Detection method for reducing off-target risk of targeting drug
By screening the optimal working concentration of the test substance and combining it with a human plasma membrane protein array library containing the fluorescent protein ZsGreen/mCherry, along with chemiluminescence and flow cytometry detection, the problem of insufficient accuracy in predicting the off-target risk of targeted drugs in membrane protein array technology was solved, thus improving the accuracy of drug safety assessment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-03-31
AI Technical Summary
Existing membrane protein array technologies are not accurate enough in predicting the off-target risk of targeted drugs, making it difficult to effectively assess drug safety issues.
By screening the optimal working concentration of the test substance, high-throughput transient transfection was performed using a human plasma membrane protein array library of fluorescent protein ZsGreen/mCherry. Positive, negative, and quality control controls were set up, and target proteins with specific binding relationships to the test substance were screened by combining chemiluminescence detection and flow cytometry detection, thereby reducing false negative results.
It improves the accuracy of predicting the off-target risk of targeted drugs, reduces the occurrence of false negative results, and enhances the reliability of drug safety assessment.
Smart Images
Figure CN121762846A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of immunoassay technology, and more specifically to a detection method that reduces the risk of off-target effects of targeted drugs. Background Technology
[0002] Membrane proteins are indispensable components of the cell membrane, playing a crucial role in communication between the cell and its external environment, including signal transduction, substance transport, and cell recognition. However, membrane protein-targeted drugs in clinical practice commonly exhibit off-target effects, and off-target toxicity of biologics remains a leading cause of failure in preclinical drug projects.
[0003] Membrane protein array technology is a high-throughput platform for analyzing membrane protein function. It utilizes living cells as expression carriers for membrane proteins, allowing them to naturally embed within the cellular phospholipid bilayer, thus maintaining their correct three-dimensional structure and biological activity. Membrane protein array technology can assess the cross-reactivity of therapeutic antibody drugs with human membrane proteins, predict potential off-target effects, and significantly improve drug safety. However, current membrane protein array technology has limitations, particularly in the accuracy of predicting off-target risks.
[0004] Therefore, providing a detection method that can reduce the risk of off-target effects of targeted drugs is a technical problem that urgently needs to be solved. Summary of the Invention
[0005] Specifically addressing the shortcomings of existing technologies, a detection method for reducing the off-target risk of targeted drugs is provided. This method includes the following steps: S1, screening for the optimal working concentration of the test substance; S2, performing high-throughput transient transfection of a human plasma membrane protein array library containing the fluorescent protein ZsGreen / mCherry in tool cells, setting up positive controls, negative controls, and quality control controls, and screening for human plasma membrane proteins with a fluorescence cut-off value ≥1; then incubating the sample with the optimal working concentration of the test substance and the second binding protein, followed by chemiluminescence detection, and preliminarily screening for human plasma membrane proteins with a binding risk to the test substance based on the screening conditions; S3, preliminarily screening for... Human plasma membrane proteins were analyzed by flow cytometry. Human plasma membrane proteins with an average fluorescence intensity (MFI) value more than twice that of the negative control were identified as target proteins with specific binding to the test substance. The detection method provided in this invention involves transfecting cells with a ZsGreen / mCherry fluorescent protein expression cassette added to the rear end of the human plasma membrane protein expression cassette. The expression of the fluorescent protein can indicate the expression status of human plasma membrane proteins. Positive controls, quality control controls, and negative controls were set up. Target proteins with specific binding to the test substance were obtained through chemiluminescence detection and flow cytometry screening, reducing the risk of false negatives and improving the accuracy of predicting the off-target risk of targeted drugs.
[0006] The objective of this invention is achieved through the following technical solution: This invention provides a detection method for reducing the off-target risk of targeted drugs, the detection method comprising the following steps: S1. Screening the optimal working concentration of the test substance; S2. The human plasma membrane protein array library containing fluorescent protein ZsGreen / mCherry was transfected in tool cells using high-throughput transient transfection. Positive control, negative control and quality control were set up to screen human plasma membrane proteins with fluorescence cut-off value ≥1. Then, the protein was incubated with the test substance and the second binding protein at the optimal working concentration. Chemiluminescence detection was then performed. Based on the screening conditions, human plasma membrane proteins that have a risk of binding with the test substance were preliminarily screened. S3. The preliminarily screened human plasma membrane proteins were subjected to flow cytometry detection. Human plasma membrane proteins with an average fluorescence intensity (MFI) value that was more than twice that of the negative control were identified as target proteins that had a specific binding relationship with the test substance.
[0007] In some specific embodiments of the present invention, the quality control control in step S2 is one or more of type I transmembrane protein CD7, type II transmembrane protein CD70, and type III transmembrane protein BCMA.
[0008] In some specific embodiments of the present invention, the optimal working concentration of the test substance in step S1 is selected by calculating the signal-to-background ratio, and the concentration corresponding to the largest signal-to-background ratio is the optimal working concentration of the test substance.
[0009] In some specific embodiments of the present invention, the transfection method described in step S2 is reverse transient transfection of lip3000.
[0010] In some specific embodiments of the present invention, the concentration of the transfected plasmid in step S2 is 500 ng to 1000 ng.
[0011] In some specific embodiments of the present invention, the transfection conditions in step S2 are: cell culture at 37°C and 5% CO2 for 36–72 h.
[0012] In some specific embodiments of the present invention, the tool cells in step S2 are HEK293T, HEK293T-BFP, or QT6.
[0013] In some specific embodiments of the present invention, the incubation conditions in step S2 are incubation at 4°C for 20 to 40 minutes in a dark environment.
[0014] In some specific embodiments of the present invention, the chemiluminescence detection method in step S2 is as follows: according to the luciferase activity detection kit, chemiluminescence Smart-ECL Super reagent I solution is added to the sample to be tested, followed by chemiluminescence Smart-ECL Super reagent II solution. After incubation in the dark for 4 minutes, the chemiluminescence value of the sample to be tested is detected by an enzyme-linked immunosorbent assay (ELISA) reader.
[0015] In some specific embodiments of the present invention, the flow cytometry detection in step S3 also needs to be verified by isotype control to exclude non-specific binding relationships.
[0016] The beneficial effects achieved by this invention are as follows: The detection method provided by this invention involves transfecting cells with a ZsGreen / mCherry fluorescent protein expression cassette added to the rear end of the human plasma membrane protein expression cassette. The expression of the fluorescent protein can indicate the expression status of human plasma membrane proteins. Positive controls, quality control controls, and negative controls are set up. Target proteins with specific binding relationships to the test substance are obtained through chemiluminescence detection and flow cytometry screening, reducing the risk of false negative results and improving the accuracy of predicting the off-target risk of targeted drugs. Attached Figure Description
[0017] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 The optimal working concentration of camrelizumab monoclonal antibody is shown in the figure. Figure 2 A preliminary screening result of human plasma membrane proteins that pose a risk of binding to camrelizumab monoclonal antibodies; Figure 3 This is a flow cytometry verification diagram of camrelizumab monoclonal antibody. Figure 4 This is a graph showing the optimal working concentration of trastuzumab monoclonal antibody. Figure 5 Figure showing the preliminary screening results of human plasma membrane protein array for trastuzumab monoclonal antibody; Figure 6 This is a flow cytometry verification diagram of trastuzumab monoclonal antibody. Figure 7 Graph showing the optimal working concentration of FMC63 scFv; Figure 8 Preliminary screening results of human plasma membrane protein array for FMC63 scFv; Figure 9 The graph shows the flow cytometry verification of FMC63 scFv. Detailed Implementation
[0019] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0020] The reagents used in the examples are as follows: Lipofectamine™ 3000 transfection reagent, brand Thermo Fisher, model L3000-015; CD7 Monoclonal Antibody, brand Thermo Fisher, model 13-0079-82; Biotin anti-human CD70 Antibody, brand Biolegend, model 355114; Biotin anti-human CD269 (BCMA) Antibody, brand Biolegend, model 357514; Smart-ECL Super, brand Changzhou Tiandi Renhe, model S32500.
[0021] Example 1 Methods: Using camrelizumab monoclonal antibody as the research subject, the off-target risk of camrelizumab monoclonal antibody was assessed.
[0022] The optimal working concentration of camrelizumab monoclonal antibody in high-throughput screening was determined. A positive group was established, with the known binding antigen PD-1 of camrelizumab monoclonal antibody as the positive group and an empty vector as the negative group. Both groups were transfected into 384-well plates using a reverse transient transfection method. Forty hours after transient transfection, four concentration gradients of camrelizumab monoclonal antibody (2 μg / mL, 4 μg / mL, 6 μg / mL, and 8 μg / mL) were established, and the signal values at each concentration were obtained using chemiluminescence detection. The signal-to-weight ratio (S / B) within the screening concentration range was calculated, and the concentration of camrelizumab monoclonal antibody corresponding to the highest S / B signal value was taken as the optimal working concentration for screening. Comparison of the chemiluminescence results at the four concentrations confirmed that 4 μg / mL was the optimal working concentration of camrelizumab monoclonal antibody. Figure 1 As shown.
[0023] A high-throughput screening of a human plasma membrane protein array containing 4321 membrane proteins was performed using 4 μg / mL camrelizumab monoclonal antibody. Membrane proteins suspected of binding to camrelizumab monoclonal antibody were screened one by one. The specific steps are as follows.
[0024] A ZsGreen / mCherry fluorescent protein expression cassette was added to the rear end of the human plasma membrane protein expression cassette to obtain a plasmid containing the fluorescent protein for transfection. 800 ng of the plasmid was added to each 12-well plate, and transfection was performed using the lip3000 transfection method at 5 × 10⁻⁶ cycles. 4 In HEK293T cells, reverse transfection was performed. After brief centrifugation, the cells were cultured for 40 hours at 37°C with 5% CO2 to obtain all membrane proteins expressing native conformation on the surface of HEK293T cells. Transfection was performed in 384-well plates. Each 384-well plate contained PD-1 as a positive control, 32 negative controls transfected with empty vector, and typical proteins from different human plasma membrane protein types. Type I transmembrane protein CD7 was used as a quality control. The fluorescence value of each well was detected by a multi-mode microplate reader to determine the transfection status. The ZsGreen / mCherry fluorescence value of each well represented the transfection effect of the plasmid indicating the protein. Wells with a cut-off value of 1 (mean and 3 times standard deviation, Meanag + 3×SDneg) higher than the fluorescence value of the negative control (empty vector transfected wells) were considered to have been successfully transfected and met the requirements for further detection.
[0025] Cells successfully transfected were subjected to chemiluminescence detection. First, the cells were incubated with 4 μg / mL of camrelizumab monoclonal antibody at 4°C in the dark for 30 min, followed by two washes. Then, the cells were incubated with the secondary antibody Goat anti-Human IgG Fc Secondary Antibody (HRP), which binds to the IgG4 Fc fragment of camrelizumab monoclonal antibody, at 4°C in the dark for 30 min, followed by two washes.
[0026] According to the luciferase activity assay kit, cells were resuspended in 25 μL / well of Smart-ECL Super Reagent I solution under dark conditions and transferred to 384-well white plates. Then, using a multi-functional microplate reader's automated sample loading program, 25 μL of Smart-ECL Super Reagent II solution was added to each well. After incubation in the dark for 4 min, the chemiluminescence detection program was started. For each well's result, a cut-off value of 2 (the sum of the mean and 3 times the standard deviation) was calculated based on the chemiluminescence value higher than the negative control. neg + 3×SD negResults below a cut-off value of 2 were considered suspected positive; results below this value were considered negative. Preliminary screening identified human plasma membrane proteins at risk of binding to camrelizumab monoclonal antibody. High-throughput screening preliminarily identified PD-1, VEGFR2, ULBP2, FZD5, DRD2, and MRGPRE proteins as potential binding targets. Figure 2 As shown.
[0027] Flow cytometry was used to titrate suspected target proteins to confirm target binding relationships. Human plasma membrane proteins suspected of being at risk in the high-throughput screening results, along with positive and negative controls, were transiently transfected. After transfection, four concentrations (2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL) of camrelizumab were incubated, followed by incubation with the fluorescent secondary antibody APC anti-human IgG Fc Antibody, which binds to the IgG4 Fc fragment of camrelizumab. After washing, flow cytometry was performed to analyze the molecular weight fraction (MFI) of all samples and perform isotype control verification to exclude non-specific binding relationships, further identifying human plasma membrane proteins that bind to camrelizumab. When the MFI value of a sample at the camrelizumab detection concentration was more than twice that of the negative control, the human plasma membrane protein was determined to be a target protein with a specific binding relationship to camrelizumab.
[0028] Flow cytometry titration was used to analyze the MFI data, ultimately confirming that the camrelizumab monoclonal antibody targets and binds to PD-1, VEGFR2, ULBP2, and FZD5 proteins, but not to DRD2 or MRGPRE proteins. Figure 3 As shown in Table 1.
[0029] Table 1. Statistical values of MFI for flow cytometry validation of camrelizumab monoclonal antibody
[0030] Conclusion: Camrelizumab monoclonal antibody shows targeted binding relationships with PD-1, VEGFR2, ULBP2, and FZD5 proteins, but no clear targeted binding relationship with 4317 other proteins.
[0031] Example 2 Methods: Trastuzumab monoclonal antibody was used as the research object to evaluate its targeting.
[0032] The optimal working concentration of trastuzumab monoclonal antibody was determined for high-throughput screening. A positive group was established, with the known binding antigen HER2 of trastuzumab monoclonal antibody as the positive group and an empty vector as the negative group. Both groups were transfected into 384-well plates using a reverse transient transfection method. Forty hours after transient transfection, four concentration gradients of trastuzumab monoclonal antibody (0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL) were established. Signal values at each concentration were obtained using chemiluminescence detection, and the signal-to-weight ratio (S / B) within the screening concentration range was calculated. The concentration of trastuzumab monoclonal antibody corresponding to the highest S / B signal value was taken as the optimal working concentration for screening. Comparison of the chemiluminescence results at the four concentrations confirmed that 0.5 μg / mL was the optimal working concentration of trastuzumab monoclonal antibody. Figure 4 As shown.
[0033] High-throughput screening of a human plasma membrane protein array containing 4868 membrane proteins was performed using trastuzumab monoclonal antibody. Membrane proteins suspected of binding to trastuzumab monoclonal antibody were screened one by one. The specific steps are as follows.
[0034] A ZsGreen / mCherry fluorescent protein expression cassette was added to the rear end of the human plasma membrane protein expression cassette to obtain a plasmid containing the fluorescent protein for transfection. 800 ng of the plasmid was added to each 12-well plate, and transfection was performed using the lip3000 transfection method at 5 × 10⁻⁶ cycles. 4 In HEK293T-BFP cells, all plasmids contained in the human plasma membrane protein array were transfected. After 40 h of transfection, all membrane proteins expressing their native conformation on the surface of HEK293T-BFP cells were obtained. Transfection was performed in 384-well plates. Each 384-well plate included HER2 as a positive control, 32 negative controls transfected with empty vector, and typical proteins from different human plasma membrane protein types. Type II transmembrane protein CD70 was used as a quality control. The fluorescence value of cells in each well was detected using a multi-mode microplate reader to determine the transfection status. The ZsGreen / mCherry fluorescence value of each well characterized the transfection effect of the plasmid indicating the protein. A cut-off value (mean value) calculated from a fluorescence value higher than that of the negative control (empty vector transfected wells) was 1 (the sum of the mean and three standard deviations). neg +3×SD neg The wells were considered to have been successfully transfected and met the requirements for further testing.
[0035] Cells successfully transfected from the wells were subjected to chemiluminescence assay. First, the cells were incubated with 0.5 μg / mL trastuzumab monoclonal antibody, followed by incubation with the secondary antibody Goat anti-Human IgG FcSecondary Antibody (HRP), which binds to the trastuzumab monoclonal antibody tag IgG1. Finally, the chemiluminescence values were detected using a Smart-ECL Super assay kit and a microplate reader. For each well, a cut-off value of 2 (the sum of the mean and three times the standard deviation) was calculated for results higher than the negative control chemiluminescence value. neg + 3×SD neg A result below a cut-off value of 2 was considered a suspected positive result; a result below this value was considered a negative result. Initial screening identified human membrane proteins that posed a risk of binding to camrelizumab monoclonal antibody. Through high-throughput screening, HER2 protein was initially identified as a suspected binding target. Figure 5 As shown.
[0036] Flow cytometry was used to titrate suspected target proteins to confirm target binding relationships. Human plasma membrane proteins suspected of being at risk in the high-throughput screening results, along with positive and negative controls, were transiently transfected. After transfection, four concentrations (1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL) of trastuzumab monoclonal antibody were incubated, followed by incubation with the fluorescent secondary antibody APC anti-human IgG Fc Antibody, which binds to the trastuzumab monoclonal antibody tag IgG1. After washing, flow cytometry was performed, and the molecular weight fraction (MFI) of all samples was analyzed to further identify human plasma membrane proteins that bind to trastuzumab monoclonal antibody. For human plasma membrane proteins that bind to trastuzumab monoclonal antibody, if the MFI value of the sample at the trastuzumab monoclonal antibody detection concentration was more than twice that of the negative control, the human plasma membrane protein was determined to be a target protein with a specific binding relationship to trastuzumab monoclonal antibody. Flow cytometry titration was used to statistically analyze the MFI data, ultimately confirming a targeted binding relationship between trastuzumab monoclonal antibody and HER2 protein. Figure 6 As shown in Table 2.
[0037] Table 2. MFI statistic for trastuzumab monoclonal antibody flow cytometry titration validation
[0038] Conclusion: Trastuzumab monoclonal antibody has a targeted binding relationship with HER2 protein, but no clear targeted binding relationship with 4867 proteins.
[0039] Example 3 Methods: Using FMC63 scFv as the research object, the targeting of FMC63 scFv was evaluated.
[0040] The optimal working concentration of FMC63 scFv in high-throughput screening was determined. The known binding antigen CD19 of FMC63 scFv was used as the positive group, and the empty vector as the negative group. Both groups were transfected in 384-well plates using a reverse transient transfection method. Forty hours after transient transfection, four FMC63 scFv concentration gradients (0.1 μg / mL, 0.25 μg / mL, 1 μg / mL, and 4 μg / mL) were established. The signal values at each concentration were obtained using chemiluminescence detection, and the signal-to-weight ratio (S / B) within the screening concentration range was calculated. The FMC63 scFv concentration corresponding to the highest S / B signal value was taken as the optimal working concentration for screening. Comparison of the chemiluminescence results at the four concentrations confirmed that 1 μg / mL was the optimal working concentration of FMC63 scFv. Figure 7 As shown.
[0041] High-throughput screening of a human plasma membrane protein array containing 4636 membrane proteins was performed using FMC63 scFv. Membrane proteins suspected of binding to FMC63 scFv were screened one by one. The specific steps are as follows.
[0042] A ZsGreen / mCherry fluorescent protein expression cassette was added to the rear end of the human plasma membrane protein expression cassette to obtain a plasmid containing the fluorescent protein for transfection. 800 ng of the plasmid was added to each 12-well plate, and transfection was performed using the lip3000 transfection method at 5 × 10⁻⁶ cycles. 4 In QT6 cells, all plasmids contained in the human plasma membrane protein array were transfected. After 40 h of transfection, all membrane proteins expressing their native conformation on the surface of QT6 cells were obtained. Transfection was performed in 384-well plates. Each 384-well plate included CD19 as a positive control, 32 negative controls transfected with empty vectors, and typical proteins from different human plasma membrane protein types. Type III transmembrane protein BCMA was used as a quality control. Furthermore, to characterize the detection of different membrane protein types in this assay system, typical proteins (CD7, CD70, and BCMA) from different human plasma membrane protein types were used as quality control controls, incubated with corresponding commercially available antibodies during detection. The fluorescence value of each well was measured using a multi-mode microplate reader to determine the transfection status. The ZsGreen / mCherry fluorescence value of each well characterized the transfection effect of the plasmid indicating the protein. A cut-off value (mean value) higher than the fluorescence value of the negative control (empty vector transfected wells) was calculated as 1 (the sum of the mean and three standard deviations). neg + 3×SD neg The wells were considered to have been successfully transfected and met the requirements for further testing.
[0043] Cells successfully transfected from the wells were subjected to chemiluminescence assay. First, the cells were incubated with the optimal working concentration of FMC63 scFv obtained in the above process. Then, they were incubated with the secondary antibody HRP Streptavidin, which binds to the FMC63 scFv tag biotin. Finally, the chemiluminescence values were detected using a Smart-ECL Super assay kit and a microplate reader. For each well, a cut-off value of 2 (the sum of the mean and three standard deviations) was calculated for values higher than the negative control chemiluminescence value. neg + 3×SD neg Results below a cut-off value of 2 were considered suspected positive; results below this value were considered negative. Preliminary screening identified human membrane proteins at risk of binding to camrelizumab monoclonal antibodies. High-throughput screening preliminarily identified suspected binding targets including CD19 (variant 1), CD19 (variant 2), DDR2, GPRC6A, and ATP6V0A1 proteins. Figure 8 As shown.
[0044] Flow cytometry was used to titrate suspected target proteins to confirm target binding relationships. Human plasma membrane proteins suspected of being at risk in the high-throughput screening results, along with positive and negative controls, were transiently transfected. After transfection, the samples were first incubated with four concentrations (2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL) of FMC63 scFv, followed by incubation with the fluorescent secondary antibody FITC Streptavidin or PE Streptavidin, which binds to the FMC63 scFv tag Biotin. After washing, flow cytometry was performed. MFI analysis of all samples was conducted. For human plasma membrane proteins binding to FMC63 scFv, if the MFI value of the sample at the detected FMC63 scFv concentration was more than twice that of the negative control, the human plasma membrane protein was identified as a target protein specifically binding to FMC63 scFv.
[0045] Flow cytometry titration was used to statistically analyze MFI data, ultimately confirming that FMC63 scFv has a targeted binding relationship with CD19 (variant 1) and CD19 (variant 2) proteins, but no clear targeted binding relationship with DDR2, GPRC6A, and ATP6V0A1 proteins. Figure 9 As shown in Table 3.
[0046] Table 3. MFI Statistical Values for FMC63 scFv Flow Cytometry Validation
[0047] Conclusion: FMC63 scFv has a targeted binding relationship with CD19 (variant 1) and CD19 (variant 2) proteins, but no clear targeted binding relationship with 4634 proteins.
[0048] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.
Claims
1. A detection method for reducing the off-target risk of targeted drugs, characterized in that, The detection method includes the following steps: S1. Screening the optimal working concentration of the test substance; S2. The human plasma membrane protein array library containing fluorescent protein ZsGreen / mCherry was transfected in tool cells using high-throughput transient transfection. Positive control, negative control and quality control were set up to screen human plasma membrane proteins with fluorescence cut-off value ≥1. Then, the protein was incubated with the test substance and the second binding protein at the optimal working concentration. Chemiluminescence detection was then performed. Based on the screening conditions, human plasma membrane proteins that have a risk of binding with the test substance were preliminarily screened. S3. The preliminarily screened human plasma membrane proteins were subjected to flow cytometry detection. Human plasma membrane proteins with an average fluorescence intensity (MFI) value that was more than twice that of the negative control were identified as target proteins that had a specific binding relationship with the test substance.
2. The detection method according to claim 1, characterized in that, The quality control in step S2 is one or more of the following: type I transmembrane protein CD7, type II transmembrane protein CD70, and type III transmembrane protein BCMA.
3. The detection method according to claim 1, characterized in that, The optimal working concentration of the test substance in step S1 is selected by calculating the signal-to-background ratio, and the concentration corresponding to the largest signal-to-background ratio is the optimal working concentration of the test substance.
4. The detection method according to claim 1, characterized in that, The transfection method described in step S2 is the reverse instantaneous transfection of lip3000.
5. The detection method according to claim 1, characterized in that, The concentration of the plasmid used for transfection in step S2 is 500 ng to 1000 ng.
6. The detection method according to claim 1, characterized in that, The transfection conditions described in step S2 are: cell culture at 37°C and 5% CO2 for 36–72 h.
7. The detection method according to claim 1, characterized in that, The tool cells mentioned in step S2 are HEK293T, HEK293T-BFP, or QT6.
8. The detection method according to claim 1, characterized in that, The incubation conditions described in step S2 are: incubation at 4°C for 20–40 minutes in a dark environment.
9. The detection method according to claim 1, characterized in that, The chemiluminescence detection method described in step S2 is as follows: according to the luciferase activity assay kit, add chemiluminescence Smart-ECL Super reagent I solution to the sample to be tested, then add chemiluminescence Smart-ECL Super reagent II solution, incubate in the dark for 4 min, and then detect the chemiluminescence value of the sample to be tested using an enzyme-linked immunosorbent assay (ELISA) reader.
10. The detection method according to claim 1, characterized in that, The flow cytometry detection described in step S3 also requires isotype control verification to exclude non-specific binding relationships.