Fusion protein for detecting anti-LGI1 antibody and application thereof

Abstract

The invention relates to a fusion protein for detecting an anti-LGI1 antibody and application of the fusion protein. The invention particularly relates to a fusion protein for detecting an anti-LGI1 antibody, the fusion protein sequentially comprises a kappa chain signal peptide, an LGI1 protein and a PDGFR beta transmembrane peptide from an N terminal to a C terminal, and the amino acid sequence of the LGI1 protein is shown as SEQ ID NO.1. The kit prepared from the fusion protein has the advantages of simplicity, accuracy, high sensitivity and the like in LGI1 antibody detection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a fusion protein for detecting anti-LGI1 antibodies and its uses. Background Technology

[0002] Autoimmune encephalitis (AE) is an immune-mediated neurological disorder mediated by autoantibodies against neurons, which can cause symptoms such as cognitive impairment, behavioral abnormalities, seizures, mental disorders, involuntary movements, and autonomic dysfunction. In 2007, Dalmau first reported a group of patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Over the following decade, a series of autoantibodies against neuronal cell surfaces or synaptic proteins were subsequently discovered.

[0003] Leucine-rich glioma-inactivated 1 protein (LGI1) encephalitis is the second most common autoimmune encephalitis after anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Anti-LGI1 encephalitis typically presents with seizures, cognitive and psychiatric disturbances. With timely detection and treatment, clinical symptoms may improve. It primarily causes relatively reversible neuronal dysfunction through humoral immune mechanisms, and early immunotherapy is effective; however, diagnosis relies on the detection of neuronal antibodies.

[0004] Currently, co-transfection with full-length wild-type LGI1 and ADAM22 / 23 is commonly used (Lai M, Huijbers MG, Lancaster E, et al. Investigation of LGI1 as the antigen in limbicencephalitis previously attributed to potassium channels: a case series. Lancet Neurol 2010;9:776–85.), and anti-LGI1 antibodies are detected by cell-based immunofluorescence assay (CBA).

[0005] However, recent reports indicate that some anti-LGI1 antibodies compete with ADAM, preventing them from binding to the co-expressed LGI+ADAM complex (Kreye J, Wenke NK, Chayka M, et al. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis. Brain 2016; 139: 2641–2652.). Furthermore, if the expressed LGI1 does not bind to ADAM, it exists as a free protein. Free LGI1 binding to antibodies is washed away, reducing the detection signal. Furthermore, the dual-plasmid system may screen for ADAM22 / 23 antibodies instead of LGI1 antibodies, which could be mistakenly identified as LGI1 antibodies (Pinfei Ni, Lin Bai, Nan Jiang et al. Case Report: Anti-ADAM23 antibody: an overlooked autoantibody against VGKC-complex in autoimmune encephalitis. Front Immunol. 2025 May 30). Therefore, existing LGI1 detection systems have certain problems, which may lead to weaker detection signals or detection errors.

[0006] Therefore, there is an urgent need for a diagnostic reagent and method that can accurately detect LGI1 antibodies. Summary of the Invention

[0007] In view of this, the main objective of the present invention is to provide a simple and accurate method for detecting LGI1 antibodies.

[0008] The specific technical solution of the present invention is as follows.

[0009] This invention provides a fusion protein for detecting anti-LGI1 antibodies, comprising, from the N-terminus to the C-terminus, a κ chain signal peptide, an LGI1 protein, and a PDGFR β transmembrane peptide, wherein the amino acid sequence of the LGI1 protein is shown in SEQ ID NO.1.

[0010] In some embodiments, the amino acid sequence of the PDGFR β transmembrane peptide is shown in SEQ ID NO.2.

[0011] In some embodiments, the amino acid sequence of the κ chain signal peptide is shown in SEQ ID NO.3.

[0012] The present invention also provides an isolated or synthesized nucleic acid molecule that encodes the fusion protein of the present invention.

[0013] In some implementations, the sequence of the nucleic acid molecule is shown in SEQ ID NO.7.

[0014] The present invention also provides a carrier comprising the nucleic acid molecule of the present invention.

[0015] In some implementations, the vector is an expression vector, which may be a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

[0016] The present invention also provides a host cell comprising the nucleic acid molecule or vector of the present invention.

[0017] The present invention also provides the use of the above-mentioned fusion protein in the preparation of a kit for detecting anti-LGI1 antibodies.

[0018] The present invention also provides the use of the above-mentioned fusion protein in screening anti-LGI1 antibodies.

[0019] The beneficial effects of this invention are that by integrating LGI1 protein with specific types of signal peptides and transmembrane regions to form a fusion protein, the resulting fusion protein becomes a transmembrane protein that can anchor itself to the cell membrane after expression. Thus, LGI1 antibody detection can be achieved without co-transferring ADAM22 / 23, reducing the cost of detection reagents and improving the convenience of detection.

[0020] Furthermore, compared to other improvements, such as removing the signal peptide or adding a glycosylphosphatidylinositol (GPI) anchoring method, the fusion protein of the present invention can more effectively identify both non-ADAM competing antibodies and ADAM competing antibodies, thereby giving the kit made from the fusion protein of the present invention higher sensitivity in LGI1 antibody detection. Attached Figure Description

[0021] Figure 1 The image shows the results of expressing LGI1 wild-type plasmid alone.

[0022] Figure 2 The image shows the results of co-transfection expression of LGI1-ADAM23 with two plasmids.

[0023] Figure 3 The image shows the results of LGI1 dSP plasmid expression.

[0024] Figure 4 This image shows the results of positive serum detection using the LGI1 dSP plasmid.

[0025] Figure 5The diagram shows the structure and expression results of the novel plasmids constructed in the experimental and control groups in Example 4.

[0026] Figure 6 Coomassie brilliant blue staining images of 12 human LGI1 monoclonal antibodies expressed and purified.

[0027] Figure 7 The results of screening ADAM competitive antibodies for the novel LGI1 protein are shown in the figure.

[0028] Figure 8 This is a diagram showing the results of using the novel LGI1 plasmid for actual clinical screening. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The following definitions supplement those in the art and relate to this application, but are not extrapolated to any relevant or unrelated circumstances, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0031] In this invention, the terms "comprising," "including," and "having" are open-ended descriptions that include the specified steps described, as well as other steps that do not substantially affect them, and are optional and not excluded.

[0032] As mentioned earlier, detecting LGI1 antibodies is a characteristic indicator for diagnosing LGI1 antibody encephalitis. Immunofluorescence-based detection methods are widely used for autoantibody detection due to their high specificity and accuracy. Conventional detection typically uses natural LGI1 expression. Since LGI1 is a secreted protein, it requires the simultaneous expression of membrane proteins ADAM22 / 23 to help transport and anchor LGI1 to the cell membrane. However, reports have shown that the presence of ADAM22 can hinder the binding of some LGI1 antibodies to LGI1, meaning that some ADAM-competing antibodies exist within LGI1 antibodies. Therefore, using traditional LGI1+ADAM detection will miss some ADAM-competing antibodies. Furthermore, when using a dual-plasmid expression system, LGI1 that is not bound to ADAM will remain free in the culture medium and be washed away during the rinsing step, reducing the detection signal. Additionally, the dual-plasmid system may screen for ADAM22 / 23 antibodies instead of LGI1 antibodies, leading to false positive results.

[0033] Therefore, to optimize the LGI1 antibody CBA method detection system, the inventors conducted extensive research and compared various improvement schemes, such as: 1) removing the signal peptide to turn LGI1 protein into an intracellular protein; 2) adding a transmembrane domain to turn LGI1 into a type I transmembrane protein; and 3) adding a glycosylphosphatidylinositol anchoring signal (GPI) to the C-terminus to turn LGI1 into a membrane-anchored protein. After verification and screening, a new LGI expression system was finally constructed to replace the traditional dual-plasmid expression detection system, thus completing this invention.

[0034] The first aspect of the present invention provides a fusion protein for detecting anti-LGI1 antibodies, comprising, from the N-terminus to the C-terminus, a κ chain signal peptide, an LGI1 protein, and a PDGFR β transmembrane peptide, wherein the amino acid sequence of the LGI1 protein is shown in SEQ ID NO.1.

[0035] SEQ ID NO.1:

[0036] MESERSKRMGNACIPLKRIAYFLCLLSALLLTEGKKPAKPKCPAVCTCTKDNALCENARSIPRTVPPDVISLSFVRSGFTEISEGSFLFTPSLQLLLFTSNSFDVISDDAFIGLPHLEYLFIENNNIKSISRHTFRGLKSLIHLSLANNNLQTLPKDIFKGLDSLTNVDLRGNSFNCDCKLKWLVEWLGHTNATVEDIYCEGPPEYKKRKINSLSSKDFDCIITEFAKSQDLPYQSLSIDTFSYLNDEYVVIAQPFTGKCIFLEWDHVEKTFRNYDNITGTSTVVCKPIVIETQLYVIVAQLFGGSHIYKRDSFANKFIKIQDIEILKIRKPNDIETFKIENNWYFVVADSSKAGFTTIYKWNGNGFYSHQSLHAWYRDTDVEYLEIVRTPQTLRTPHLILSSSSQRPVIYQWNKATQLFTNQTDIPNMEDVYAVKHFSVKGDVYICLTRFIGDSKVMKWGGSSFQDIQRMPSRGSMVFQPLQINNYQYAILGSDYSFTQVYNWDAEKAKFVKFQELNVQAPRSFTHVSINKRNFLFASSFKGNTQIYKHVIVDLSA

[0037] In some embodiments, the nucleotide sequence of the LGI1 protein is as shown in SEQ ID NO. 4.

[0038] SEQ ID NO.4:

[0039]

[0040] In this invention, by adding strong signal peptides and transmembrane regions to both ends of the LGI1 protein, the constructed fusion protein can become a single-transmembrane protein, thereby enhancing the expression of LGI1 when it is used for detection and effectively avoiding the spatial shielding effect of ADAM22 / 23, thus improving the detection efficiency.

[0041] In some embodiments, the amino acid sequence of the PDGFR β transmembrane peptide is shown in SEQ ID NO.2.

[0042] SEQ ID NO.2:

[0043] AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0044] In some implementations, the nucleotide sequence of the PDGFR β transmembrane peptide is shown in SEQ ID NO.5.

[0045] SEQ ID NO.5:

[0046] GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT

[0047] In some embodiments, the amino acid sequence of the κ chain signal peptide is shown in SEQ ID NO.3.

[0048] SEQ ID NO.3:

[0049] METDTLLLWVLLLWVPGSTGD

[0050] In some implementations, the nucleotide sequence of the κ chain signal peptide is shown in SEQ ID NO.6.

[0051] SEQ ID NO.6:

[0052] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC

[0053] In some embodiments, the amino acids of the fusion protein can be conserved variant sequences that do not affect its function, generated by mutation based on the above-described amino acid sequence. For example, it can be a conserved substitution of one or more amino acids in the above-described amino acid sequence, or the addition of one or more amino acids that do not affect its function (e.g., adding a linker peptide, protein tag sequence, etc.) to the N-terminus or C-terminus of the above-described amino acid sequence.

[0054] In some implementations, the protein tag sequence includes, but is not limited to, HA tag, Myc tag, His tag, GST tag, MBP tag, etc.

[0055] In some implementations, the linker peptide can be a flexible linker peptide rich in GS.

[0056] In some embodiments, the amino acids of the fusion protein can be derived sequences obtained by one or more modifications based on the above-mentioned amino acid sequence. For example, these modifications may include phosphorylation, PEGylation, amidation, glycosylation, biotinylation, and coupling or fusion with antibodies, vectors, ligands, albumin, Fc fragments, etc.

[0057] A second aspect of the present invention provides an isolated or synthesized nucleic acid molecule that encodes the fusion protein of the present invention.

[0058] Based on the amino acid sequence and codon rules of the fusion protein provided by this invention, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned fusion protein. Due to the degeneracy of codons, the nucleotide sequence encoding a single amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the above-mentioned fusion protein are within the protection scope of this invention.

[0059] In some implementations, the sequence of the nucleic acid molecule is shown in SEQ ID NO.7.

[0060] SEQ ID NO.7:

[0061]

[0062] In some implementations, nucleic acid molecules include DNA or RNA.

[0063] The present invention also provides a carrier comprising the nucleic acid molecule of the present invention.

[0064] In some implementations, the vector is an expression vector, which may be a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

[0065] In some embodiments, the viral vector includes, but is not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Preferably, the vector can transfer the nucleic acid molecules of the present invention into cells, such as T cells.

[0066] The present invention also provides a host cell comprising the nucleic acid molecule or vector of the present invention.

[0067] In some implementations, the host cell is a mammalian cell. For example, the host cell is a human cell. However, the host cell can be any cell type, can originate from any type of tissue, and can be a cell at any developmental stage.

[0068] In some implementations, methods for transfecting host cells include PEI transfection, lipofectamin 2000 transfection, lipofectamin 3000 transfection, other liposome transfection, electroporation, or other suitable transfection methods.

[0069] The present invention also provides the use of the above-mentioned fusion protein in the preparation of a kit for detecting anti-LGI1 antibodies.

[0070] In some embodiments, the kit includes the fusion protein of the present invention linked to a detectable marker.

[0071] In some implementations, the kit includes additional antigens or a second antibody.

[0072] In some implementations, the kit also includes a blocking solution and a working concentrate.

[0073] In some implementations, the blocking solution is goat serum and / or fetal bovine serum.

[0074] In some implementations, an additional antigen or secondary antibody is attached with a detectable marker.

[0075] In some implementations, the detectable marker is selected from luciferin or enzyme.

[0076] In some implementations, the second antibody is selected from one or more of the following: goat anti-human antibody, rat anti-human antibody, mouse anti-human antibody, pig anti-human antibody, donkey anti-human antibody, sheep anti-human antibody, chicken anti-human antibody, horse anti-human antibody, rabbit anti-human antibody, hamster anti-human antibody, dog anti-human antibody, or bovine anti-human antibody.

[0077] In some embodiments, the fluorescein is selected from one or more of fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, Texas Red fluorescent dye, phycoerythrin, propidium iodide, Alexa Fluor series fluorescent dyes, Dylight series fluorescent dyes, or iFluor series fluorescent dyes.

[0078] In some implementations, the working concentrate is selected from one or more of PBS, Triton 100, and Tween 20.

[0079] The present invention also provides the use of the above-mentioned fusion protein in screening anti-LGI1 antibodies.

[0080] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0081] Example 1 Expression of LGI1 wild-type plasmid

[0082] 1. Gene synthesis

[0083] In this embodiment, the LGI1 gene plasmid was synthesized by General Biotech.

[0084] LGI1 was synthesized according to the Uniprot: O95970 sequence, consisting of 557aa. After adding the N-terminal signal peptide, an HA tag was added to construct the pEGFP-N1 vector. The restriction enzyme sites were NheI and NotI, and the C-terminal GFP tag was removed. After successful sequencing, the plasmid was extracted for subsequent transfection.

[0085] 2. Cell transfection of LGI1 plasmid

[0086] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0087] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-60%.

[0088] The N1-LGI1 wild-type expression plasmid and the pMcherry-N1 (empty vector control) plasmid were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 48 h.

[0089] 3. Observe fluorescence expression under a microscope

[0090] 24 hours after cell transfection, cells were fixed with 1% PFA for 5 minutes, washed three times with PBS, and their expression was verified using anti-HA monoclonal antibody. Immunofluorescence staining results were observed using a Nikon TS2 microscope under a 20X objective lens. The results are as follows. Figure 1 As shown.

[0091] from Figure 1 As can be seen, when only LGI1 is transfected, most of the LGI1 expressed alone is in the cell, or the secreted LGI1 is released into the culture medium because ADAM22 / 23 is not present.

[0092] Example 2: LGI1 dual plasmid expression system

[0093] 1. Construction of LGI1 helper plasmid ADAM23

[0094] In this embodiment, the ADAM23 plasmid was synthesized by General Biotech.

[0095] ADAM23 was synthesized into 832aa cells according to the Uniprot: O75077 sequence. A Flag tag was added to the C-terminus, and the cells were constructed into the pEGFP-N1 vector. NheI and NotI restriction enzymes were used for restriction, and the C-terminal GFP tag was removed. After successful sequencing, the plasmid was extracted for subsequent transfection.

[0096] 2. Cell transfection with LGI1 dual plasmids

[0097] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0098] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-60%.

[0099] The N1-LGI1 and ADAM23 expression plasmids and the pMcherry-N1 (empty vector control) plasmid were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 48 h.

[0100] 3. Observe fluorescence expression under a microscope

[0101] 24 hours after cell transfection, cells were fixed with 1% PFA for 5 minutes and washed three times with PBS. The fixed cells were then used to verify expression using anti-HA and anti-Flag monoclonal antibodies. Immunofluorescence staining results were observed using a Nikon TS2 microscope under a 20X objective lens. The results are as follows: Figure 2 As shown.

[0102] from Figure 2 As can be seen, in the presence of ADAM23, LGI1 can be secreted and transported to the cell membrane together with its receptor ADAM23, and has good co-localization with ADAM23.

[0103] Example 3: Novel LGI1 Expression System - Intracellular Protein

[0104] 1. Construction of a new expression plasmid for LGI1

[0105] The LGI1 gene vector in Example 1 was modified by removing the LGI1 signal peptide (MESERSKRMGNACIPLKRIAYFLCLLSALLLTEG) using genetic engineering techniques (application number: CN202110163385.3). After successful sequencing, the plasmid was extracted for subsequent transfection.

[0106] 2. Cell transfection

[0107] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0108] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-50%.

[0109] The new LGI1 expression plasmids LGI1 dSP and pMcherry-N1 (empty vector control) were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 24-48 h.

[0110] 3. Observe fluorescence expression under a microscope

[0111] 24 hours after cell transfection, cells were fixed with 1% PFA for 5 minutes, washed three times with PBS, and the fixed cells were then used to verify expression using anti-HA monoclonal antibody. The results are as follows. Figure 3 As shown.

[0112] from Figure 3 As can be seen, the LGI1 protein expressed using the LGI1 dSP plasmid is located in the cytoplasm and has a strong expression signal.

[0113] 4. Clinical detection of intracellularly expressed LGI1 dSP

[0114] After confirming intracellular expression of LGI1, further testing was conducted to determine its suitability for clinical detection. Four LGI1-positive sera samples (stored in the Xuanwu Hospital laboratory) were tested; the specific testing procedure is as follows:

[0115] Take approximately 150-200 μL of anti-LGI1 antibody dilution buffer and add it to a 48-well plate transfected with LGI1 dSP plasmid. Incubate at 37°C for 1 hour in the dark. Wash 5 times with PBS. Add secondary antibody conjugated with Alexa Fluor 488 anti-human IgG and incubate for 1 hour. Wash 5 times with PBS. Observe and photograph the fluorescence under a fluorescence microscope (Nikon TS2) with a 20X objective lens. The results are as follows: Figure 4 As shown.

[0116] from Figure 4 As can be seen, none of the four positive samples were detected using LGI1 dSP. These results indicate that LGI1 protein requires further protein modification via the normal secretion pathway to achieve good antigenicity. Therefore, the secretion and processing of LGI1 protein are essential for its antigenicity. Simply removing the signal peptide from LGI1 protein is not sufficient for the detection of autoantibodies.

[0117] Example 4: Novel LGI1 Expression System - Membrane Surface Protein

[0118] The results of Example 3 show that the secretion and processing of LGI1 protein are very important for the antigenicity of the protein. Therefore, a new secretion expression scheme was designed in this example to make it exist on the cell membrane through different pathways.

[0119] 1. Construction of a new expression plasmid for LGI1

[0120] Experimental group: LGI1 transmembrane expression plasmid (LGI1-pDisplay plasmid, denoted as LGI1-TM, structure as follows) Figure 5 As shown in Figure A): The LGI1 gene vector in Example 1 was modified by replacing its own signal peptide with a κ chain signal peptide (nucleotide sequence as shown in SEQ ID NO. 6), and a transmembrane region of PDGFR β (nucleotide sequence as shown in SEQ ID NO. 5) was added to the C-terminus of LGI1. The restriction enzyme sites were SmaI and SalI. The inserted LGI1 gene had a Myc tag at the N-terminus and an HA tag at the C-terminus. After successful sequencing, the plasmid was extracted for subsequent transfection.

[0121] Control group: LGI1 membrane-anchored expression plasmid (LGI1-GPI plasmid, denoted as LGI1-GPI, structure as shown) Figure 5 (As shown in B): The gene in N1-LGI1 from Example 1 was modified by replacing its own signal peptide with a κ chain signal peptide (nucleotide sequence as shown in SEQ ID NO. 6). A GPI sequence (amino acid sequence as shown in SEQ ID NO. 8: SRSDPVTLNVRYESVQASSPDLSAGTAVSIMIGVLAGMALI, nucleotide sequence as shown in SEQ ID NO. 9: AGCCGCAGTGACCCAGTCACCCTGAATGTCCGCTATGAGTCAGTACAAGCAAGTTCACCTGACCTCTCAGCTGGGACCGCTGTCAGCATCATGATTGGAGTACTGGCTGGGATGGCTCTGATA) was added to the C-terminus. The C-terminus also contained an HA tag. After correct sequencing, the plasmid was extracted for subsequent transfection.

[0122] 2. Cell transfection

[0123] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0124] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-50%.

[0125] Two new LGI1 expression plasmids (LGI1-TM and LGI1-GPI) and pMcherry-N1 (empty vector control) from the experimental and control groups were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 24-48 h.

[0126] 3. Observe fluorescence expression under a microscope

[0127] 24 hours after cell transfection, cells were fixed with 1% PFA for 5 minutes, washed three times with PBS, and the fixed cells were then used to verify expression using anti-HA monoclonal antibody. The results are as follows. Figure 5 As shown in C.

[0128] Depend on Figure 5 It can be seen that LGI1 can be efficiently expressed in both new plasmids and exhibits typical membrane localization, indicating that LGI1 in both plasmids can be transported to the cell membrane via the secretory pathway and has the potential for LGI1 antibody detection.

[0129] Example 5: Screening of LGI1 human monoclonal antibodies

[0130] In this embodiment, monoclonal antibody screening is performed by referring to a previously established mature human B-cell antibody screening platform (e.g., patent applications with application numbers CN202410804339.0, CN202411526031.0, CN202311275512.4, and CN202410095226.8).

[0131] First, patients who were positive for LGI1 autoantibodies were screened using cell immunofluorescence technology, and the BCR sequences of the patients were obtained using BCR sequencing technology.

[0132] Patient inclusion: Patients from Xuanwu Hospital who tested positive for LGI1 antibodies were selected for cerebrospinal fluid cell collection. All patients signed informed consent forms.

[0133] LGI1 antibody-positive cerebrospinal fluid samples were sent to Novogene for single-cell sequencing to obtain antibody sequences with light and heavy chains. Based on the characteristics of the antibody amino acid sequence and single-cell distribution, different types of antibodies (12 groups in total) such as IgG1, IgG2, IgG3, or IgG4 were selected for further expression and validation.

[0134] The 12 pairs of heavy and light chains selected above were synthesized by completing human IgG1 κ / λ and optimizing the codons. The resulting gene was then inserted into the eukaryotic expression vector pTT5 (synthesized and subcloned by General Biotech) to obtain the heavy and light chain expression plasmid.

[0135] The specific method is as follows:

[0136] Expression of human LGI1 antibody

[0137] FreeStyle™ CHO-S cells were cultured to a density of 2.5-3.0 × 10⁶ cells / year. 6 Cells / mL, ready for transfection;

[0138] Add the human anti-LGI1 antibody expression plasmid (heavy and light chain plasmids, transfected at a ratio of 1:2) to 3 mL of OptiMEM and mix well;

[0139] Add 120µL of PEI MAX 40K (1mg / mL) to the above OptiMEM and mix well. Let stand at room temperature for 10-15 minutes.

[0140] Add the well-mixed plasmid and PEI mixture to 30 mL of FreeStyle™ CHO-S cells and transfect by shaking culture at 37°C.

[0141] 24 hours after transfection, VPA and glucose solution were added to final concentrations of 1 mM and 2 g / L, respectively.

[0142] Continue shaking culture for 4 days, then harvest the culture.

[0143] Purification of human anti-LGI1 antibody:

[0144] Centrifuge the collected FreeStyle™ CHO-S cell culture at 15,000 rpm and 4°C for 10 minutes, and collect the supernatant to remove dead cells and cell debris;

[0145] Add the appropriate volume of Protein A beads to the supernatant according to the instructions for use of Protein A beads, and rotate the supernatant and Protein A beads to combine and incubate.

[0146] After incubation for 1 hour, rinse Protein A beads with washing buffer 3-5 times to remove impurities that are weakly bound to beads or antibodies.

[0147] The antibodies bound to Protein A beads were then eluted with acidic glycine buffer (pH 3.0-3.5), and the elution buffer was then neutralized with 1 / 10 volume of 1M Tris-HCl pH 9.0 in acidic glycine buffer.

[0148] After Protein A affinity chromatography, the eluent was concentrated to a volume of 0.5 mL using an Amicon Ultra 4 mL ultrafiltration tube. A second molecular sieve chromatography step was performed using a BioCore SEC-300 column. The protein peak flow-through was collected and concentrated by ultrafiltration to obtain approximately 1 mL of human LGI1 antibody.

[0149] 2 μg of human LGI1 antibody was added to SDS-loading buffer with and without mercaptoethanol (β-ME) to prepare samples. SurePAGE™ Bis-Tris SDS-PAGE 4-12% precast gels were used for electrophoresis at 100V for 1 hour. After removing the gel, the samples were stained with Coomassie Brilliant Blue for 20 minutes. Protein bands of the human LGI1 antibody were then visible. Results are shown below. Figure 6 As shown.

[0150] The protein concentration of approximately 1 mL of human LGI1 antibody obtained in step 5 was determined using a nanodrop A280. Each antibody was diluted to a final concentration of 1 μg / mL for subsequent CBA assay testing to screen for LGI1 positive antibodies.

[0151] Testing of human anti-LGI1 monoclonal antibodies:

[0152] Take approximately 150-200 μL of anti-LGI1 antibody dilution buffer and add it to a 48-well plate transfected with either a single or double plasmid (the double plasmid in Example 2 and the plasmids from the experimental and control groups in Example 4, respectively). Incubate at 37°C for 1 hour in the dark. Wash 5 times with PBS. Add secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour. Wash 5 times with PBS. Observe and photograph the fluorescence under a fluorescence microscope (Nikon TS2) with a 20X objective lens. The results are as follows: Figure 7 As shown.

[0153] If the monoclonal antibody is a specific LGI1 antibody, it will produce a distinct green fluorescent signal after reacting with transfected LGI1 cells. Figure 7As can be seen, among the 12 expressed antibodies, 2 dual-plasmid (LGI1+ADAM23) transfected cells produced obvious positive signals, but 4 experimental and control groups showed obvious green fluorescence signals in cells expressing the new LGI1 plasmid. These results indicate that the LGI1 protein expressed by the two new plasmids can fully expose the LGI1 antigenic epitope, enabling the screening of both non-competitive and competitive ADAM antibodies.

[0154] Furthermore, compared to the detection performance of LGI1-GPI, the detection using LGI1-TM plasmid results in less cytotoxicity, fewer dead cells caused by transfection (represented as brighter green spheres in the image), and a stronger positive fluorescence signal, which is beneficial for result interpretation and thus more conducive to detection.

[0155] Example 6: LGI1 Single Plasmid Detection Kit for Clinical Testing

[0156] Patients with LGI1 antibodies and NMDAR have a high proportion of encephalitis patients and are classic targets for detecting autoimmune antibodies in encephalitis. These are essential screening tests for encephalitis patients, and generally, patients suspected of having encephalitis will undergo corresponding antibody screening. Previous reports have shown that anti-LGI1 encephalitis accounts for approximately 20% to 30% of all antibody-positive adverse events (AEs), second only to NMDAR at approximately 40% to 50%.

[0157] The detection kit prepared using the LGI1 single plasmid expression system of the present invention was used to screen patients clinically suspected of having autoimmune encephalitis. Samples from 100 suspected autoimmune encephalitis patients (samples from patients suspected of having autoimmune encephalitis and submitted for testing at Xuanwu Hospital) were selected for screening.

[0158] 1. Cell transfection

[0159] The cultured Hep2 cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0160] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-50%.

[0161] The prepared LGI1 expression plasmids (6 μg each of the dual plasmids in Example 2 and the plasmids of the experimental and control groups in Example 4) and pMcherry-N1 (empty vector control) plasmids were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 48 h.

[0162] 2. Prepare a CBA detection kit

[0163] Cells transfected for 24-48 hours were fixed using PFA, and then the cell slides were prepared into freeze-dried cell detection slide matrix using freeze-drying technology (refer to the patent application number CN202210855857.6). This matrix, along with other detection-related reagents, was used to form a detection kit for later use.

[0164] 3. Sample screening

[0165] For the 100 clinically suspected patients with autoimmune encephalitis, screening was performed using the test kit prepared in step 2. Blood samples were diluted 1:10 (cerebrospinal fluid samples were used directly), and cell smears were incubated for 0.5-1 h; washed 3 times with PBS, 2 min each time; incubated with fluorescently labeled secondary antibody Alexa Fluor 488 (purchased from Thermo Fisher Scientific) for 30 min; washed 3 times with PBS, 2 min each time; the immunofluorescence staining results were observed and photographed using a Nikon TS2 microscope under a 20X objective.

[0166] The results are as follows Figure 8 As shown, using a novel reagent kit for immunofluorescence detection, 4 LGI1 antibody-positive samples were obtained from 100 suspected autoimmune encephalopathy patients (the results of the remaining 96 negative cases are not shown).

[0167] Similarly, compared to the detection effect of LGI1-GPI, the detection using LGI1-TM plasmid has less cytotoxicity, fewer dead cells caused by transfection (shown as brighter green spheres in the figure), stronger positive fluorescence signal, and is more conducive to the interpretation of results.

[0168] In summary, the results of the above embodiments demonstrate that the novel LGI1 cell detection matrix of the present invention can fully expose the antigenic epitopes of LGI1 and simultaneously recognize both competitive and non-competitive antibodies against ADAM, resulting in superior sensitivity in practical detection. Furthermore, an LGI1 antibody detection kit has been prepared, which can better meet the practical needs of auxiliary clinical diagnosis.

[0169] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A fusion protein for detecting anti-LGI1 antibodies, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, a κ chain signal peptide, an LGI1 protein, and a PDGFR β transmembrane peptide. The amino acid sequence of the LGI1 protein is shown in SEQ ID NO.

1.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the PDGFR β transmembrane peptide is shown in SEQ ID NO.

2.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the κ chain signal peptide is shown in SEQ ID NO.

3.

4. A nucleic acid molecule that has been isolated or synthesized, characterized in that, The nucleic acid molecule combination encodes the fusion protein according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

7.

6. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4 or 5.

7. The carrier according to claim 6, characterized in that, The vector is an expression vector, which is a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

8. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 4 or 5 or the vector of claim 6 or 7.

9. Use of the fusion protein according to any one of claims 1 to 3 in the preparation of a kit for detecting anti-LGI1 antibodies.

10. Use of the fusion protein according to any one of claims 1 to 3 in screening for anti-LGI1 antibodies.

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