CN105891496A - Tyrosine kinase inhibitor targeted medication guidance antibody chip and detection method - Google Patents
Tyrosine kinase inhibitor targeted medication guidance antibody chip and detection method Download PDFInfo
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Abstract
The invention discloses two kinds of antibody chips used for activity detection of related target proteins to tyrosine kinase inhibitor antitumor drugs. The antibody chips comprise substrate-based solid phase antibody chips and microsphere-based liquid suspension chips, and detection indexes contained in the antibody chips cover the listed related targets of the tyrosine kinase inhibitor antitumor drugs at present. The invention also discloses four detection methods by using the antibody chips. The detection methods comprise a sample labeling-based single-colored fluorescence detection method, a sample labeling-based double-colored fluorescence detection method, a double antibody sandwich detection method and a detection method by utilizing the liquid phase suspension chips. According to the chips and detection methods disclosed by the invention, excison samples of tumor patients are detected, and medication guidance of personalized effective treatment is realized.
Description
Technical field
The present invention relates to biochemical pharmaceutical technology field, particularly relate to tyrosine kinase inhibitor series antineoplastic medicament personalized treatment and refer to
The antibody chip of the drug target protein active detection led and detection method.
Background technology
Protein tyrosine kinase (PTKs, protein tyrosine kinases) or tyrosine kinase (TKs, tyrosine kinases),
Specifically by some the tyrosine residue phosphorylation on protein substrate, thus regulate the enzyme of its function.Can be divided three classes: be 1. subject to
Body tyrosine kinase, for single pass transmembrane albumen；2. cytoplasmic tyrosine kinase, as Src family, Tec family, ZAP70 family,
JAK family etc.；3. tyrosine kinase in core, such as Abl and Wee.Whether it is cell-membrane receptor according to PTK, is divided into receptor type
And non-receptor type.
Receptor tyrosine kinase (receptor tyrosine kinases, RTKs) is maximum class of enzymes connection receptor, and it is receptor,
It is again kinases, it is possible to be combined with part, and by the tyrosine residue phosphorylation of target protein.All of RTKs is by three portions
Be grouped into: extracellular domain containing ligand-binding site point, the hydrophobic alpha helical region of single pass transmembrane, containing tyrosine protein
The intracellular domain of kinase activity.Mainly there is epidermal growth factor (EGF) receptor, including EGFR, HER2, ErbB4
Deng member；PDGF (PDGF) receptor and colony-stimulating factor 1 (CSF-1) receptor, including PDGFRA,
The members such as PDGFRB, CSF1R, KIT；Insulin and insulin-like growth factor-i (IGF-1) receptor, including IGF1R
Deng member；Nerve growth factor (NGF) receptor, fibroblast growth factor (FGF) receptor, vascular endothelial growth factor
Son (VEGF) receptor and hepatocyte growth factor (HGF) receptor, including VEGFR1 (FLT-1), VEGFR2 (KDR),
The members such as VEGFR3 (FLT-4).
Most oncoproteins being belonging to oncornavirus in the protein tyrosine kinase found so far, it is possible to by vertebrates
Proto-oncogene produce.Proto-oncogene and oncoprotein more than 50% all have protein tyrosine kinase activity, and regulation is normal
The signal transmission of cell and growing, also with the propagation of tumor cell, break up, migrate and apoptosis is closely related.The mistake of PTK function
Tune can cause the activation of signal path downstream, and then causes cell proliferation regulation disorder, leads oncogenic formation.Therefore, with
PTK is one of medicament research and development focus having become as the research of current antineoplastic medicine of target spot.
Currently mainly having two kinds of approach can terminate the signal transduction pathway that tyrosine kinase is mediated, a class is monoclonal antibody, as
Herceptin (Trastuzumab), Cetuximab (cetuximab)；Another kind of is tyrosine kinase inhibitor class (Tyrosine
Kinase Inhibitors, TKIs) small-molecule drug.The TKI series antineoplastic medicament having been approved by listing includes imatinib
(Imatinib), gefitinib (Gefitinib), Erlotinib (Erlotinib), Sorafenib (Sorafenib), Dasatinib
(Dasatinib), Sutent (Sunitinib), AMN107 (Nilotinib), Lapatinib (Lapatinib), handkerchief azoles handkerchief
Buddhist nun (Pazopanib), Luso profit for Buddhist nun (Ruxolitinib), gram azoles for Buddhist nun (Crizotinib), Wei Luofeini (Vemurafenib),
ZD6474 (Vandetanib), Conmana (Icotinib), Ponatinib (Ponatinib), card ripple for Buddhist nun (Cabozantinib),
Rui Gefeini (Regorafenib), Bosutinib (Bosutinib), Axitinib (Axitinib), draw many for Buddhist nun (Radotinib),
Afatinib (Afatinib), Sibutramine Hydrochloride replace Buddhist nun (Trametinib), dabrafenib (Dabrafenib), replace Buddhist nun (Ibrutinib) according to Shandong
Totally 24 kinds, it is shown in Table 1, including respective pharmaceutically-active target spot.The heavy pound new drug Ah handkerchief of permanent auspicious medicine can for Buddhist nun (Apatinib)
Suppression VEGFR tyrosine kinase activity, the signal after blocking VEGF combines conducts, and causes tumor-blood-vessel growth to suppress.This
Being to be gone through 10 years with national pharmacy corporation by oncologist team of China, the little molecule in first, the whole world of common innovation research and development is anti-angiogenic
Generating targeted drug, this medicine gastric cancer the most late has been proved definite efficacy and saferry.Enter three-in-one integrated at present
Evaluate, granted entrance countdown.
The TKIs antitumor drug that table 1 lists at present[1]
[1], action target spot inquiry is from DrugBank (http://www.drugbank.ca/) and Therapeutic target database
(http://bidd.nus.edu.sg/group/TTD/ttd.asp).
Though the curative effect of TKI class targeted drug is widely recognized, but the drug effect individual variation after patient medication is the biggest.Substantial amounts of face
Bed research it turned out, and the curative effect/toxic and side effects of TKIs is directly related with the activity of some tyrosine kinase in the patient, is shown in Table 1.
Therefore, before patient accepts antitumor drug, the activity of these tyrosine kinase is carried out qualification and will assist in clinician and sentence
Other patient, if appropriate for using this type of medicine, reduces the generation of poisonous side effect of medicine, avoids the waste of medical resource simultaneously.
Meanwhile, tumor is as the disease of a kind of complexity, and its growth and survival do not depend solely on a kind of receptor or signal path, and this makes
The medicine that must act on single target spot can not kill tumor cell completely.Research shows that different being used in combination of target drug can be pressed down
Make multiple signal transduction pathway, the apoptosis of inducing tumor cell, block the generation of new vessels, the propagation of suppression tumor cell.
Therefore, before patient accepts antitumor drug, the activity of these tyrosine kinase is carried out qualification also contribute to doctor and differentiate be
No need drug combination.
At present, the activity affecting tyrosine kinase includes that the detection of its expression and modification level is based primarily upon immunoblotting
(Western blot, WB).But WB method is time-consuming, laborious, it is difficult to carry out the detection of batch albumen, be not suitable for simultaneously
Clinical practice.Although some medicine is to design for the sudden change of target spot, but these sudden changes are the most also simulation active sites
The modification of point, or promote the modification of avtive spot.Therefore, tyrosine-kinase expression of enzymes can be detected in the urgent need to one simultaneously and repair
The high-throughput techniques of decorations level.
Antibody chip technology is similar with gene chip, utilizes the principle that antigen-antibody combines, on substrate by corresponding antibody according to
Certain regular stationary arrangement, possesses high-throughout feature.Although Full is Moon, RayBio, R&D, CST, Merck
Company has the similar products for scientific research, but the Testing index comprised only includes the effect of a small amount of TKI series antineoplastic medicament
Target spot, product does not focus on TKI class action target spot, and does not the most include the detection of decorating site, it is difficult to be applied to clinic (table
2)。
The product that table 2 is similar in the market
Summary of the invention
The related target albumen that present invention provide for the tyrosine kinase inhibitor series antineoplastic medicament that personalized treatment instructs is lived
Property detection antibody chip and detection method.This antibody chip utilizes the principle that antigen-antibody combines, and can be used for detection the most
The protein active of antitumor drug related target of listing, the expression of i.e. following 46 kinds of medicine related target albumen and (or)
Modification level: Bcr-Abl, ABL1, ABL2, ALK, BTK, CSF1R, DDR1, DDR2, EGFR, EPHA1,
EPHA2、EPHA3、EPHA4、EPHA5、EPHA6、EPHA7、EPHA8、ERBB2、ERBB3、ERBB4、
FGFR1、FGFR2、FGFR3、FGFR4、FLT1、FLT3、FLT4、FRK、FYN、HCK、ITK、JAK1、
JAK2、JAK3、KDR、KIT、LCK、LYN、MET、NTRK1、PDGFRA、PDGFRB、PTK6、RET、
SRC, TEK, YES1, BRAF, RAF1, MAPK11, MAP2K1, MAP2K2, MAP3K2 (comprise 6 kinds
Serine/threonine kinase, is shown in Table 3).
The Testing index that table 3 present invention is comprised
To achieve these goals, a kind of technical scheme of the present invention is:
Use insolubilized antibody chip, i.e. respective antibody corresponding for above-mentioned Testing index selected system on substrate by chip point sample instrument,
Every kind of antibody arranges repetition.The internal reference antibody of some system is β-actin, α-tubulin and GAPDH etc..Select fluorophor
As positive control, spotting buffer is as negative control (table 4).
Preferentially, described antibody includes: for monoclonal antibody and (or) the polyclonal antibody of These parameters.
Described antibody also includes: the antibody of the protein expression level of detection These parameters, and (or) detects everybody of These parameters
The antibody of some phosphorylation modification level.
Described substrate includes: aldehyde radical, epoxy resin, polysaccharide or other macromolecule modified slide formula or membrane type substrate.
The design (comprising each decorating site antibody) of table 4 antibody chip of the present invention
| Antibody Name | Swiss Prot | Antibody Name | Swiss Prot |
| Positive Marker | P | HCK(Ab-522) | P08631 |
| Empty | E | HCK(Phospho-Tyr522) | P08631 |
| Bcr-Abl(Ab-177) | A9UF02 | ITK(Ab-512) | Q08881 |
| Bcr-Abl(Phospho-Tyr177) | A9UF02 | ITK(Phospho-Tyr512) | Q08881 |
| Bcr-Abl(Ab-245) | A9UF02 | JAK1(Ab-1022) | P23458 |
| Bcr-Abl(Phospho-Tyr245) | A9UF02 | JAK1(Phospho-Tyr1022) | P23458 |
| Abl1(Ab-204) | P00519 | JAK2(Ab-1007) | O60674 |
| Abl1(Ab-754/735) | P00519 | JAK2(Ab-221) | O60674 |
| Abl1(Phospho-Thr754/735) | P00519 | JAK2(Phospho-Tyr1007) | O60674 |
| Abl1(Phospho-Tyr204) | P00519 | JAK2(Phospho-Tyr221) | O60674 |
| Abl1(Ab-412) | P00519 | JAK3(Ab-785) | P52333 |
| Abl1(Ab-245) | P00519 | JAK3(Ab-904) | P52333 |
| Abl1(Phospho-Tyr412) | P00519 | JAK3(Ab-939) | P52333 |
| Abl1(Phospho-Tyr245) | P00519 | JAK3(Ab-981) | P52333 |
| ABL2(Ab-261) | P42684 | JAK3(Phospho-Tyr785) | P52333 |
| ABL2(Ab-439) | P42684 | JAK3(Phospho-Tyr904) | P52333 |
| ABL2(Phospho-Tyr261) | P42684 | JAK3(Phospho-Tyr939) | P52333 |
| ABL2(Phospho-Tyr439) | P42684 | JAK3(Phospho-Tyr981) | P52333 |
| ALK(Ab-1507) | Q9UM73 | VEGFR2(Ab-1054) | P35968 |
| ALK(Ab-1604) | Q9UM73 | VEGFR2(Phospho-Tyr1054) | P35968 |
| ALK(Phospho-Tyr1507) | Q9UM73 | VEGFR2(Ab-1059) | P35968 |
| ALK(Phospho-Tyr1604) | Q9UM73 | VEGFR2(Phospho-Tyr1059) | P35968 |
| BTK(Ab-222) | Q06187 | VEGFR2(Ab-1175) | P35968 |
| BTK(Ab-550) | Q06187 | VEGFR2(Ab-1214) | P35968 |
| BTK(Phospho-Tyr222) | Q06187 | VEGFR2(Ab-951) | P35968 |
| BTK(Phospho-Tyr550) | Q06187 | VEGFR2(Phospho-Tyr1175) | P35968 |
| CSFR(Ab-561) | P07333 | VEGFR2(Phospho-Tyr1214) | P35968 |
| CSFR(Phospho-Tyr561) | P07333 | VEGFR2(Phospho-Tyr951) | P35968 |
| CSFR(Ab-809) | P07333 | KIT(Ab-703) | P10721 |
| CSFR(Phospho-Tyr809) | P07333 | KIT(Ab-936) | P10721 |
| DDR1(Ab-513) | Q08345 | KIT(Phospho-Tyr703) | P10721 |
| DDR1(Ab-796) | Q08345 | KIT(Phospho-Tyr936) | P10721 |
| DDR1(Phospho-Tyr513) | Q08345 | KIT(Ab-721) | P10721 |
| DDR1(Phospho-Tyr796) | Q08345 | KIT(Phospho-Tyr721) | P10721 |
| DDR2(Ab-471) | Q16832 | LCK(Ab-192) | P06239 |
| DDR2(Ab-740) | Q16832 | LCK(Ab-393) | P06239 |
| DDR2(Phospho-Tyr471) | Q16832 | LCK(Ab-504) | P06239 |
| DDR2(Phospho-Tyr740) | Q16832 | LCK(Ab-59) | P06239 |
| EGFR(Ab-1016) | P00533 | LCK(Phospho-Ser59) | P06239 |
| EGFR(Ab-1069) | P00533 | LCK(Phospho-Tyr192) | P06239 |
| EGFR(Ab-678) | P00533 | Lck(Phospho-Tyr393) | P06239 |
| EGFR(Ab-693) | P00533 | LCK(Phospho-Tyr504) | P06239 |
| EGFR(Ab-998) | P00533 | LYN(Ab-507) | P07948 |
| EGFR(Phospho-Tyr998) | P00533 | LYN(Phospho-Tyr507) | P07948 |
| EGFR(Phospho-Thr678) | P00533 | Met(Ab-1003) | P08581 |
| EGFR(Phospho-Thr693) | P00533 | Met(Phospho-Tyr1003) | P08581 |
| EGFR(Phospho-Tyr1016) | P00533 | Met(Ab-1234) | P08581 |
| EGFR(Phospho-Tyr1069) | P00533 | Met(Ab-1349) | P08581 |
| EGFR(Ab-1070) | P00533 | Met(Phospho-Tyr1234) | P08581 |
| EGFR(Ab-1092) | P00533 | Met(Phospho-Tyr1349) | P08581 |
| EGFR(Ab-1110) | P00533 | NTRK1(Ab-496) | P04629 |
| EGFR(Ab-1172) | P00533 | NTRK1(Ab-680) | P04629 |
| EGFR(Ab-1197) | P00533 | NTRK1(Ab-681) | P04629 |
| EGFR(Ab-869) | P00533 | NTRK1(Ab-701) | P04629 |
| EGFR(Phospho-Ser1070) | P00533 | NTRK1(Ab-791) | P04629 |
| EGFR(Phospho-Tyr1092) | P00533 | NTRK1(Phospho-Tyr496) | P04629 |
| EGFR(Phospho-Tyr1110) | P00533 | NTRK1(Phospho-Tyr680) | P04629 |
| EGFR(Phospho-Tyr1172) | P00533 | NTRK1(Phospho-Tyr681) | P04629 |
| EGFR(Phospho-Tyr1197) | P00533 | NTRK1(Phospho-Tyr701) | P04629 |
| EGFR(Phospho-Tyr869) | P00533 | NTRK1(Phospho-Tyr791) | P04629 |
| EphA2(Ab-588) | P29317 | PDGF R alpha(Ab-849) | P16234 |
| EphA2(Ab-594) | P29317 | PDGF R alpha(Phospho-Tyr849) | P16234 |
| EphA2(Ab-735) | P29317 | PDGF R beta(Ab-1009) | P09619 |
| EphA2(Ab-930) | P29317 | PDGF R beta(Ab-1021) | P09619 |
| EphA2(Phospho-Tyr588) | P29317 | PDGF R beta(Ab-740) | P09619 |
| EphA2(Phospho-Tyr594) | P29317 | PDGF R beta(Ab-751) | P09619 |
| EphA2(Phospho-Tyr735) | P29317 | PDGF R beta(Phospho-Tyr1009) | P09619 |
| EphA2(Phospho-Tyr930) | P29317 | PDGF R beta(Phospho-Tyr1021) | P09619 |
| FRK(Ab-387) | P42685 | PDGF R beta(Phospho-Tyr740) | P09619 |
| FRK(Phospho-Tyr387) | P42685 | PDGF R beta(Phospho-Tyr751) | P09619 |
| HER2(Ab-686) | P04626 | PTK6(Ab-342) | Q13882 |
| HER2(Phospho-Thr686) | P04626 | PTK6(Ab-447) | Q13882 |
| HER2(Ab-1112) | P04626 | PTK6(Phospho-Tyr342) | Q13882 |
| HER2(Phospho-Tyr1112) | P04626 | PTK6(Phospho-Tyr447) | Q13882 |
| HER2(Ab-1221/1222) | P04626 | Ret(Ab-905) | P07949 |
| HER2(Ab-1248) | P04626 | Ret(Phospho-Tyr905) | P07949 |
| HER2(Ab-877) | P04626 | Src(Ab-418) | P12931 |
| HER2(Phospho-Tyr1221/1222) | P04626 | Src(Ab-529) | P12931 |
| HER2(Phospho-Tyr1248) | P04626 | Src(Phospho-Tyr418) | P12931 |
| HER2(Phospho-Tyr877) | P04626 | Src(Phospho-Tyr529) | P12931 |
| HER3(Ab-1222) | P21860 | Src(Ab-75) | P12931 |
| HER3(Ab-1289) | P21860 | Src(Ab-216) | P12931 |
| HER3(Phospho-Tyr1222) | P21860 | Src(Phospho-Ser75) | P12931 |
| HER3(Phospho-Tyr1289) | P21860 | Src(Phospho-Tyr216) | P12931 |
| HER4(Ab-1056) | Q15303 | TEK(Ab-1102) | Q02763 |
| HER4(Phospho-Tyr1056) | Q15303 | TEK(Ab-1108) | Q02763 |
| HER4(Ab-1284) | Q15303 | TEK(Phospho-Tyr1102) | Q02763 |
| HER4(Phospho-Tyr1284) | Q15303 | TEK(Phospho-Tyr1108) | Q02763 |
| FGFR1(Ab-154) | P11362 | Yes(Ab-537) | P07947 |
| FGFR1(Ab-654) | P11362 | Yes(Phospho-Tyr537) | P07947 |
| FGFR1(Ab-766) | P11362 | B-RAF(Ab-446) | P15056 |
| FGFR1(Phospho-Tyr154) | P11362 | B-RAF(Ab-598) | P15056 |
| FGFR1(Phospho-Tyr654) | P11362 | B-RAF(Ab-601) | P15056 |
| FGFR1(Phospho-Tyr766) | P11362 | B-RAF(Phospho-Ser446) | P15056 |
| FGFR2(Ab-769) | P21802 | B-RAF(Phospho-Ser601) | P15056 |
| FGFR2(Ab-782) | P21802 | B-RAF(Phospho-Thr598) | P15056 |
| FGFR2(Phospho-Tyr769) | P21802 | Raf1(Ab-259) | P04049 |
| FGFR2(Phospho-Ser782) | P21802 | Raf1(Ab-289) | P04049 |
| FGFR3(Ab-724) | P22607 | Raf1(Ab-338) | P04049 |
| FGFR3(Ab-760) | P22607 | Raf1(Ab-341) | P04049 |
| FGFR3(Phospho-Tyr724) | P22607 | Raf1(Ab-621) | P04049 |
| FGFR3(Phospho-Tyr760) | P22607 | Raf1(Phospho-Ser259) | P04049 |
| FGFR4(Ab-642) | P22455 | Raf1(Phospho-Ser289) | P04049 |
| FGFR4(Ab-643) | P22455 | Raf1(Phospho-Ser338) | P04049 |
| FGFR4(Phospho-Tyr642) | P22455 | Raf1(Phospho-Tyr341) | P04049 |
| FGFR4(Phospho-Tyr643) | P22455 | Raf1(Phospho-Ser621) | P04049 |
| VEGFR-1(Ab-1213) | P17948 | MAPK11(Ab-182) | Q15759 |
| VEGFR-1(Phospho-Tyr1213) | P17948 | MAPK11(Phospho-Tyr182) | Q15759 |
| VEGFR-1(Ab-1333) | P17948 | MEK1(Ab-217) | Q02750 |
| VEGFR-1(Phospho-Tyr1333) | P17948 | MEK1(Ab-221) | Q02750 |
| FLT3(Ab-599) | P36888 | MEK1(Ab286) | Q02750 |
| FLT3(Ab-842) | P36888 | MEK1(Ab-291) | Q02750 |
| FLT3(Ab-969) | P36888 | MEK1(Ab-298) | Q02750 |
| FLT3(Phospho-Tyr599) | P36888 | MEK1(Phospho-Ser217) | Q02750 |
| FLT3(Phospho-Tyr842) | P36888 | MEK1(Phospho-Ser221) | Q02750 |
| FLT3(Phospho-Tyr969) | P36888 | MEK1(Phospho-Ser298) | Q02750 |
| FLT4(Ab-1063) | P35916 | MEK1(Phospho-Thr286) | Q02750 |
| FLT4(Ab-1230) | P35916 | MEK1(Phospho-Thr291) | Q02750 |
| FLT4(Ab-1231) | P35916 | MEK-2(Ab-394) | P36507 |
| FLT4(Ab-1337) | P35916 | MEK-2(Phospho-Thr394) | P36507 |
| FLT4(Phospho-Tyr1063) | P35916 | MAP3K2(Ab-520) | Q9Y2U5 |
| FLT4(Phospho-Tyr1230) | P35916 | MAP3K2(Ab-522) | Q9Y2U5 |
| FLT4(Phospho-Tyr1231) | P35916 | MAP3K2(Ab-524) | Q9Y2U5 |
| FLT4(Phospho-Tyr1337) | P35916 | MAP3K2(Phospho-Ser520) | Q9Y2U5 |
| Fyn(Ab-420) | P06241 | MAP3K2(Phospho-Thr522) | Q9Y2U5 |
| Fyn(Phospho-Tyr420) | P06241 | MAP3K2(Phospho-Thr524) | Q9Y2U5 |
| Fyn(Ab-531) | P06241 | Beta actin | A |
| Fyn(Phospho-Tyr531) | P06241 | Alpha tubulin | T |
| HCK(Ab-411) | P08631 | GAPDH | G |
| HCK(Phospho-Tyr411) | P08631 | Negative control | N |
Another kind of technical scheme of the present invention is:
Using liquid phase suspension chip to realize the detection of above-mentioned multiple indexes, the substrate of liquid phase suspension chip includes diameter 1-10 micron
With different fluorescently-labeled microballons, owing to microballon carries fluorescence difference, can be used for preparing the microballon up to 1000 kinds of chip.Each
Plant the respective antibody that on the microballon of color, the species specific above-mentioned Testing index of coupling one is corresponding.Specific antibody is micro-by coupling
Pearl and the albumino reaction in sample, add detection antibody and detect.
Another aspect of the present invention, additionally provides the method utilizing above-mentioned antibody chip to detect.
Detecting including one-color fluorescence based on sample labeling method, i.e. each sample carries out biotin labeling respectively, respectively with respectively
From chip hybridization, detecting finally by adding fluorescently-labeled (strepto-) Avidin ((strept) avidin), step is as follows:
The TBST solution room temperature comprising defatted milk powder closes chip, washing；
Then with biotin labeled sample and chip incubated at room, washing；
Again with fluorescently-labeled (strepto-) Avidin and chip incubated at room, washing, dry；
Finally scan chip with chip scanner, detect respective fluorescence intensity, and utilize software to read data, it is thus achieved that antitumor
The expression of medicine related target albumen and (or) the level of modification.By cancer chip-count other with cancer in tumour patient operation sample
According to comparison, the target point protein of the differential expression level screened and modification level is carried out expert's deciphering, helps clinic doctor
Take root and formulate personalized medicine scheme according to the individual variation of patient, to improve curative effect of medication, reduce the generation of poisonous side effect of medicine.
Preferentially, described biotin labeled sample, labelling groups is not limited to biotin, it is also possible to be fluorophor or Radix Cochleariae officinalis mistake
Oxide enzyme etc..
Described fluorescently-labeled (strepto-) Avidin, fluorescence is not limited to Cy3, Cy5, it is also possible to be AlexaFluor fluorescence etc..
Or, use Two Colour Fluorescence based on sample labeling method to carry out detecting (Fig. 1), i.e. each sample carries out biotin mark respectively
Note, each adds different fluorescently-labeled (strepto-) Avidin, takes equal portions and hybridize with same chip, and step is as follows:
Biotin labeling sample；
Different fluorescently-labeled (strepto-) Avidins and respective biotin labeling sample incubation；
The TBST solution room temperature comprising defatted milk powder closes chip, washing；
Take equal portions sample to hatch with same chip, washing, dry；
Finally scan chip, fluorescence intensity with chip scanner, and utilize software to read data, it is thus achieved that antitumor drug phase
Close expression and (or) the modulation situation of the level of modification of target point protein.
Preferentially, described biotin labeling sample, labelling groups is not limited to biotin, it is also possible to be fluorophor etc..
Or, use double-antibody method to detect, i.e. sample is hatched with chip, adds the mixing of biotin labeled detection antibody
Thing, detects finally by adding fluorescently-labeled (strepto-) Avidin, and step is as follows:
The TBST solution room temperature comprising defatted milk powder closes chip, washing；
Then sample and chip incubated at room, washing；
Add biotin labeled detection mixtures of antibodies, with chip incubated at room, washing；
Again with fluorescently-labeled (strepto-) Avidin and chip incubated at room, washing, dry；
Finally scan chip with chip scanner, detect respective fluorescence intensity, utilize software to read data, it is thus achieved that antineoplastic agent
The expression of thing related target albumen and (or) the modulation situation of the level of modification.
Wherein, detection antibody is the mixtures of antibodies that identify another epi-position corresponding with the capture antibody on chip.
Present invention also offers the method utilizing above-mentioned liquid phase suspension chip detection, comprise the following steps:
Adding microballon in reacting hole, magnetic force plate washing device washs；
Add sample, stick sealed membrane, be placed on plate shaker vibration, lucifuge incubated at room, washing；
Add biotin labeled detection antibody, stick sealed membrane, be placed on plate shaker vibration, lucifuge incubated at room, wash
Wash；
Add (strepto-) Avidin of phycoerythrin (phycoerythrin) labelling, stick sealed membrane, be placed on plate shaker
Vibration, lucifuge incubated at room, washing；
Resuspended with reaction buffer, stick sealed membrane, lucifuge shaken at room temperature；
Send into readings in the liquid-phase chip analyser that corrected, it is thus achieved that the expression of antitumor drug related target albumen and (or)
The modulation situation of modification level.
Main advantages of the present invention are:
(1) present invention can realize the detection of running simultaneously of bulk drug target spot in primary first-order equation, it is achieved in real high flux height
Contain detection.
(2) present invention can not only detect the protein expression level of each drug target, and can detect each drug target protein loci
Modification level.
(3) the detection energy drug of tumor sample is researched and developed and is provided novel targets, new approaches by the present invention.
(4) present invention is simple to operate, detection is quick, it is easy to clinical practice.
(5) present invention is specifically directed to the albumen of the tyrosine kinase inhibitor series antineoplastic medicament related target that detection has listed at present
Activity, the personalized treatment that can be used for tumor patient medication instructs.
(6) detection of tumor sample can be provided for drug combination and instruct by the present invention.
Accompanying drawing explanation
The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings.
Fig. 1 is present invention Two Colour Fluorescence based on sample labeling method Cleaning Principle figure；
Fig. 2 is the antibody chip result scanogram of the embodiment of the present invention 2；
Fig. 3 is the gray-scale map of the antibody chip application of results immunoblotting checking of the embodiment of the present invention 2.
Detailed description of the invention
In the following example, the experimental technique of unreceipted actual conditions, the most routinely condition, as " fine works molecular biology is real
Test guide " (F.M. Ao Sibai, R.E. James Kingston, J.G. Sai Deman etc. edits, and Ma Xuejun, Su Yuelong etc. translates school.Beijing:
Science Press, 2004) method described in is carried out.
The preparation of embodiment 1 antibody chip
1. immunoblot experiment: self-corresponding candidate antibodies each to drug target carries out specificity and titer is identified, screens high-quality
Antibody；
2. antibody purification: by proteinA/G pillar and dialysis antibody purification, it is 2mg/mL that ultrafiltration adjusts antibody concentration,
5 μ L subpackages ,-20 DEG C of preservations；
3. prepare point template: 5 μ L antibody add 5 μ L 2 × spotting buffer, suck point template, it is to avoid produce bubble；With
Sample, 10 μ L positive controls (include that 0.25mg fluorophor labelled protein, 0.75mg BSA and 1 × point sample are slow
Rush liquid) and 10 μ L negative controls (including 5 μ L2 × spotting buffer and 5 μ L PBS) also suck point template；
Whirlpool device mixes, and 1,000 × g2min is centrifuged, 4 DEG C of preservations；
4. point sample: operate requirement according to chip point sample instrument, carries out and prepares before point sample, including washing liquid, humidity, temperature, spotting needle,
Substrate and point template etc., point sample meron proceeds to 4 DEG C and overnight protects after putting at least 2 hours temporarily under the humidity of 50%
Deposit.
Embodiment 2 applies the antibody chip detection to clinical sample
One, Protein Extraction
Tumor patient excision sample, carries out paraffin section making, hematoxylin-eosin staining (hematoxylin-eosin staining,
HE dyes), by the interpretation of pathology expert, separate cancer beside organism's part and tumor tissues part, utilize Tissue lysates with even
Slurry device extracts its total protein respectively.
Two, biotin labeling
1. every 1mg biotin adds 100 μ L DMF (N, N-Dimethylformamide), final concentration of 10 μ g/ μ L, mark
It is designated as Biotin/DMF；
The most each sample respectively takes 100 μ g protein samples and is marked, point two pipes, often adds 3 μ L in pipe 50 μ g protein sample
Biotin/DMF is marked, and final volume is 70 μ L；
3. mix homogeneously, concussion reaction 2h under room temperature；
4. add 30 μ L stop buffers, concussion reaction 30min under room temperature；
Enter next step chip detection or sample is stored in-80 DEG C.
Three, chip detection
A. close
1. the antibody chip of above-mentioned point is taken out from refrigerator, equilibrium at room temperature 45min；
2. in 100 × 15mm culture dish, add 30mL lock solution, chip is totally submerged.Guarantee chip surface
Upwards, being put on shaking table by culture dish, room temperature 55rpm closes 1h；
3. use Milli-Q water to wash according to below step:
A) chip is put into 50mL circle centrifuge tube, add 45mL Milli-Q water, screw lid；
B) turn upside down with hands and shake pipe 10s, outwell Milli-Q water；
C) centrifuge tube changes to new Milli-Q water, shake pipe 10s, outwell Milli-Q water；
D) it is repeated 10 times；
4. get rid of the Milli-Q water that chip surface is unnecessary, immediately enter next step reaction.
B. hybridize
1., in a test tube, add 6mL hybridization buffer, add biotin labeled albumen (50 μ g), mixing
Uniformly.It is labeled as Protein Coupling Mix；
2. chip is put into clean culture dish, chip surface upwards, slowly by 6mL Protein Coupling Mix
Being added to chip surface, be totally submerged by chip, cover Coupling Chamber, room temperature 35rpm hatches 2h；
3. chip is moved on in a 100 × 15mm culture dish filling 30mL 1 × Wash Solution, room temperature 55
Rpm washes 10min, outwells washing liquid, repeats this step 3 time；
4. use Milli-Q water to wash according to below step:
A) chip is put into 50mL circle centrifuge tube, add 45mL Milli-Q water, screw lid；
B) turn upside down with hands and shake pipe 10s, outwell Milli-Q water；
C) centrifuge tube changes to new Milli-Q water, shake pipe 10s, outwell Milli-Q water；
D) it is repeated 10 times；
5. get rid of the Milli-Q water that chip surface is unnecessary, immediately enter next step reaction.
C. detect
1. in 60mL Detection Buffer, add 60 μ L Cy3-Streptavidin (0.5mg/mL)；
2. in 100 × 15mm culture dish, add 30mL above-mentioned Cy3-Streptavidn Solution；
Chip is submerged into Cy3-Streptavidin solution.Room temperature lucifuge, on shaking table, 55rpm reacts 20min；
3. chip is moved in a new 100 × 15mm culture dish filling 30mL 1 × Wash Solution；Room temperature
On shaking table, 55rpm washes 10min.Repeat this step 3 time；
4. use Milli-Q water to wash according to below step:
A) chip is put into 50mL circle centrifuge tube, add 45mL Milli-Q water, screw lid；
B) turn upside down with hands and shake pipe 10s, outwell Milli-Q water；
C) centrifuge tube changes to new Milli-Q water, shake pipe 10s, outwell Milli-Q water；
D) it is repeated 10 times；
5. being placed in by chip in chip compact centrifuge, centrifugal 2min dries；
6. lucifuge, GenePix 4000B scanner scans chip (Fig. 2) at 532nm.
Four, data analysis
Using GenePix Pro software to read initial data, the image obtained by scanning finds have Multiple Antibodies to detect on chip
Fluorescence signal, represents protein corresponding containing these antibody in this sample, and the power of fluorescence signal has reacted the content of protein.
This experiment, by the comparison of chip data other to cancer in tumour patient excision sample and cancer, finds the eggs such as EGFR, ALK
Phosphorylation level strengthens in vain, and prompting not only needs to use the antitumor drug for EGFR, in addition it is also necessary to use resisting for ALK
Tumour medicine, reaches the purpose of therapeutic alliance targetedly.
Five, immunoblotting result verification
The cancer beside organism extracted part is utilized to carry out immunoblot experiment (Fig. 3) with tumor tissues Partial Protein sample.Result confirms
Not only the phosphorylation level of EGFR (Y869) strengthens, and the phosphorylation level of ALK (Y1604) also strengthens.Meanwhile, under
Trip signaling pathway protein JAK1 (Y1022), the phosphorylation level of Akt1 (S473) and MEK2 (T394) strengthen equally.
Embodiment described above only have expressed embodiments of the present invention, and it describes more concrete and in detail, but can not therefore and
It is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, do not taking off
On the premise of present inventive concept, it is also possible to make some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. two kinds of antibody chips for the detection of tyrosine kinase inhibitor series antineoplastic medicament related target protein active, its feature
Being, described antibody chip includes insolubilized antibody chip based on substrate and liquid suspending chip based on microballon.
Antibody chip the most according to claim 1, it is characterised in that described substrate includes: aldehyde radical, epoxy resin, poly-
Sugar or other macromolecule modified slide formula or membrane type substrates.
Antibody chip the most according to claim 1, it is characterised in that the Testing index that described antibody chip includes covers
The related target of the tyrosine kinase inhibitor series antineoplastic medicament listed at present, the expression of i.e. 46 kinds medicine related target albumen
Level and (or) the level of modification.
Antibody chip the most according to claim 3, it is characterised in that described 46 kinds of medicine related target albumen comprise:
Bcr-Ab1、ABL1、ABL2、ALK、BTK、CSF1R、DDR1、DDR2、EGFR、EPHA2、ERBB2、
ERBB3、ERBB4、FGFR1、FGFR2、FGFR3、FGFR4、FLT1、FLT3、FLT4、FRK、FYN、
HCK、ITK、JAK1、JAK2、JAK3、KDR、KIT、LCK、LYN、MET、NTRK1、PDGFRA、
PDGFRB、PTK6、RET、SRC、TEK、YES1、BRAF、RAF1、MAPK11、MAP2K1、MAP2K2、
MAP3K2。
Antibody chip the most according to claim 3, it is characterised in that on described antibody chip, the antibody of set comprises: inspection
Survey medicine related target protein expression level and (or) the monoclonal antibody of each site phosphorylation modification level and (or) polyclone
Antibody.
6. the method that an one-color fluorescence based on sample labeling method carries out detecting, it is characterised in that comprise the following steps:
Sample is carried out biotin labeling；
With the antibody chip described in claim 1, the sample of labelling is detected；
Detect with fluorescently-labeled (strepto-) Avidin again；
According to the antibody of fluorescence signal being detected, determine medicine related target albumen in testing sample expression and (or)
Modification level.
7. the method that a Two Colour Fluorescence based on sample labeling method carries out detecting, it is characterised in that comprise the following steps:
Sample is carried out respectively biotin labeling；
Different fluorescently-labeled (strepto-) Avidins and respective biotin labeling sample incubation；
With the antibody chip described in claim 1, the mixture of equal portions marker samples is detected；
According to the antibody of fluorescence signal being detected, determine medicine related target albumen in testing sample expression and (or)
The modulation situation of modification level.
8. one kind uses the method that double-antibody method carries out detecting, it is characterised in that comprise the following steps:
With the antibody chip described in claim 1, sample is detected；
Add biotin labeled detection mixtures of antibodies；
Detect with fluorescently-labeled (strepto-) Avidin again；
According to the antibody of fluorescence signal being detected, determine medicine related target albumen in testing sample expression and (or)
Modification level.
9. the application of antibody chip described in claim 1, it is characterised in that for tumor patient excision sample is examined
Survey, it is achieved the medication guide of personalized effectively treatment.
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Cited By (1)
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| WO2020140927A1 (en) * | 2019-01-02 | 2020-07-09 | Crownmab Biotech Inc. | Cancer treatment using multi-targeted kinase inhibitor in combination of protein kinase biomarkers |
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