T cell for detecting anti-Fc antibody and kit and detection method thereof

Abstract

The invention relates to a T cell for detecting an anti-Fc antibody as well as a kit and a detection method thereof. The T cell expresses the chimeric antigen receptor, and the chimeric antigen receptor comprises an Fc region, a CD8 alpha transmembrane region and a CD3z intracellular signal transduction region; the T cell further comprises an NFAT-luciferase reporter gene, the NFAT-luciferase reporter gene comprises a luciferase coding gene and a promoter for regulating and controlling the coding gene, and the promoter comprises an NFAT binding site. According to the present invention, the brand new anti-Fc antibody detection principle is provided, the Fc region of the chimeric antigen receptor is adopted, the anti-Fc antibody is specifically combined, the detection signal is converted into the T cell activation signal, the T cell activation signal is transmitted to the NFAT-luciferase reporter gene, and the anti-Fc antibody is detected by detecting the luciferase expression so as to provide characteristics of sensitivity, high efficiency and the like.

Description

Technical Field

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

[0002] In the process of drug development, the study of immunogenicity has become an essential and key part. For most drugs, especially antibody drugs, the production of immunogenicity is mainly mediated by humoral immunity, so anti-drug antibodies (ADA) have always been the main criterion for defining the immunogenicity of such drugs; among them, anti-Fc antibodies targeting the Fc region of antibody drugs belong to a common type of anti-drug antibodies. On the one hand, the presence of anti-drug antibodies may neutralize the biological activity of the drug and significantly reduce its efficacy. On the other hand, it may cross-react with endogenous proteins, leading to new immune responses, which are seriously life-threatening.

[0003] At present, the commonly used ADA detection methods on the market are the direct method and the bridge method developed based on the 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 it is species-specific. Although the bridge method is not species-specific, it is easily interfered by drug concentration and soluble targets, and cannot detect low-affinity ADA. Therefore, it is particularly important to develop reliable and effective detection methods for 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 test results are not accurate enough. Moreover, this detection method is usually not suitable for samples with ADA concentrations higher than 1μg / ml, and the detection range is limited. In order to better evaluate the immunogenicity of drugs, it is urgent to develop more accurate and simple analysis methods. Summary of the invention

[0005] The present invention provides a T cell, a corresponding kit and a detection method thereof, aiming to provide a new anti-Fc antibody detection method based on cell signaling, which has the advantages of being sensitive, efficient and suitable for detecting high-concentration anti-Fc antibody samples.

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

[0007] In a first aspect, the present invention provides a T cell, wherein the T cell expresses a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an Fc region, a CD8α transmembrane region, and a CD3z intracellular signaling region;

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

[0009] The T cells of the present invention utilize the Fc region to bind to the anti-Fc antibody and activate the chimeric antigen receptor, converting the signal of the binding of Fc to the anti-Fc antibody into a T cell activation signal, and the T cell activation signal stimulates NFAT to bind to the promoter of the NFAT-luciferase reporter gene to start the expression of the luciferase gene. At the cellular level, the present invention cleverly converts the detection of anti-Fc antibodies into the detection of luciferase activity through the chimeric antigen receptor and the NFAT-luciferase reporter gene, and constructs an efficient and sensitive analysis system for detecting anti-Fc antibodies.

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

[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 comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7.

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

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

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

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

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

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

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

[0020] In some specific embodiments, the promoter comprises a plurality of NFAT binding sites, for example, the number of the 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 an 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 collection number CCTCC No: C202519.

[0024] In a second aspect, the present invention provides a kit, comprising the T cell according to 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, wherein 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 an anti-Fc antibody detection method, 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 of anti-Fc antibodies in the sample or the level of their content by the luciferase expression level.

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

[0030] Beneficial Effects

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

[0032] (1) The present invention proposes a new anti-Fc antibody detection principle, which utilizes the Fc region of the chimeric antigen receptor to specifically bind to the anti-Fc antibody, 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, the presence of anti-Fc antibodies or their content level in the sample is detected.

[0033] (2) The present invention also provides a highly sensitive Jurkat-C8 cell line obtained by screening. Compared with other engineered Jurkat cell lines, the Jurkat-C8 cell line showed a higher signal-to-noise ratio in the detection and a lower expression level of the chimeric antigen receptor on its surface, indicating that the 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 a 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 described in the present invention are sensitive and efficient, and are also suitable for the detection of high-concentration anti-Fc antibody samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

[0040] Figure 4 :Jurkat / 161 / hFc-CAR cell pool validation results;

[0041] Figure 5 :Jurkat / 161 / hFc-CAR cell monoclonal screening-Assay results;

[0042] Figure 6 :Jurkat / 161 / hFc-CAR cell monoclonal screening-FACS results;

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

[0044] Figure 8 : The dose-effect curve results of the positive protein in Example 6. DETAILED DESCRIPTION

[0045] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0046] Example 1. Construction of lentiviral vector

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

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

[0049] Example 2. Lentiviral particle packaging

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

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

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

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

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

[0055] 5. After incubation, add the above mixture dropwise into the 6-well plate, mix gently, and 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, discard all the culture 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 and culture for 72 hours. After the culture, collect the lentiviral supernatant and centrifuge at 2000 rpm for 5 minutes to remove cell debris.

[0058] 8. After centrifugation, the virus supernatant was divided into 1.5 ml centrifuge tubes and stored in a -80°C refrigerator.

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

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

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

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

[0063] 3. Use the limiting dilution method to plate the Jurkat / 161 cell pool into a 96-well plate for single cloning. The 96-well plate is cultured continuously in a cell culture incubator for 14 to 21 days.

[0064] 4. Selection of monoclonal clones: 1E4 cells / well of monoclonal cells or cell pool cells were plated in a 96-well plate, mixed with 1640 complete medium or 1640 complete medium with OKT-3 (final concentration of OKT-3: 100 ng / mL), and incubated for 24 hours. The 96-well plate was centrifuged at 2000rpm for 3 minutes. Then 25μL of cell supernatant was taken to a 96-well detection plate, 25μL of luciferase reaction substrate (Promega, N1150) was added, and after incubation for 5 minutes, the fluorescence value was detected. Among them, the fluorescence value of the cell supernatant stimulated by OKT3 was used as the signal value (Signal, S), and the fluorescence signal value of the cell supernatant incubated only by 1640 complete medium without OKT3 stimulation was the background value (Background, B). The signal value / background value (Signal / Background) was used as a bar graph to preferentially select monoclonal cells that were higher than the cell pool (pool). Among the total 44 monoclones, 9 monoclones showed signal values / background values ​​higher than the cell pool; some test results are as follows Figure 3 As shown, several single cell clones showed significantly higher signal values / background values ​​than the cell pool, such as clones 1, 3, 5, 8, 10 and 13; Figure 3 Except for clones 1-20 shown, the signal / background values ​​of the remaining clones were lower than those of the cell pool and were not included in the Figure 3 Shown in.

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

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

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

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

[0069] 3. The Jurkat / 161 / hFc-CAR cell pool cells were plated at 1E5 cells / mL, 0.1mL / well in a 96-well plate, and goat anti-human IgG Fc antibody (abcam #ab97221) with gradient concentrations of 1640 complete medium or an equal volume of 1640 complete medium was added at 0.1mL / well and incubated for 24 hours. The 96-well plate was centrifuged at 2000rpm for 3 minutes. Then 25μL of cell supernatant was taken to a 96-well detection plate, 25μL of luciferase reaction substrate (Promega, N1150) was added, and the fluorescence value was detected after incubation for 5 minutes. Among them, the fluorescence value of the cell supernatant stimulated by goat anti-human IgG Fc antibody was used as the signal value (S), and the fluorescence signal value of the cell supernatant incubated only by 1640 complete medium and without stimulation was the background value (B). The signal group / background value (S / B) was used as the signal-to-noise ratio to evaluate whether the cell pool was successfully constructed. The results are as follows Figure 4 As shown, the Jurkat / 161 / hFc-CAR cell pool was able to detect high concentrations of anti-human Fc antibodies, and the cell pool was successfully constructed.

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

[0071] 1) The cells in the cell pool were plated in a 96-well plate by limiting dilution method for single clones. The 96-well plate was cultured in a cell culture incubator for 14 to 21 days.

[0072] 2) The positive monoclonal cells were plated into 96-well plates, and an equal volume of 0.5 μg / mL goat anti-human IgGFc antibody (abcam #ab97221) or an equal volume of 1640 complete medium (1640 basal medium + 10% FBS + 1% P / S) was added and incubated for 24 hours.

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

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

[0075] 5) Based on the results of monoclonal cell Assasy and flow cytometry, clone 8 (C8) was selected as the fixed clone of Jurkat / 161 / hFC-CAR for subsequent testing.

[0076] The C8 cell line was sent to the China Center for Type Culture Collection for biological material preservation for patent procedures. The preservation information is as follows: Deposited in the China Center for Type Culture Collection, the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072, classified and named as 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 Jurkat / 161 / hFc-CAR cell line (C8)

[0078] Jurkat / 161 / hFc-CAR cell line (C8) was used to detect 19 normal monkey serum samples. The test results showed that the detection system can be used for the detection of monkey serum, and the detection threshold of the system is defined as S / B value Mean±3SD, that is, the sample group S / B value exceeding 3.2 is defined as anti-Fc antibody positive.

[0079] 1. Dilute the positive control (goat anti-human IgGFc antibody (abcam #ab97221) with 1640 complete medium according to Top Conc. = 5 μg / ml, DF = 5, 8 dose conditions as 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% as the monkey negative serum sample working solution.

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

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

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

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

[0086] The raw data were exported and processed using Office Excel. The fluorescence values ​​of the positive control wells and sample wells were taken as the signal values ​​(Signal, S), the fluorescence values ​​of the blank wells were taken as the background values ​​(Background, B), and the signal-to-noise ratio of the positive control wells or sample wells was S / B.

[0087] The test results of the positive control wells are as follows Figure 7 As shown. Figure 7 As shown in the test, the present invention can be used for the detection of high concentration anti-Fc antibodies. The test results of the sample well (monkey negative serum sample) are shown in Table 1. The results show that no false positives occur in the negative serum sample 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 with higher concentrations.

[0088] Table 1

[0089]

[0090] Example 6. Detection of whether serum samples contain ADA

[0091] The detection system of the present invention was used to detect whether multiple monkey serum samples contained anti-Fc antibodies, and the detection results were compared with the detection results of other companies. The detection results showed that the detection system of the present invention can accurately detect serum samples positive for anti-Fc antibodies.

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

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

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

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

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

[0097] 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 cell supernatant to the 96-well assay plate.

[0098] 6. Take out the test reagents in advance and restore them to room temperature. Prepare the test reagent working solution according to the kit instructions.

[0099] 7. Transfer 25 μl / well detection reagent working solution to the corresponding wells of the 96-well detection plate and shake gently. Incubate at room temperature in the dark for 5 minutes, and detect chemiluminescence (Luminescence) on the detection instrument. Export the raw data and use Office Excel to process the data. Among them, the fluorescence value of the positive control well, the sample well, and the negative control well is 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 or the sample well or the negative control well is S / B. According to the standard set in Example 5 (Mean ± 3SD), the threshold is calculated according to the S / B of the negative control well (the detection results of the negative control well are shown in Table 2). The S / B value of the positive control well or the sample well> the threshold value is judged as positive.

[0100] Table 2

[0101]

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

[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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, 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] Amino acid sequence of hFc-CAR (SEQ ID NO:6):

[0118] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

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

[0120] EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0121] Amino acid sequence of the transmembrane region of CD8α (SEQ ID NO:8):

[0122] ETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL。

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

[0124] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

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

[0126] GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGCGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTC。

Claims

1. A T cell, which is an acute T-cell leukemia cell line Jurkat-C8 strain, wherein the Jurkat-C8 strain is deposited in China Center for Type Culture Collection with a 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-Fc antibody, and the negative control does not contain an anti-Fc antibody. The kit according to claim 4 , which is used for detecting a serum sample from an organism.

6. A method for detecting anti-Fc antibodies, wherein the method comprises co-incubating the T cells of claim 1 with a serum sample to be tested, and then detecting the luciferase expression level of the T cells, and the luciferase expression level indicates the presence or absence of anti-Fc antibodies in the sample or the level of their content.

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