Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1 |
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