WO2017107541A1 - 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒 - Google Patents

一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒 Download PDF

Info

Publication number
WO2017107541A1
WO2017107541A1 PCT/CN2016/097197 CN2016097197W WO2017107541A1 WO 2017107541 A1 WO2017107541 A1 WO 2017107541A1 CN 2016097197 W CN2016097197 W CN 2016097197W WO 2017107541 A1 WO2017107541 A1 WO 2017107541A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
receptor tyrosine
tyrosine kinase
detecting
membrane
Prior art date
Application number
PCT/CN2016/097197
Other languages
English (en)
French (fr)
Inventor
黄若磐
黄若春
Original Assignee
广州瑞博奥生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州瑞博奥生物科技有限公司 filed Critical 广州瑞博奥生物科技有限公司
Publication of WO2017107541A1 publication Critical patent/WO2017107541A1/zh

Links

Images

Classifications

    • 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
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • the invention belongs to the field of biotechnology, and relates to a phosphorylated antibody chip kit, in particular to a phosphorylated antibody chip kit for human receptor tyrosine kinase.
  • Protein phosphorylation refers to the fact that intracellular proteins are stimulated by protein kinases at specific amino acid sites under the stimulation of external signals, and phosphorylated proteins are produced, which in turn activate downstream signaling molecules through cascades. Achieve biological effects.
  • a protein can be phosphorylated by multiple protein kinases at one or more sites.
  • the cell membrane receptor EGFR family includes four different proteins, EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. Under normal physiological conditions, ErbB receptors play a crucial role in regulating signal transduction of cell proliferation, differentiation, movement and apoptosis.
  • the EGF receptor family shows significant differences in different receptors and also shows large crossover.
  • ErbB1 has the most partners in family members and has the highest ratio of receptor tyrosine residues that bind multiple partners; ErbB3 is characterized by multiple phosphatidylinositol-3-kinase (PI3K) binding sites ErbB2 has few associated partners, and Shc is the most common; ErbB1 and ErbB4 have multiple phosphate receptor tyrosine residues that bind to Grb2 or Grb2 and Shc, and show a larger range of spouses than ErbB2 and ErbB3. ErbB1 and ErbB2 are usually overexpressed or amplified in cancer cells, making them important target proteins in drug use or development.
  • PI3K phosphatidylinositol-3-kinase
  • Tyrosine phosphorylation of proteins and enzymes can only be detected by very dangerous and time consuming radioactivity experiments in the absence of anti-tyrosine phosphorylation antibodies.
  • anti-tyrosine phosphorylation antibodies With anti-tyrosine phosphorylation antibodies, phosphorylation signals can be easily detected by Western Blot or other immunological methods.
  • Conventional assays include detection of endogenous or exogenously expressed phosphorylated proteins on Western Blots with anti-tyrosine phosphorylated antibodies. If the content of the target protein is low, the phosphorylated tyrosine protein can be first enriched by immunoprecipitation, and the level of the target protein can be detected.
  • the object of the present invention is to provide a phosphorylated antibody chip kit for human receptor tyrosine kinase (RTK), which can rapidly, easily and accurately determine 71 RTK pathways through one experiment.
  • RTK human receptor tyrosine kinase
  • the phosphorylated antibody chip kit for detecting human receptor tyrosine kinase of the present invention comprises: a solid phase carrier which is a standard membrane for coating specific antibodies; and a washing solution comprising 20X concentration containing 0.1% Tween 20 Washing solution and dilution thereof; sample diluent; dilution for diluting antibody and HRP-streptavidin; biotinylated detection antibody mixture; 300X concentrated fluorescein-streptavidin solution; sample treatment solution, 2X cell lysate; wherein the specific antibody is an antibody against 71 proteins selected from the group consisting of: ABL1, ACK1, ALK, Ax1, Blk, BMX, Btk, Csk, Dtk, EGFR, EphA1, EphA1, EphA2, EphA3 , EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, Er
  • the solid phase carrier is a porous fiber membrane selected from the group consisting of a nitrocellulose membrane, a fiber filtration membrane, and a polyvinylidene fluoride.
  • the porous membrane is 0.05 to 0.1% of a surfactant or is tightly bound to a specific antibody at 20 to 26 ° C and 40% humidity after forming a hydrophilic group under strong oxidizing conditions.
  • the porous membrane is a PVDF membrane treated with a nonionic surfactant.
  • the solid phase carrier is a slide.
  • the slide is intimately bound to the specific antibody at 20 to 26 ° C after treatment with glutaraldehyde or polylysine, alkyl glycoside or APES adhesive.
  • the slide is a slide treated with a hydrophilic reagent alkyl glycoside.
  • the hydrophilic agent is an alkyl glycoside having a mass fraction of 0.01 to 0.2%, 0.01 to 0.1% of glycerin, and 0.01 to 0.05% of an ultrapure aqueous solution of polyethylene glycol 4000; a hydrophilic reagent-treated slide
  • the method is to soak the slide in the hydrophilic reagent for 3 to 5 minutes and dry it.
  • the hydrophilic agent is an alkyl glycoside having a mass fraction of 0.1%, 0.05% glycerol, and 0.01% poly
  • test antibody mixture is spotted onto a solid support at 24 ° C, 40% humidity.
  • a further feature of the phosphorylated antibody chip kit for detecting a human receptor tyrosine kinase according to the present invention is a RIPA buffer comprising a mixture of a protease and a phosphatase inhibitor.
  • the phosphatase inhibitor is selected from the group consisting of Na 3 VO 4 , NaF.
  • the antibody chip kit of the present invention can measure RTK phosphorylated protein by a detection method similar to a sandwich ELISA.
  • the operation steps include:
  • the slides with the Cy3 channel were scanned and imaged.
  • the appropriate laser scanning parameters were used to make the highest signal on the chip nearly saturated, and the resulting image was stored as a tiff file.
  • the fluorescence signal at each point is then converted to a digital signal using chip reading software.
  • the signal of each sample is plotted by the digital signal of the diluted RTK phosphorylated protein reference, and then the expression level of the RTK phosphorylated protein in the sample is judged by the corresponding standard reference value.
  • the result is determined according to the following calculation method:
  • X(Ny) X(y)*P1/P(y), after subtracting the background signal value, where P1 represents the signal intensity of the positive control in the reference group, and P(y) represents the positive control in the reaction group.
  • the signal intensity, X(y) represents the signal intensity of the sample in the reaction set and represents the detected value of the sample in the reaction set. When the detected value is 1.5 times larger than the reference value or 0.65 times smaller than the reference value, it may be judged as positive or negative, respectively.
  • the antibody chip kit of the present invention will have a specific antibody.
  • the step of immobilization on the slide is greatly simplified, and the operation steps of the active ingredient on the slide after spotting are not commonly used in the prior art (immunoprecipitation or ordinary ELISA).
  • a fully automatic spotting instrument manufactured by Bio-Rad Corporation or Perkin Elmer Company of the United States is used.
  • the specific antibody chip arrays are arranged on the membrane, and in the specific operation process, the arrangement of each specific antibody can be adjusted according to the experimental design, and the automatic antibody sampling is controlled according to different antibody chip arrays. Instrument to prepare the required intermediate products.
  • the phosphorylated antibody chip kit for human receptor tyrosine kinase (RTK) of the present invention has the following advantages: (1) simultaneous detection of 71 protein phosphorylation levels, and parallelization of multiple samples and multiple indicators Detection, gram It has the defects of cumbersome operation, single detection index, low cost instrument and low sensitivity. It has the advantages of low cost, convenience, sensitivity, accuracy, high throughput, low sample consumption, and can be promoted and scaled in ordinary laboratories; (2)
  • the phosphorylated antibody chip used in the present invention is suitable for spotting with a fully automatic spotting instrument, and thus has high specificity and multi-site specificity; (3)
  • the present invention preferably uses a porous fiber membrane as a solid phase carrier. The membrane is optimized during the spotting and coating process, thereby improving the performance, accuracy, high throughput and the like of the antibody chip kit.
  • the humanized receptor tyrosine kinase (RTK) phosphorylated antibody chip kit of the present invention is particularly suitable for use in biology, medicine, drug development, disease treatment, and related fields.
  • Fig. 1 is a spot-like array diagram of a membrane chip (using a membrane as a solid phase carrier) for detecting a phosphorylated antibody chip kit for human receptor tyrosine kinase according to the present invention.
  • Fig. 2 is a spot-like array diagram of a glass chip (using a slide as a solid phase carrier) for detecting a phosphorylated antibody chip kit for human receptor tyrosine kinase according to the present invention.
  • Figure 3 shows the results of A431 cells on the chip; the left panel shows the results of EGF-induced A431 cells on the chip; the right panel shows the results of EGF-induced A431 cells on the chip.
  • This embodiment employs a film chip.
  • Figure 4 is a graph showing the results of detection of Cont-ODN and gal-1-ODN-treated trophoblast cells by the human RTK phosphorylated antibody chip kit of the present invention. This embodiment employs a glass chip.
  • Example 1 Screening of optimal antibody chip vectors.
  • the membrane In order to immobilize the antibody on the surface of the membrane, we screened the membrane for different methods, as shown in the table below. The luminescent liquid is then spotted on the surface of the diaphragm using a fully automated spotter and then read with a UV scanner.
  • Group 1 nitrocellulose membranes, purchased from FISHER, Inc., serial number LC2009.
  • PVDF Polyvinylidene fluoride
  • Group 3 Soak the PVDF membrane in Group 2 at 0.02% to 0.075% of nonionic surfactant Tween80 In an aqueous solution.
  • Group 4 The PVDF membrane in the second group was added to the catalyst-containing hydrogen peroxide to carry out a strong oxidation reaction, and then H 2 SO 4 was added to form a hydrophilic group.
  • the specific antibodies were diluted to the above four groups of membranes, and then the secondary antibody and the substrate were added for detection. According to the pore size uniformity, detection limit and background, the most suitable spotting membrane was selected. See Table 1 below.
  • the PVDF membrane treated by the surfactant has higher uniformity and the lowest detection limit.
  • the detection limit of the PVDF membrane treated by the strong oxidant is lower than that of the untreated one, but the background is clear.
  • the ordinary NC membrane has poor protein adsorption and low sensitivity.
  • the present invention preferentially selects a PVDF membrane treated with a nonionic surfactant as a solid phase carrier.
  • Group 1 aminated slides purchased from Corning Incorporated under the trade designation UltraGAPS 40019.
  • Group 2 Aldylated Slides The slides of Group 1 were soaked in glutaraldehyde for 40 minutes to make aldehydeylated slides.
  • Group 3 APES slides A common slide was added to acetone-diluted APES for 0.5 to 1 minute, and then washed with pure acetone to make an APES slide.
  • Group 4 polylysine slide The ordinary slide was immersed in PBS diluted polylysine for 0.5 to 1 hour, and then washed with pure water to make a polylysine slide.
  • Group 5 hydrophilic-treated slides The ordinary slides were treated with a hydrophilic reagent, and the hydrophilic reagent was 0.01 to 0.1% of alkyl glycosides.
  • the spotting effect of various slides is not the same.
  • the effect of the slides added with the hydrophilic coating is more obvious, and the spotting effect is higher than that of the untreated ones.
  • the slides which have been subjected to amination and hydrophilic treatment have higher effects than other types. Under the condition of 20 to 26 degrees, the film glass/slices have clearer effect and the background value is low. Under the condition of 27 to 30 degrees, no matter what kind of film/slide film spotting effect, the diffusion phenomenon occurs, and the temperature and humidity are more High, the more obvious the diffusion.
  • a slide treated with a hydrophilic reagent alkyl glycoside was selected as a solid phase carrier.
  • Example 2 Preparation of a kit.
  • the antibody chip kit of the present invention comprises the following components:
  • Solid phase carrier A standard membrane coated with antibodies.
  • Washing solution 20X concentrated washing solution containing Tween 20.
  • the 1X washing solution was pH 7.2, containing 0.1% Tween 20, 0.1 mol/L phosphate buffer.
  • Diluent 2 bottles of 15 ml 5X concentrated diluent D for dilution of the sample, 1 bottle for 15 ml of 5X concentrated dilution B for dilution of antibody and HRP-streptavidin.
  • 1X Diluent B is 15 mM, p H7.4 in PBS buffer, solute and its mass concentration or molar concentration or volume concentration in the dilution B are as follows: 0.5% casein, 2-4% sucrose, 150 mM NaCl .
  • 1X Diluent D is 15 mM, pH 6.5 in PBS buffer, the solute and its mass concentration or molar concentration or volume concentration in the sample dilution is as follows: 2-4% sucrose, 150 mM NaCl.
  • Detection antibody biotinylated detection antibody mixture.
  • Sample treatment solution 10 ml of 2X cell lysate containing 10 mM pH 7.5, Tris.HCl, 25 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, containing phosphatase and protease inhibitor.
  • the specific antibody coated on the solid phase carrier is an antibody against 71 proteins selected from the following (see Table 3). These antibodies are commercially available, for example, from companies such as R&D Systems, PeproTech, Raybiotech, and the like.
  • the antibody chip is arranged in a lattice on the membrane, and the membrane has 10 to 100 specific antibodies per square centimeter.
  • the arrangement of each specific antibody can be adjusted according to the needs of the experimental design.
  • the intermediate product can be prepared by controlling the automatic spotting instrument.
  • the patch also includes a positive control and a negative control.
  • the positive control wells are biotinylated IgG antibodies of corresponding concentration corresponding to specific antibodies, and the concentrations of biotinylated IgG antibodies per reaction are consistent to facilitate standardized detection.
  • Negative control wells are biotinylated, irrelevant antibodies with corresponding concentrations, and the concentration of biotinylated, irrelevant antibodies per reaction is consistent to facilitate standardized detection.
  • the solid phase carrier was a porous fiber membrane comprising: a nitrocellulose membrane, a fibrous filtration membrane, a glass fiber substrate, a polyvinylidene fluoride membrane (PVDF) or a nylon membrane.
  • PVDF polyvinylidene fluoride membrane
  • the fiber membrane is easily hydrolyzed under high temperature, high pressure and acid-base conditions, and is also easily decomposed by microorganisms. It is easy to lose water and dry for a long time, and is charged and brittle, and has poor durability. Therefore, the present invention passes 0.05 to 0.1% of a surfactant, 0.1
  • the porous membrane was treated with 0.5% sodium azide and tightly bound to specific antibodies at 18 to 22 ° C, and the resulting spotted wells were clear and intact.
  • the automatic spotting instrument is a product produced by American Bole Company or Platinum Elmer Company; the glass piece is a product of Corning Company of the United States.
  • the adoption of the instruments and materials is not limited to the enumeration of the embodiment, but is based on the technical problem capable of solving the present invention and achieving the corresponding technical effects.
  • Example 3 Detection of RTK phosphorylated protein using the kit of Example 2
  • the bottom film is placed in the matching box. Since the bottom film of the embodiment has a plurality of chip lattices distributed thereon, in the embodiment, the square box is provided with 8 squares, and the squares between the square boxes will be The chip array is divided into mutually independent reaction zones. In this embodiment, 8 square square boxes are arranged, and 2 ml of blocking solution is added to each square, and then placed at room temperature for 30 minutes, and then sequentially. The following steps:
  • Washing solution I cleaning Aspirate the sample from the square, wash it with 1 to 5 ml of 1 times washing solution I, then shake it on a shaker at room temperature for 5 minutes, and repeat the washing step twice.
  • Washing liquid II cleaning The residual liquid is aspirated from the square, washed with 1 to 5 ml of 1 times washing liquid II, and then shaken at room temperature for 5 minutes on a shaker, and the washing step is repeated once.
  • HRP-Streptavidin horseradish peroxidase-labeled streptavidin
  • HRP-Streptavidin is commercially available from BD (Product No. 554066) and requires a 20,000-fold dilution with a blocking solution prior to the experiment.
  • Tris buffer pH 7.5: Trizma Base 484 g, purified water 1.3 L, adjusted pH to 7.5, and purified water was added to 2 L.
  • the preparation method of the blocking solution is as follows: firstly, 20 ⁇ PBS (KCl 16 g, NaCl 640 g, KH 2 PO 4 16 g, Na 2 HPO 4 92 g, dissolved in 2.6 L of purified water, and then purified water to 4 liters) is diluted into 1 ⁇ PBS (20 ⁇ PBS 200 ml, purified water 3800 ml), then 10% BSA (BSA 400 g, 1 ⁇ PBS added to 4 liters) was prepared, and finally a blocking solution (10% BSA 4 liters, Casein 4 liters, and mixed) was prepared.
  • 20 ⁇ washing solution II (20 ⁇ TBS) distribution is as follows: 2M Tris buffer (pH 7.5) 800ml, 5M NaCl 4800ml (NaCl 1461g, purified water 3.3 liters, dissolved water after adding to 5 liters), after mixing, Purified water was added to 8 liters.
  • the 20 ⁇ washing solution can be diluted by a factor of two.
  • the 20 ⁇ washing solution I (2% Tween/20 ⁇ TBS) was distributed as follows: 20 ⁇ washing solution II 1 L, Tween 20 ml, and mixing. When used, the 20 ⁇ washing solution can be diluted by a factor of two.
  • Scale epidermal cell carcinoma A431 was treated with serum starvation overnight and exposed to 37 ° C, 100 ng/ml EGF for 10 minutes, and the control cells were also starved but not exposed to EGF. The two groups of cells were lysed and added to the antibody chip for incubation, and then biotinylated anti-phosphorylated receptor tyrosine antibody was added to detect the phosphorylated receptor tyrosine on the activated receptor. After the action of the fluorescent dye, the signal is detected by a laser scanner.
  • the left panel of Figure 3 shows the results of EGF-induced A431 cells assayed on the chip; the right panel of Figure 3 shows the results of EGF-induced A431 cells assay on the chip.
  • the human RTK phosphorylated antibody chip kit of the present invention detects Cont-ODN and gal-1-ODN-treated trophoblast cells.
  • GAL-1 plays a key role in maternal immune regulation during pregnancy by regulating the expression of HLA-G on embryonic trophoblasts. GAL-1 levels can be used as a predictor of abortion.
  • the human receptor tyrosine kinase phosphorylation antibody chip monitors the effect of GAL-1 on the level of cellular protein phosphorylation.
  • Embryonic trophoblast cells (Cont-ODN) and trophoblast cells (gal-1-ODN) that inhibit GAL-1 expression are lysed and added to human receptor tyrosine kinase phosphorylation antibody chip, fully washed, and then added Biotinylated anti-phosphotyrosine antibody, HRP marker streptomycin and luminescent substrate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种人受体酪氨酸激酶(RTK)的磷酸化抗体芯片试剂盒，包括：固相载体，为包被特异性抗体的标准膜片；洗涤液，包括含0.1％吐温20的20X浓缩洗涤液以及其稀释液；样本稀释液；用于稀释抗体和HRP-链亲和素的稀释液；生物素化的检测抗体混合物；300X浓缩荧光素-链亲和素溶液；样本处理液，为2X细胞裂解液；该特异性抗体是针对71种蛋白质的抗体。该RTK磷酸化抗体芯片试剂盒能够通过一次实验迅速、简捷和准确地测定RTK通路中71个蛋白质的磷酸化情况，通过监测实验模型系统中的蛋白磷酸化变化，便能快速检测到细胞中的途径激活作用，而不需要经过繁琐的免疫沉淀法与印迹法。

Description

一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒 技术领域
本发明属于生物技术领域，涉及一种磷酸化抗体芯片试剂盒，尤其是涉及一种人受体酪氨酸激酶的磷酸化抗体芯片试剂盒。
背景技术
蛋白质磷酸化是指细胞内蛋白质在外界信号刺激下、在特定的氨基酸位点上由蛋白激酶催化发生磷酸化发生、而产生磷酸化的蛋白将依次激活下游各级信号分子、通过级联反应最终实现生物学效应。一个蛋白质可以在一个或者多个位点上，由多个蛋白激酶产生磷酸化。细胞膜受体EGFR家族包括EGFR/ErbB1/HER1、ErbB2/HER2、ErbB3/HER3和ErbB4/HER4四种不同蛋白。在正常的生理状态下，ErbB受体对于调节细胞增殖、分化、运动与凋亡的信号转导起着至关重要的作用。EGF受体家族在不同的受体中显示着明显差异，也显示大的交叉性。其中，ErbB1在家族成员中最多配偶体，并具有最高比率的结合多个配偶体的受体酪氨酸残基；ErbB3特征化为多个磷脂酰肌醇-3-激酶(PI3K)结合位点；ErbB2具有很少的关联配偶体，Shc是最常的一个；ErbB1与ErbB4具有结合Grb2或Grb2和Shc多种磷酸受体酪氨酸残基，并显示着比ErbB2与ErbB3更大范围的配偶体；ErbB1与ErbB2通常在癌细胞中过表达或放大，使得它们在药物使用或开发中成为重要的目标蛋白。磷酸化的正常与否，也常常预示着细胞信号传导是否出现异常，而异常的传导通路经常又导致一些疾病。因此，对磷酸化的研究呈现出很重要的生物和临床价值。由于磷酸化位点的多样性，如何来快速有效地了解细胞内的磷酸化状态，一直是困扰广大科研工作者的一个难题。
在没有抗酪氨酸磷酸化抗体之前，蛋白质和酶的酪氨酸磷酸化只能通过非常危险的并且很费时的放射性实验来检测。而利用抗酪氨酸磷酸化抗体，则可以通过Western Blot或其它免疫学方法轻松地检测到磷酸化信号。常规的检测方法包括：用抗酪氨酸磷酸化抗体在Western Blot上检测内源或外源表达的磷酸化蛋白。如果目标蛋白的含量较低，也可利用免疫沉淀的方法先富集发生磷酸化的酪氨酸蛋白，再检测目标蛋白的水平。可见，检测受体酪氨酸激酶的磷酸化操作繁琐，成本高；另外，由于科学家研究的通常是采用新的磷酸化位点，而制备蛋白特定位点的磷酸化抗体往往比一般抗体的制备更加费时费力，效果也不好。
发明内容
本发明的目的在于提供一种人受体酪氨酸激酶(RTK)的磷酸化抗体芯片试剂盒，该RTK磷酸化抗体芯片试剂盒能够通过一次实验迅速、简捷和准确地测定RTK通路中71个蛋白质的磷酸化情况，通过监测实验模型系统中的蛋白磷酸化变化，便能快速检测到细胞中的途径激活作用，而不需要经过繁琐的免疫沉淀法与印迹法(Western Blotting)。
本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒包括：固相载体，为包被特异性抗体的标准膜片；洗涤液，包括含0.1％吐温20的20X浓缩洗涤液以及其稀释液；样本稀释液；用于稀释抗体和HRP-链亲和素的稀释液；生物素化的检测抗体混合物；300X浓缩荧光素-链亲和素溶液；样本处理液，为2X细胞裂解液；其中，所述特异性抗体是针对选自以下71种蛋白质的抗体：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型)、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。
根据本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒的进一步特征，所述固相载体为多孔纤维膜，选自：硝酸纤维素膜、纤维过滤膜、聚偏氟乙烯膜(PVDF)或者尼龙膜。
优选地，所述多孔膜是0.05至0.1％的表面活性剂或者在强氧化条件下形成亲水基团后在20至26℃，40％湿度条件下与特异性抗体紧密结合的。
更优选地，所述多孔膜是经过非离子表面活性剂处理的PVDF膜。
根据本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒的进一步特征，所述固相载体为玻片。
优选地，所述玻片是通过戊二醛或多聚赖氨酸、烷基糖苷或者APES粘附剂处理后在20至26℃条件下与特异性抗体紧密结合的。
更优选地，所述玻片是经过亲水试剂烷基糖苷处理的玻片。
更优选地，所述亲水试剂为质量分数为0.01～0.2％的烷基糖苷、0.01～0.1％的甘油，0.01～0.05％的聚乙二醇4000的超纯水溶液；亲水试剂处理玻片的方法为将玻片在亲水试剂中浸泡3～5分钟，晾干即可。
最优选地，所述亲水试剂为质量分数为0.1％的烷基糖苷、0.05％的甘油，0.01％的聚 乙二醇4000的超纯水溶液；亲水试剂处理玻片的方法为将玻片在亲水试剂中浸泡3分钟，晾干即可。
更优选地，所述检测抗体混合物在24℃、40％的湿度条件下点样到固相载体上。
根据本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒的进一步特征，所述样本处理液为包含蛋白酶和磷酸酶抑制剂混合物的RIPA缓冲液。
优选地，所述磷酸酶抑制剂是选自：Na3VO4、NaF。
本发明所述的抗体芯片试剂盒可通过类似于夹心法ELISA的检测方法来测定RTK磷酸化蛋白。其操作步骤包括：
1)用样本稀释液稀释相应浓度的样本于反应槽中孵育；
2)洗涤，加入稀释的生物素化的检测抗体孵育；
3)洗涤，加入稀释的荧光素标记的链亲和素溶液孵育；
4)洗涤，于激光扫描仪对玻片进行成像并判断结果。
用含Cy3通道的激光扫描仪对反应结束的玻片进行扫描成像，选用合适的激光扫描参数使芯片上最高信号接近饱和，所得图像存储为tiff文件。然后用芯片读数软件将每个点的荧光信号转化为数码信号。通过稀释的RTK磷酸化蛋白参照物的数码信号绘制每个样本的信号，然后通过相应的标准参照值判断出RTK磷酸化蛋白在样品中的表达水平。
优选地，结果判定根据以下计算方式：
X(Ny)＝X(y)*P1/P(y),减去背景信嗓值后，其中P1表示阳性对照在参考组中的信号强度，P(y)表示阳性对照在反应组中的信号强度，X(y)表示样本在反应组中的信号强度，表示样本在反应组中的检测值。当检测值大于参考值1.5倍或者小于参考值0.65倍，可分别判定为阳性或者阴性。
由于本发明采用点样液的优良特性，即特异性抗体与含有0.5至1.5％酪蛋白的pH7.4磷酸盐缓冲液混合形成抗体混合物，使本发明所述的抗体芯片试剂盒将特异性抗体固定于玻片的步骤得到极大的简化，无须现有技术(免疫沉淀法或者普通的ELISA法)中普遍采用的点样后封闭玻片上的有效成分的操作步骤。
在本发明的一个实施例中，步骤(1)的点样操作中，采用美国伯乐(Bio-Rad)公司或铂金艾尔默(Perkin Elmer)公司生产的全自动点样仪器。各特异性抗体芯片点阵排布于膜片，而在具体的操作过程中，各特异性抗体的排布可以按照实验设计需要进行调整，根据不同的抗体芯片排布阵列，控制全自动点样仪，制备出所需要的中间产品。
本发明所述的人受体酪氨酸激酶(RTK)的磷酸化抗体芯片试剂盒，具有以下优点：(1)能够同时检测出71项蛋白磷酸化水平，并且能够实现多样本多指标的并行检测，克 服了现有技术操作繁琐、检测指标单一、需有费仪器、灵敏度低等缺陷，具有廉价、便利、灵敏、准确、高通量、标本用量少、能在普通实验室推广和规模化；(2)本发明所采用的磷酸化抗体芯片适合于采用全自动点样仪来点样，因此具有高通量、多位点的特定；(3)本发明优选采用多孔纤维膜作为固相载体，并在点样和包被过程中对膜片进行了优化处理，从而提高了该抗体芯片试剂盒在灵敏度、准确性、高通量等方面的性能。
综上所述，本发明所述的人受体酪氨酸激酶(RTK)的磷酸化抗体芯片试剂盒特别适用于生物学、医学、药物研发、疾病治疗及其相关领域检测。
附图说明
图1为本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒的膜芯片(采用膜片作为固相载体)的点样阵列图。
图2为为本发明所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒的玻璃芯片(采用玻片作为固相载体)的点样阵列图。
图3为A431细胞在芯片的检测结果；左图显示EGF未诱导的A431细胞在芯片的检测结果；右图显示EGF已诱导的A431细胞在芯片的检测结果。此实施例采用的是膜芯片。
图4为本发明所述的人RTK磷酸化抗体芯片试剂盒检Cont-ODN和gal-1-ODN处理的滋养层细胞的结果图。此实施例采用的是玻璃芯片。
具体实施方式
实施例1：最佳抗体芯片载体的筛选。
常规的抗体芯片多以玻片为载体，由于玻片易碎且检测设备昂贵，不适合小规模应用，也为诊断领域的试剂运输带来难题。而传统的硝酸纤维素膜易碎背景高，结果波动大。我们筛选了市面上不用活性方法处理的膜片及玻片，经过大量的筛查得出无论是膜片还是玻片，温度及表面的活性试剂成分才是决定点样效果的关键。
(1)膜片的筛选
为了使抗体固定在膜片表面，我们筛选了不同方法处理膜片，如下表。再用全自动点样仪把发光液点在在膜片的表面，然后用UV扫描仪来读数。
各组膜片的处理方法如下：
第1组：硝酸纤维素膜片，购于FISHER公司，货号为LC2009。
第2组：聚偏氟乙烯(PVDF)膜，购于WHATMAN公司，货号为10485289。
第3组：将第2组中的PVDF膜浸泡于0.02％至0.075％的非离子表面活性剂Tween80 的水溶液中。
第4组：将第2组中的PVDF膜加入含催化剂的双氧水进行强氧化反应，再加入H2SO4形成亲水基团。
分别将特异性抗体按梯度稀释点于上述4组膜片上，然后加入二抗及底物进行检测，根据点样的孔径均一性，检测限及背景情况选择选择最合适的点样膜，结果见下表1。
表1：不同处理方法膜片性能的比较
膜的类型 均一性 背景 最低检测限
1 点样孔均一性较差 较清晰 0.4ng/ml
2 点样孔径均一无扩散 有杂点 0.2ng/ml
3 点样孔径均一无扩散 清晰 0.2ng/ml
4 点样孔径均一 清晰 0.4ng/ml
由上表可见，经过表面活性剂处理的PVDF膜具有较高的均一性及最低的检测限，经过强氧化剂处理的PVDF膜的检测限较未处理的低，但背景较清晰。而普通NC膜的蛋白吸附性差，灵敏度较低。综合以上指标，本发明优先选择经过非离子表面活性剂处理的PVDF膜作为固相载体。
(2)玻片的筛选
各组玻片的处理方法如下：
第1组的氨基化玻片：购自康宁公司，货号为UltraGAPS40019。
第2组的醛基化玻片：将第1组的玻片加入戊二醛浸泡40分钟制作醛基化玻片。
第3组的APES玻片：将普通玻片加入丙酮稀释的APES中浸泡0.5至1分钟，再用纯丙酮清洗制成APES玻片。
第4组的多聚赖氨酸玻片：将普通玻片加入PBS稀释的多聚赖氨酸浸泡0.5至1个小时，再用纯水清洗制成多聚赖氨酸玻片。
第5组的经亲水试剂处理的玻片：将普通玻片用亲水试剂处理，亲水试剂为0.01至0.1％的烷基糖苷。
各种玻片的点样效果不尽相同，加入亲水涂层的玻片点样效果较明显，点样效果较未处理过的高。其中经过氨基化及亲水处理的玻片较其他的类型效果较高。在20至26度条件下，膜玻/片点样效果较清晰，背景值低；而在27至30度条件下，无论何种膜/玻片的点样效果都出现扩散现象，温度湿度越高，扩散越明显。
各组的玻片结合抗体后加入二抗及底物，在不同的温度及湿度条件下的灵敏度(ng/ml)筛选结果如下表2。
表2：
Figure PCTCN2016097197-appb-000001
综合以上指标，选择经过亲水试剂烷基糖苷处理的玻片作为固相载体。
实施例2：试剂盒的制备。
本发明所述的抗体芯片试剂盒包括以下组份：
固相载体：包被抗体的标准膜片。
洗涤液：含吐温20的20X浓缩洗涤液。1X洗涤液为pH 7.2，含0.1％吐温20、0.1mol/L的磷酸盐缓冲液。
稀释液：2瓶用于稀释样本的15ml 5X浓缩稀释液D，1瓶用于稀释抗体和HRP-链亲和素的15ml 5X浓缩稀释液B。1X稀释液B为15mM,p H7.4的PBS缓冲液，溶质及其在所述稀释液B中的质量浓度或摩尔浓度或体积浓度如下：0.5％酪蛋白，2-4％蔗糖，150mM NaCl。1X稀释液D为15mM,pH6.5的PBS缓冲液，溶质及其在所述标本稀释液中的质量浓度或摩尔浓度或体积浓度如下：2-4％蔗糖，150mM NaCl。
检测抗体：生物素化的检测抗体混合物。
200μl 300X浓缩荧光素-链亲和素溶液。
样本处理液：2X细胞裂解液10ml，1X细胞裂解液包含10mM pH 7.5，Tris.HCl，25mM NaCl，1％脱氧胆酸钠，1％Triton X-100，包含磷酸酶及蛋白酶抑制剂。
在固相载体上包被的特异性抗体为针对选自如下71种蛋白质的抗体(参见表3)。这些抗体均可商业购买，例如购自R&D Systems、PeproTech、Raybiotech等公司。
表3：特异性抗体所针对的抗原蛋白质名称
Figure PCTCN2016097197-appb-000002
Figure PCTCN2016097197-appb-000003
Figure PCTCN2016097197-appb-000004
Figure PCTCN2016097197-appb-000005
这些特异性抗体通过全自动点样仪完成点样操作，将特异性抗体固定于点样膜的方法包括以下步骤：
1)特异性抗体与含有0.5至1.5％酪蛋白的pH7.4磷酸盐缓冲液混合形成抗体混合物；
2)于抗体混合物中将每个抗体含量以0.01至2ng固定于所述点样孔，每个抗体设置2至4个重复孔；
3)抗体芯片点阵排布于膜片，膜片每平方厘米点10至100个特异性抗体。各特异性抗体的排布可以按照实验设计需要进行调整，根据不同的抗体芯片排布阵列，通过控制全自动点样仪，制备出所需要的中间产品。
特异性抗体的一种点阵顺序可参见图2。
所述膜片还包括阳性对照和阴性对照。阳性对照孔为对应特异性抗体的具有相应浓度的生物素化IgG抗体，每个反应的生物素化的IgG抗体的浓度一致，以便于标准化检测。阴性对照孔为具有相应浓度的生物素化的无关抗体，每个反应的生物素化的无关抗体的浓度一致，以便于标准化检测。
根据实施例1的筛选结果，固相载体采用多孔纤维膜，包括：硝酸纤维素膜，纤维过滤膜，玻璃纤维基片，聚偏氟乙烯膜(PVDF)或者尼龙膜。
纤维膜在高温，高压及酸碱条件下易水解，也易被微生物分解，长时间保存也易失水而干燥，带电荷并变脆，耐久性较差。因此，本发明通过0.05至0.1％的表面活性剂，0.1 至0.5％的叠氮化钠处理多孔膜,在18至22℃条件下与特异性抗体紧密结合，最终形成的点样孔清晰完整。
本实施例中，全自动点样仪为美国伯乐公司或铂金艾尔默公司生产的产品；玻片为美国康宁公司产品。当然，在发明技术方案的上述步骤中，仪器和材料的采用并不局限于本实施例的列举，而是以能够解决本发明的技术问题，并实现相应的技术效果为依据。
实施例3：用实施例2的试剂盒检测RTK磷酸化蛋白
将底膜放于配套方盒内，由于本实施例的底膜上分布有多个芯片点阵，故而在本实施例中方盒设置有8个方格，通过方盒之间的方格将每个芯片点阵划分为相互独立的反应区，本实施例中采用的设置有8个方格方盒，向每个方格内加2毫升封闭液后放置于室温下培养30分钟，而后依次进行如下各步骤的操作：
1、加样
吸出每个方格中的封闭液，将100微升～5毫升经封闭液稀释过的样品放入有膜的方格内，然后放在摇床上室温下摇动1至2小时，或者亦可在4℃下反应12-18小时。
2、洗膜
洗涤液I清洗：从方格内吸出样品，用1～5毫升1倍的洗涤液I清洗，之后放在摇床上室温摇动5分钟，再重复此清洗步骤两次。
洗涤液II清洗：从方格内吸出残液，用1～5毫升1倍的洗涤液II清洗，之后放在摇床上室温摇动5分钟，再重复此清洗步骤一次。
3、加入标记有生物素的抗体混合液
向每个方格加入生物素标记的,混合有针对表1中所列抗原蛋白质的特异性抗体的混合液500微升～2毫升,然后放在摇床上室温摇动1至2小时。亦可在4℃下反应12-18小时。而后从方格内吸出标记有生物素的抗体混合液，重复步骤2的洗膜步骤。
4、加入HRP-Streptavidin
向每个方格加入500微升～2毫升稀释过的辣根过氧化物酶标记的抗生物素蛋白链菌素(HRP-Streptavidin),然后放在摇床上室温条件下摇动1至2小时，亦可在4℃下反应12-18小时。而后从方格内吸出HRP—Streptavidin，重复步骤2的洗膜步骤。
HRP—Streptavidin商购自BD公司(产品号554066)，实验前，需要用封闭液进行20,000倍稀释。
需要说明的是，在本实施的步骤1至步骤4中，用到如下容易，其成分可配制方法如下：
2M Tris缓冲液(pH7.5)：Trizma Base 484g、纯化水1.3L，调节酸碱度至7.5，纯化水加至2L。
封闭液的配制方法如下：先将20×PBS(KCl 16g,NaCl 640g,KH2PO4 16g,Na2HPO492g,溶解于2.6L纯化水后,再加纯化水至4升)稀释成1×PBS(20×PBS 200ml，纯化水3800ml)，然后配制10％BSA(BSA 400g，1×PBS加至4升)，最后配制封闭液(10％BSA 4升，Casein 4升，混匀)。
20×洗涤液II(20×TBS)配分如下：2M Tris缓冲液(pH7.5)800ml，5M NaCl 4800ml(NaCl 1461g,纯化水3.3升，溶解后纯化水加至5升)，混匀后，纯化水加至8升。使用时，将20×洗涤液II倍比稀释即可。
20×洗涤液I(2％Tween/20×TBS)配分如下：20×洗涤液II 1L，Tween 20ml,混匀。使用时，将20×洗涤液II倍比稀释即可。
5、检测
用镊子夹出底膜并置于垂直状态使多余的液体滴干；而后将膜放在清洁的塑料片上，保证底膜固定有抗体的一面朝上；加入500微升配制好的(A:B＝1:1)发光液(商购自BIOFX公司，产品号LUMB-0500-01)到每张底膜上并在室温下放置2分钟，此过程必须确保发光液完全覆盖到整张膜；再用另一张干净的塑料片覆盖于底膜表面，小心地将塑料片中的气泡挤压出去，在将塑料片中的气泡挤压出去的过程中应当避免在膜上用力。上述步骤后，将底膜放在室温下5到10秒成像，拍摄时采用低温CCD，并采用相配套的UV扫描仪中读取数据。
实施例4：试剂盒的应用
1)鳞表皮细胞癌细胞A431经血清饥饿过夜处理后暴露于37℃，100ng/ml的EGF10分钟，对照细胞同样经饥饿处理但不暴露于EGF中。两组细胞经裂解后加入抗体芯片中孵育，再加入生物素标记的抗磷酸化受体酪氨酸抗体来检测激活受体上的磷酸化受体酪氨酸。经过荧光染料作用后，采用激光扫描仪检测信号。
图3的左图显示EGF未诱导的A431细胞在芯片的检测结果；图3的右图显示EGF已诱导的A431细胞在芯片的检测结果。
结果显示：经过EGF诱导的A431细胞的EGFR,ErbB2,ErbB3蛋白的表达量比未诱导的高。
2)本发明所述的人RTK磷酸化抗体芯片试剂盒检测Cont-ODN和gal-1-ODN处理的滋养层细胞。
GAL-1通过调节胚胎滋养细胞上HLA-G的表达，在妊娠期母体免疫调节中发挥关键作用。GAL-1水平可以作为预测流产的因子。
人受体酪氨酸激酶磷酸化抗体芯片可监控GAL-1对细胞蛋白磷酯化水平的作用。胚胎滋养细胞细胞(Cont-ODN)及其抑制GAL-1表达的滋养层细胞(gal-1-ODN)经过裂解并分别加入人受体酪氨酸激酶磷酸化抗体芯片，充分洗涤后，再加入生物素标记的抗磷酸酪氨酸抗体，HRP标记物链霉素及发光底物。
结果显示：FGFR2、SRMS、TXK蛋白的表达受到不同程度的抑制。

Claims (10)

  1. 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于，包括：
    固相载体，为包被特异性抗体的标准膜片；
    洗涤液，包括含0.1％吐温20的20X浓缩洗涤液以及其稀释液；
    样本稀释液；
    用于稀释抗体和HRP-链亲和素的稀释液；
    生物素化的检测抗体混合物；
    300X浓缩荧光素-链亲和素溶液；
    样本处理液，为2X细胞裂解液；
    其中，所述特异性抗体是针对选自以下71种蛋白质的抗体：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型)、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. 根据权利要求1所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述固相载体为多孔纤维膜，选自：硝酸纤维素膜、纤维过滤膜、聚偏氟乙烯膜(PVDF)或者尼龙膜。
  3. 根据权利要求2所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述多孔膜是0.05至0.1％的表面活性剂或者在强氧化条件下形成亲水基团后在20至26℃，40％湿度条件下与特异性抗体紧密结合的。
  4. 根据权利要求3所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述多孔膜是经过非离子表面活性剂处理的PVDF膜。
  5. 根据权利要求1所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述固相载体为玻片。
  6. 根据权利要求5所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述玻片是通过戊二醛或多聚赖氨酸、烷基糖苷或者APES粘附剂等亲水试剂处理后在20至26℃条件下与特异性抗体紧密结合的。
  7. 根据权利要求6所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述玻片是经过亲水试剂烷基糖苷处理的玻片。
  8. 根据权利要求1所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于：所述样本处理液为包含蛋白酶和磷酸酶抑制剂混合物的RIPA缓冲液。
  9. 根据权利要求8所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，其特征在于，所述磷酸酶抑制剂是选自：Na3VO4、NaF。
  10. 根据权利要求1所述的检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒，，所述亲水试剂为质量分数为0.01～0.2％的烷基糖苷、0.01～0.1％的甘油，0.01～0.05％的聚乙二醇4000的超纯水溶液；亲水试剂处理玻片的方法为将玻片在亲水试剂中浸泡3～5分钟，晾干即可。
PCT/CN2016/097197 2015-12-25 2016-08-29 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒 WO2017107541A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201511005672.2 2015-12-25
CN201511005672.2A CN106918698B (zh) 2015-12-25 2015-12-25 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒

Publications (1)

Publication Number Publication Date
WO2017107541A1 true WO2017107541A1 (zh) 2017-06-29

Family

ID=59088917

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/097197 WO2017107541A1 (zh) 2015-12-25 2016-08-29 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒

Country Status (2)

Country Link
CN (1) CN106918698B (zh)
WO (1) WO2017107541A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10618959B2 (en) 2016-01-20 2020-04-14 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US10758556B2 (en) 2017-08-07 2020-09-01 Nbe-Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability
CN114076823A (zh) * 2020-08-13 2022-02-22 深圳迈瑞生物医疗电子股份有限公司 固相组分的制备方法及所制备的固相组分
US11845793B2 (en) 2015-10-30 2023-12-19 Nbe-Therapeutics Ag Anti-ROR1 antibodies

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110095605B (zh) * 2018-04-27 2020-10-20 四川大学华西医院 一种肺癌的筛查试剂盒
CN109212214B (zh) * 2018-09-25 2019-08-06 四川大学华西医院 一种肺癌筛查试剂盒
CN109957139A (zh) * 2019-03-14 2019-07-02 天韧膜科技(苏州)有限公司 一种用于硝化纤维素膜的后处理方法
CN115097129B (zh) * 2022-08-24 2023-03-10 山东子峰生物技术有限公司 一种用于胎盘生长因子和可溶性fms样酪氨酸激酶-1的检测试剂组合

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102077091A (zh) * 2008-06-30 2011-05-25 积水医疗株式会社 用于结合测定的多孔性固相和使用所述多孔性固相的结合测定法
CN102341708A (zh) * 2009-03-16 2012-02-01 湖西大学校产学协力团 表皮生长因子受体酪氨酸激酶抑制剂的筛选方法及由其筛选的抑制剂
CN102901815A (zh) * 2012-11-05 2013-01-30 武汉远征世纪制药有限公司 一种检测TrkB受体816/817位酪氨酸位点活性的ELISA试剂盒及其使用方法
CN102944674A (zh) * 2012-11-05 2013-02-27 武汉远征世纪制药有限公司 一种检测TrkB受体pan-Tyr位点活性的ELISA试剂盒及其使用方法
CN103278649A (zh) * 2013-05-28 2013-09-04 深圳市博锐德生物科技有限公司 精子酪氨酸磷酸化的检测试剂盒及其检测方法
CN104111329A (zh) * 2013-04-17 2014-10-22 广州瑞博奥生物科技有限公司 一种改良的同时定量检测多个细胞因子的抗体芯片试剂盒
CN104569418A (zh) * 2013-10-12 2015-04-29 广州瑞博奥生物科技有限公司 一种用于生物标记物筛选的抗体芯片试剂盒
CN104655851A (zh) * 2013-11-22 2015-05-27 广州瑞博奥生物科技有限公司 一种同时定量检测多个受体的抗体芯片试剂盒
WO2015149903A1 (en) * 2014-03-31 2015-10-08 Merck Patent Gmbh Method for detecting protein modifications using specific antibodies
WO2015193739A2 (en) * 2014-04-22 2015-12-23 The Governors Of The University Of Alberta Biomarker for identification of drugable cancers

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2827894A1 (en) * 2011-02-22 2012-08-30 Caris Life Sciences Luxembourg Holdings, S.A.R.L. Circulating biomarkers
CN104204803B (zh) * 2012-02-09 2018-08-07 米密德诊断学有限公司 用于诊断感染的标记和决定因素和其使用方法
CN103869068B (zh) * 2012-12-18 2016-03-09 广州瑞博奥生物科技有限公司 一种用于多种肿瘤诊断的抗体芯片试剂盒

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102077091A (zh) * 2008-06-30 2011-05-25 积水医疗株式会社 用于结合测定的多孔性固相和使用所述多孔性固相的结合测定法
CN102341708A (zh) * 2009-03-16 2012-02-01 湖西大学校产学协力团 表皮生长因子受体酪氨酸激酶抑制剂的筛选方法及由其筛选的抑制剂
CN102901815A (zh) * 2012-11-05 2013-01-30 武汉远征世纪制药有限公司 一种检测TrkB受体816/817位酪氨酸位点活性的ELISA试剂盒及其使用方法
CN102944674A (zh) * 2012-11-05 2013-02-27 武汉远征世纪制药有限公司 一种检测TrkB受体pan-Tyr位点活性的ELISA试剂盒及其使用方法
CN104111329A (zh) * 2013-04-17 2014-10-22 广州瑞博奥生物科技有限公司 一种改良的同时定量检测多个细胞因子的抗体芯片试剂盒
CN103278649A (zh) * 2013-05-28 2013-09-04 深圳市博锐德生物科技有限公司 精子酪氨酸磷酸化的检测试剂盒及其检测方法
CN104569418A (zh) * 2013-10-12 2015-04-29 广州瑞博奥生物科技有限公司 一种用于生物标记物筛选的抗体芯片试剂盒
CN104655851A (zh) * 2013-11-22 2015-05-27 广州瑞博奥生物科技有限公司 一种同时定量检测多个受体的抗体芯片试剂盒
WO2015149903A1 (en) * 2014-03-31 2015-10-08 Merck Patent Gmbh Method for detecting protein modifications using specific antibodies
WO2015193739A2 (en) * 2014-04-22 2015-12-23 The Governors Of The University Of Alberta Biomarker for identification of drugable cancers

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11845793B2 (en) 2015-10-30 2023-12-19 Nbe-Therapeutics Ag Anti-ROR1 antibodies
US10618959B2 (en) 2016-01-20 2020-04-14 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US11242388B2 (en) 2016-01-20 2022-02-08 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US10758556B2 (en) 2017-08-07 2020-09-01 Nbe-Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability
CN114076823A (zh) * 2020-08-13 2022-02-22 深圳迈瑞生物医疗电子股份有限公司 固相组分的制备方法及所制备的固相组分
CN114076823B (zh) * 2020-08-13 2024-05-17 深圳迈瑞生物医疗电子股份有限公司 固相组分的制备方法及所制备的固相组分

Also Published As

Publication number Publication date
CN106918698A (zh) 2017-07-04
CN106918698B (zh) 2019-11-22

Similar Documents

Publication Publication Date Title
WO2017107541A1 (zh) 一种检测人受体酪氨酸激酶的磷酸化抗体芯片试剂盒
Li et al. Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis
US7771955B2 (en) Affinity membrane for capture of a target biomolecule and formation thereof by site-directed immobilization of a capture biomolecule
KR101233837B1 (ko) 비특이 반응이 억제된 면역 측정 방법 및 시약
CN212228961U (zh) 一种检测血清异常脱羧凝血酶原的蛋白芯片
US20200271643A1 (en) Methods and systems for the detection of analyte molecules
US20090088334A1 (en) Method of high sensitive immunoassay
JP2013511032A (ja) 較正試薬およびその使用
US20090131267A1 (en) Use of polymers for increasing the signal intensity when carrying out detection reactions
KR102433648B1 (ko) 항인간 헤모글로빈 모노클로날 항체 또는 항체 키트, 항인간 헤모글로빈 모노클로날 항체 고정화 불용성 담체 입자, 및 이들을 이용한 측정 시약 또는 측정 방법
JP4534817B2 (ja) 蛋白質の検出方法及びペプチドの検出方法
TW201307848A (zh) 糖化血紅素之偵測晶片及其偵測方法
US20190324019A1 (en) Calibration reagent and method
CN114308154A (zh) 一种双环氧基修饰的生物芯片基片的制备方法
US20070172904A1 (en) Method for quantitative detection of biological toxins
US20170261481A1 (en) Protein adsorption inhibitor and method for inhibiting protein adsorption
CN110498844B (zh) 小反刍兽疫诊断试剂盒
JP2008044917A (ja) タンパク質の固定化方法
US20150369802A1 (en) Biomolecule Binding Composite Surfaces, Methods Of Making Such Surfaces, Devices Incorporating Such Surfaces, And Methods Of Using Such Surfaces In Biomolecule Binding Assays, And Devices Therefor
RU2320994C1 (ru) Способ количественного обнаружения биологических токсинов
JP2020112486A (ja) 複合体の検出方法、並びにそれに用いる担体及び検出キット
CN115993452B (zh) 一种流感病毒疫苗血凝素含量测定方法及试剂盒
JP2005069788A (ja) リン酸化蛋白質の検出方法
JP2017173113A (ja) ＴＲＡｂ測定試薬およびその製造方法
ES2268874T3 (es) Mejora de ensayos de fijacion mediante analisis multiepitopico.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16877353

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16877353

Country of ref document: EP

Kind code of ref document: A1