Citrullinated autoantigen specific cellular immune function detection kit, detection method and application

Abstract

The invention provides a citrullinated autoantigen specific cell immune function detection kit, a detection method and application, and relates to the technical field of medical biology, in the embodiment of the invention, a specific citrullinated polypeptide pool is used for evaluating and monitoring specific T cell response of RA patients. In the embodiment of the invention, not only can quantitative data about specific Th1 and Th2 cellular immune responses be provided, but also more accurate evaluation can be realized in ACPA or RF negative patients. Therefore, in the embodiment of the invention, the immune function state of the specific cells of the RA patient can be accurately monitored and evaluated, especially the specific T cell response aiming at the citrullinated antigen, and the kit has important significance for realizing an individualized treatment scheme.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of pharmaceutical biotechnology, and in particular to a citrullinated autoantigen-specific cellular immune function detection kit, a detection method and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Current treatments rely on global immunosuppression, which is often not targeted and may lead to unwanted side effects.

[0003] In the related art, serological tests (such as the determination of anti-cyclic citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF)) are generally used to screen and assist in the early diagnosis of RA.

[0004] However, this method has a certain rate of missed diagnosis, especially in RA patients with negative ACPA or RF, and cannot comprehensively evaluate the disease activity and progression of RA patients, especially the dynamic changes at the level of specific cellular immune function, which limits the in-depth understanding and effective monitoring of the disease status. Summary of the invention

[0005] The embodiments of the present invention provide a citrullinated autoantigen-specific cellular immune function detection kit, detection method and application, so as to at least partially solve the problems existing in the related art.

[0006] A first aspect of an embodiment of the present invention provides a citrullinated autoantigen-specific cellular immune function detection kit, the detection kit comprising: at least one citrullinated antigen T cell epitope polypeptide pool for patients with positive anti-cyclic citrullinated peptide antibodies and at least one citrullinated T cell epitope polypeptide pool for patients with negative anti-cyclic citrullinated peptide antibodies.

[0007] Optionally, the citrullinated antigen T cell epitope polypeptide pool for patients positive for anti-cyclic citrullinated peptide antibodies includes one or more of the following citrullinated T cell epitope polypeptides: TSVTPVLRGQPIYIQ, TMQSLNDRLASYLDR, STFSTNYRSLGSVQA, SRTTGRQRTSHSEST, SRTTGRQRSSHSESS, SESIVPERHGTTHGQ, SESIVHERHGTIHGQ, SESIVDERHGTTHGQ, SESIIHDRHRITHGQ, QHKFQERREVELRAK, PKSGSTVRRRQGTTH, PELLLGARHYTKAID, NSTQSRIREQKLGSS, NGQLLFDRPMHVKMD, ML DRDHDRRLDFTEF, LSGLGGPRKSGPRGA, KSHISPSRESGEEYE, GSRISVSRSTSFRGG, GQTINTTRHSQSGQG, GHTYGQARSQHGESG, FKIIEDLRAQIFANT, ETTLTELRRTVQSLE, EQYINNLRRQLDSIV, E NDIHGLRKVIDDTN, EIPVVAIRTAKGEKF, EIDLDSMRNLKASLE, EEEMKDLRNVSTGDV, EAEIATYRRLLEDGE, CSQSYSQRGYGAREN, ASYLDRVRSLETENR, ANTVDNARIVLQIDN, AKIMADIRAQYDELA.

[0008] Optionally, the pool of citrullinated antigen T cell epitope polypeptides for anti-cyclic citrullinated peptide antibody-negative patients includes one or more of the following citrullinated T cell epitope polypeptides: ALKFLYKRGANVNLR, EIQPSTFRCVYVRSA, ENYIGDLRRQVDLLS, IEEMPSERLCRSTRQ, KTILDDLRAEDHFSV, LLIYKQGRKLFPIKL, MFLYEYARRHPDYSV, PGQTVKFRVVSMDEN, QDVVNAVRQLTLLGG, RMTLDDFRIKFEMEQ, SISDAEQRGENALKD, and VSLALTQRGVKVFAV.

[0009] Optionally, the citrullinated antigen T cell epitope polypeptide pool includes: at least one acidic polypeptide, at least one basic polypeptide and at least one neutral polypeptide.

[0010] Optionally, the detection kit further comprises: an ELISPOT microplate, an IFN-γ capture antibody, an IL4 capture antibody, biotinylated IFN-γ and IL4 detection antibodies, a buffer for incubation and culture, a lymphocyte separation solution and a 1640 culture medium.

[0011] Optionally, the detection kit further comprises: HRP-streptavidin and AEC display agent.

[0012] The second aspect of the embodiment of the present invention provides a detection method based on the kit described in the first aspect, the method comprising:

[0013] Peripheral blood was collected from whole blood samples, and PBMCs were isolated based on density gradient centrifugation;

[0014] Wash PBMCs twice with PBS;

[0015] Resuspend PBMC cells in culture medium, count and adjust cell concentration for later use;

[0016] Place the PBMCs with adjusted concentration into the wells of the ELISPOT microplate;

[0017] Add citrullinated antigen T cell epitope peptide pool molecules and incubate at room temperature. During the incubation process, specific T cells recognize and bind to the epitope peptide-HLA complex, activate T cells and secrete specific cytokines IFN-γ or IL-4;

[0018] Based on ELISPOT technology, detect specific cytokines secreted by cells;

[0019] An automated reader is used to count the positive spots secreting cytokines IFN-γ or IL-4 in the microplate; the number of spots represents the frequency of activated positive specific T cells, and the size of the spots represents the level of specific cytokines secreted by T cells.

[0020] Optionally, the method further comprises:

[0021] According to the detected cytokine levels, the patient's specific cellular immune function status was determined by comparison with the control group data. Among them, high expression of IFN-γ indicates Th1 response, and high expression of IL-4 indicates Th2 response;

[0022] The control group data are obtained by detecting cytokines in unstimulated PBMC or PBMC stimulated with other non-specific peptides.

[0023] The third aspect of the embodiments of the present invention provides the use of the citrullinated autoantigen-specific cellular immune function detection kit described in the first aspect, which is used in preparing a rheumatoid arthritis diagnosis kit.

[0024] The fourth aspect of the embodiments of the present invention provides the use of the citrullinated autoantigen-specific cellular immune function detection kit described in the first aspect, which is used to prepare a rheumatoid arthritis treatment response detection kit.

[0025] In the embodiment of the present invention, a specific citrullinated polypeptide pool is used to evaluate and monitor the specific T cell response of RA patients. In the embodiment of the present invention, not only can quantitative data on specific Th1 and Th2 cellular immune responses be provided, but more accurate evaluation can also be achieved in patients with negative ACPA or RF. Therefore, in the embodiment of the present invention, the specific cellular immune function status of RA patients, especially the specific T cell response to citrullinated antigens, can be accurately monitored and evaluated, which is of great significance for realizing individualized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 It is a schematic diagram of the screening process of citrullinated antigen T cell epitope polypeptides in the citrullinated autoantigen-specific cellular immune function detection kit provided in an embodiment of the present invention;

[0028] Figure 2A Schematic diagram of predicting HLA-II molecule restricted T cell epitopes based on citrullinated short peptides (n=53) reported in literature provided in an embodiment of the present invention;

[0029] Figure 2B Schematic diagram of predicting T cell epitopes based on mass spectrometry identification of citrullinated short peptides (n=41) in patients with anti-cyclic citrullinated peptide antibodies positive RA_ACPA(+) in the embodiment of the present invention;

[0030] Figure 2C Schematic diagram of predicting T cell epitopes based on mass spectrometry identification of citrullinated short peptides (n=43) from RA_ACPA(-) patients in an embodiment of the present invention;

[0031] Figure 3 It is a schematic diagram of the operation flow of the detection method of the citrullinated autoantigen-specific cellular immune function detection kit provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] The citrullinated autoantigen-specific cellular immune function detection kit provided in the embodiments of the present invention aims to solve the current problem of insufficient monitoring of cellular immune function and disease status assessment in rheumatoid arthritis, as follows:

[0034] Missed detection problem: In related technologies, serological markers often lack effective evaluation indicators in RA patients with negative ACPA or RF, resulting in a high missed detection rate.

[0035] Insufficient assessment of disease activity: Relevant technologies are unable to comprehensively assess the disease activity and progression of RA patients, especially the dynamic changes at the level of specific cellular immune function, which limits the in-depth understanding and effective monitoring of the disease status.

[0036] Lack of detection of specific T cell responses: There is a lack of effective methods in the relevant technology to monitor specific T cell responses in RA patients.

[0037] In view of these problems, the present invention provides a new technology based on enzyme-linked immunosorbent spot (ELISpot) test. In the present invention, a specific citrullinated peptide pool is used to evaluate and monitor the specific T cell response of RA patients. In the present invention, not only can quantitative data on specific Th1 and Th2 cell immune responses be provided, but also more accurate evaluation can be achieved in patients with negative ACPA or RF.

[0038] Therefore, in the embodiments of the present invention, the specific cellular immune function status of RA patients, especially the specific T cell response to citrullinated antigens, can be accurately monitored and evaluated, which is of great significance for realizing individualized treatment plans.

[0039] Specifically, the citrullinated autoantigen-specific cellular immune function detection kit provided in the embodiment of the present invention comprises: at least one citrullinated antigen T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody-positive patients and at least one citrullinated T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody-negative patients.

[0040] In the embodiment of the present invention, the following steps can be specifically adopted: screening and identifying T cell epitope peptides of citrullinated antigens closely related to RA:

[0041] S1, mass spectrometry technology is used to separate proteins and peptides from serum and synovial tissue samples of RA patients; combined with immunoprecipitation, it ensures that the screened peptides are immunogenic and related to the pathological process of RA.

[0042] S2, bioinformatics tools were used to analyze the sequences of these peptides to identify citrullinated antigens with potential T cell epitopes. The epitope peptides finally identified showed a high correlation with known RA autoimmune responses.

[0043] In the embodiment of the present invention, any currently mature bioinformatics prediction tool, such as NetMHCIIpan, can be used to analyze the HLA-II molecule restriction of the screened polypeptides. In this step, it is ensured that the selected epitope peptides can present immune responses in different patient groups.

[0044] In the embodiments of the present invention, a wide range of HLA typing databases can also be integrated to ensure that the predicted T cell epitope peptides can cover a variety of population genetic backgrounds, thereby enhancing the universality and effectiveness of the application of the kit.

[0045] Specifically, if Figure 1 As shown, it shows a schematic diagram of the screening process of citrullinated antigen T cell epitope polypeptides in the citrullinated autoantigen-specific cellular immune function detection kit provided in an embodiment of the present invention. Specifically, in an embodiment of the present invention, chemical methods and immunoprecipitation techniques can be used to separate circulating immune complexes (CICs), synovial tissue antigens, and synovial deposited immune complex ICs in serum. Mass spectrometry is then used to identify the citrulline modification levels in serum CICs, synovial tissue antigens, and synovial deposited immune complex antigens, and RA-specific citrullinated modified peptides and proteins are identified based on database and bioinformatics analysis.

[0046] In the embodiments of the present invention, a variety of prediction tools can be used to virtually predict the spectrum of citrullinated T cell epitopes presented by dominant HLA-DR molecules for RA-specific citrullinated autoantigens.

[0047] Furthermore, the patient's PBMCs were co-cultured with each candidate epitope peptide in the T cell epitope spectrum, and the immunogenicity of the candidate epitope peptides was verified by ELISPOT experiments, and the relatively specific T cell epitope peptides for RA were identified.

[0048] In the embodiment of the present invention, the T cell epitope peptides of the citrullinated antigen closely related to RA determined in the above steps are used as the polypeptide pool of the citrullinated autoantigen specific cellular immune function detection kit for citrullinated autoantigen specific T cells (Cit_Pro-AST) detection.

[0049] In the embodiment of the present invention, a variety of prediction tools are used to predict the spectrum of citrullinated T cell epitopes presented by dominant HLA-DR molecules for RA-specific citrullinated autoantigens, as shown in Table 1 below:

[0050] Table 1 Websites of online epitope peptide prediction tools

[0051]

[0052]

[0053] Specifically, in the embodiment of the present invention, the amino acid sequence of the protein corresponding to each RA-specific and differentially expressed citrullinated modified polypeptide is obtained through databases such as UniProt / NCBI, and the citrullinated modified site is used as the center, and 7 amino acids are extended before and after, and 53 citrullinated modified short peptides composed of 15 amino acids are sorted out, which are closely related to RA and show different degrees of proliferation response to self-T cells. T cell epitope prediction was performed in advance based on the restriction of 6 allele types of MHC-II molecules. The epitope peptide prediction was derived from the globally recognized SYFPEITHI database (website http: / / www.syfpeithi.de / ). The results were scored greater than or equal to 20 (Score ≥ 20) as T cell epitope peptides and entered into the candidate library. A total of 22 candidate citrullinated antigen T cell epitope peptides were screened, such as Figure 2A As shown, it shows a schematic diagram of predicting HLA-II molecule restricted T cell epitopes based on literature reports of citrullinated short peptides (n=53) in an embodiment of the present invention.

[0054] In the embodiment of the present invention, for each citrulline modified peptide fragment screened by mass spectrometry identification, the above prediction tool was used to virtually predict the T cell epitope peptides presented by 6 DRB1 molecules in the Chinese population. As a result, Cit_ epitope polypeptides with a score greater than or equal to 20 (Score ≥ 20) were used as epitope polypeptides with strong immunogenicity and were included in the candidate library. Among them, the peptides with a score ≥ 20 identified from RA_ACPA(+) patients, such as Figure 2B As shown in FIG. 1 , which shows a schematic diagram of predicting T cell epitopes based on mass spectrometry identification of citrullinated short peptides (n=41) in patients with anti-cyclic citrullinated peptide antibodies positive RA_ACPA(+) in an embodiment of the present invention, peptides with score ≥ 20 identified from patients with anti-cyclic citrullinated peptide antibodies negative RA_ACPA(-) are shown in FIG. Figure 2C As shown, it shows a schematic diagram of predicting T cell epitopes based on mass spectrometry identification of RA_ACPA(-) patient citrullinated short peptides (n=43) in an embodiment of the present invention) wherein the result uses Score≥20 as the criterion for judging candidate peptides of epitope peptide T cell epitopes. The R (arginine) in the middle position of a short peptide with a length of 15 amino acids is Cit (citrulline) that can be modified and substituted. Specifically, in an embodiment of the present invention, the short peptides identified based on mass spectrometry and the scores are shown in Table 2 below.

[0055] Table 2 Short peptides identified based on mass spectrometry and their scores

[0056]

[0057]

[0058]

[0059]

[0060] In the embodiment of the present invention, the HLA-II molecule matching result can be output and displayed in the form of a score heat map of the epitope peptide binding to the HLA-II molecule. The binding affinity of the polypeptide is predicted according to the HLA-II typing of the patient. The HLA-DR molecule restricted epitope peptide is set to 15-mer, which meets the scoring criteria of at least two or more prediction methods, and then its hydrophobicity and solubility are analyzed, and polypeptides that are easier to synthesize and dissolve and have higher affinity are selected as candidate epitope peptides.

[0061] In the embodiments of the present invention, the specific T cell epitope peptides contained in the polypeptide pool can be used as PBMC stimulators to stimulate specific T cell responses of cells to be detected.

[0062] In the embodiments of the present invention, citrullinated T cell epitope polypeptides with RA-ACPA(+) specificity and citrullinated T cell epitope polypeptides with both RA-ACPA(+) specificity and RA-ACPA(-) specificity can be used to form a citrullinated antigen T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody-positive patients. Correspondingly, citrullinated T cell epitope polypeptides with RA-ACPA(-) specificity and citrullinated T cell epitope polypeptides with both RA-ACPA(+) specificity and RA-ACPA(-) specificity can be used to form a citrullinated antigen T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody-negative patients.

[0063] In the embodiment of the present invention, in the actual application process, a plurality of citrullinated antigen T cell epitope polypeptides can be formed into a polypeptide pool. Specifically, in order to cover most people in the detection process, each polypeptide pool should contain acidic polypeptides, basic polypeptides and neutral polypeptides at the same time. Specifically, HLA-DR molecule-restricted positive epitope peptides can be formulated into a mixed polypeptide pool containing at least 3 polypeptides (try to cover a more comprehensive HLA-DRB allele of the population) according to different combinations of acidic / alkaline / neutral, for extensive stimulation of T cells.

[0064] In the embodiment of the present invention, polypeptide pools can be set up for anti-cyclic citrullinated peptide antibody-positive patients and anti-cyclic citrullinated peptide antibody-negative patients respectively to perform immune function tests respectively.

[0065] In an embodiment of the present invention, the detection kit further comprises: an ELISPOT microplate, an IFN-γ capture antibody, an IL4 capture antibody, biotinylated IFN-γ and IL4 detection antibodies, a buffer for incubation and culture, a lymphocyte separation fluid and a 1640 culture medium for ELISPOT detection.

[0066] In the embodiment of the present invention, the ELISPOT microplate may be a PVDF membrane 96-well microplate.

[0067] In the embodiment of the present invention, the detection kit further comprises: HRP-streptavidin and AEC display agent, which are used in ELISPOT experiment and are suitable for enzymatic reaction and pigment substrate for spot detection.

[0068] In the embodiments of the present invention, advanced bioinformatics tools and databases are used to improve the accuracy and efficiency of specific T cell epitope peptide screening. Not only the experimental design is optimized, but also the experimental cycle is shortened, and the overall research efficiency is improved. In the embodiments of the present invention, ELISPOT technology is used to achieve high-sensitivity detection of RA-related specific T cell responses. Not only the accuracy of the detection is improved, but also a new means is provided for evaluating the activity and progression of the disease by directly monitoring the specific cellular immune response.

[0069] The existing technology mainly relies on indicators at the humoral immunity level (such as autoantibody determination), while the embodiments of the present invention provide the level of cytokine secretion and the frequency of positive cells at the single cell level. It is an evaluation and effective monitoring method based on specific cellular immune response, which can provide a deeper understanding of the immune regulatory mechanism of the occurrence and development of RA.

[0070] In the embodiments of the present invention, a more comprehensive and accurate laboratory index can be provided for the individualized treatment of RA patients through a detailed analysis of the specific T cell response, which helps doctors to formulate more targeted treatment strategies according to the specific immune response of the patient.

[0071] The kit provided in the embodiment of the present invention can be used as an effective assessment of the cellular immune function of elderly RA or early RA patients (especially ACPA or RF negative patients). This method reduces the risk of inadequate monitoring of traditional humoral immune responses.

[0072] In an embodiment of the present invention, a detection method based on a citrullinated autoantigen-specific cellular immune function detection reagent is also provided. Specifically, the method comprises the following steps:

[0073] S31, peripheral blood was collected from whole blood samples, and PBMCs were separated based on density gradient centrifugation; PBMCs were washed twice with PBS; PBMC cells were resuspended in culture medium, counted and the cell concentration was adjusted for later use.

[0074] Specifically, in the embodiment of the present invention, PBMC can be isolated by the following steps: First, peripheral blood is collected from a whole blood sample of an RA patient. Using density gradient centrifugation, the blood is mixed with an equal volume of lymphocyte separation solution, and the mixed blood is slowly inverted in a centrifuge tube containing Ficoll separation medium. Centrifuge at 800g for 30 minutes at room temperature. Carefully collect the middle layer, i.e., the PBMC layer, and transfer it to a new centrifuge tube.

[0075] In the embodiment of the present invention, the specific T cell response of PBMC is detected based on the above-mentioned citrullinated autoantigen-specific cellular immune function detection kit.

[0076] S32, placing the PBMCs with adjusted concentration into the wells of the ELISPOT microplate.

[0077] S33, adding citrullinated antigen T cell epitope peptide pool molecules and incubating at room temperature. During the incubation process, specific T cells recognize and bind to the epitope peptide-HLA complex, activate T cells and secrete specific cytokines IFN-γ or IL-4.

[0078] S34, based on ELISPOT technology, detects specific cytokines secreted by cells.

[0079] S35, using an automated reading device to count the positive spots secreting cytokines IFN-γ or IL-4 in the microplate; wherein the number of spots represents the frequency of activated positive specific T cells, and the size of the spots represents the level of specific cytokines secreted by T cells.

[0080] In the present invention, the analysis results can be standardized and interpreted by a control group (unstimulated PBMC or PBMC stimulated with other non-specific peptides) to understand the specific cellular immune response characteristics of RA patients and evaluate the immunogenicity of T cell epitopes and the response to treatment.

[0081] Specifically, in the embodiments of the present invention, a schematic diagram of the operation flow of the detection method based on the citrullinated autoantigen-specific cellular immune function detection kit is also provided, such as Figure 3 As shown, specifically, in the embodiment of the present invention, relevant reagent preparation and buffer solution configuration can be performed first, the experiment can be designed, and the reagents and reactants to be added to each well can be made into cards, as follows:

[0082] (1) Antibody coating: Add 15 μl of 70% ethanol to each well of a PVDF membrane 96-well microplate to soak the membrane, evaporate to semi-dryness within 5 min, and add 100 μl of sterile deionized water to wash twice. Add 50 μl of citrullinated antigen T cell epitope peptide pool molecules of appropriate concentration under sterile conditions. After overnight at 4°C, wash with PBS and pat dry on sterile absorbent paper. Add 200 μl of blocking solution, block at 37°C for 1 h, and pour off the blocking solution.

[0083] (2) Inoculation of cells: Under sterile conditions, inoculate different concentrations of cells to be tested in the coated and sealed culture wells, 100 μl / well, and add 10 μl / well of activator (specific antigen, mitogen), cover the cover, and culture in a CO2 incubator at 37°C for 18-24 hours. Each peptide pool combination has 2-4 replicate wells, and a group of positive control (PHA stimulator), test cell control (no stimulator) and background negative control (no cells, only culture medium and all detection reagents) are set up.

[0084] (3) Post-cell culture detection operation: Pour off the cells and culture medium in the wells, add 200 μl of ice-cold deionized water to each well, ice bath for 10 min, wash 10 times with PBST, and pat dry; add 100 μl of biotinylated detection antibody to each well, incubate at 37°C for 1 h, wash 5 times with 200 μl PBST, and pat dry; add 100 μl of working concentration of enzyme-labeled avidin to each well, incubate at 37°C for 1 h, and wash as above; add 100 μl of color development solution (enzyme substrate / chromogen), let stand at room temperature in the dark for 25 min to form colored spots that grow to an appropriate size, then wash twice with deionized water to terminate the color development process, dry at room temperature, and count using a spot reader.

[0085] Specifically, in the embodiment of the present invention, the detection reagent includes:

[0086] A) RPMI-1640 contains 2 mM glutamin and antibiotics, 5% HI-FCS, Hepes buffer, and needs to be sterilized by filtration before use;

[0087] B) For the positive control, use a culture medium containing phytohemagglutinin (PHA) at 4-8 μg / ml or a culture medium containing PHA at 1.25-10 μg / ml, filter sterilize (it can be prepared into a 2-fold concentrated solution and diluted 2-fold when used).

[0088] C) PBS, sterilize, adjust pH to about 7.4.

[0089] D) Sterile PBS containing 0.05% Tween-20 (PBST).

[0090] E) Sterile PBS containing 1% BSA (PBS-BSA).

[0091] Specifically, in the embodiment of the present invention, the detailed steps of antibody coating may include:

[0092] A) Add sterile deionized water to the coated antibody bottle, mix for 15 seconds, and let stand at room temperature for 5 minutes.

[0093] B) Take out 50 μl, add 5 ml of sterile PBS, mix well, and add to ELISPOT plate, 50 μl per well.

[0094] C) Add 50 μl PBS / well, cover the plate, and incubate at 37°C for 1 hour or at 4°C overnight.

[0095] D) Aliquot other coating antibodies into 50 μl / bottle and store at -20℃.

[0096] Specifically, in the embodiment of the present invention, the detailed steps of inoculating cells may include:

[0097] A) Take out the ELISPOT microplate, remove the liquid, and wash 5 times with sterile PBST. (You can use a tray and do this in a clean bench)

[0098] B) Add 200 μl / well of sterile PBS-BSA and incubate at 37°C for 1 hour or at 4°C overnight.

[0099] C) Remove liquid without washing the plate.

[0100] D) Add 100 μl of prepared cells, with a total cell count of 1 to 4 × 10 5 / well (For the first test, a preliminary test of stimulant concentration gradient and / or cell count gradient should be performed). Incubate in a 37°C 5% CO2 incubator for 5-20 hours (the specific time can be determined according to the type of cytokine detected and whether the cells are pre-incubated).

[0101] In the embodiment of the present invention, the detailed steps of detection may include:

[0102] A) Add 500 μl of sterile deionized water to the freeze-dried biotin-labeled antibody bottle and leave it at room temperature for 5 minutes. Take out 100 μl and add it to 10 ml of PBS-BSA and mix well.

[0103] B) Take out the ELISPOT plate, remove the cultured cells, add 200 μl / well of ice-cold deionized water, place the plate on ice for 10 minutes, and wash the plate 10 times with PBST.

[0104] C) Add biotin-labeled detection antibody, 100 μl / well, at 37°C for 1 hour or at 4°C overnight.

[0105] D) Wash the plate 5 times with PBST. Add 500μl of sterile deionized water to the enzyme marker bottle (GABA) and leave it at room temperature for 5 minutes. Take out 100μl and add it to 5ml of PBS-BSA and mix well. (The rest of the enzyme marker can be stored in aliquots at -20℃).

[0106] E) Add 50 μl / well of enzyme-labeled antibody (GABA) and incubate at 37°C for 1 hour or at 4°C overnight.

[0107] F) Wash the plate 5 times. (PBS can be used for the last wash to avoid the influence of Tween20 on the color developer).

[0108] G) Take out 1.5 ml of Color Developer I and 1.5 ml of Color Developer II and mix them in the dark at 4°C.

[0109] H) Add 30 μl / well of freshly prepared color developer I / II, place in a dark place, and incubate at room temperature for 20-30 minutes (until spots can be observed under a microscope; if no obvious spots are observed within 30 minutes, add more color developer).

[0110] I) Remove the color developing solution and wash the ELISPOT plate with deionized water.

[0111] J) Dry the ELISPOT plate at room temperature and observe the results under an inverted microscope or use an automated enzyme-linked spot image analyzer for analysis.

[0112] In the embodiment of the present invention, the specific cellular immune function state of the patient can be determined based on the detected cytokine levels by comparing with the control group data, wherein high expression of IFN-γ represents a Th1 response, and high expression of IL-4 represents a Th2 response; wherein the control group data is obtained by detecting cytokines analyzed on unstimulated PBMCs or PBMCs stimulated with other non-specific peptides.

[0113] In the embodiments of the present invention, standardized data analysis and interpretation process for interpreting T cell response detection results are also provided. Standardized analysis and accurate interpretation of data help to deeply understand the role of citrullinated antigen-specific T cell response in RA and provide a scientific basis for the diagnosis and treatment of the disease, including:

[0114] 1) Counting and recording:

[0115] The positive spots were counted using an ELISpot scanner, with each spot representing a cell producing a specific cytokine.

[0116] 2) Standardization:

[0117] A) Calculate the stimulation index (SI): Compare the number of spots in the sample stimulated with the number of spots in the unstimulated control. [SI = number of spots (antigen stimulation) / number of spots (unstimulated control)]

[0118] B) Consider background noise: Make sure the number of spots is significantly above the background level.

[0119] C) Statistical analysis: The number of spots between different samples was compared using t-test or non-parametric test to determine the statistical significance of the differences.

[0120] 3) Interpretation of results:

[0121] A) Determine T cell subtype: Determine T cell subtype based on the detected cytokines (for example, high expression of IFN-γ indicates Th1 response, and high expression of IL-4 indicates Th2 response).

[0122] B) Clinical relevance: Analyze the correlation between T cell response stimulated by specific antigens and the patient's clinical manifestations, such as disease activity, clinical prognosis, etc.

[0123] C) Comparative studies: Compare the data of RA patients with healthy controls or other disease states to explore the special role and significance of specific T cell responses in RA.

[0124] 4) Notes:

[0125] A) Control variables: Ensure that all experimental conditions (such as cell number, antigen concentration, incubation time) are consistent between the control group and the experimental group to reduce experimental errors.

[0126] B) Repeatability: Increase the repeatability of the experiment to ensure the reliability and consistency of the results.

[0127] The citrullinated autoantigen-specific cellular immune function detection kit provided in the embodiment of the present invention can be used to evaluate the disease state of RA patients during treatment, so as to provide patients with a more suitable personalized treatment plan based on the evaluation results.

[0128] Specifically, the kit of the present invention is used to detect the response of the patient's T cells to the RA-related specific epitope peptide / mixed polypeptide pool during the treatment process, and the change of disease activity is observed by comparing the data in time series.

[0129] The citrullinated autoantigen-specific cellular immune function detection kit provided in the embodiment of the present invention can be used to diagnose rheumatoid arthritis. Specifically, whole blood samples can be collected from suspected RA or confirmed RA patients. PBMCs are separated according to standard operating procedures for subsequent testing. Using the kit provided in the embodiment of the present invention, a specific epitope peptide / mixed polypeptide pool is added to the PBMC sample. The ELISPOT technology is used to detect the response of T cells to specific epitope peptides, thereby evaluating their activation state and number. The cytokine release is analyzed, and the control group data is compared to understand the patient's specific cellular immune function state (Th1 / Th2 balance), providing experimental data to support clinicians in disease status assessment and patient management decisions.

[0130] The citrullinated autoantigen-specific cellular immune function detection kit provided in an embodiment of the present invention can also be used for regular follow-up of confirmed RA patients. Specifically, blood samples from confirmed RA patients can be collected regularly. Similarly, PBMCs are separated by standardized procedures for further testing. The kit provided in an embodiment of the present invention is used to detect the response of T cells to RA-related specific epitope peptides / mixed polypeptide pools. By comparing the data in the time series, the changes in disease activity are observed. By analyzing the changes in T cell activity, the patient's response to the ongoing treatment regimen is evaluated. Provide important information about the level of disease control and the potential need to adjust the treatment strategy.

[0131] Based on the implementation of the above two application scenarios, the kit provided in the embodiments of the present invention can not only help doctors evaluate RA more accurately in clinical laboratories, but also provide valuable data on disease activity and treatment response during regular follow-up of patients, thereby optimizing treatment plans and improving the quality of life of patients.

[0132] In summary, in the embodiments of the present invention, a new specific cellular immune function evaluation method was developed based on the kit provided by the present invention, which can accurately monitor and analyze the T cell (Cit_Pro-AST) response corresponding to specific citrullinated antigens in patients with rheumatoid arthritis (RA).

[0133] Traditional RA diagnosis relies on a wide range of inflammatory indicators, while in the embodiments of the present invention, based on the application of highly specific biomarkers, the focus is on detecting T cells activated by specific antigenic epitopes that are particularly related to RA pathology, using a citrullinated peptide pool as a stimulator, directly targeting the immune response that may occur in RA patients, and evaluating their specific cellular immune function status. Compared with traditional nonspecific immune monitoring techniques (such as flow cytometry analysis of T cell subsets), it can more accurately reflect the immunological status of the disease.

[0134] The kit provided by the embodiment of the present invention can monitor the response of T cells to citrullinated antigens in real time, detect specific T cells using ELISpot technology, and quantitatively analyze cytokine release at the single cell level, with high sensitivity and resolution. It provides real-time data on disease activity and individual response to treatment, which is superior to traditional technologies.

[0135] The kit provided based on the embodiment of the present invention can provide a detailed understanding of the immune function status of each patient (dual cytokine detection: simultaneous monitoring of the secretion of IFN-γ and IL-4 can more comprehensively evaluate the specific Th1 and Th2 immune responses), which is of great significance for understanding the immune regulation mechanism of RA. It can also help doctors develop more personalized treatment plans, optimize treatment effects, and reduce unnecessary drug side effects.

[0136] In the embodiment of the present invention, by detecting the T cell response of the patient to a specific citrullinated antigen, a more personalized treatment plan can be provided for the patient. The kit provided in the embodiment of the present invention can also be used to evaluate the effect of a new drug on the specific immune response of RA patients as a biomarker of drug effect. The kit provided in the embodiment of the present invention can also be used to screen high-risk populations, which helps to identify disease risks early and achieve early intervention.

[0137] The kit provided by the embodiment of the present invention can improve the diagnostic accuracy of early-onset or atypical RA, especially ACPA or RF-negative RA diseases, reduce missed detection and provide pathophysiological basis.

[0138] By using the kit provided in the embodiment of the present invention, by detecting specific T cell responses, specific Th1 and Th2 immune responses are more comprehensively evaluated, providing new laboratory indicators for individualized treatment of RA. By using the kit provided in the embodiment of the present invention, through accurate disease monitoring and early functional evaluation, effective intervention means can be provided for patients to improve the prognosis and quality of life of patients.

[0139] In summary, in clinical practice, the kit provided by the embodiment of the present invention can be used to accurately monitor and evaluate the condition of patients with rheumatoid arthritis, especially in the early stages of the disease or atypical RA patients. The kit provided by the embodiment of the present invention can be used as a tool for precision medicine. This technology helps to achieve personalized disease management and formulate personalized treatment plans based on the patient's specific immune response. In addition, it can also help doctors adjust drug treatment plans to achieve the best therapeutic effect. In clinical studies, the kit provided by the embodiment of the present invention can be used to study the pathological mechanism of rheumatoid arthritis, especially in terms of citrullinated antigens and T cell activation, which helps to develop new treatment methods and drugs.

[0140] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments, and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0141] The above is a detailed introduction to the citrullinated autoantigen-specific cellular immune function detection kit, detection method and application provided by the present invention. The principles and implementation methods of the present invention are described in detail using specific examples herein. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A citrullinated autoantigen-specific cellular immune function detection kit, characterized in that: The detection kit comprises: at least one citrullinated antigen T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody positive patients and at least one citrullinated T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody negative patients.

2. The citrullinated autoantigen-specific cellular immune function detection kit according to claim 1, characterized in that: The citrullinated antigen T cell epitope polypeptide pool for anti-cyclic citrullinated peptide antibody-positive patients includes one or more of the following citrullinated T cell epitope polypeptides: TSVTPVLRGQPIYIQ, TMQSLNDRLASYLDR, STFSTNYRSLGSVQA, SRTTGRQRTSHSEST, SRTTGRQRSSHSESS, SESIVPERHGTTHGQ, SESIVHERHGTIHGQ, SESIVDERHGTTHGQ, SESIIHDRHRITHGQ, QHKFQERREVELRAK, PKSGSTVRRRQGTTH, PELLGARHYTKAID, NSTQSRIREQKLGSS, NGQLLFDRPMHVKMD, MLDRDHDRRLDFTEF, LSGLGGPRKSGPRGA, KSHISPSRESGEEYE, GSRISVSRSTSFRGG, GQTINTTRHSQSGQG, GHTYGQARSQHGESG, FKIIEDLRAQIFANT, ETTLTELRRTVQSLE, EQYINNLRRQLDSIV, ENDIHGLRKVIDDTN, EIPVVAIRTAKGEKF,EIDLDSMRNLKASLE,EEEMKDLRNVSTGDV,EAEIATYRRLLEDGE,CSQSYSQRGYGAREN,ASYLDRVRSLETENR,ANTVDNARIVLQIDN,AKIMADIRAQYDELA.

3. The citrullinated autoantigen-specific cellular immune function detection kit according to claim 1, characterized in that: The pool of citrullinated antigen T cell epitope polypeptides for patients with negative anti-cyclic citrullinated peptide antibodies includes one or more of the following citrullinated T cell epitope polypeptides: ALKFLYKRGANVNLR, EIQPSTFRCVYVRSA, ENYIGDLRRQVDLLS, IEEMPSERLCRSTRQ, KTILDDLRAEDHFSV, LLIYKQGRKLFPIKL, MFLYEYARRHPDYSV, PGQTVKFRVVSMDEN, QDVVNAVRQLTLLGG, RMTLDDFRIKFEMEQ, SISDAEQRGENALKD, and VSLALTQRGVKVFAV.

4. The citrullinated autoantigen-specific cellular immune function detection kit according to claim 1, characterized in that: The citrullinated antigen T cell epitope polypeptide pool includes: at least one acidic polypeptide, at least one basic polypeptide and at least one neutral polypeptide.

5. The citrullinated autoantigen-specific cellular immune function detection kit according to claim 1, characterized in that: The detection kit also includes: an ELISPOT microplate, an IFN-γ capture antibody, an IL4 capture antibody, biotinylated IFN-γ and IL4 detection antibodies, a buffer for incubation and culture, a lymphocyte separation fluid and a 1640 culture medium.

6. The citrullinated autoantigen-specific cellular immune function detection kit according to claim 1, characterized in that: The detection kit also includes: HRP-streptavidin and AEC display agent.

7. A detection method based on the kit according to any one of claims 1 to 6, characterized in that: The method comprises: Peripheral blood was collected from whole blood samples, and PBMCs were isolated based on density gradient centrifugation; Wash PBMCs twice with PBS; Resuspend PBMC cells in culture medium, count and adjust cell concentration for later use; Place the PBMCs with adjusted concentration into the wells of the ELISPOT microplate; Add citrullinated antigen T cell epitope peptide pool molecules and incubate at room temperature. During the incubation process, specific T cells recognize and bind to the epitope peptide-HLA complex, activate T cells and secrete specific cytokines IFN-γ or IL-10; Based on ELISPOT technology, detect specific cytokines secreted by cells; An automated reader is used to count the positive spots secreting cytokines IFN-γ or IL-4 in the microplate; the number of spots represents the frequency of activated positive specific T cells, and the size of the spots represents the level of specific cytokines secreted by T cells.

8. The detection method according to claim 7, characterized in that: The method further comprises: According to the detected cytokine levels, the patient's specific cellular immune function status was determined by comparison with the control group data. Among them, high expression of IFN-γ indicates Th1 response, and high expression of IL-4 indicates Th2 response; The control group data are obtained by detecting cytokines in unstimulated PBMC or PBMC stimulated with other non-specific peptides.

9. Use of the citrullinated autoantigen-specific cellular immune function detection kit according to any one of claims 1 to 6, characterized in that: Used in the preparation of rheumatoid arthritis diagnostic kits.

10. Use of the citrullinated autoantigen-specific cellular immune function detection kit according to any one of claims 1 to 6, characterized in that: Used in the preparation of rheumatoid arthritis treatment response detection kit.

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