CN116240261A - Kit for detecting activity of SHP2 inhibitor in PBMC and method thereof - Google Patents
Kit for detecting activity of SHP2 inhibitor in PBMC and method thereof Download PDFInfo
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Abstract
The invention relates to a kit for detecting SHP2 inhibitor activity in PBMC and a method thereof, wherein the kit comprises a stimulator GM-CSF and a reagent for detecting total ERK and phosphorylated ERK. The invention finds a proper stimulator GM-CSF which can promote the phosphorylation signal of ERK in PBMC in a larger window, and can sensitively reflect the inhibition effect of an SHP2 inhibitor on phosphorylated ERK by detecting the change of the phosphorylation level of ERK protein so as to indirectly evaluate the activity of the SHP2 inhibitor. Solves one pain point in the study of SHP2 inhibitor solid tumor: namely, how to monitor the efficacy of the SHP2 inhibitor conveniently and timely without surgery or puncture of tumor tissue.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a kit for detecting the activity of an SHP2 inhibitor in PBMC (peripheral blood mononuclear cells, peripheral blood mononuclear cell) and a method thereof, and particularly relates to a kit for detecting the inhibition rate of the SHP2 inhibitor in PBMC on ERK (extracellular signal-regulated kinase) phosphorylation and a method thereof.
Background
SHP2 is known as Src homology 2 domain-containing protein tyrosine phosphatase and is encoded by the gene PTPN 11. The activity of SHP2 phosphatase is regulated and controlled by the change of the conformation of the SHP2 phosphatase, and in the ground state, the N-SH2 domain is combined with the PTP domain and directly blocks the active site of the PTP domain, so that the self-inhibition conformation is maintained; upstream receptor tyrosine kinase (Receptor Tyrosine Kinase, RTK) activation triggers binding of the pTyr residues on the upstream signaling factor to both SH2 domains of SHP2, altering the conformation of SHP2, exposing the active site on the PTP domain, releasing SHP2 from the self-inhibiting state. SHP2 acts downstream of various RTKs, mediating cascade activation of downstream RAS/MAPK signaling pathways resulting from RTK activation. SHP2 is involved in post-dephosphorylation modification of proteins and plays an important role in a number of signaling pathways involved in controlling cancer progression. Cytokines and the like bind to receptors on the surface of the cell membrane, induce the formation of SHP-2 complex, and further activate RAS-RAF-MEK-ERK signaling pathway. In addition, almost all Receptor Tyrosine Kinases (RTKs) activate RAS in a major, and even the only, way to activate SHP 2. Preclinical studies have shown that decreasing SHP2 expression levels or inhibiting their phosphatase activity significantly inhibits MAPK signaling pathway activity as well as cell proliferation in a variety of cancer cells, particularly in RTK-dependent or cancers that carry RAS pathway specific mutations (e.g., KRASG12C mutations, BRAFClass3 mutations, NF1 inactivating mutations, etc.). In addition, SHP2 acts downstream of PD-1 in T cells, mediating tumor immunosuppression resulting from activation of the PD-L1/PD-1 pathway. Therefore, SHP2 can promote the occurrence and development of tumors by regulating multiple mechanisms, and the development of selective SHP2 small molecule inhibitors is expected to treat various cancers.
SHP2 inhibitors are commonly used in cancer therapy, and thus most of their research is focused on advanced solid tumors such as non-small cell lung cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gastrointestinal stromal tumor and colorectal cancer, and how to go to them conveniently and to be able to monitor the efficacy of SHP2 inhibitors in time becomes a difficulty, because it is relatively difficult to obtain fresh tumor tissue clinically at different administration time points, and is painful for patients to frequently operate or puncture. Thus, researchers have generally suggested indirect methods such as drawing human peripheral blood and isolating mononuclear cells to indirectly assess the effect of SHP2 inhibitors by assessing the level of ERK protein phosphorylation in PBMCs.
However, since in solid tumors, human peripheral blood PBMCs belong to a normal tissue relative to cancer tissue, the level of ERK phosphorylation in normal PBMCs is very low, substantially below the detection limit achievable by many current detection means.
Therefore, how to accurately evaluate the activity of the SHP2 inhibitor in the PBMC, so that the efficacy of the SHP2 inhibitor can be conveniently and timely monitored under the condition of no surgery or puncture of tumor tissue, is still a problem to be solved by the technicians in the field,
disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a kit for detecting the activity of an SHP2 inhibitor in PBMC and a method thereof, in particular to a kit for detecting the inhibition rate of the SHP2 inhibitor in PBMC to ERK phosphorylation and a method thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a kit for detecting SHP2 inhibitor activity in PBMC, the kit comprises a stimulator GM-CSF (granulocyte-macrophage colony stimulating factor) and a reagent for detecting total ERK and phosphorylated ERK.
The invention creatively discovers that the stimulator GM-CSF can obviously improve the background signal of ERK phosphorylation in PBMC, and simultaneously makes the SHP2 inhibitor sensitive to the signal so as to fully inhibit, thus the inhibition rate of the SHP2 inhibitor to ERK phosphorylation is estimated by comparing the signal value changes before and after administration, and the drug effect is indirectly estimated.
In a second aspect, the present invention provides a method of detecting SHP2 inhibitor activity in PBMCs, the method comprising detecting using a kit as described in the first aspect, comprising in particular the steps of:
mixing a PBMC sample to be tested with a stimulator GM-CSF, incubating, centrifuging, collecting PBMC cell sediment, splitting, detecting ERK total signal value and phosphorylated ERK signal value in the splitting solution, and calculating the ratio of phosphorylated ERK;
the same procedure as for the PBMC test sample was performed on the PBMC control sample containing no SHP2 inhibitor;
according to the detection results of the sample to be detected and the control sample, the inhibition rate of the SHP2 inhibitor on ERK phosphorylation is calculated, and the higher the inhibition rate is, the stronger the activity of the SHP2 inhibitor in the sample to be detected is.
Preferably, the ratio of PBMC to GM-CSF in the mixed system is (0.5-5). Times.10 7 Individual cells/(1-100 ng).
The above (0.5-5). Times.10 7 Specific values in individual cells, e.g. 5X 10 6 Individual cells, 8×10 6 Individual cells, 1×10 7 Individual cells, 1×10 7 Individual cells, 1.5X10 7 Individual cells, 2×10 7 Individual cells, 2.5X10 7 Individual cells, 3×10 7 Individual cells, 3.5X10 7 Individual cells, 4×10 7 Individual cells, 4.5X10 7 Individual cells, 5×10 7 Individual cells, etc.
Specific values in the above (1-100) ng are, for example, 1ng, 5ng, 10ng, 20ng, 30ng, 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, etc.
Preferably, the ratio of PBMC to GM-CSF in the mixed system is (2-5). Times.10 7 Individual cells/(10-100 ng).
Preferably, the incubation temperature is 10-30 ℃, e.g., 10 ℃,15 ℃, 20 ℃,25 ℃,30 ℃, etc.
Preferably, the incubation time is 3-8min, e.g., 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, etc.
The incubation time may be selected to be within the above range, and if too long, the signal values will decay, and too short will not be stimulated.
Preferably, the speed of the centrifugation is 300-800g, e.g. 300g, 350g, 400g, 450g, 500g, 550g, 600g, 650g, 700g, 750g, 800g etc. for a period of 1-5min, e.g. 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min etc.
Preferably, the centrifugation is performed at 10-30 ℃, e.g., 10 ℃,15 ℃, 20 ℃,25 ℃,30 ℃, etc.
Preferably, the cleavage specifically comprises: the cell pellet was mixed with the cell lysate.
Preferably, the mixing in the cleavage is performed at 0℃to 4℃such as 0℃1℃2℃3℃4℃for 20 minutes or more such as 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes and the like.
Preferably, the method further comprises centrifuging at 10000-15000g for 8-15min, and collecting supernatant to obtain lysate.
Specific values among 10000 to 15000g are 10000g, 10500g, 11000g, 11500g, 12000g, 12500g, 13000g, 13500g, 14000g, 14500g, 15000g, and the like.
The specific value of the above 8-15min is 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.
Preferably, the centrifugation in the collection lysate is performed at 0-8 ℃, e.g. 0 ℃,1 ℃,2 ℃,3 ℃,4 ℃,5 ℃, 6 ℃, 7 ℃, 8 ℃ etc.
In a third aspect, the present invention provides a method of using a kit for detecting SHP2 inhibitor activity in PBMCs according to the first aspect, the method of using comprising:
mixing a PBMC sample to be tested with a stimulator GM-CSF, incubating, centrifuging, collecting PBMC cell sediment, splitting, detecting ERK total signal value and phosphorylated ERK signal value in the splitting solution, and calculating the ratio of phosphorylated ERK;
the same procedure as for the PBMC test sample was performed on the PBMC control sample containing no SHP2 inhibitor;
according to the detection results of the sample to be detected and the control sample, the inhibition rate of the SHP2 inhibitor on ERK phosphorylation is calculated, and the higher the inhibition rate is, the stronger the activity of the SHP2 inhibitor in the sample to be detected is.
Preferably, the ratio of PBMC to GM-CSF in the mixed system is (0.5-5). Times.10 7 Individual cells/(1-100 ng).
Preferably, the ratio of PBMC to GM-CSF in the mixed system is (2-5). Times.10 7 Individual cells/(10-100 ng).
Preferably, the temperature of the incubation is 10-30 ℃.
Preferably, the incubation time is 3-8min.
Preferably, the speed of centrifugation is 300-800g for 1-5min.
Preferably, the cleavage specifically comprises: the cell pellet was mixed with the cell lysate.
Preferably, the mixing in the cleavage is performed at 0℃to 4℃for more than 20 min.
Preferably, the method further comprises centrifuging at 10000-15000g for 8-15min, and collecting supernatant to obtain lysate.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the following beneficial effects:
the present invention creatively discovers a suitable stimulator GM-CSF and a corresponding stimulation method, which can promote ERK phosphorylation in PBMCs within a larger window, and simultaneously, the SHP2 inhibitor is sensitive to the signal, so as to fully inhibit the signal. Therefore, by detecting the change of the phosphorylation level of the ERK protein, the inhibition effect of the SHP2 inhibitor on the phosphorylated ERK can be reflected very sensitively, so that the activity of the SHP2 inhibitor can be estimated indirectly.
The kit and the method can be used for directly detecting the activity of the SHP2 inhibitor (namely the inhibition rate of the SHP2 inhibitor on ERK phosphorylation) in the PBMC, wherein the PBMC can be extracted by any conventional method in the art, and therefore, the kit and the method can be applied to basic research related to the SHP2 inhibitor and ERK phosphorylation. In addition, the kit and the method can monitor the drug effect of the SHP2 inhibitor by collecting blood of a patient and separating PBMC, and solve one pain point in the study of the SHP2 inhibitor solid tumor: namely, how to monitor the efficacy of the SHP2 inhibitor conveniently and timely without surgery or puncture of tumor tissue.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following exploration experiments and examples, all reagents and consumables were purchased from the conventional reagent manufacturers in the art unless specifically stated otherwise; unless otherwise indicated, all methods and techniques used are those conventional in the art.
GM-CSF is available from R & D SYSTEMS under the trade designation 215-GM;
SHP2 inhibitor TNO155 was purchased from Selleck China under the trade designation S8987.
Investigation experiment
(1) The signal value excitation condition of different stimulators on ERK phosphorylation and the inhibition result of the SHP2 inhibitor on the ERK phosphorylation signal value are explored:
test stimulants were selected: PMA, M-CSF, GM-CSF, G-CSF, then divided into 3-4 groups: blank PBMC group (PBMC), PBMC plus SHP2 inhibitor group (pbmc+shp2i 40nm,2 h), PBMC plus stimulator group, PBMC plus SHP2 inhibitor plus stimulator group. pERK (phosphorylated ERK, phospho-ERK) and Total-ERK (Total ERK) were simultaneously detected by ECL-MSD kit, and the results are shown in tables 1-5.
TABLE 1 PMA stimulators and SHP2 inhibitors
Note that: the sample loading amount of the experimental group is larger, so the background signal value is higher.
Table 2.M-CSF stimulators and SHP2 inhibitors
TABLE 3G-CSF stimulators and SHP2 inhibitors
TABLE 4 GM-CSF stimulators and SHP2 inhibitors
TABLE 5 PMA+GM-CSF stimulators and SHP2 inhibitors
From the detection results in the above tables, it can be seen that: the inhibitory effect of SHP2 inhibitors on ERK phosphorylation was undetectable without the addition of stimulators. Of the various stimulators, only the stimulators GM-CSF and PMA were able to stimulate a large window of change in pERK signal values, but the PMA-stimulated pERK signal values were not inhibited by the SHP2 inhibitor, with an inhibition rate of 0%. And pERK signal value after GM-CSF stimulation can be inhibited by SHP2 inhibitor, and inhibition rate can be up to 97%. The pERK stimulation signal values of the M-CSF and the G-CSF are lower, and the inhibition rate can reach about 44% -48%. In addition, as shown in Table 5, experiments using PMA and GM-CSF in combination were also performed in this example, and it was found that the signal value of pERK was able to be stimulated effectively, but the inhibition rate after the addition of SHP2 inhibitor did not reach the inhibition effect of GM-CSF, only 40.449%, probably because SHP2 inhibitor inhibited only the phosphorylation by GM-CSF stimulator, and the phosphorylation by PMA stimulator was not inhibited, and the results were consistent with those of the PMA stimulator alone. Thus, in combination, GM-CSF was chosen to be most suitable as the stimulator in the SHP2 inhibitor study.
(2) Concentration selection of GM-CSF stimulators
PBMC were first isolated from human peripheral blood and then conditioned to a cell density of 1X 10 7 /mL, then divided into 6 groups: blank PBMC (PBMC), PBMC-added GM-CSF stimulant 1ng/mL, PBMC-added GM-CSF stimulant 5ng/mL, PBMC-added GM-CSF stimulant 10ng/mL, PBMC-added GM-CSF stimulant 50ng/mL, PBMC-added GM-CSF stimulantThe excitation agent is 100ng/mL, each group is stimulated for 5min, and then the subsequent operations such as centrifugation and cracking are carried out. pERK and Total ERK were finally detected simultaneously by ECL-MSD kit, the results are shown in Table 6.
TABLE 6 GM-CSF stimulators at different concentrations
The results show that GM-CSF stimulators are able to stimulate ERK phosphorylation at lower concentrations, with a 9.2 increase in phosphorylated ERK, but at 50ng/mL the signal value of pERK is highest. Thus, subsequent experiments used a concentration of 50ng/mL to stimulate PBMC.
(3) Cell density selection of PBMC
PBMCs were first isolated from human peripheral blood and then conditioned to different cell densities: 3X 10 7 /mL、2×10 7 /mL、1×10 7 /mL、5×10 6 Per mL, each cell density group was then divided into 3 groups: blank PBMC (0), PBMC with 50ng/mL of GM-CSF stimulator after SHP2 inhibitor, and GM-CSF for 5min were subjected to subsequent centrifugation and lysis. pERK and Total ERK were finally detected simultaneously by ECL-MSD kit, and the results are shown in Table 7.
TABLE 7 cell densities of different PBMC
The results show that: when the cell density of PBMC was 5.0X10 6 In the above cases, the signal value of pERK can meet the requirements. But the cell density reached 2.0X10 7 Or above, the signal value of pERK increases more significantly, and the fold change of pERK can be 23 times or more. Thus, it is suggested that PBMC cell densities for GM-CSF stimulation treatment may be at 2.0X10 7 at/mL or above, more pronounced pERK results can be obtained.
(4) Comparison of different preservation modes after PBMC sample collection stimulation
PBMC were first isolated from human peripheral blood and then conditioned to a cell density of 2X 10 7 /mL, then divided into 3 groups: blank PBMC group (0), PBMC adding GM-CSF stimulant 50ng/mL, PBMC adding SHP2 inhibitor and GM-CSF stimulant 50ng/mL, GM-CSF stimulation for 5min, then subsequent centrifugation, separation of the centrifuged PBMC precipitate into 2 kinds of operation modes, freezing the PBMC cell precipitate directly at-80 ℃ or-20 ℃ in one kind of refrigerator, direct lysis of the PBMC precipitate in the other kind of refrigerator, and freezing the obtained cell lysate at-80 ℃ or-20 ℃. pERK and Total ERK were finally detected simultaneously by ECL-MSD kit, the results are shown in Table 8.
Table 8 comparison of different modes of preservation after stimulation of PBMC sample collection
From the above results, it was found that the signal value of pERK cleaved immediately after the PBMC sample collection treatment (GM-CSF) was the highest, the signal value of PBMC lysate could not be reduced by freezing at-20℃or-80℃for 2 weeks, and the signal value obtained by re-lysing the PBMC cell pellet frozen at-20℃was significantly reduced. The freezing storage at the temperature of minus 80 ℃ has little influence. Therefore, after the PBMC are collected and separated, the stimulation of GM-CSF is carried out as soon as possible, and then the GM-CSF is cracked as soon as possible, and the obtained cell lysate is frozen for detection; PBMC cell pellets can also be frozen at-80 ℃ and lysed within 2 weeks.
Example 1
Whole blood of healthy volunteers is selected and divided into a plurality of groups, SHP2 inhibitors with different concentrations are added to simulate the drugs to enter human blood, and then pERK detection is carried out according to the method of the invention to evaluate the effect of the SHP2 inhibitors. The following are specific operational steps.
Whole blood incubation with SHP2 inhibitor
Whole blood of healthy volunteers was collected using EDTAK2 anticoagulated blood collection tubes, and then divided into 10 groups of 4mL of whole blood on average, and SHP2 inhibitor was added at concentrations of 0nM, 0.013nM, 0.064nM, 0.32nM, 1.6nM, 8.0nM, 40nM, 200nM, 1000nM, respectively, and incubated at 37℃for 2 hours. While a set of whole blood without any reagent was left as a control.
PBMC isolation and GM-CSF stimulation and lysis
2.1 PBMC isolation
Whole blood after incubation of the drug and control group were transferred to a mononuclear cell separation tube (i.e., CPT tube, BD company), placed in a horizontal rotor (out-opening head) centrifuge, carefully balanced, for 180 g,25 min,25 ℃ and centrifuged without brake (i.e., slow ramp).
After centrifugation, the mononuclear cells will be in the white layer, next to the upper plasma layer. The 2/3 of the upper plasma was carefully aspirated using a Pasteur pipette, taking care not to destroy the underlying cell layer. The upper plasma layer can be disposed of according to the regulations related to biomedical waste. A new Pasteur pipette is then carefully removed to transfer the white layer of cells (lymphocytes and monocytes) into a 15mL pointed centrifuge tube, and the corresponding sample information is marked on the tube wall with a marker.
PBMC washing: at least 10 PBMC volumes of PBS solution were added each time 300g,15min,25 ℃. Washing at least 2 times.
Transfer of PBMCs: after centrifugation, the supernatant was carefully and as much as possible aspirated without the cell pellet being blown up. Cells were resuspended using 200 μl of resuspension (RPMI 1640+10% fbs, ensuring that room temperature had been restored (10 ℃ -30 ℃) and cell counted and transferred to 1 1.5mL centrifuge tubes.
2.2 stimulation of PBMC by GM-CSF
Stimulation of PBMC: mu.L of the stimulator GM-CSF (5. Mu.g/mL) was added to 200. Mu.L of PBMC resuspension (cell density ca. 2.0X10) 7 The final concentration of the stimulator is 50ng/mL, the mixture is immediately blown and evenly mixed, and the mixture is incubated for 5 minutes at room temperature (10 ℃ C. -30 ℃ C.) (the incubation time is strictly controlled).
Immediately, the PBMC suspension was centrifuged using a centrifuge in a 1.5mL centrifuge tube at 500g for 3 minutes at 18℃to 25 ℃. The supernatant was removed as much as possible after centrifugation was complete (ensuring that less than 20. Mu.L of liquid remained on the cell pellet).
2.3 lysis of PBMC
After centrifugation, 100. Mu.L of cell lysate (MSD Complete Lysis Buffer) was immediately added, and the mixture was stirred and homogenized on ice for at least 30 minutes.
After the completion of the pyrolysis, a 1.5mL centrifuge tube is centrifuged at 12000g,10min and 4 ℃, the supernatant is transferred to a new tube, marked and stored in a refrigerator at-65 ℃ to-90 ℃ for standby.
2.4BCA method protein quantification
Taking out the cell lysate from the refrigerator at the temperature of between 65 ℃ below zero and 90 ℃ below zero, melting the cell lysate on wet ice, and detecting the protein concentration of each group by using the BCA protein quantitative kit. The purpose is to adjust the loading amounts of each group in ERK detection to be consistent.
3pERK and Total ERK detection
The whole lysate after protein quantification was diluted to a concentration of 1mg/mL using the cell lysate (MSD Complete Lysis Buffer).
Detection was performed according to the instructions of the MSD kit Phospho/Total ERK1/2Whole Cell Lysate Kit (MSD; CAT#. K15107D).
4. Interpretation and description of results
The pERK and Total ERK measurements (Table 9) show that GM-CSF is able to significantly stimulate the pERK signal values in PBMC up to 14-fold. In addition, the SHP2 inhibitor can inhibit pERK stimulation signal value caused by GM-CSF at different concentrations in whole blood, and the pERK inhibition rate also tends to decrease along with the decrease of the drug concentration, and the result is good in linearity.
TABLE 9 inhibition of SHP2 inhibitors at various concentrations in whole blood
In summary, the invention provides a suitable stimulator GM-CSF and a corresponding method, which can effectively evaluate the efficacy of SHP2 inhibitors by detecting the change in the phosphorylation level of ERK protein. And solves one difficulty: i.e. how to go to a solid tumor and to be able to monitor the efficacy of SHP2 inhibitors in a timely manner, is relatively difficult to obtain fresh tumor tissue clinically at different administration time points, and is painful for the patient to frequently perform surgery or puncture. Thus, the present invention employs an indirect method to indirectly evaluate the effect of SHP2 inhibitors by evaluating the efficacy of phosphorylation of ERK proteins in PBMCs.
The invention finally finds the GM-CSF and the corresponding stimulation method by screening several potential stimulators (such as PKC activators PMA, colony stimulating factors M-CSF, G-CSF and GM-CSF), which can raise the phosphorylation level of ERK in PBMC in a larger window and can sensitively reflect the inhibition effect of SHP2 inhibitors on phosphorylated ERK.
The applicant states that the present invention is illustrated by the above examples as a kit and method for detecting SHP2 inhibitor activity in PBMCs, but the invention is not limited to, i.e. it is not meant that the invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A kit for detecting SHP2 inhibitor activity in PBMCs, comprising the stimulator GM-CSF and reagents for detecting total ERK and phosphorylated ERK.
2. A method for detecting SHP2 inhibitor activity in PBMCs, said method comprising the step of detecting using the kit of claim 1, comprising in particular the steps of:
mixing a PBMC sample to be tested with a stimulator GM-CSF, incubating, centrifuging, collecting PBMC cell sediment, splitting, detecting ERK total signal value and phosphorylated ERK signal value in the splitting solution, and calculating the ratio of phosphorylated ERK;
the same procedure as for the PBMC test sample was performed on the PBMC control sample containing no SHP2 inhibitor;
according to the detection results of the sample to be detected and the control sample, the inhibition rate of the SHP2 inhibitor on ERK phosphorylation is calculated, and the higher the inhibition rate is, the stronger the activity of the SHP2 inhibitor in the sample to be detected is.
3. The method of detecting SHP2 inhibitor activity in PBMCs according to claim 2, wherein the ratio of PBMCs to GM-CSF in the mixed system is (0.5-5) x 10 7 Individual cells/(1-100 ng).
4. The method of detecting SHP2 inhibitor activity in PBMCs according to claim 3, wherein the ratio of PBMCs to GM-CSF in the mixed system is (2-5) x 10 7 Individual cells/(10-100 ng).
5. The method of detecting SHP2 inhibitor activity in PBMCs according to any one of claims 2 to 4, wherein the incubation is at a temperature of 10 to 30 ℃.
6. The method of detecting SHP2 inhibitor activity in PBMCs according to any one of claims 2 to 5, wherein the incubation is for a period of 3 to 8 minutes.
7. The method of detecting SHP2 inhibitor activity in PBMCs according to any one of claims 2 to 6, wherein the centrifugation is at a speed of 300 to 800g for a period of 1 to 5 minutes.
8. The method of detecting SHP2 inhibitor activity in PBMCs according to any one of claims 2 to 7, wherein the lysing specifically comprises: the cell pellet was mixed with the cell lysate.
9. The method of detecting SHP2 inhibitor activity in PBMCs of claim 8, wherein the mixing during lysis is performed at 0 ℃ to 4 ℃ for more than 20 minutes.
10. The method of detecting SHP2 inhibitor activity in PBMCs according to any one of claims 2 to 9, further comprising centrifugation at 10000 to 15000g for 8 to 15min after the lysis, and collecting the supernatant as a lysate.
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