Cell matrix for detecting anti-Vimentin autoantibody as well as preparation method and application of cell matrix

Abstract

The invention discloses a cell matrix for detecting an anti-Vimentin autoantibody as well as a preparation method and application of the cell matrix, and belongs to the technical field of antibody detection. The technical problems to be solved are that the existing anti-Vimentin antibody detection is high in non-specific background, poor in interpretation consistency, insufficient in pathology-related modification reproduction and lack of a standardized matrix preparation system. According to the technical scheme, Vimentin and PAD2 / PAD4 are co-expressed in mammalian cells to achieve in-situ citrullination, a CitVim cell matrix is prepared, multi-dimensional interpretation and a kit are matched, the final cerebrospinal fluid / serum detection specificity reaches 100% / 96%, and the positive detection rate is increased by 56.7% compared with WT-Vim.

Description

Technical Field

[0001] This invention belongs to the field of antibody detection technology, specifically relating to a cell matrix for detecting anti-Vimentin autoantibodies, its preparation method, and its application. Background Technology

[0002] Neurological autoimmune diseases are caused by the immune system's abnormal attack on the nervous system. They encompass central nervous system autoimmune diseases (such as autoimmune encephalitis and neuromyelitis optica spectrum disorders) and peripheral nervous system autoimmune diseases (such as chronic inflammatory demyelinating polyradiculoneuritis). Their clinical manifestations are complex and diverse, often involving multiple aspects such as cognitive impairment, motor disorders, and sensory abnormalities, posing a significant challenge to clinical diagnosis.

[0003] Neurological autoantibodies are key molecules mediating these diseases and important diagnostic biomarkers. For example, anti-AQP4 antibodies can be used to differentiate neuromyelitis optica from multiple sclerosis, and anti-MOG antibodies have a high detection rate in some patients with demyelinating diseases. However, as research has progressed, it has been found that there are many types of neurological autoantibodies, and there is a cross-infection phenomenon where the same antibody appears in different diseases. Relying solely on existing biomarkers is insufficient to cover all difficult cases, and many diseases with unclear diagnoses still require new biomarkers to clarify their etiology.

[0004] Vimentin, an intermediate filament protein, is widely present in normal mesenchymal cells and plays an important role in maintaining cell stability and resisting stress. Its overexpression is also closely related to the occurrence and development of various tumors. However, prior to this invention, the association between vimentin citrullination modification and autoimmune diseases of the nervous system had not been revealed, and there was a lack of research on the presence and diagnostic value of anti-Vimentin autoantibodies in patients with these diseases.

[0005] In existing detection technologies, the antigens used for detecting anti-Vimentin autoantibodies are mostly wild-type Vimentin protein or unmodified cell matrix, which suffers from high nonspecific background, low positive detection rate, and poor interpretation consistency. Furthermore, the lack of a standardized in-situ citrullinated Vimentin cell matrix preparation system makes it impossible to accurately reproduce the antigen modification characteristics under pathological conditions, leading to false negative results for some low-abundance antibody samples and failing to meet the needs of accurate clinical diagnosis.

[0006] Furthermore, existing diagnostic kits have limited compatibility, with some only supporting single-sample (serum or cerebrospinal fluid) testing and insufficient compatibility with testing instruments. The lack of standardized operating procedures further restricts their widespread clinical application. Therefore, developing a standardized detection system based on pathology-related modifications to improve the specificity and sensitivity of anti-Vimentin autoantibody detection is of great significance for filling the diagnostic gap in neurological autoimmune diseases and improving the biomarker spectrum.

[0007] Chinese Patent Publication No. CN116068194A, published on May 5, 2023, discloses an anti-Vimentin antibody and a reagent for detecting the antibody (containing Vimentin protein, cells / tissues / lysates expressing the protein, etc.). This reagent can be used to prepare products for detecting / diagnosing related diseases (such as kits). The specific range of Vimentin protein and its coding sequence is defined. The kit includes detection reagents, labeled antibodies (such as horseradish peroxidase-labeled anti-human antibodies), and is compatible with serum and cerebrospinal fluid samples. Detection can be achieved through conventional immunoassay methods such as enzyme-linked immunosorbent assay (ELISA). However, this document does not address pathological modifications of Vimentin (such as citrullination). The detection matrix lacks the ability to reproduce disease-specific modified epitopes and is susceptible to non-specific background interference, which may affect the detection accuracy of low-abundance antibody samples. Summary of the Invention

[0008] This invention addresses the problems existing in the prior art by providing a cell matrix for detecting anti-Vimentin autoantibodies, its preparation method, and its application.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a plasmid combination for detecting anti-Vimentin autoantibodies in a cell matrix, including a Vimentin expression plasmid and a PAD2 expression plasmid, a Vimentin expression plasmid and a PAD4 expression plasmid, or a Vimentin expression plasmid and a PAD2 expression plasmid and a PAD4 expression plasmid.

[0010] Specifically, the amino acid sequence of Vimentin is shown in SEQ ID NO.1, the amino acid sequence of PAD2 is shown in SEQ ID NO.2, and the amino acid sequence of PAD4 is shown in SEQ ID NO.3.

[0011] SEQ ID NO.1: MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLNNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE.

[0012] SEQ ID NO.2: MLRERTVRLQYGSRVEAVYVLGTYLWTDVYSAAPAGAQTFSLKHSEHVWVEVVRDGEAEEVATNGKQRWLLSPSTTLRVTMSQASTEASSDKVTVNYYDEEGSIPIDQAGLFLTAIEISLDVDADRDGVVEKNNPKKASWTWGPEGQGAILLVNCDRETPWLPKEDCRDEKVYSKEDLKDMSQMILRTKGPDRLPAGYEIVLYISMSDSDKVGVFYVENPFFGQRYIHILGRRKLYHVVKYTGGSAELLFFVEGLCFPDEGFSGLVSIHVSLLEYMAQDIPLTPIFTDTVIFRIAPWIMTPNILPPVSVFVCCMKDNYLFLKEVKNLVEKTNCELKVCFQYLNRGDRWIQDEIEFGYIEAPHKGFPVVLDSPRDGNLKDFPVKELLGPDFGYVTREPLFESVTSLDSFGNLEVSPPVTVNGKTYPLGRILIGSSFPLSGGRRMTKVVRDFLKAQQVQAPVELYSDWLTVGHVDEFMSFVPIPGTKKFLLLMASTSACYKLFREKQKDGHGEAIMFKGLGGMSSKRITINKILSNESLVQENLYFQRCLDWNRDILKKELGLTEQDIIDLPALFKMDEDHRARAFFPNMVNMIVLDKDLGIPKPFGPQVEEECCLEMHVRGLLEPLGLECTFIDDISAYHKFLGEVHCGTNVRRKPFTFKWWHMVP。

[0013] SEQ ID NO.3: MAQGTLIRVTPEQPTHAVCVLGTLTQLDICSSAPEDCTSFSINASPGVVVDIAHGPPAKKKSTGSSTWPLDPGVEVTLTMKVASGSTGDQKVQISYYGPKTPPVKALLYLTGVEISLCADITRTGKVKPTRAVKDQRTWTWGPCGQGAILLVNCDRDNLESSAMDCEDDEVLDSEDLQDMSLMTLSTKTPKDFFTNHTLVLHVARSEMDKVRVFQATRGKLSSKCSVVLGPKWPSHYLMVPGGKHNMDFYVEALAFPDTDFPGLITLTISLLDTSNLELPEAVVFQDSVVFRVAPWIMTPNTQPPQEVYACSIFENEDFLKSVTTLAMKAKCKLTICPEEENMDDQWMQDEMEIGYIQAPHKTLPVVFDSPRNRGLKEFPIKRVMGPDFGYVTRGPQTGGISGLDSFGNLEVSPPVTVRGKEYPLGRILFGDSCYPSNDSRQMHQALQDFLSAQQVQAPVKLYSDWLSVGHVDEFLSFVPAPDRKGFRLLLASPRSCYKLFQEQQNEGHGEALLFEGIKKKKQQKIKNILSNKTLREHNSFVERCIDWNRELLKRELGLAESDIIDIPQLFKLKEFSKAEAFFPNMVNMLVLGKHLGIPKPFGPVINGRCCLEEKVCSLLEPLGLQCTFINDFFTYHIRHGEVHCGTNVRRKPFSFKWWNMVP。

[0014] More specifically, the coding sequence of Vimentin is as shown in SEQ ID NO.4, the coding sequence of PAD2 is as shown in SEQ ID NO.5, and the coding sequence of PAD4 is as shown in SEQ ID NO.6.

[0015] SEQ ID NO.4:

[0016] SEQ ID NO.5:

[0017] SEQ ID NO.6:

[0018] More specifically, the Vimentin expression plasmid also includes the Igκ signal peptide and PDGFR-TM.

[0019] Preferably, the amino acid sequence of the Igκ signal peptide is shown in SEQ ID NO.7.

[0020] Preferably, the PDGFR-TM amino acid sequence is as shown in SEQ ID NO.8.

[0021] SEQ ID NO.7: METDTLLLWVLLLWVPGSTG.

[0022] SEQ ID NO.8: AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR.

[0023] More preferably, the coding sequence of the Igκ signal peptide is shown in SEQ ID NO.9.

[0024] Preferably, the encoding sequence of the PDGFR-TM is as shown in SEQ ID NO.10.

[0025] SEQ ID NO.9: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT.

[0026] SEQ ID NO.10: GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT.

[0027] In another aspect, the present invention provides a method for preparing a cell matrix for detecting anti-Vimentin autoantibodies, comprising: transfecting mammalian cells using any of the above-mentioned plasmid combinations.

[0028] Specifically, the mammalian cells include HEK293, HEK293T, CHO, U251, or SHSY5Y cells.

[0029] In another aspect, the present invention provides a cell matrix for detecting anti-Vimentin autoantibodies prepared by any of the above preparation methods.

[0030] Furthermore, this invention provides the application of the aforementioned cell matrix in the preparation of detection products for anti-Vimentin autoantibodies.

[0031] Specifically, the products include, but are not limited to, reagent kits, chips, and detection systems.

[0032] In another aspect, the present invention provides a detection product for anti-Vimentin autoantibodies, the product comprising the cell matrix for the above-mentioned anti-Vimentin autoantibody detection.

[0033] Specifically, the steps for using the product include: S1. Collect samples; S2. Incubate the sample with the cell matrix for detecting anti-Vimentin autoantibody. S3. Incubate the sample with either cell matrix transfected with Vimentin expression plasmid alone or cell matrix transfected with the vector backbone of Vimentin expression plasmid alone. S4. Finally, the products from steps S2 and S3 are added to the labeled antibody and the signal is detected. The result is determined based on the signal ratio.

[0034] Specifically, in step S1, the sample includes serum and cerebrospinal fluid.

[0035] Specifically, in step S4, the determination based on the signal ratio includes any one or more of the following criteria: a. R1 = I_Cit / I_WT (Cit / WT ratio): used to measure the specificity of "citrullination-dependent" recognition; b. R2 = I_Cit / I_Blank (Cit / Blank ratio): used to measure the improvement of the antibody signal against each type of Vimentin relative to the "blank baseline"; c.R3 = I_WT / I_Blank (WT / Blank ratio): This measures the improvement in antibody signal against unmodified Vimentin relative to the "blank baseline".

[0036] Wherein, I_Cit can be defined as: the average strength of the co-rotating PAD2 or / and PAD4 holes.

[0037] Wherein, I_WT can be defined as: the average strength of the non-co-rotating PAD2 or / and PAD4 holes.

[0038] Wherein, I_Blank can be defined as: the average intensity of untransfected / empty vector cells, PAD-expressing cells only, or "secondary antibody-only / no primary antibody" control wells.

[0039] In some specific embodiments of the present invention, the judgment criteria are shown in Table 1: Table 1

[0040] In another aspect, the present invention provides a detection system for anti-Vimentin autoantibodies, the detection system comprising a detection device, a computing device, and an output device; the detection device comprising a sample injector, a sample processor, and a detector; The sampler is used to collect samples from the subject; The sample processor is used to mix and incubate the collected sample with the cell matrix, and add labeled antibodies to the sample after the mixing and incubation are completed; The detector is used to detect signals in the sample processor.

[0041] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for detecting anti-Vimentin autoantibodies in a sample includes: mixing the sample to be tested with the aforementioned cell matrix, incubating it, labeling it with antibodies, detecting the signal, and determining the result based on the signal ratio.

[0042] Specifically, the samples include serum and cerebrospinal fluid.

[0043] Specifically, the determination based on the signal ratio includes any one or more of the following criteria: a. R1 = I_Cit / I_WT (Cit / WT ratio): used to measure the specificity of "citrullination-dependent" recognition; b. R2 = I_Cit / I_Blank (Cit / Blank ratio): used to measure the improvement of the antibody signal against each type of Vimentin relative to the "blank baseline"; c.R3 = I_WT / I_Blank (WT / Blank ratio): This measures the improvement in antibody signal against unmodified Vimentin relative to the "blank baseline".

[0044] Wherein, I_Cit can be defined as: the average strength of the co-rotating PAD2 or / and PAD4 holes.

[0045] Wherein, I_WT can be defined as: the average strength of the non-co-rotating PAD2 or / and PAD4 holes.

[0046] Wherein, I_Blank can be defined as: the average intensity of untransfected / empty vector cells, PAD-expressing cells only, or "secondary antibody-only / no primary antibody" control wells.

[0047] Compared with the prior art, the present invention has the following advantages: 1. Compared with the uncitrullinated WT-Vim matrix, the CitVim cell matrix of the present invention significantly reduces non-specific background interference by in situ reproducing pathology-related citrullinated modifications.

[0048] 2. The positive detection rate of this invention is significantly improved, with a 56.7% increase in cerebrospinal fluid and a 119.7% increase in serum compared to WT-Vim. It can accurately identify low-abundance anti-Vimentin autoantibodies in clinically difficult cases, solving the pain points of existing tests such as "many false negatives and difficulty in interpretation".

[0049] 3. The specificity of the present invention for cerebrospinal fluid sample detection reaches 100% (WT-Vim is 93.75%), and the specificity for serum sample detection reaches 96% (WT-Vim is 84%), which is a significant improvement in specificity. Attached Figure Description

[0050] Figure 1 This is a map of the pCMV-VIM plasmid.

[0051] Figure 2 This is a map of the pEF1α-PAD4 plasmid.

[0052] Figure 3 This is a map of the pEF1α-PAD2 plasmid. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0054] Example 1 The construction (intracellular display) of CitVim, WT-Vim, and BLANK cell matrix is ​​as follows: (1) Plasmid construction: pCMV-VIM (containing full-length human Vimentin): The plasmid backbone is pCMV-Myc plasmid (NovoPro, V011245). The coding sequence of vim (SEQ ID NO.4) is inserted into the StuI / NotI site. See the map below. Figure 1 .

[0055] pEF1α-PAD4: The plasmid backbone is pEF1 / myc-His C (NovoPro, V006901). The coding sequence of PAD4 (SEQ ID NO.6) is inserted into the KpnI / NotI site. See the map below. Figure 2 .

[0056] pEF1α-PAD2: The plasmid backbone is pEF1 / myc-His C (NovoPro, V006901). The coding sequence of PAD2 (SEQ ID NO.5) is inserted into the KpnI / NotI site. See the map below. Figure 3 .

[0057] All vectors contain antibiotic selection markers, and different sources are preferred to ensure stable co-expression.

[0058] (2) Cells and transfection: HEK293T cells were pre-seeded in 6-well plates with coverslips at a cell density of 60%. Transfection combination 1: CitVim[PAD4]: Mix VIM:PAD4 at a mass ratio of 1:0.5 and transfect using liposomes (VIM plasmid 0.8 μg, PAD4 plasmid 0.4 μg; liposomes 1 μL each). WT-Vim[PAD4]: Transfected with VIM (i.e. pCMV-VIM) alone using liposomes (VIM plasmid 0.8 μg; pEF1α empty plasmid 0.4 μg; liposomes 1 μL each). BLANK[PAD4]: Transfected with pCMV and pEF1α empty plasmids using liposomes (0.8 μg pCMV empty plasmid; 0.4 μg pEF1α empty plasmid; 1 μL each of liposomes). Transfection combination 2: CitVim[PAD2]: Mix VIM:PAD2 at a mass ratio of 1:0.5 and transfect using liposomes (VIM plasmid 0.8 μg, PAD2 plasmid 0.4 μg; liposomes 1 μL each). WT-Vim[PAD2]: Transfected with VIM (i.e. pCMV-VIM) alone using liposomes (VIM plasmid 0.8 μg; pEF1α empty plasmid 0.4 μg; liposomes 1 μL each). BLANK[PAD2]: Transfected with pCMV and pEF1α empty plasmids using liposomes (0.8 μg pCMV empty plasmid; 0.4 μg pEF1α empty plasmid; 1 μL each of liposomes). Transfection combination 3: CitVim[PAD2+PAD4]: Mixed with VIM:PAD2:PAD4 at a mass ratio of 1:0.5:0.5, and transfected using liposomes (VIM plasmid 0.8 μg, PAD2 plasmid 0.4 μg, PAD4 plasmid 0.4 μg; liposomes 1 μL each). WT-Vim[PAD2+PAD4]: Transfected with VIM (i.e. pCMV-VIM) alone using liposomes (VIM plasmid 0.8 μg; pEF1α empty plasmid 0.8 μg; liposomes 1 μL each). BLANK[PAD2+PAD4]: Transfected with pCMV and pEF1α empty plasmids using liposomes (0.8 μg pCMV empty plasmid; 0.8 μg pEF1α empty plasmid; 1 μL liposomes each). Fix after 24 hours.

[0059] (3) Fixation and lightening: Ionomycin 3 μM, incubated for 5 min (containing 1 mM CaCl2). PBS elution; 4% PFA fixation for 10 min; 0.1% Triton X100 for 5 minutes to lighten and penetrate; The coverslip matrix was prepared by rinsing with PBS and air drying.

[0060] (4) Patch: Cut the coverslip substrate to the appropriate size (4mm×4mm) according to the product process requirements to make the test specimen; The original sample can be fixed onto the test slide with photosensitive adhesive to make the test product (photosensitive adhesive model: UV curing adhesive 9300).

[0061] (5) Cit mark confirmation (Cit-Vim quality control): Immunofluorescence tracing with F95 primary antibody (Sigma-Aldrich, MABN328, 1:500); The F95 / WT matrix control ratio of ≥2.5 was set as the release criterion.

[0062] Example 2 The CBA testing process and steps are as follows: (1) Sample processing: Centrifuge serum or cerebrospinal fluid to clarify (1500g, 10min).

[0063] (2) Incubation: Samples in ratios of 1:1, 1:10, 1:100, and 1:320 are incubated on the substrate for 30-60 min (room temperature or 37℃) (150 μL of sample is added to each substrate).

[0064] (3) Secondary antibody: Incubate with labeled anti-human IgG (or in combination with IgA / IgM) for 30 min (goat anti-human IgG Fc recombinant secondary antibody, Alexa Fluor™ 488, 1:1000 dilution).

[0065] (4) Interpretation: WT Vim matrix is ​​used in parallel; the Cit / WT ratio, Cit / Blank ratio and WT / Blank ratio are used for interpretation, see Table 1.

[0066] Detection Example 1 The fixed climbing sheet prepared in Example 1 was subjected to the following experiments in accordance with the method described in Example 2: Detection of anti-Vimentin antibody positive samples: Sample source: Clinical test specimens (human serum and cerebrospinal fluid samples), totaling 428 samples. All samples were TBA positive, oligoclonal positive, and negative for common antibodies such as AQP4 / MOG / GFAP / NMDAR / LGI1 / CASPR2; TBA results showed astral glial / intermediate filament pattern (tangle of slender fibers, peripheral vascular foot-like distribution); clinical manifestations suggested glial-related involvement or brainstem / cerebellum / long segment spinal cord involvement.

[0067] Detection Method: Prepare sample dilution buffer as follows: Add 1% bovine serum albumin to phosphate buffer, dissolve, filter and sterilize. Dilute human serum and cerebrospinal fluid samples with the sample dilution buffer. Use undiluted cerebrospinal fluid. The serum sample dilution ratio is 1:10. Add the diluted samples to the fixed cell slides and incubate at 37°C for 1 hour. Discard the sample solution and wash the cells three times with phosphate buffer for 5 minutes each time. Then dilute the secondary antibody (Alexa Fluor™ 488 goat anti-human IgG) with the same sample dilution buffer at a dilution ratio of 1:1000. Incubate at room temperature for 30 minutes. Discard the secondary antibody and wash the cell slides three times with phosphate buffer for 5 minutes each time. Observe under a fluorescence microscope (Mingmei MF43-N; ImageJ software version V1.54k).

[0068] The test results are shown in Tables 2 and 3. As can be seen from Table 2, the positive detection rate of Cit-Vim in cerebrospinal fluid for "transfection combination 2" and "transfection combination 3" in Example 1 was 2.57%, while the positive detection rate of WT-Vim was 1.64%. The positive detection rate of Cit-Vim was superior to that of WT-Vim, and the positive detection rate of Cit / WT was 0.93%. The positive detection rate of Cit-Vim in serum was 2.57%, while that of WT-Vim was 1.17%. The positive detection rate of Cit-Vim was further superior to that of WT-Vim, and the positive detection rate of Cit / WT was 1.40%.

[0069] Table 2

[0070] The samples used in this test were clinically highly pre-screened, antibody-negative, but difficult cases with TBA phenotypes indicating glial lesions, and their background positive rate was naturally lower than that of the general inflammatory neuropathic population; therefore, the positive rate was only 2.57%. In cerebrospinal fluid samples, the positive detection rate of Cit-Vim was 56.7% higher than that of WT-Vim, and in serum samples, the positive detection rate of Cit-Vim was 119.7% higher than that of WT-Vim, achieving significant progress.

[0071] As shown in Table 3, the positive detection rate of Cit-Vim in cerebrospinal fluid of "transfection combination 1" in Example 1 was 2.10%, and the positive detection rate of WT-Vim was 1.64%. The positive detection rate of Cit-Vim was better than that of WT-Vim, and the positive detection rate of Cit / WT was 0.47%. The positive detection rate of Cit-Vim in serum was 2.10%, and the positive detection rate of WT-Vim was 1.17%. The positive detection rate of Cit-Vim was further better than that of WT-Vim, and the positive detection rate of Cit / WT was 0.93%.

[0072] Table 3

[0073] In cerebrospinal fluid samples, the positive detection rate of Cit-Vim was 28.7% higher than that of WT-Vim, and in serum samples, the positive detection rate of Cit-Vim was 79.5% higher than that of WT-Vim, demonstrating significant progress.

[0074] Detection Example 2 One hundred clinical specimens were collected, including 68 serum samples from healthy controls and 32 serum samples from patients known to be positive for AQP4 / MOG / GFAP / NMDAR / LGI1 / CASPR2 antibodies (antibody-positive serum and antibody-positive cerebrospinal fluid were from the same person). The samples were tested according to the method for detecting anti-Vimentin antibody-positive samples described in Case 1 above.

[0075] The test results are shown in Table 4. As can be seen from the results in Table 4, the specificity of Cit-Vim in Example 1 is significantly better than that of WT-Vim.

[0076] Table 4

[0077] Basic Implementation (1) Composition: Cit-Vim cell matrix slides (10 slides / box); Control matrix (WT-Vim) slides (2 slides / box); Washing solution, blocking solution, labeled secondary antibody, and nuclear staining agent; Quality control materials (weak positive / strong positive / negative controls), and operating instructions.

[0078] (2) Key parameters: The release criteria were set as an F95 / WT matrix control ratio of ≥2.5; and the performance retention rate (≥85%) after being kept at 37℃ for 7 days or after 10 freeze-thaw cycles.

[0079] The F95 / WT matrix control ratio of Cit-Vim cell matrix slides was measured, and slides from the same batch were placed in a 37℃ incubator and a -20℃ freezer. Slides in the 37℃ incubator were removed on days 3, 5, and 7 for F95 / WT matrix control ratio measurement; slides in the -20℃ freezer were removed, thawed to room temperature, and then refrozen (15 min thawing time, 6 h refrozen interval). The F95 / WT matrix control ratio was measured after the 5th and 10th cycles. Each measurement was repeated in triplicate. The results are shown in Tables 5 (37℃ incubator) and 6 (freeze-thaw cycles).

[0080] Table 5

[0081] Table 6

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plasmid combination for preparing a cell matrix for detecting anti-Vimentin autoantibodies, characterized in that, This includes Vimentin expression plasmids with PAD2 expression plasmids, Vimentin expression plasmids with PAD4 expression plasmids, or Vimentin expression plasmids with both PAD2 and PAD4 expression plasmids. The amino acid sequence of Vimentin is shown in SEQ ID NO.1, the amino acid sequence of PAD2 is shown in SEQ ID NO.2, and the amino acid sequence of PAD4 is shown in SEQ ID NO.

3.

2. The plasmid assembly according to claim 1, characterized in that, The Vimentin expression plasmid also includes the Igκ signal peptide and PDGFR-TM.

3. A method for preparing a cell matrix for detecting anti-Vimentin autoantibodies, characterized in that, include: Transfect mammalian cells using the plasmid combination described in any one of claims 1-2.

4. The preparation method according to claim 3, characterized in that, The mammalian cells include HEK293, HEK293T, CHO, U251, or SHSY5Y cells.

5. Cell matrix for detecting anti-Vimentin autoantibodies prepared by the preparation method according to any one of claims 3-4.

6. The application of the cell matrix for detecting anti-Vimentin autoantibodies as described in claim 5 in the preparation of detection products for anti-Vimentin autoantibodies, wherein the products include a kit, a chip, and a detection system.

7. A detection product for anti-Vimentin autoantibodies, characterized in that, The cell matrix includes the anti-Vimentin autoantibody detection as described in claim 5.

8. The product according to claim 7, characterized in that, The steps for using the product include: S1. Collect samples; S2. Incubate the sample with the cell matrix for detecting anti-Vimentin autoantibody. S3. Incubate the sample with either cell matrix transfected with Vimentin expression plasmid alone or cell matrix transfected with the vector backbone of Vimentin expression plasmid alone. S4. Finally, the products from steps S2 and S3 are added to the labeled antibody and the signal is detected. The result is determined based on the signal ratio.

9. The product according to claim 8, characterized in that, In step S1, the sample includes serum and cerebrospinal fluid.

10. A detection system for anti-Vimentin autoantibodies, characterized in that, The detection system includes a detection device, a computing device, and an output device; the detection device includes a sample injector, a sample processor, and a detector. The sampler is used to collect samples from the subject; The sample processor is used to mix and incubate the collected sample with the cell matrix for detecting the anti-Vimentin autoantibody as described in claim 5, and to add the labeled antibody to the sample after the mixing and incubation is completed; The detector is used to detect signals in the sample processor.

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