A detection kit for improving the diagnostic efficiency of autoimmune encephalitis antibody and a preparation method thereof

Abstract

This invention relates to a detection kit and preparation method for enhancing the detection efficacy of autoimmune encephalitis (CBA) antibodies. The kit comprises genetically engineered 293T cells stably integrating and co-expressing the eIF2α S52A mutant and HSP70, and transiently transfected with CBA-associated antigens. The preparation method includes constructing a co-expression vector, establishing a stable transfection cell line, stress preconditioning treatment, antigen transfection, and kit assembly. A method for assessing antibody affinity maturation using this kit is also provided. By synergistically modifying detection cells at three levels—translational regulation, protein folding, and anti-apoptosis—the invention significantly increases antigen expression, reduces apoptosis, and improves detection sensitivity and specificity. Furthermore, it integrates antibody affinity assessment into CBA detection for the first time, providing auxiliary information for clinical staging.

Description

Technical Field

[0001] This invention relates to the field of cell immunofluorescence detection technology, specifically to a detection kit and preparation method for improving the diagnostic efficacy of autoimmune encephalitis antibodies. Background Technology

[0002] In the detection of autoimmune encephalitis antibodies (such as NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, GAD65, etc.), cell-based immunofluorescence assay (CBA) holds a crucial position and is one of the mainstream laboratory immunological detection techniques. Immunofluorescence, with its unique fluorescent labeling and detection mechanism, can more accurately locate autoimmune encephalitis-related antibodies, offering significant advantages in qualitative detection. Nevertheless, the CBA method still requires continuous optimization and improvement in practical applications, such as further enhancing detection sensitivity and specificity, and optimizing its adaptability to complex samples. Therefore, there is an urgent need to develop new detection kits to effectively improve detection performance and overcome the limitations of existing technologies.

[0003] 293T cells are commonly used as tool cells in CBA assay kits, but their basic state suffers from low target protein expression and weak anti-apoptotic ability. Current main techniques involve using 293T or HEK293 cells as tool cells, introducing the target antigen gene into the cells through transient or stable transfection, and then using immunofluorescence to detect the binding of autoantibodies in patient serum. For example, existing patents have used GABABR recombinant protein to construct stable expression cell lines for antibody detection by transfecting CHO or HEK293 cells; other studies have established a two-color indirect immunofluorescence method based on HEK293 cells for the detection of autoantibodies in neuroimmunological diseases. The aforementioned techniques have recognized the advantages of 293T cells, such as high transfection efficiency and ease of operation, and have already resulted in commercialized products. Based on this, this invention will reveal the synergistic mechanism of eIF2α S52A + HSP70, enhancing the overall performance of cells and improving the diagnostic efficacy of autoimmune encephalitis antibodies.

[0004] However, the existing CBA technology still has the following unresolved technical issues:

[0005] Cells used for detection are prone to stress and apoptosis during transfection and detection, leading to unstable antigen expression levels and increased background noise. 293T cells face stress from transfection reagents such as liposomes and PEI, as well as subsequent fixation and washing procedures, which can induce endoplasmic reticulum stress, translational inhibition, and apoptosis. This results in significant fluctuations in target antigen expression levels, making it easy to miss weakly positive samples. While current technologies have addressed this issue, they only optimize transfection conditions or stabilize gene expression, failing to provide a systematic solution at the fundamental level of cellular stress tolerance.

[0006] Current technologies do not systematically address the impact of the basic physiological state of the cells on detection efficacy. Current solutions focus only on antigen gene construction and transfection condition optimization, failing to recognize the comprehensive influence of factors such as cellular translation regulation, protein folding, and apoptosis tendency on antigen expression stability and detection signal quality. While some literature discusses the application of eIF2α S52A mutants in improving recombinant protein production—for example, Biogen Idec MA Inc.'s patent application discloses polynucleotides encoding the spliced ​​form of XBP-1, ATF6, and eIF2α S52A, whose expression vectors increase specific cellular productivity by enhancing the unfolded protein response in cells; Chinese patent application CN202410237067 also relates to the application of eIF2α mutants in protein expression—these solutions all focus on general recombinant protein production systems and do not systematically improve diagnostic efficacy by synergistically introducing eIF2α S52A with HSP70 into CBA detection cells. Meanwhile, although the application of HSP70 as a molecular chaperone has been widely documented, there are no publicly reported technical solutions for synergistically introducing both into the same CBA detection cell for the diagnostic purpose of autoimmune encephalitis antibodies.

[0007] Current CBA technology cannot provide information on antibody affinity maturation. Routine CBA tests only report antibody "positive" or "negative," failing to assess the affinity of autoantibodies in a patient's serum. However, clinical studies have shown that high-affinity antibodies are generally associated with more severe clinical presentation and poorer prognosis, while low-affinity antibodies may appear in the early stages of disease or during the recovery phase. Current technology cannot provide this crucial information for clinical staging and treatment decisions. Although the determination of antibody affinity using thiocyanate elution has been reported in ELISA systems, a technical approach integrating it into the CBA testing process for assessing antibody affinity maturation in autoimmune encephalitis has not been publicly disclosed.

[0008] Therefore, there is an urgent need to develop a novel detection kit and method that can systematically improve the stress tolerance of CBA detection cells, stabilize antigen expression, and provide antibody affinity information. Summary of the Invention

[0009] To address the above problems, this invention provides a method for preparing a detection kit that enhances the detection efficacy of autoimmune encephalitis antibodies, comprising the following steps:

[0010] (1) Construct a recombinant expression vector that co-expresses the eIF2α S52A mutant and heat shock protein 70 (HSP70), wherein the eIF2α S52A mutant is obtained by mutating serine at position 52 of wild-type eIF2α to alanine;

[0011] (2) The recombinant expression vector was introduced into 293T cells, and stable transfected cell lines co-expressing eIF2α S52A mutant and heat shock protein 70 (HSP70) were screened to obtain stable transfection cell lines.

[0012] (3) Before antigen transfection, the stable transfected cell lines obtained in step (2) are subjected to stress preconditioning treatment;

[0013] (4) Transiently transfect the gene encoding the autoimmune encephalitis-associated antigen into a stable transfected cell line that has undergone stress preconditioning treatment, so that the cells express the antigen;

[0014] (5) The cells expressing the antigen were fixed and blocked, and the detection kit was assembled.

[0015] The construction of the recombinant expression vector in step (1) includes: cloning the coding sequences of eIF2α S52A and HSP70 into a lentiviral expression vector by tandem T2A sequence through overlap extension PCR.

[0016] Furthermore, the stress preconditioning treatment is as follows: heat shock temperature 40-43℃, time 30-60 min; H2O2 concentration 30-100 μM, treatment time 15-45 min; serum deprivation time 0.5-2 h; high serum recovery time 2-6 h; resting recovery time 8-16 h.

[0017] The preferred option is as follows:

[0018] (a) Heat shock treatment at 42℃ for 45 min, followed by incubation at 37℃ for 2 h;

[0019] (b) Add H2O2 to the culture medium to a final concentration of 50 μM, treat for 30 min, and then replace with fresh culture medium;

[0020] (c) Incubate in serum-free medium for 1 h, then replace with complete medium containing 20% ​​fetal bovine serum and incubate for 4 h;

[0021] (d) Incubate at rest for 12 h in a 37℃, 5% CO2 incubator.

[0022] Furthermore, the lentiviral expression vector is pLVX-EF1α-IRES-Puro.

[0023] Further, the importation in step (2) includes: co-transfecting 293T cells with the recombinant expression vector, packaging plasmid psPAX2 and envelope plasmid pMD2.G to package lentivirus, infecting 293T cells with the obtained lentivirus, and then selecting stable transfected cell lines by puromycin.

[0024] Further, the autoimmune encephalitis-related antigen mentioned in step (4) is selected from at least one of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and GAD65.

[0025] Further, the fixation treatment in step (5) is performed using a 3% paraformaldehyde solution. The fixation is performed using 3% paraformaldehyde for 15 min.

[0026] The cell fixation method of the detection kit includes the following steps:

[0027] Mild crosslinking: Cells were treated with a mixture of 0.5% glutaraldehyde and 0.1% paraformaldehyde for 5 min;

[0028] Post-repair: Cells were treated for 15 min with a repair solution containing 0.5% glycine, 0.1% sodium borohydride, 1% BSA and 0.05% Triton X-100.

[0029] The patient was washed twice with PBS between the mild crosslinking step and the post-repair step.

[0030] The concentration range of glutaraldehyde is 0.2%-1.0%, and the concentration range of paraformaldehyde is 0.05%-0.5%.

[0031] Further, in step (5), after cell fixation, the cells were incubated at 4°C for 30 min using the treatment solution, followed by blocking. The treatment solution was an antigen conformation stabilizing solution, and its composition by weight / volume percentage was: 0.5%-2.0% trehalose, 0.1%-0.5% betaine, 0.05%-0.2% polyethylene glycol 400, 0.01%-0.05% sodium azide, with the remainder being PBS buffer (pH 7.4).

[0032] This invention provides a detection kit prepared by the aforementioned method, comprising:

[0033] Genetically engineered 293T cells, wherein the eIF2α S52A mutant and HSP70 are stably integrated and co-expressed, and wherein the cells are transiently transfected or stably integrated with the encoding gene of autoimmune encephalitis-related antigen.

[0034] In addition to cell fixative, blocking solution, negative and positive control sera, and fluorescently labeled secondary antibodies.

[0035] The detection kit also includes a cell fixation posttreatment solution, which is an antigen conformation stabilizing solution. Its composition, by weight / volume percentage, is: 0.5%-2.0% trehalose, 0.1%-0.5% betaine, 0.05%-0.2% polyethylene glycol 400, 0.01%-0.05% sodium azide, with the balance being PBS buffer (pH 7.4).

[0036] The trehalose content is 1.0%, betaine content is 0.3%, and polyethylene glycol 400 content is 0.1%.

[0037] The genetically engineered 293T cells in the test kit are present in a lyophilizable test cell preparation. The lyophilizable test cell preparation contains genetically engineered 293T cells expressing antigens, and a lyophilization protectant solution. The lyophilization protectant solution consists of 10% trehalose, 5% sucrose, 2% mannitol, 1% polyvinylpyrrolidone, and 0.5% gelatin.

[0038] The formulation is prepared by the following method: cells are incubated at 5 × 10 5 Cells / mL were suspended in a lyophilization protectant and pre-frozen at -40°C at a rate of 1°C / min. Then, they were dried at -30°C and 10 Pa vacuum for 24 h, and then dried at 20°C and 1 Pa vacuum for 6 h.

[0039] The formulation can be stored at room temperature. When used, it can be incubated at 37°C for 15 min with pre-warmed refolding solution (DMEM containing 10% FBS) to restore its activity.

[0040] Furthermore, this application provides a method for evaluating antibody affinity using the aforementioned detection kit, comprising the following steps:

[0041] Step (i) Incubate and wash the serum to be tested with the genetically engineered 293T cells expressing antigens from the test kit;

[0042] Step (ii) divides the incubated cells into a control group and a dissociation group. The dissociation group is treated with a solution containing potassium thiocyanate (KSCN), while the control group is treated with a solution without KSCN.

[0043] Step (iii) Add fluorescently labeled secondary antibody to both groups of cells for detection and obtain fluorescence intensity;

[0044] Step (iv) calculates the KSCN concentration that results in 50% antibody dissociation as the affinity index.

[0045] Further, in step (ii), the concentration of the KSCN solution is selected from at least two of 0.5 M, 1.0 M, 1.5 M and 2.0 M.

[0046] Furthermore, the treatment time of the KSCN solution in step (ii) is 10 min.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] By stably co-expressing the eIF2α S52A mutant and HSP70, translational inhibition under stress conditions is relieved, protein folding is promoted correctly, and apoptosis is inhibited. The modified 293T cells of this invention, after transfection with the target antigen, showed a 3.2 ± 0.5-fold increase in target protein expression compared to ordinary 293T cells. Compared to existing technologies that use eIF2α S52A or anti-apoptotic genes alone, the synergistic effect of the factors in this invention results in an additional 1.8-fold increase in expression.

[0049] Flow cytometry analysis showed that after transfection with the target antigen, the apoptosis rate of the modified cells of this invention was only 7.2% ± 1.8%, while the apoptosis rate of ordinary 293T cells reached 23.5% ± 3.6%. Cell viability analysis using the CCK-8 assay showed that the modified cells had a viability of 82.3% ± 4.5%, while ordinary cells had a viability of only 54.1% ± 6.2%. The anti-apoptotic effect of existing technologies is far lower than the synergistic effect of the two components in this invention, indicating that the synergistic effect of eIF2α S52A and HSP70 has unexpected technical benefits.

[0050] The assay was validated using 20 positive serum samples from clinically diagnosed autoimmune encephalitis patients and 20 negative serum samples from healthy controls. The modified cell group kit of this invention detected 20 / 20 positive samples, while the ordinary cell group detected only 17 / 20. The modified cell group correctly interpreted 20 / 20 negative serum samples, compared to 18 / 20 in the ordinary cell group. Both sensitivity and specificity are improved.

[0051] The modified cell line was tested using three independently prepared batches. The inter-batch coefficient of variation (CV) was 8.5%, while the CV for the ordinary cell group was 19.3%, indicating improved reproducibility. This demonstrates that the cell system of the present invention has high stability, significantly reduced inter-batch variability, and is more suitable for industrial production and clinical application.

[0052] This invention integrates a potassium thiocyanate (KSCN) dissociation assay into routine antibody affinity assays (CBA) to calculate the antibody affinity index (AI). Clinical validation shows that the average AI value for patients in the acute phase is 1.45 ± 0.23 M, with all values ​​≥ 1.2 M; the average AI value for patients in the recovery phase is 0.65 ± 0.18 M, with all values ​​< 0.8 M. Using an AI ≥ 1.2 M as the criterion for determining the acute phase, both sensitivity and specificity reach 100%. Existing CBA techniques only report positive / negative results and cannot provide this crucial clinical information. This invention successfully integrates the thiocyanate elution method from the traditional ELISA system into the CBA testing process for the first time, filling the technological gap in antibody affinity assessment during CBA testing.

[0053] Through synergistic modification, the basic state of cells is improved, antigen presentation density is increased, and conformation is homogeneous. The detection rate of low-titer (1:32) weakly positive samples is increased from 78% in the prior art to 96%, effectively reducing the risk of false negatives. Compared with the existing technology that reduces false negatives by membrane-anchored peptides, this invention fundamentally solves the problem of missed detection of weak positives by improving the overall performance of cells.

[0054] Due to improved cell stability, the kit's pass rate increased from 92% to 99.5%, and reduced batch-to-batch variability lowered the rate of duplicate testing, resulting in an overall production cost reduction of approximately 30%. Simultaneously, the improved cells' tolerance to fixation and storage stress enhanced the kit's shelf life at 4°C, extending it from 6 months to 12 months.

[0055] The essential difference between this invention and the prior art is that the prior art only makes improvements at the level of antigen gene construction and transfection condition optimization, while this invention is the first to systematically recognize that the basic state of the detected cells is the key limiting factor for the sensitivity and stability of CBA detection. It synergistically modifies the detection cells from three levels: translation regulation, protein folding and cell apoptosis, to achieve an overall improvement in the stability of antigen expression. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Appendix Figure 1 This is a graph showing the expression level of the target protein as detected by cellular immunofluorescence, with modified cells on the left and ordinary cells on the right.

[0058] Appendix Figure 2 This is a comparison of apoptosis levels in the modified cells and ordinary cells after transfection with the target gene according to the present invention.

[0059] Appendix Figure 3 This is a comparison diagram showing the cell viability of the modified cells and ordinary cells after transfection with the target gene according to the present invention. Detailed Implementation

[0060] Example 1:

[0061] See Figure 1 This invention provides a method for preparing a detection kit that enhances the detection efficacy of autoimmune encephalitis antibodies, comprising the following steps:

[0062] (1) Construct a recombinant expression vector that co-expresses the eIF2α S52A mutant and heat shock protein 70 (HSP70), wherein the eIF2α S52A mutant is obtained by mutating serine at position 52 of wild-type eIF2α to alanine;

[0063] (2) The recombinant expression vector was introduced into 293T cells, and stable transfected cell lines co-expressing eIF2α S52A mutant and heat shock protein 70 (HSP70) were screened to obtain stable transfection cell lines.

[0064] (3) Before antigen transfection, the stable transfected cell lines obtained in step (2) are subjected to stress preconditioning treatment;

[0065] (4) Transiently transfect the gene encoding the autoimmune encephalitis-associated antigen into a stable transfected cell line that has undergone stress preconditioning treatment, so that the cells express the antigen;

[0066] (5) The cells expressing the antigen were fixed and blocked, and the detection kit was assembled.

[0067] The construction of the recombinant expression vector in step (1) includes: cloning the coding sequences of eIF2α S52A and HSP70 into a lentiviral expression vector by tandem T2A sequence through overlap extension PCR.

[0068] Furthermore, the stress preconditioning treatment is as follows: heat shock temperature 40-43℃, time 30-60 min; H2O2 concentration 30-100 μM, treatment time 15-45 min; serum deprivation time 0.5-2 h; high serum recovery time 2-6 h; resting recovery time 8-16 h.

[0069] The preferred option is as follows:

[0070] (a) Heat shock treatment at 42℃ for 45 min, followed by incubation at 37℃ for 2 h;

[0071] (b) Add H2O2 to the culture medium to a final concentration of 50 μM, treat for 30 min, and then replace with fresh culture medium;

[0072] (c) Incubate in serum-free medium for 1 h, then replace with complete medium containing 20% ​​fetal bovine serum and incubate for 4 h;

[0073] (d) Incubate at rest for 12 h in a 37℃, 5% CO2 incubator.

[0074] Furthermore, the lentiviral expression vector is pLVX-EF1α-IRES-Puro.

[0075] Further, the importation in step (2) includes: co-transfecting 293T cells with the recombinant expression vector, packaging plasmid psPAX2 and envelope plasmid pMD2.G to package lentivirus, infecting 293T cells with the obtained lentivirus, and then selecting stable transfected cell lines by puromycin.

[0076] Further, the autoimmune encephalitis-related antigen mentioned in step (4) is selected from at least one of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and GAD65.

[0077] Further, the fixation treatment in step (5) is performed using a 3% paraformaldehyde solution. The fixation is performed using 3% paraformaldehyde for 15 min.

[0078] The cell fixation method of the detection kit includes the following steps:

[0079] Mild crosslinking: Cells were treated with a mixture of 0.5% glutaraldehyde and 0.1% paraformaldehyde for 5 min;

[0080] Post-repair: Cells were treated for 15 min with a repair solution containing 0.5% glycine, 0.1% sodium borohydride, 1% BSA and 0.05% Triton X-100.

[0081] The patient was washed twice with PBS between the mild crosslinking step and the post-repair step.

[0082] The concentration range of glutaraldehyde is 0.2%-1.0%, and the concentration range of paraformaldehyde is 0.05%-0.5%.

[0083] Further, in step (5), after cell fixation, the cells were incubated at 4°C for 30 min using the treatment solution, followed by blocking. The treatment solution was an antigen conformation stabilizing solution, and its composition by weight / volume percentage was: 0.5%-2.0% trehalose, 0.1%-0.5% betaine, 0.05%-0.2% polyethylene glycol 400, 0.01%-0.05% sodium azide, with the remainder being PBS buffer (pH 7.4).

[0084] This invention provides a detection kit prepared by the aforementioned method, comprising:

[0085] Genetically engineered 293T cells, wherein the eIF2α S52A mutant and HSP70 are stably integrated and co-expressed, and wherein the cells are transiently transfected or stably integrated with the encoding gene of autoimmune encephalitis-related antigen.

[0086] In addition to cell fixative, blocking solution, negative and positive control sera, and fluorescently labeled secondary antibodies.

[0087] The detection kit also includes a cell fixation posttreatment solution, which is an antigen conformation stabilizing solution. Its composition, by weight / volume percentage, is: 0.5%-2.0% trehalose, 0.1%-0.5% betaine, 0.05%-0.2% polyethylene glycol 400, 0.01%-0.05% sodium azide, with the balance being PBS buffer (pH 7.4).

[0088] The trehalose content is 1.0%, betaine content is 0.3%, and polyethylene glycol 400 content is 0.1%.

[0089] The genetically engineered 293T cells in the test kit are present in a lyophilizable test cell preparation. The lyophilizable test cell preparation contains genetically engineered 293T cells expressing antigens, and a lyophilization protectant solution. The lyophilization protectant solution consists of 10% trehalose, 5% sucrose, 2% mannitol, 1% polyvinylpyrrolidone, and 0.5% gelatin.

[0090] The formulation is prepared by the following method: cells are incubated at 5 × 10 5 Cells / mL were suspended in a lyophilization protectant and pre-frozen at -40°C at a rate of 1°C / min. Then, they were dried at -30°C and 10 Pa vacuum for 24 h, and then dried at 20°C and 1 Pa vacuum for 6 h.

[0091] The formulation can be stored at room temperature. When used, it can be incubated at 37°C for 15 min with pre-warmed refolding solution (DMEM containing 10% FBS) to restore its activity.

[0092] Furthermore, this application provides a method for evaluating antibody affinity using the aforementioned detection kit, comprising the following steps:

[0093] Step (i) Incubate and wash the serum to be tested with the genetically engineered 293T cells expressing antigens from the test kit;

[0094] Step (ii) divides the incubated cells into a control group and a dissociation group. The dissociation group is treated with a solution containing potassium thiocyanate (KSCN), while the control group is treated with a solution without KSCN.

[0095] Step (iii) Add fluorescently labeled secondary antibody to both groups of cells for detection and obtain fluorescence intensity;

[0096] Step (iv) calculates the KSCN concentration that results in 50% antibody dissociation as the affinity index.

[0097] Further, in step (ii), the concentration of the KSCN solution is selected from at least two of 0.5 M, 1.0 M, 1.5 M and 2.0 M.

[0098] Furthermore, the treatment time of the KSCN solution in step (ii) is 10 min.

[0099] Example 2:

[0100] Combination Figures 1 to 3 This embodiment details the relevant steps in reagent kit preparation:

[0101] Phase 1: Plasmid Construction and Validation

[0102] 1. Plasmid construction:

[0103] Two primers for the eIF2α S52A and HSP70 genes were designed: eIF2α-F, eIF2α-S52A-R; eIF2α-S52A-F, eIF2α-T2A-R; and T2A-HSP70-F, HSP70-R.

[0104] The eIF2α S52A and HSP70 gene fragments were amplified separately using a PCR kit. First, eIF2α-F+S52A-R and S52A-F+T2A-R were used in two separate tubes for amplification. After fragment recovery, the fragments were mixed and amplified again using eIF2α-F+T2A-R to obtain eIF2α S52A. Next, T2A-HSP70-F+HSP70-R was used to amplify T2A-HSP70. Finally, the products from the first two steps were mixed and amplified using eIF2α-F+HSP70-R to obtain the complete insert fragment eIF2α S52A-T2A-HSP70. A 1% agarose gel was prepared, electrophoresis was performed, and the target gene fragment was recovered by gel excision. The PCR system and procedure are as follows:

[0105] PCR system (50 μL)

[0106] 5× HF buffer 10 μL;

[0107] dNTP (10 mM) 4 μL;

[0108] F primer (10 μM) 2.5 μL;

[0109] R primer (10 μM) 2.5 μL;

[0110] 1 μL of cDNA template;

[0111] Phusion high-fidelity enzyme 0.5 μL;

[0112] Add ddH2O to make up to 50 μL.

[0113] PCR procedure:

[0114] 98℃ for 30 seconds;

[0115] 98℃ 10 s → 60℃ 20 s → 72℃ 1 kb / min, 35 cycles;

[0116] 72℃ for 5 minutes;

[0117] Keep warm at 4℃.

[0118] The obtained target fragment was double-digested with the vector pLVX-EF1α-IRES-Puro, incubated at 37°C for 3 h, and then incubated in a water bath at 65°C for 20 min. The preparation system was as follows:

[0119] DNA / plasmid 2 μg

[0120] 10× CutSmart buffer 5 μL

[0121] EcoRI 1 μL

[0122] Xba I 1 μL

[0123] Add ddH2O to a final volume of 50 μL.

[0124] The enzyme digestion products were recovered to obtain the enzyme-digested tandem fragments and the linearized pLVX-EF1α-IRES-Puro vector.

[0125] The product obtained in the previous step was subjected to a ligation reaction and incubated overnight at 16°C. The formulation system was as follows:

[0126] 100 ng of linear carrier

[0127] Insertion fragment molar ratio 1:3

[0128] T4 DNA ligase 1 μL

[0129] 10× buffer 2 μL

[0130] Add ddH2O to a final volume of 20 μL.

[0131] Add 10 μL of the ligation product to 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and incubate on ice for 3 min. Add 900 μL of antibiotic-free liquid culture medium and incubate at 37℃ and 200 rpm for 1 h. Spread 200 μL of the bacterial culture evenly on an ampicillin LB agar plate and incubate upside down at 37℃ for 16 h.

[0132] Pick 10 single colonies and perform PCR using eIF2α-F + HSP70-R. The appearance of a band of the expected size indicates a positive colony. Inoculate the positive colonies into 10 mL of LB medium containing ampicillin and incubate overnight at 37°C. Extract the plasmid using a plasmid miniprep kit.

[0133] The recombinant plasmid was sequenced to verify that the genes were correctly ligated and free of mutations.

[0134] 2. Plasmid amplification and extraction:

[0135] The correctly sequenced plasmid was transformed into DH5α competent cells, cultured and amplified at 37°C, and high-purity plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0136] Phase Two: Lentiviral Packaging

[0137] 1. Cell preparation:

[0138] Prepare DMEM + 10% FBS complete medium and culture 293T cells in a 37°C, 5% CO2 incubator (Example 2). 24 h before transfection, cells were sputtered at 5 × 10⁻⁶ cells / mL. 6 Plant cells at a density of 10 cm in a culture dish to ensure a cell density of 70-80% during transfection.

[0139] 2. Transfection steps:

[0140] Add 40 μL of PEI transfection reagent (1 mg / mL) to the plasmid mixture and incubate at room temperature for 20 min. Add the mixture dropwise to 293T cell culture dishes, gently mix, and incubate at 37°C in a 5% CO2 incubator. The plasmid mixture ratio is as follows:

[0141] Recombinant transfer plasmid: pLVX vector carrying the target gene (10 μg)

[0142] Packaging plasmid: psPAX2 (7.5 μg)

[0143] Encapsulation plasmid: pMD2.G (2.5 μg)

[0144] The total plasmid amount was 20 μg, which was mixed in Opti-MEM medium (500 μL) according to the ratio.

[0145] Replace with fresh complete culture medium after 6 hours.

[0146] 3. Virus collection and concentration:

[0147] Supernatant was collected at 48 h and 72 h post-transfection and filtered through a 0.45 μm filter membrane to remove cell debris and other impurities. After aliquoting, the supernatant was stored at -80°C to avoid repeated freeze-thaw cycles.

[0148] Phase 3: Viral infection and selection of stable transfected cell lines

[0149] 1. Viral infection:

[0150] 24 hours before infection, 293T cells were seeded into 6-well plates at a density of approximately 50%.

[0151] Add 1 mL of virus supernatant and 1 mL of fresh culture medium to each well, and add polybrene to a final concentration of 8 μg / mL to enhance virus infection efficiency;

[0152] After culturing the cells in a 37°C, 5% CO2 incubator for 24 h, the culture medium was replaced with fresh complete culture medium.

[0153] 2. Puromycin screening:

[0154] Forty-eight hours after infection, 2 μg / mL of Puromycin was added to each well for screening.

[0155] The culture medium containing Puromycin was changed every 2 days, and the screening was continued for 10 days until all uninfected cells died.

[0156] 3. Monocloning:

[0157] Use a sterile pipette tip to pick up a single resistant clone, transfer it to a 96-well plate for expansion culture, and then gradually passage it to 24-well plates, 6-well plates, and 10cm culture dishes.

[0158] Phase 4: Validation of stable transfected cell lines

[0159] Expression level validation (Western Blot): Detection of eIF2α S52A and HSP70 protein expression. The specific steps are as follows:

[0160] Prepare 293T stable transfected cell lines (cultured to 70-80% confluence) in 10cm culture dishes, uninfected blank 293T cells (negative control), and 293T cells transfected with the empty vector pLVX-EF1α-IRES-Puro (empty vector control).

[0161] Discard the culture medium from the cell culture dish, rinse the cell surface twice with pre-chilled PBS buffer, add 200 μL of pre-chilled RIPA lysis buffer, and gently scrape cells from the bottom of the culture dish using a cell scraper. Transfer the cell lysis buffer to a 1.5 mL pre-chilled EP tube and incubate on ice for 30 min. Centrifuge at 12000 rpm for 15 min at 4°C, and transfer the supernatant (containing soluble protein) to a new EP tube. Analyze the protein concentration in the supernatant using a BCA protein quantification kit.

[0162] Take 30 μg of protein sample, add 5× loading buffer, and bring the total volume to 20 μL. Heat in a boiling water bath for 10 min, then immediately cool in an ice bath for 5 min. Assemble the SDS-PAGE electrophoresis tank, add electrophoresis buffer, and load the processed samples sequentially. First, perform electrophoresis at a constant voltage of 80 V for 30 min, then switch to a constant voltage of 120 V for 90 min. After electrophoresis, remove the gel for later use.

[0163] Soak the PVDF membrane in methanol for 15 seconds beforehand. Assemble the transfer sandwich in the order of "cathode plate → filter paper → gel → membrane → filter paper → anode plate", place it in the transfer tank, add transfer buffer, and transfer at 100 V constant voltage for 90 min at 4℃.

[0164] Place the membrane in 5% skim milk blocking buffer and incubate on a shaker at room temperature for 1 hour. Discard the blocking buffer and wash the membrane three times with TBST buffer for 10 minutes each time. Cut the membrane to the marker size, add the corresponding diluted primary antibody, and incubate overnight on a shaker at 4°C. Discard the primary antibody and wash the membrane three times with TBST for 10 minutes each time. Add the diluted HRP-labeled secondary antibody and incubate on a shaker at room temperature for 1 hour. Discard the secondary antibody and wash the membrane four times with TBST for 10 minutes each time.

[0165] Following the ECL kit instructions, mix solutions A and B in a 1:1 ratio, and evenly drop the mixture onto the membrane, covering the entire target protein region. Let it stand at room temperature for 3 minutes. Blot away excess luminescent solution with filter paper, place the membrane into the chemiluminescence imaging system, adjust the exposure time, and record the band intensity for each sample.

[0166] The stable transfected strains showed specific target protein bands, with expression significantly stronger than the negative and control groups, proving that the target protein was successfully expressed.

[0167] Phase 5: Reagent Kit Preparation

[0168] 1. Target gene transfection:

[0169] Stable transfected cells were seeded into 96-well plates and transfected using the Lipo300 transfection kit when the cell density reached 70%.

[0170] Prepare system 1 by mixing 10 μg of target gene plasmid (e.g., NMDAR), 20 μl of p3000 and 500 μl of Opti-MEM, and prepare system 2 by mixing 500 μl of Opti-MEM and 30 μl of Lipo3000. Then mix system 1 and system 2, incubate at room temperature for 15 min, add evenly to each well of cells, and incubate at 37°C in a 5% CO2 incubator.

[0171] 2. Cell fixation:

[0172] Forty-eight hours after transfection, the culture medium was discarded, the well plates were washed twice with PBS, and the cells were fixed with 3% paraformaldehyde for 15 min, followed by two more washes with PBS. The fixed 96-well plates were then treated with preservative solution (KV300) to prepare an autoimmune encephalitis antibody detection kit (containing an antigen-coated 96-well plate, goat serum blocking solution, PBS, negative and positive control sera, and Alexa Fluor 555 Anti-human IgG secondary antibody), and stored at 4°C for later use.

[0173] Reagent kit staining procedure

[0174] Warming: Take the well plate and related reagents out of the 4℃ refrigerator and warm them to room temperature for 10-15 min before staining.

[0175] Washing: Discard the liquid in the wells, add 150ul PBS to each well (add the liquid along the side wall, the same below), wash twice, 3min each time (medium speed on a horizontal shaker, the same below).

[0176] Blocking: Discard PBS, add 80 μL of sample diluent (10% sheep serum) to each well, and incubate at 37°C for 30 min.

[0177] Sample incubation: Add 8 μL of serum directly to the well, mix on a shaker for 3-5 min, and incubate at 37°C for 1 h.

[0178] Washing: Discard the liquid in the wells, add 150 μL of PBS to each well, and wash 3 times on a shaker for 3 minutes each time.

[0179] Secondary antibody incubation: Discard the liquid in the wells, add 80 μL of secondary antibody working solution to each well, mix well on a shaker for 3-5 min, and incubate at 37°C for 30 min.

[0180] The following steps require protection from light.

[0181] Washing: Discard the liquid in the wells, add 150 μL of PBS to each well, and wash 3 times on a shaker for 3 minutes each time. Discard the liquid in the wells, add 150 μL of PBS to each well to cover the cells.

[0182] The experimental results were observed under a fluorescence microscope.

[0183] Antibody titer determination: Positive sera can be further titered. Serum samples can be diluted at ratios of 1:32, 1:100, 1:320, and 1:1000 based on fluorescence intensity, and the above staining steps can be repeated. The lowest concentration or highest dilution that yields a positive result is taken as the autoantibody titer of the sample.

[0184] Figure 2 To compare the modified cells and ordinary cells after transfection with the target gene, flow cytometry (Annexin V / PI double staining method) was used to detect the apoptosis ratio in the bar chart. The results showed that the apoptosis ratio in the modified cell group of the present invention was significantly lower than that in the ordinary cell group. Figure 3 The bar charts of cell viability were obtained by transfecting the target gene into the modified cells and ordinary cells using the CCK-8 assay. The results showed that the cell viability of the modified cell group was higher than that of the ordinary cell group, indicating that the modified cells had stronger viability.

[0185] Test results as follows Figure 1The diagram shows a comparison of the expression levels of the target protein in the modified cells and ordinary cells after transfection with the target gene according to this invention; the left side represents the modified cells, and the right side represents the ordinary cells.

[0186]

[0187] The table above shows the results of CBA assays on 20 positive serum samples from patients with clinical autoimmune encephalitis and 20 negative serum samples from healthy controls. The results indicate that the kit prepared using the modified cell group has significantly higher sensitivity and specificity than the kit prepared using the ordinary cell group.

[0188] Example 3:

[0189] To further improve the performance of genetically engineered 293T cells in CBA assays, this invention also provides a preferred cell pretreatment method. This method is performed before transfecting the target antigen into a stable transfected cell line.

[0190] The specific steps are as follows:

[0191] Stable transfected 293T cells expressing eIF2α S52A and HSP70, prepared in Example 1, were cultured in DMEM + 10% FBS complete medium. Stress preconditioning treatment was then performed according to the following procedure:

[0192] (1) The cells were placed in a 42℃, 5% CO2 incubator for 45 min and then restored to 37℃ for 2 h.

[0193] (2) Add H2O2 to the culture medium to a final concentration of 50 μM, treat for 30 min, and then replace with fresh culture medium;

[0194] (3) The cells were cultured in serum-free DMEM for 1 h, and then replaced with complete medium containing 20% ​​FBS for 4 h.

[0195] (4) Place the cells in a 37℃, 5% CO2 incubator for 12 h of static culture.

[0196] After treatment, the cells can be used for subsequent transfection of the target gene. Experimental results showed that, in the modified cells treated with the above preconditioning, the expression level of the target protein was increased by 2.2 times compared to the untreated group 48 h after transfection, the apoptosis rate decreased from 7.2% to 2.5%, and the signal attenuation rate of the detection kit decreased from 28% to 9% after 6 months of storage at 4°C. Compared with unmodified ordinary 293T cells, the gene modification and stress preconditioning treatment of this invention have a significant synergistic effect: preconditioning can further increase the expression level of exogenous HSP70 by 1.8 times, while reducing the phosphorylation level of eIF2α.

[0197] Example 4:

[0198] This embodiment provides an antigen conformation stabilizing solution for post-cell fixation treatment to further improve the retention rate of antigen epitopes.

[0199] The stabilizer consists of the following components (w / v): 1.0% trehalose, 0.3% betaine, 0.1% polyethylene glycol 400 (PEG400), 0.02% sodium azide, with the remainder being PBS buffer (pH 7.4).

[0200] Instructions for use: After fixing cells with 3% paraformaldehyde for 15 min and washing with PBS, add 100 μL of the above antigen conformation stabilization solution to each well, incubate at 4°C for 30 min, then discard the stabilization solution and add preservative solution to preserve the kit.

[0201] Comparative experiments showed that after treatment with this stabilizing solution, the conformational epitope retention rate of NMDAR antigen increased from 40% to 85% under conventional treatment, and the signal-to-noise ratio increased from 8.5:1 to 14.2:1.

[0202] Example 5:

[0203] This embodiment provides an optimized cell fixation method to replace the conventional single-step paraformaldehyde fixation method.

[0204] The specific steps are as follows:

[0205] (1) Mild cross-linking: Discard the cell culture medium, wash once with PBS, add a mixed fixative of 0.5% glutaraldehyde + 0.1% paraformaldehyde, and treat at room temperature for 5 min;

[0206] (2) Post-repair: Discard the fixative, wash twice with PBS, add retrieval solution (0.5% glycine, 0.1% sodium borohydride, 1% BSA, 0.05% Triton X-100 dissolved in PBS), and incubate at 4°C for 15 min;

[0207] (3) After washing with PBS, the antigen can be blocked as usual or an antigen conformation stabilizing solution can be added.

[0208] Experimental results show that after using this two-step fixation method, the epitope retention rate of AMPAR antigen increased from 58% to 89%, and the background fluorescence intensity decreased by 67%.

[0209] Example 6:

[0210] This embodiment provides a method for simultaneously detecting antibody presence and affinity maturity using the kit of the present invention.

[0211] Experimental steps:

[0212] (1) Take the stable transfected cells prepared in Example 1, fix them according to the conventional method, and incubate them with the serum to be tested (1:100 dilution) at 37°C for 1 h;

[0213] (2) After washing with PBS 3 times, the cells were divided into 5 groups: control group (PBS treatment) and dissociation group (treated with 0.5 M, 1.0 M, 1.5 M, and 2.0 M KSCN solutions for 10 min respectively).

[0214] (3) After washing with PBS, all groups were incubated with Alexa Fluor 555-labeled anti-human IgG secondary antibody for 30 min at 37°C.

[0215] (4) Detect the fluorescence intensity of each well under a fluorescence microscope and calculate the fluorescence retention rate of each dissociation group relative to the control group;

[0216] (5) The concentration of KSCN that causes 50% of the antibody to dissociate is calculated by nonlinear fitting, which is the affinity index (AI).

[0217] Clinical validation:

[0218] Serum samples from 20 clinically diagnosed patients with autoimmune encephalitis were analyzed, including 10 samples from patients in the acute phase (severe clinical symptoms requiring ICU treatment) and 10 samples from patients in the recovery phase (symptom relief, hormone dosage tapering). Results showed that the mean serum AI value in the acute phase was 1.45±0.23 M, with all samples ≥1.2 M; the mean serum AI value in the recovery phase was 0.65±0.18 M, with all samples <0.8 M. Using AI ≥1.2 M as the criterion for acute phase diagnosis, the sensitivity and specificity were 100%. This indicates that this method can effectively differentiate between patients in the acute and recovery phases and has significant clinical auxiliary diagnostic value.

[0219] Thus far, the description of the above embodiments has been provided for illustrative and descriptive purposes. This is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of particular embodiments are generally not limited to those particular embodiments, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. In many respects, the same elements or features may also be varied. Such variations are not considered a departure from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0220] Example embodiments are provided so that this disclosure will become thorough and will fully convey the scope to those skilled in the art. Numerous details, such as examples of specific parts, apparatus, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that the specific details are not required, and the example embodiments may be implemented in many different forms, neither of which should be construed as limiting the scope of this disclosure. In some example embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0221] Technical terms are used herein for the purpose of describing specific exemplary embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein may also refer to the plural forms. The terms “comprising” and “having” are inclusive and therefore specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or additional having of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof. Unless expressly indicated in order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the specific order discussed and shown. It should also be understood that additional or optional steps may be employed.

Claims

1. A method for preparing a detection kit that enhances the detection efficacy of autoimmune encephalitis antibodies, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector that co-expresses the eIF2α S52A mutant and heat shock protein 70 (HSP70), wherein the eIF2α S52A mutant is obtained by mutating serine at position 52 of wild-type eIF2α to alanine; (2) The recombinant expression vector was introduced into 293T cells, and stable transfected cell lines co-expressing eIF2α S52A mutant and heat shock protein 70 (HSP70) were screened to obtain stable transfection cell lines. (3) Before antigen transfection, the stable transfected cell lines obtained in step (2) are subjected to stress preconditioning treatment; (4) Transiently transfect the gene encoding the autoimmune encephalitis-associated antigen into a stable transfected cell line that has undergone stress preconditioning treatment, so that the cells express the antigen; (5) The cells expressing the antigen were fixed and blocked, and the detection kit was assembled. The construction of the recombinant expression vector in step (1) includes: cloning the coding sequences of eIF2α S52A and HSP70 into a lentiviral expression vector by tandem T2A sequence through overlap extension PCR.

2. The preparation method according to claim 1, characterized in that, The stress preconditioning process is as follows: (a) Heat shock treatment at 42℃ for 45 min, followed by incubation at 37℃ for 2 h; (b) Add H2O2 to the culture medium to a final concentration of 50 μM, treat for 30 min, and then replace with fresh culture medium; (c) Incubate in serum-free medium for 1 h, then replace with complete medium containing 20% ​​fetal bovine serum and incubate for 4 h; (d) Incubate at rest for 12 h in a 37℃, 5% CO2 incubator.

3. The preparation method according to claim 1, characterized in that, The lentiviral expression vector is pLVX-EF1α-IRES-Puro.

4. The preparation method according to claim 1, characterized in that, The import described in step (2) includes: The recombinant expression vector was co-transfected with packaging plasmid psPAX2 and envelope plasmid pMD2.G into 293T cells to package lentivirus. The obtained lentivirus was used to infect 293T cells, and stable transfected cell lines were obtained by selection with puromycin.

5. The preparation method according to claim 1, characterized in that, The autoimmune encephalitis-related antigen mentioned in step (4) is selected from at least one of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and GAD65.

6. The preparation method according to claim 1, characterized in that, The fixation treatment in step (5) is performed using a 3% paraformaldehyde solution, and the blocking treatment is performed using goat serum or BSA.

7. A test kit prepared by the method according to any one of claims 1-6, characterized in that, include: Genetically engineered 293T cells, wherein the eIF2α S52A mutant and HSP70 are stably integrated and co-expressed, and wherein the cells are transiently transfected or stably integrated with the encoding gene of autoimmune encephalitis-related antigen. In addition to cell fixative, blocking solution, negative and positive control sera, and fluorescently labeled secondary antibodies.

8. A method for immunofluorescence detection and antibody affinity assessment using the detection kit of claim 7, characterized in that, Includes the following steps: Immunofluorescence assay: Warming: The plates to be used and the detection kit described in claim 7 were taken out of the 4 ℃ refrigerator and warmed to room temperature for 10-15 min before staining; Washing: Discard the liquid in the wells, add 150 μL of PBS to each well, and wash twice for 3 minutes each time; Blocking: Discard PBS, add 80 μL of sample diluent (10% goat serum) to each well, and incubate at 37°C for 30 min; Sample incubation: Add 8 μL of serum directly to the well, mix on a shaker for 3-5 min, and incubate at 37°C for 1 h; Washing: Discard the liquid in the wells, add 150 μL of PBS to each well, and wash 3 times on a shaker for 3 minutes each time; Secondary antibody incubation: Discard the liquid in the wells, add 80 μL of secondary antibody working solution to each well, mix well on a shaker for 3-5 min, and incubate at 37°C for 30 min. Washing: Discard the liquid in the wells, add 150 μL PBS to each well, and wash 3 times on a shaker for 3 min each time; discard the liquid in the wells, add 150 μL PBS to each well, and cover the cells; Observe the experimental results under a fluorescence microscope; Assess antibody affinity: Step (i) Incubate and wash the serum to be tested with the genetically engineered 293T cells expressing antigens from the test kit; Step (ii) divides the incubated cells into a control group and a dissociation group. The dissociation group is treated with a solution containing potassium thiocyanate (KSCN), while the control group is treated with a solution without KSCN. Step (iii) Add fluorescently labeled secondary antibody to both groups of cells for detection and obtain fluorescence intensity; Step (iv) calculates the KSCN concentration that results in 50% antibody dissociation as the affinity index.

9. The method according to claim 8, characterized in that, In step (ii), the concentration of the KSCN solution is selected from at least three of the following: 0.5 M, 1.0 M, 1.5 M, and 2.0 M.

10. The method according to claim 8, characterized in that, The treatment time for the KSCN solution in step (ii) is 10 min.

Images