A T cell for detecting anti-Fc antibodies, a kit thereof, and a detection method

Abstract

The present invention relates to a T cell for detecting anti-Fc antibodies, a kit thereof, and a detection method. The T cell of the present invention expresses a chimeric antigen receptor, and the chimeric antigen receptor includes an Fc region, a CD8α transmembrane region, and a CD3z intracellular signaling region; the T cell further includes an NFAT-luciferase reporter gene, and the NFAT-luciferase reporter gene includes a luciferase encoding gene and a promoter that regulates the encoding gene, and the promoter includes an NFAT binding site. The present invention proposes a completely new detection principle for anti-Fc antibodies. It utilizes the Fc region of the chimeric antigen receptor to specifically bind to anti-Fc antibodies, converts the detection signal into a T cell activation signal, and transmits it to the NFAT-luciferase reporter gene. By detecting the expression of luciferase, anti-Fc antibodies are detected, which has the characteristics of sensitivity, high efficiency, etc.

Description

Technical Field

[0001] The present invention belongs to the field of anti-drug antibody detection. Specifically, it relates to a T cell for detecting anti-Fc antibody, a kit thereof, and a detection method. Background Art

[0002] During the drug development process, the study of immunogenicity has become an essential key part. For most drugs, especially antibody drugs, the generation of immunogenicity is mainly mediated by humoral immunity. Therefore, anti-drug antibody (ADA) has always been the main criterion for defining the immunogenicity of such drugs. Among them, anti-Fc antibody against the Fc region of antibody drugs belongs to a common type of anti-drug antibody. The presence of anti-drug antibody may, on the one hand, neutralize the biological activity of the drug and significantly reduce the drug efficacy. On the other hand, it may cross-react with endogenous proteins, leading to new immune responses and seriously endangering life.

[0003] Currently, the commonly used ADA detection methods on the market are the direct method and the bridging method developed based on enzyme-linked immunosorbent assay technology. Both methods have advantages and disadvantages in terms of detection sensitivity, detection range, ability to detect different immunoglobulins, and availability of detection reagents. For example, the direct method can detect low-affinity ADA, but there is species specificity. Although the bridging method has no species specificity, it is easily interfered by drug concentration and soluble targets and cannot detect low-affinity ADA. Therefore, it is particularly important to develop a reliable method for effectively evaluating ADA.

[0004] The above detection methods developed based on enzyme-linked immunosorbent assay technology are simple to operate and suitable for large-scale screening, but there are many interfering factors and the detection results are not accurate enough. Moreover, this detection method is usually not applicable to samples with ADA concentration higher than 1 μg / ml, and the detection range is limited. In order to better evaluate the immunogenicity of drugs, it is urgent to develop a more accurate and simple analysis method. Summary of the Invention

[0005] The present invention provides a T cell, a corresponding kit thereof, and a detection method, aiming to provide a new detection method for anti-Fc antibody based on cell signal transduction, which has the advantages of sensitivity, high efficiency, and suitability for detecting high-concentration samples of anti-Fc antibody.

[0006] To achieve the object of the invention, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a T cell, which expresses a chimeric antigen receptor, and the chimeric antigen receptor includes an Fc region, a CD8α transmembrane region, and a CD3z intracellular signal transduction region;

[0008] The T cell also includes an NFAT-luciferase reporter gene, which includes a luciferase-encoding gene and a promoter that regulates the encoding gene, and the promoter includes an NFAT binding site.

[0009] The T cell of the present invention binds to an anti-Fc antibody using the Fc region and activates the chimeric antigen receptor, converting the signal of the binding of Fc to the anti-Fc antibody into a T cell activation signal. The T cell activation signal stimulates NFAT to bind to the promoter of the NFAT-luciferase reporter gene, initiating the expression of the luciferase gene. At the cellular level, the present invention ingeniously converts the detection of anti-Fc antibody into the detection of luciferase activity through the chimeric antigen receptor and the NFAT-luciferase reporter gene, constructing a highly efficient and sensitive analytical system for detecting anti-Fc antibody.

[0010] In some specific embodiments, after the chimeric antigen receptor binds to the anti-Fc antibody, the expression of the NFAT-luciferase reporter gene is activated.

[0011] In some specific embodiments, the Fc region is a human Fc region, preferably a human IgG Fc region.

[0012] In some specific embodiments, the Fc region includes a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:7.

[0013] In some specific embodiments, the CD8α transmembrane region includes a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:8.

[0014] In some specific embodiments, the CD3z intracellular signaling region includes a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:9.

[0015] In some specific embodiments, the chimeric antigen receptor includes a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:6.

[0016] In some specific embodiments, the chimeric antigen receptor is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:5.

[0017] In some specific embodiments, the luciferase comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:3.

[0018] In some specific embodiments, the luciferase-encoding gene comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4.

[0019] In some specific embodiments, the NFAT binding site comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2.

[0020] In some specific embodiments, the promoter comprises a plurality of NFAT binding sites. For example, the number of NFAT binding sites is 6. Preferably, the promoter comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:10.

[0021] In some specific embodiments, the NFAT-luciferase reporter gene comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1.

[0022] In some specific embodiments, the T cell is the acute T cell leukemia cell line Jurkat.

[0023] In some specific embodiments, the T cell is the acute T cell leukemia cell line Jurkat-C8 strain, and the Jurkat-C8 strain is deposited at the China Center for Type Culture Collection with the deposit number CCTCC No: C202519.

[0024] In a second aspect, the present invention provides a kit, which comprises the T cells described in the first aspect of the present invention.

[0025] In some specific embodiments, the kit further comprises a luciferase detection reagent.

[0026] In some specific embodiments, the luciferase detection reagent comprises a cell lysate, a luciferase substrate, and a reaction buffer.

[0027] In some specific embodiments, the kit further comprises a positive control and a negative control. The positive control contains an anti-human Fc antibody, and the negative control does not contain an anti-human Fc antibody.

[0028] In a third aspect, the present invention further provides a method for detecting an anti-Fc antibody, which comprises co-incubating and culturing the T cells described in the first aspect of the present invention with a sample to be tested, and then detecting the luciferase expression level of the T cells, and indicating the presence or absence or the content level of the anti-Fc antibody in the sample through the luciferase expression level.

[0029] In some specific embodiments, the sample is a blood sample, such as a serum sample.

[0030] Advantageous Effects

[0031] The T cells, kit, and detection method of the present invention have at least one of the following advantages:

[0032] (1) The present invention proposes a completely new principle for detecting anti-Fc antibodies. It uses the Fc region of the chimeric antigen receptor to specifically bind to anti-Fc antibodies, converts the detection signal into a T cell activation signal, and transmits it to the NFAT-luciferase reporter gene. By detecting the expression of luciferase, it detects whether there is an anti-Fc antibody or its content level in the sample.

[0033] (2) The present invention also provides a highly sensitive Jurkat-C8 cell line obtained by screening. Compared with other engineered Jurkat cell lines, it shows a higher signal-to-noise ratio in detection, and at the same time, the expression level of the chimeric antigen receptor on its surface is lower, indicating that this cell line has high sensitivity.

[0034] (3) Compared with the ELISA method (fixing the Fc protein on the surface of a solid-phase carrier), the present invention displays the Fc region on the cell surface through the chimeric antigen receptor, eliminating the negative impact of Fc protein fixation on its binding to anti-Fc antibodies.

[0035] (4) The T cells, kit, and detection method of the present invention are sensitive and efficient, and are also applicable to the detection of samples with a high concentration of anti-Fc antibodies. Brief Description of the Drawings

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0037] Figure 1 It is the map of the lentiviral vector of NFAT-luciferase reporter gene (161 lentiviral plasmid);

[0038] Figure 2 It is the map of the lentiviral expression vector of hFc-CAR (358 lentiviral plasmid);

[0039] Figure 3 It is the detection result of the fluorescence signal of Jurkat / 161 monoclonal cells;

[0040] Figure 4 : Verification result of Jurkat / 161 / hFc-CAR cell pool;

[0041] Figure 5 : Results of monoclonal screening - Assay of Jurkat / 161 / hFc-CAR cells;

[0042] Figure 6 : Results of monoclonal screening - FACS of Jurkat / 161 / hFc-CAR cells;

[0043] Figure 7 : Results of the dose-effect curve of the positive protein in Example 5;

[0044] Figure 8 : Results of the dose-effect curve of the positive protein in Example 6. Specific Embodiments

[0045] The following will describe the implementation schemes of the present invention in detail in combination with the embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0046] Example 1. Construction of Lentiviral Vector

[0047] Through molecular biology methods and genetic engineering, construct as Figure 1The lentiviral vector of NFAT (Nuclear Factor of Activated T Cells)-luciferase reporter gene shown is named 161 lentiviral plasmid. The nucleic acid sequence of the NFAT-luciferase reporter gene is as shown in SEQ ID NO:1, which includes a promoter containing 6 NFAT binding sites and a Nano-Luciferase (Nluc) encoding gene. Among them, the nucleic acid sequence of the NFAT binding site is as shown in SEQ ID NO:2. The amino acid sequence of Nano-Luciferase is as shown in SEQ ID NO:3, and the Nano-Luciferase encoding gene is as shown in SEQ ID NO:4.

[0048] By molecular biology methods and genetic engineering, construct the hFc-CAR lentiviral expression vector as Figure 2 shown, named 358 lentiviral plasmid. hFc-CAR from the N-terminus to the C-terminus includes hFc, CD8a transmembrane region, and CD3z intracellular signaling region. Among them, the nucleic acid sequence encoding hFc-CAR is as shown in SEQ ID NO:5, and the amino acid sequence of hFc-CAR is as shown in SEQ ID NO:6, where the amino acid sequences corresponding to hFc, CD8a transmembrane region, and CD3z intracellular signaling region are as shown in SEQ ID NO:7, 8, and 9 respectively.

[0049] Example 2. Lentivirus particle packaging

[0050] Using 293T cells (commercial human kidney epithelial cells) with a three-plasmid lentivirus packaging system, package the 161 and 358 lentiviral plasmids constructed in Example 1 into the corresponding lentiviruses respectively. The specific steps are as follows:

[0051] 1. Seed 293T cells at 1E6 cells / well, 2 ml of DMEM complete medium (DMEM basal medium + 10% FBS + 1% P / S) / well in a 6-well plate, and culture overnight in a 37°C, 5% CO2 incubator for 12 - 22 hours.

[0052] 2. The next day, replace the complete medium in the 6-well plate with lentivirus packaging medium (DMEM basal medium + 5% FBS), 1.5 ml / well, and place it in the incubator to equilibrate for 1 - 2 hours.

[0053] 3. During cell equilibration, use Opti-MEM medium (Gibco) to prepare a suspension of the transfer plasmid (161 lentiviral plasmid or 358 lentiviral plasmid), packaging plasmid (psPAX2), and envelope plasmid (pMD2.G) in a ratio of 5:3:2 according to the three-plasmid packaging system.

[0054] 4. According to the instructions of the Lipo3000 kit, add the corresponding volume of P3000 to the above suspension, and prepare the working solution of Lipo3000 using Opti-MEM Media. After incubating each for 5 minutes, mix them well and incubate at room temperature for 15 minutes.

[0055] 5. After the incubation, add the above mixture dropwise to a 6-well plate. After gently mixing, continue to place the 6-well plate in a 37°C, 5% CO2 cell culture incubator for 5 - 6 hours.

[0056] 6. After the incubation, aspirate all the medium in the 6-well plate and replace it with 3 mL / well of fresh lentivirus packaging medium (DMEM basal medium + 10% FBS + 10 mM sodium pyruvate + 10 mM HEPES).

[0057] 7. Place the 6-well plate in a 37°C, 5% CO2 cell culture incubator for 72 hours. After the culture, collect the lentivirus supernatant, centrifuge at 2000 rpm for 5 minutes to remove cell debris.

[0058] 8. Aliquot the centrifuged virus supernatant into 1.5 ml centrifuge tubes and store them in an -80°C refrigerator.

[0059] Example 3. Construction of Jurkat / 161 cells

[0060] Use 161 lentivirus to infect commercial Jurkat cells to construct a Jurkat / 161 cell pool and select monoclonal cells. Specifically, the steps are as follows:

[0061] 1. Seed Jurkat cells at 1E5 cells / mL, 0.5 mL in a 24-well plate, and add 500 μL of the 161 lentivirus suspension packaged in Example 2. Centrifuge at 2000 rpm for 40 minutes. After centrifugation, place the 24-well plate in a 37°C, 5% CO2 cell culture incubator for 48 hours.

[0062] 2. After the culture, transfer the cells in the 24-well plate to a 6-well plate and add Puromycin at a final concentration of 1 μg / mL, and continue to culture for 3 - 5 days to obtain the Jurkat / 161 cell pool.

[0063] 3. Plate the Jurkat / 161 cell pool for monoclonal cells in a 96-well plate using the limiting dilution method. Continuously culture the 96-well plate in a cell culture incubator for 14 - 21 days.

[0064] 4. Monoclonal selection: Seed 1E4 cells / well of monoclonal cells or cell pool cells in a 96-well plate, and mix them with complete RPMI 1640 medium or complete RPMI 1640 medium supplemented with OKT-3 (final concentration of OKT-3: 100 ng / mL) respectively, and co-incubate for 24 hours. Centrifuge the 96-well plate at 2000 rpm for 3 minutes. Then, take 25 μL of cell supernatant into a 96-well detection plate, add 25 μL of luciferase reaction substrate (Promega, N1150), and after incubating for 5 minutes, detect its fluorescence value. Among them, the fluorescence value of the cell supernatant stimulated by OKT3 is used as the signal value (Signal, S), while the fluorescence signal value of the cell supernatant incubated only with complete RPMI 1640 medium without OKT3 stimulation is the background value (Background, B). Make a bar graph of Signal / Background, and preferentially select monoclonal cells with a Signal / Background higher than that of the cell pool. Among a total of 44 monoclonal cells, 9 monoclonal cells showed a Signal / Background higher than that of the cell pool; some detection results are shown as Figure 3 below. Multiple single-cell clones showed a significantly higher Signal / Background than that of the cell pool, such as clones 1, 3, 5, 8, 10, and 13; except for Figure 3 the clones 1-20 shown as Figure 3 below, the Signal / Background of the remaining monoclonal cells was lower than that of the cell pool and was not shown in

[0065] Example 4. Construction of Jurkat / 161 / hFc-CAR functional cell line

[0066] Use lentivirus 358 to infect Jurkat / 161 monoclonal cells, and verify whether the cell line is successfully constructed and select monoclonal cells. The specific steps are as follows:

[0067] 1. Seed the Jurkat / 161 clone 10 screened in Example 3 at 1E5 cells / mL, 0.5 mL in a 24-well plate, add 500 μL of the lentivirus 358 suspension packaged in Example 2 thereto, and centrifuge at 2000 rpm for 40 minutes. After centrifugation, place the 24-well plate in a 37 °C, 5% CO2 cell culture incubator and culture for 48 hours.

[0068] 2. After the culture is completed, transfer the cells in the 24-well plate to a 6-well plate, and add Blasticidin with a final concentration of 10 μg / mL, and continuously culture for 7 days to obtain a Jurkat / 161 / hFc-CAR cell pool.

[0069] 3. Seed the Jurkat / 161 / hFc-CAR cell pool cells at a density of 1E5 cells / mL, 0.1 mL / well into a 96-well plate, and add 0.1 mL / well of goat anti-human IgG Fc antibody (abcam #ab97221) at gradient concentrations in complete 1640 medium or an equal volume of complete 1640 medium. Incubate for 24 hours. Centrifuge the 96-well plate at 2000 rpm for 3 minutes. Then, take 25 μL of cell supernatant into a 96-well detection plate, add 25 μL of luciferase reaction substrate (Promega, N1150), and after incubating for 5 minutes, measure the fluorescence value. Among them, the fluorescence value of the cell supernatant stimulated by goat anti-human IgG Fc antibody is used as the signal value (S), while the fluorescence signal value of the cell supernatant incubated only with complete 1640 medium without stimulation is the background value (B). The signal-to-background ratio (S / B) is used to evaluate whether the cell pool is successfully constructed. The results are as Figure 4 shown. The Jurkat / 161 / hFc-CAR cell pool can detect high concentrations of anti-human Fc antibody, and the cell pool is successfully constructed.

[0070] 4. Select Jurkat / 161 / hFc-CAR monoclonal cells. The specific steps are as follows:

[0071] 1) Seed the above cell pool cells into a 96-well plate for monoclonal culture using the limiting dilution method. Continuously culture the 96-well plate in a cell culture incubator for 14 - 21 days.

[0072] 2) Seed the positive monoclonal cells into 96-well plates respectively, and add an equal volume of 0.5 μg / mL goat anti-human IgG Fc antibody (abcam #ab97221) or an equal volume of complete 1640 medium (1640 basal medium + 10% FBS + 1% P / S). Incubate for 24 hours.

[0073] 3) After incubation, take the cell supernatant and measure its fluorescence signal value to reflect the response window of each monoclonal cell (the detection method and data processing method refer to the fluorescence signal detection of the Jurkat / 161 / hFc-CAR cell pool in point 3 of this example). The results are as Figure 5 shown. Among them, clone 8 (C8) shows a significantly higher signal-to-noise ratio than other monoclonal cells.

[0074] 4) Take another aliquot of positive monoclonal cells and perform flow cytometry to detect the expression level of human IgG Fc-CAR on the cell membrane using a PE-labeled anti-human IgG Fc recombinant antibody (Biolegend # 366904), and calculate the corresponding expression fold. The results are as Figure 6 shown. The expression of hFc-CAR on the surface of clone 8 is significantly lower than that of other monoclonal cells. Combining Figure 5It can be seen that the detection of 8 pairs of anti-hFc antibodies by cloning has high sensitivity and is suitable for constructing detection assays.

[0075] 5) Combining the results of monoclonal cell Assasy and flow cytometry, clone 8 (C8) was selected as the established clone of Jurkat / 161 / hFC-CAR for subsequent detection use.

[0076] The C8 cell line was sent to the China Center for Type Culture Collection for the preservation of biological materials for patent procedures. The preservation information is as follows: preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the postal code is 430072, the classification name is acute T cell leukemia cell line Jurkat-C8, the preservation date is January 23, 2025, and the preservation number is CCTCC NO: C202519.

[0077] Example 5. Detection of negative serum samples using the Jurkat / 161 / hFc-CAR cell line (C8)

[0078] Nineteen normal monkey serum samples were detected using the Jurkat / 161 / hFc-CAR cell line (C8). The detection results showed that this detection system can be used for the detection of monkey serum, and the detection threshold of this system was defined as the S / B value Mean±3SD, that is, the S / B value of the sample group exceeding 3.2 was defined as positive for anti-Fc antibody.

[0079] 1. Dilute the positive control (goat anti-human IgGFc antibody (abcam #ab97221)) according to the conditions of Top Conc.= 5 μg / ml, DF=5, 8 doses with 1640 complete medium to prepare the positive control working solution.

[0080] 2. Dilute the monkey negative serum sample with 1640 complete medium to a final concentration of 3% or 1% to prepare the monkey negative serum sample working solution.

[0081] 3. Adjust the C8 cell line to a density of 1E5 cells / mL with 1640 complete medium. Use a multi-channel pipette to transfer 100 μL / well of the cell suspension to a 96-well flat bottom plate.

[0082] 4. Transfer 100 μL / well of the positive control working solution, monkey negative serum sample working solution, and 1640 complete medium to the corresponding wells of the 96-well plate as the positive control well, sample well, and blank well, respectively.

[0083] 5. Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and incubate for 24 hours. After incubation, centrifuge the 96-well plate at 2000 rpm for 3 minutes. After centrifugation, transfer 25 μl / well of the cell supernatant to a 96-well detection plate.

[0084] 6. Take out the detection reagent (Promega, N1150) in advance and let it return to room temperature. Prepare the working solution of the detection reagent freshly according to the kit instructions.

[0085] 7. Transfer 25 μl / well of the working solution of the detection reagent to the corresponding wells of the 96-well detection plate and gently shake. Incubate at room temperature in the dark for 5 minutes and perform chemiluminescence detection on the detection instrument.

[0086] Export the original data and process the data using Office Excel. Among them, the fluorescence values of the positive control wells and the sample wells are used as the signal values (Signal, S), the fluorescence value of the blank well is used as the background value (Background, B), and the signal-to-noise ratio of the positive control well or the sample well is S / B.

[0087] The detection results of the positive control wells are as Figure 7 shown. According to Figure 7 the detection shown, the present invention can be used for the detection of high-concentration anti-Fc antibodies. The detection results of the sample wells (monkey negative serum samples) are shown in Table 1. The results show that no false positives occurred in the negative serum samples using the method of the present invention at either 3% or 1% concentration, and the present invention can be used for the detection of serum samples at relatively high concentrations.

[0088] Table 1

[0089]

[0090] Example 6. Detect whether the serum sample contains ADA

[0091] Use the detection system of the present invention to detect whether multiple monkey serum samples contain anti-Fc antibodies, and compare the detection results with those of other companies. The detection results show that the detection system of the present invention can accurately detect serum samples positive for anti-Fc antibodies.

[0092] 1. Dilute the positive control (goat anti-human IgGFc antibody (abcam #ab97221)) according to the Top Conc. = 5 μg / ml, DF = 5, 8-dose conditions with 1640 complete medium to prepare the working solution of the positive control.

[0093] 2. Dilute the monkey serum sample to be tested with 1640 complete medium to a final concentration of 3% to prepare the working solution of the monkey serum sample to be tested.

[0094] 3. Dilute the negative monkey serum with complete RPMI 1640 medium to a final concentration of 3% to prepare the working solution of the negative monkey serum sample.

[0095] 4. Adjust the density of the C8 cell line to 1E5 cells / mL using complete RPMI 1640 medium. Transfer 100 μL / well of the cell suspension to a 96-well flat-bottom plate using a multi-channel pipette.

[0096] 5. Transfer 100 μL / well of the positive control product working solution, the monkey serum sample working solution to be tested, the negative monkey serum sample working solution, and complete RPMI 1640 medium to the corresponding wells of the 96-well plate, serving as the positive control well, sample well, negative control well, and blank well, respectively.

[0097] 5. Place the 96-well plate in a 37 °C, 5% CO2 cell incubator and incubate for 24 hours. After incubation, centrifuge the 96-well plate at 2000 rpm for 3 minutes. After centrifugation, transfer 25 μl / well of the cell supernatant to a 96-well detection plate.

[0098] 6. Take out the detection reagent in advance and let it return to room temperature. Prepare the working solution of the detection reagent as needed according to the kit instructions.

[0099] 7. Transfer 25 μl / well of the detection reagent working solution to the corresponding wells of the 96-well detection plate and gently shake. Incubate at room temperature in the dark for 5 minutes and perform chemiluminescence detection on the detection instrument. Export the original data and process the data using Office Excel. Among them, the fluorescence values of the positive control well, sample well, and negative control well are used as the signal value (Signal, S), the fluorescence value of the blank well is used as the background value (Background, B), and the signal-to-noise ratio of the positive control well, sample well, or negative control well is S / B. According to the standard (Mean ± 3SD) set in Example 5, calculate the threshold based on the S / B of the negative control well (the detection results of the negative control well are shown in Table 2). If the S / B value of the positive control well or sample well > the threshold, it is judged as positive.

[0100] Table 2

[0101]

[0102] The detection results of the positive control well are as Figure 8As shown, it indicates that the method of the present invention can be used to detect high-concentration anti-Fc antibodies. The detection results of the sample wells (monkey serum samples) are shown in Table 3. In Table 3, other companies used the ELISA method, and the judgment criterion was: the average OD value of the sample to be tested > 2NC (negative control) was considered ADA positive. The 2NC value in this test was 0.1487. The results in Table 3 show that the detection results of the present invention are consistent with those obtained by other companies using ELISA detection.

[0103] Table 3

[0104]

[0105] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

[0106] Sequence Listing:

[0107] NFAT-luciferase reporter gene (SEQ ID NO:1):

[0108]

[0109] NFAT binding site (SEQ ID NO:2):

[0110] GGAGGAAAAACTGTTTCATACAGAAGGCGT。

[0111] Amino acid sequence of small luciferase (SEQ ID NO:3):

[0112] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA。

[0113] Coding gene of small luciferase (SEQ ID NO:4):

[0114] GTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG。

[0115] Nucleic acid sequence of hFc-CAR (SEQ ID NO:5):

[0116]

[0117] hFc-CAR amino acid sequence (SEQ ID NO:6):

[0118] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0119] hFc amino acid sequence (SEQ ID NO:7):

[0120] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0121] CD8α transmembrane region amino acid sequence (SEQ ID NO:8):

[0122] ETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL。

[0123] CD3z intracellular signaling region amino acid sequence (SEQ ID NO:9):

[0124] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0125] 6x NFAT binding site (SEQ ID NO: 10):

[0126] GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGCGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTC。

Claims

1. A T cell, which is the acute T cell leukemia cell line Jurkat-C8 strain, and the Jurkat-C8 strain is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC No: C202519.

2. A kit, wherein the kit comprises the T cell according to claim 1 and a luciferase detection reagent.

3. The kit according to claim 2, wherein the luciferase detection reagent comprises a cell lysate, a luciferase substrate and a reaction buffer.

4. The kit according to claim 2 or 3, wherein the kit further comprises a positive control and a negative control, the positive control contains an anti-human IgG1 Fc antibody, and the negative control does not contain an anti-human IgG1 Fc antibody.

5. The kit according to claim 4, which is used for detecting a serum sample from an organism.

6. A detection method for an anti-human IgG1 Fc antibody for non-diagnostic purposes, wherein the method comprises co-incubating and culturing the T cell according to claim 1 with a sample to be tested, and then detecting the luciferase expression level of the T cell, and indicating the presence or absence or the content level of the anti-human IgG1 Fc antibody in the sample through the luciferase expression level; calculating a threshold according to the signal-to-noise ratio of the negative control well, and if the signal-to-noise ratio value of the sample well is greater than the threshold, it is determined that the sample to be tested is positive for the anti-human IgG Fc antibody.

Images