Anti-LRP4 antibody and application thereof

Abstract

The invention discloses an anti-LRP4 antibody and an application thereof. The heavy chain CDR1-3 is shown as SEQ ID NO: 2-4 in sequence, and the light chain CDR1-3 is shown as SEQ ID NO: 6-8 in sequence. The anti-LRP4 antibody can be effectively used for cell or tissue sample LRP4 antigen detection (such as immunofluorescence detection) or immunoblotting analysis, and can be used as a positive standard product of the anti-LRP4 antibody detection kit, the same set of anti-human fluorescent secondary antibody is directly used, and the component composition of the detection kit is simplified.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-LRP4 antibody and its applications. Background Technology

[0002] Low-density lipoprotein receptor-related protein 4 (LRP4) is a multifunctional member of the low-density lipoprotein receptor family and the only protein in this family with a transmembrane structure. LRP4 consists of 1905 amino acids and its gene is located on human chromosome 11p11.2-p12. This protein contains multiple functional domains, including an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signal transduction domain, enabling it to interact with various ligands and play a crucial role in cell signal transduction.

[0003] The structural features of LRP4 determine its function as a signaling hub, mediating intercellular communication and tissue development. This protein is widely expressed in the postsynaptic membrane of the neuromuscular junction and in motor neurons of the brain and spinal cord, particularly in the hippocampus, olfactory bulb, cerebellum, and cerebral cortex. LRP4 participates in regulating physiological processes such as neuromuscular junction formation, bone metabolism, and central nervous system development by binding to signaling molecules such as Agrin and Wnt through its extracellular domain. Abnormal LRP4 expression is associated with various diseases, including myasthenia gravis, amyotrophic lateral sclerosis (ALS), temporal lobe epilepsy, Alzheimer's disease, and osteoporosis.

[0004] Currently, anti-LRP4 antibodies are commonly used as auxiliary diagnostic biomarkers for the aforementioned diseases, with primary detection methods including cell-based immunofluorescence assay (CBA) and enzyme-linked immunosorbent assay (ELISA). However, standardized positive standards are still lacking in existing anti-LRP4 antibody diagnostic kits. Commercially available LRP4 antibodies are mostly rabbit or mouse-derived and cannot be directly used in human autoantibody detection systems because they require a matching secondary antibody system, limiting their application in clinical testing.

[0005] Therefore, there is an urgent need in this field for a positive standard suitable for the detection of human anti-LRP4 antibodies, in order to improve the standardization and reliability of the test kits and further promote the clinical diagnosis and scientific research application of related diseases. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an anti-LRP4 antibody and its application. This invention successfully screened and obtained a human anti-LRP4 antibody, which can be effectively used for LRP4 antigen detection (such as immunofluorescence detection) or immunoblotting analysis of cell or tissue samples. Furthermore, it can be used as a positive standard in anti-LRP4 antibody detection kits, directly using the same anti-human fluorescent secondary antibody, thus simplifying the composition of the detection kit.

[0007] In a first aspect, the present invention provides an anti-LRP4 antibody comprising: heavy chain CDR1-3 as shown in SEQ ID NO: 2-4 and light chain CDR1-3 as shown in SEQ ID NO: 6-8.

[0008] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if the other such substances, components, elements, or method steps have the same function as the specified ones.

[0009] In some embodiments of the present invention, the light chain variable region CDR and the heavy chain variable region CDR are defined by antibody numbering systems such as Kabat, AbM, Chothia, Contact, or IMGT.

[0010] In this invention, the relevant antibody numbering system can be referenced from: Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997); Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Department of Health and Human Services, National Institutes of Health (1987), etc.

[0011] In some embodiments of the present invention, the light chain variable region CDR and the heavy chain variable region CDR are defined by IMGT. It should be understood that although the scope of protection claimed by the present invention is based on the sequence defined by the IMGT rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0012] In some embodiments of the present invention, the anti-LRP4 antibody comprises: (1) The heavy chain variable region as shown in SEQ ID NO: 3 and the light chain variable region as shown in SEQ ID NO: 5; or (2) A heavy chain variable region and a light chain variable region having at least 80% sequence identity with at least one of SEQ ID NO: 3 and 5, and still retaining the anti-LRP4 antibody variants of heavy chain CDR1-3 and light chain CDR1-3 as described above.

[0013] In this invention, the term "variable region" refers to the functional region in an antibody or immunoglobulin molecule that is responsible for specifically recognizing and binding antigens.

[0014] In some embodiments of the invention, the identity is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0015] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0016] In some embodiments of the present invention, the anti-LRP4 antibody further includes a constant region.

[0017] In this invention, the term "constant region" refers to a region in an antibody or immunoglobulin molecule where the amino acid sequence is highly conserved, typically located at the carboxyl terminus (C-terminus) of the heavy chain (CH) and light chain (CL) of the antibody.

[0018] In some embodiments of the invention, the constant region is connected to the variable region, either via a linking peptide or not.

[0019] In some embodiments of the present invention, the linker peptide is selected from rigid linker peptides or flexible linker peptides.

[0020] In some embodiments of the present invention, the anti-LRP4 antibody comprises: (1) The heavy chain sequence as shown in SEQ ID NO: 9 and the light chain sequence as shown in SEQ ID NO: 10; or (2) A heavy chain sequence and a light chain sequence having at least 80% sequence identity with at least one of SEQ ID NO: 9 and 10, and still retaining the anti-LRP4 antibody variants of the heavy chain CDR1-3 and light chain CDR1-3 described above.

[0021] In some embodiments of the invention, the identity is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0022] In some embodiments of the present invention, the anti-LRP4 antibody further contains chemical modifications and / or is linked with functional groups.

[0023] In some embodiments of the present invention, the chemical modification includes, but is not limited to, at least one of methylation, acetylation, phosphorylation, and glycosylation.

[0024] In some embodiments of the present invention, the functional gene includes, but is not limited to, at least one of: reporter group, protein tag, carrier and lipid group.

[0025] In some embodiments of the present invention, the reporter group includes, but is not limited to, at least one of: chromogenic enzyme, fluorescent labeling group, chemiluminescent labeling group, isotope or magnetic group.

[0026] In some embodiments of the present invention, the chromogenic enzyme includes, but is not limited to, at least one of: peroxidase and alkaline phosphatase.

[0027] In some embodiments of the present invention, the fluorescent labeling group includes, but is not limited to, at least one of the following: fluorescent protein groups, rhodamine groups, fluorescein groups, anthocyanin dyes, cyanine dyes, Alexa Fluor dyes, and / or quantum dot fluorescent groups.

[0028] In some embodiments of the present invention, the isotopes include, but are not limited to, radioactive nuclides.

[0029] In some embodiments of the present invention, the radionuclides include, but are not limited to, those used in radioimmunoassay and radioimmunotherapy.

[0030] In some embodiments of the present invention, the magnetic group includes, but is not limited to, groups that enable magnetic resonance imaging or alter relaxation efficiency.

[0031] In some embodiments of the present invention, the protein tags include, but are not limited to: affinity tags, such as GST, His, MBP, etc.; epitope tags, such as HA, Myc, Flag, etc.; fluorescent tags, such as GFP, mCherry, etc.; and other tags, such as SNAP and Avi, etc.

[0032] In some embodiments of the present invention, the carrier includes, but is not limited to, carrier proteins, such as hemocyanin, serum albumin, ovalbumin, etc.

[0033] In some embodiments of the present invention, the lipid groups include, but are not limited to, fatty acid chains (such as palmitic acid) to form lipopeptides.

[0034] In some embodiments of the present invention, the antibody includes, but is not limited to, at least one of: monoclonal antibody (mAb), polyclonal antibody, chimeric antibody, multispecific antibody, antibody fragment (such as Fab fragment, F(ab')2 and single-domain antibody (VHH)) and conjugate antibody (such as antibody-drug conjugate (ADC)).

[0035] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-LRP4 antibody described above.

[0036] A third aspect of the present invention provides an expression cassette containing nucleic acid molecules as described above.

[0037] A fourth aspect of the invention provides a transformant containing a nucleic acid molecule and / or expression cassette as described in the above aspects.

[0038] In some embodiments of the present invention, the transformant does not involve plant or animal reproductive materials.

[0039] In some embodiments of the present invention, the expression term includes viruses, plasmids, and bacteriophages.

[0040] In some embodiments of the present invention, the transformants include bacteria, fungi, and plant and animal cells.

[0041] In some embodiments of the present invention, the construction of the expressants and transformants can be achieved based on any conventional techniques in the art.

[0042] A fifth aspect of the present invention provides a conjugate containing the anti-LRP4 antibody described above.

[0043] In some embodiments of the present invention, the conjugate further includes a conjugation portion comprising at least one of a detectable marker, a drug, a toxin, biotin, a spin label, an enzyme, a nanoparticle, or a magnetic nanoparticle.

[0044] In some embodiments of the present invention, radioactive isotopes include, but are not limited to, those mentioned above. 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I or 131 At least one of I.

[0045] In some embodiments of the present invention, the toxin includes, but is not limited to, at least one of diphtheria toxin, ricin, or cholera toxin.

[0046] In some embodiments of the present invention, the enzyme includes, but is not limited to, at least one of peroxidase, alkaline phosphatase, horseradish peroxidase, or acetylcholinesterase.

[0047] A sixth aspect of the present invention provides a pharmaceutical composition comprising the anti-LRP4 antibody and / or conjugate described in the above aspects.

[0048] In some embodiments of the present invention, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0049] In some embodiments of the present invention, the pharmaceutically acceptable excipients are rationally selected based on factors such as the product form of the pharmaceutical composition, the intended use, and the route of administration, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA), etc.

[0050] A seventh aspect of the present invention provides the use of at least one of the anti-LRP4 antibody, nucleic acid molecule, expression cassette, transformant and conjugate described above in LRP4 detection.

[0051] In some embodiments of the present invention, the use does not involve the diagnosis of diseases.

[0052] In some embodiments of the present invention, the LRP4 detection can be achieved by any quantitative or qualitative biological or chemical method, including but not limited to: flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay (RIA), chemiluminescent immunoassay, agglutination assay, precipitation reaction, antibody screening precipitation assay, neutralization assay, complement fixation assay, Western blotting, etc.

[0053] An eighth aspect of the invention provides the use of at least one of the anti-LRP4 antibodies, nucleic acid molecules, expression cassettes, transformants, conjugates, and pharmaceutical compositions described above in the preparation of products for diagnosing, preventing, and / or treating LRP4-related diseases.

[0054] In some embodiments of the present invention, the product includes a drug, a solid-phase carrier, a detection chip, a detection reagent, or a test strip.

[0055] In some embodiments of the present invention, the LRP4-related diseases include: myasthenia gravis, temporal lobe epilepsy, Alzheimer's disease, and osteoporosis.

[0056] The beneficial effects of this invention are: (1) The present invention has developed a human anti-LRP4 antibody with good performance and high specificity, which can be effectively used for immunofluorescence detection of LRP4 antigen in cell or tissue samples and immunoblotting analysis of LRP4 protein.

[0057] (2) The human anti-LRP4 antibody in this invention can be used as a positive standard in the anti-LRP4 antibody detection kit. The same set of anti-human fluorescent secondary antibody is used, which effectively simplifies the composition of the kit. Attached Figure Description

[0058] Figure 1 The images are fluorescence images based on cell immunofluorescence assay, where, from left to right, they are LRP4-GFP, Anti-IgG, and Merge.

[0059] Figure 2 This is an SDS-PAGE image of 12 human B cell sequencing antibodies obtained in the embodiments of the present invention, stained with Coomassie brilliant blue.

[0060] Figure 3 This is an immunofluorescence image of some representative human B-cell sequencing antibodies against LRP4 obtained in the embodiments of the present invention.

[0061] Figure 4The image shows the Coomassie Brilliant Blue staining of the purified human LRP4 antibody, where M represents the protein marker (20-250 kDa). The lanes are: 1 for flow-through buffer; 2 for wash buffer I; 3 for wash buffer II; and 4 for elution buffer.

[0062] Figure 5 Immunofluorescence images of human anti-LRP4 antibody (LR-5) at different dilutions reacting with transformants transfected with LRP4.

[0063] Figure 6 Immunofluorescence image of mouse muscle tissue containing human anti-LRP4 antibody (LR-5), from left to right: α-BTX, Anti-IgG, and Merge.

[0064] Figure 7 A comparison of ELISA results between human anti-LRP4 antibody (LR-5) and LRP4 extracellular domain protein. Detailed Implementation

[0065] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0066] Experimental equipment and materials In the following embodiments, the relevant equipment used, as well as the source information of the biological materials and reagents, are shown below.

[0067] HEK293T cells were purchased from ATCC (Catalog No. CRL-3216). pCMV6-AC-GFP-LRP4 plasmid was purchased from Origene (Catalog No. RG217609). Alexa Fluor 568 Anti-human IgG was purchased from ThermoFisher (Catalog No. A-21090). Alexa Fluor 488 Anti-human IgG was purchased from ThermoFisher (Catalog No. A-11013). Bovine serum albumin (BSA) was purchased from Beijing Dingguo Changsheng Co., Ltd. (Catalog No. FA016-1G). Horseradish enzyme-labeled goat anti-human IgG (H+L) was purchased from Beijing Dingguo Changsheng Co., Ltd. (Catalog No. IH-0021). ECL chemiluminescence solution was obtained from the ECL high-performance chemiluminescence kit, purchased from Wuhan Saiveibao Co., Ltd. (Catalog No. GE2301-100ML). Fetal bovine serum (FBS) was purchased from Shanghai Sangon Biotech Co., Ltd., catalog number: E600001-0500. DMEM cell culture medium was purchased from Gibco, Inc. (USA), catalog number: 11965092. Triton was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., catalog number: P0874. PEI MAX 40K transfection reagent was purchased from Polysciences, catalog number: 24765-100. TurboFectin 8.0 transfection reagent was purchased from Origene, catalog number: TF81001. Protein G beads were purchased from MCE Corporation (Japan), catalog number: HY-K0214. Amicon Ultra 15 mL ultrafiltration tubes were purchased from Merck Millipore, catalog number: UFC901096. SDS-PAGE loading buffer (5×) was purchased from Wuhan Saivei Biotech Co., Ltd., catalog number: G2075-10ML. DAPI staining solution was purchased from Wuhan Saiweibao Co., Ltd., product number: G1012-10ML.

[0068] The washing buffer contains 20 mM Na2HPO4 and 0.15 M NaCl at a final concentration of pH 7.0.

[0069] Acidic glycine buffer (pH 2.5-3.5) contains 0.1 M glycine.

[0070] Neutralizing Tris-HCl contains 1 M Tris-HCl at pH 8.5.

[0071] The preparation method of PBS buffer is as follows: Take 16.00 g NaCl, 2.88 g Na2HPO4, 0.40 g KCl, and 0.484 g KH2PO4, transfer them to a 2 L beaker, add 900 mL of ultrapure water, stir until completely dissolved, adjust the pH of the solution to 7.4 with NaOH granules, bring the volume to 2 L, filter using a 0.22 μm vacuum filter, and place at 4℃.

[0072] The preparation method for 0.25% Triton X-100 solution is as follows: Take 400 mL of PBS buffer, add 1 mL of Triton X-100, place in a 37℃ incubator for 30-50 min to fully dissolve, and then store at room temperature in the dark. The preparation method for 0.5% Triton X-100 solution is the same as above, except that 2 mL of Triton X-100 is added.

[0073] The preparation method of 5% BSA is as follows: Weigh 2.5g of BSA, add it to 50 mL of 0.5% Triton X-100 solution, and dissolve it completely to obtain the product.

[0074] The multi-functional microplate reader is an Infinite M1000 Pro. The benchtop high-speed refrigerated centrifuge is a Q2048R. The electronic analytical balance is an AR223CN. The fluorescence microscope is a TS100. The ultrapure water system is an IQ-7000. The clean bench is a SW-CJ-1F. The -80℃ ultra-low temperature freezer is an MDF-682VE. The 37℃ constant temperature CO2 incubator is an IL-161CT. The peristaltic pump is a DB3000A. The diaphragm vacuum pump is a GM-0.5B.

[0075] Example 1 In this embodiment, a method for preparing a human anti-LRP4 antibody is provided.

[0076] Specifically, the following steps are included: (1) Screening of human anti-LRP4 antibody positive samples: In this embodiment, subjects were enrolled according to the following inclusion criteria. All subjects signed clinical informed consent forms.

[0077] LRP4-Inclusion criteria for myasthenia gravis (MG) patients: (1) Patients exhibiting MG symptoms, including limb weakness, ptosis, blurred vision, dysarthria, dysphagia, etc.; (2) Positive LRP4-Ab test results (serum anti-LRP4 antibody measured by cellular immunofluorescence assay (IF)).

[0078] Exclusion criteria were: (1) patients with other autoimmune diseases; (2) patients with abnormal expression or impaired consciousness or unstable vital signs; (3) patients with severe mental illness symptoms who could not cooperate; (4) patients or their families who could not sign the informed consent form; and (5) other patients who could not cooperate with this study.

[0079] Whole blood was collected from the subjects to obtain serum samples for later use.

[0080] (2) Preparation of anti-LRP4 antibody for detection of cell matrix: HEK293T cells were seeded into 48-well plates and cultured in DMEM cell culture medium containing 10% FBS at 37°C in a 5% CO2 incubator. When the cell density reached 50%-60%, the pCMV6-AC-GFP-LRP4 plasmid was transfected into HEK293T cells using Invigentech transfection reagent. After 36-48 hours of transfection, the cell density was increased to 80%-95%.

[0081] After transfection, cells were fixed and permeabilized. Specifically, after transfection, the culture medium was discarded, and the cells were washed once with PBS. Then, in a fume hood, 100 μL of 4% formaldehyde solution was added to each well, and the cells were fixed at 4°C for 30 min. The solution was discarded, and the cells were washed three times with PBS for 5 min each time. 200 μL of blocking buffer (5% BSA solution diluted with 0.5% Triton X-100) was added, and the cells were blocked at 4°C. Permeabilization was then performed for 12–16 h.

[0082] (3) Immunofluorescence detection: The production of anti-LRP4 antibody was verified using immunofluorescence (IF). Specifically, the serum sample obtained in step (1) was diluted with 0.5% Triton X-100 solution at a volume ratio of 1:10, and then 100 μL was added to each well of the 48-well plate described above. The plate was incubated at 37°C for 1.5 h. The liquid in the wells was discarded, and 200 μL of 0.25% Triton X-100 solution was added to each well. After standing for 5 min, the solution was discarded. This process was repeated 3-5 times. Secondary antibody conjugated with Alexa Fluor 568 Anti-human IgG (ThermoFisher, catalog number: A-21090, volume ratio: 1:1000) diluted with 0.5% Triton X-100 solution was added and incubated for 1.5 h. The liquid in the wells was discarded, and 200 μL of 0.25% Triton X-100 solution was added to each well. After standing for 5 min, the solution was discarded. This process was repeated 3-5 times. Add 100 μL of PBS to each well to prevent drying, observe under a 20× objective lens of a fluorescence microscope, and take fluorescence photographs.

[0083] The results are as follows Figure 1 As shown.

[0084] It was found that the subjects' serum did indeed contain anti-LRP4 antibodies, and HEK293T cells transfected with LRP4 were able to react and produce obvious red fluorescent signals upon contact.

[0085] (4) Obtaining anti-LRP4 antibody: Samples that tested positive for anti-LRP4 antibody were sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for single-cell (B cell) sequencing to obtain a certain number of light and heavy chain paired antibody sequences. Based on the characteristics of the antibody amino acid sequence and B cell distribution, different types of antibodies, such as IgG1, IgG2, or IgG3, were selected for further expression and validation.

[0086] Specifically, 10 pairs of paired heavy and light chains were selected, and after human IgG1 κ / λ completion, the resulting fragments were directly inserted into the eukaryotic secretory expression vectors pFUSE-CHIg-hG1-3, pFUSE2-CLIg-hL2, and pFUSE2-CLIg-hk (all purchased from Invivogen) to obtain light and heavy chain expression plasmids. The obtained light and heavy chain expression plasmids were transfected into HEK293T cells according to the method described in the above examples to produce anti-LRP4 antibody.

[0087] More specifically, following the method described in the above embodiments, HEK293T cells were seeded into 6-well plates, with 2 mL of DMEM cell culture medium containing 10% FBS added to each well. The plates were then incubated overnight at 37°C in a CO2 incubator. Plasmid transfection was performed when the cell density reached approximately 80%. Specifically, 2 µg of paired antibody light and heavy chain expression plasmids (light chain plasmid ratio 3:2) and 250 µL of DMEM cell culture medium containing 2.5 µL of PEI MAX 40K (2 mg / mL) were added to 250 µL of DMEM cell culture medium. The plates were incubated at room temperature for 15-25 minutes. The medium was then changed to DMEM cell culture medium. 500 µL of plasmid-transfection reagent mixture was added to each well, gently mixed, and incubated at 37°C in a CO2 incubator for 8-12 hours. The culture medium was then replaced with DMEM cell culture medium containing 10% FBS, and cultured for another two days. The culture medium was taken from two of the replacements and concentrated by ultracentrifugation using a centrifuge tube to obtain the crude product of B cell sequencing antibody.

[0088] Take 2 μg of the crude B-cell sequencing antibody product prepared in the above steps and add it to SDS-PAGE loading buffer containing DTT to prepare samples. Use 12% SDS-PAGE separating gel and SDS-PAGE stacking gel for electrophoresis at a constant voltage of 90 V for 35 min, then adjust to 120 V and continue electrophoresis for about 1 hour. Remove the gel and stain with Coomassie Brilliant Blue for 20-40 minutes to see the corresponding B-cell sequencing antibody protein bands.

[0089] The results are as follows Figure 2 As shown.

[0090] These B-cell sequencing antibodies were screened using immunofluorescence assay. Specifically, crude B-cell sequencing antibody products were prepared according to the methods described in the above examples. 100 μL of the crude B-cell sequencing antibody product was added to a 48-well plate containing transformants transfected with LRP4 and incubated at 37°C for 1.5 h. After incubation, the liquid in the wells was discarded, and 200 μL of 0.25% Triton X-100 solution was added to each well. After standing for 5 min, the solution was discarded, and this process was repeated 3-5 times. Then, secondary antibody Alexa Fluor 488 Anti-human IgG (ThermoFisher, catalog number: A-11013, dilution ratio 1:1000) diluted with 0.5% Triton X-100 solution was added and incubated for 1.5 h. The liquid in the wells was discarded, and 200 μL of 0.25% Triton X-100 solution was added to each well. After standing for 5 min, the solution was discarded, and this process was repeated 3-5 times. Add 100 μL of PBS to each well to prevent drying, observe under a 20× objective lens of a fluorescence microscope, and take fluorescence photographs.

[0091] The results are as follows Figure 3 As shown.

[0092] Based on immunofluorescence experiments, if the obtained B-cell sequencing antibody is an anti-LRP4 antibody, it will produce a clear red fluorescent signal after reacting with transformants transfected with LRP4. The merged image will show a superposition of green and red fluorescence. If the merged image shows a yellow color due to the superposition of red and green fluorescence, it indicates that the B-cell sequencing antibody has specifically bound to LRP4, i.e., a positive result.

[0093] In this embodiment, each of the 12 obtained B-cell sequencing antibodies was tested to determine which B-cell sequencing antibodies could specifically recognize LRP4 and were therefore specific anti-LRP4 antibodies. The results showed that the B-cell sequencing antibody numbered LR-5 produced a significant red fluorescent signal after reacting with the transformant transfected with LRP4, indicating that the B-cell sequencing antibody numbered LR-5 is a human anti-LRP4 antibody.

[0094] The LR-5 was sequenced and compared with IMGT IgBlast to determine its antibody sequence information, as shown in the table below.

[0095] Table 1. Sequence information of human anti-LRP4 antibody (LR-5)

[0096] Example 2 In this embodiment, the human anti-LRP4 antibody obtained in the above embodiments was purified.

[0097] The specific purification method is as follows: Collect the culture supernatant from LR-5 transformants (HEK293T cells) and filter it through a 0.22 μm filter membrane to remove dead cells and cell debris. Add the appropriate volume of Protein G beads to the filtered culture supernatant according to the Protein G beads' instructions, rotate, and incubate overnight at 4°C. Wash the Protein G beads with 15-20 column volumes of wash buffer, 3-5 times, to remove impurities that bind weakly to Protein G beads or antibodies.

[0098] Elute the antibody bound to Protein G beads with acidic elution buffer (pH 2-3) and collect the eluent. Neutralize the eluent with 1M Tris-HCl (pH 8.5) at 1 / 10 volume of acidic elution buffer. Concentrate the neutralized eluent to approximately 0.5 mL using an ultrafiltration tube, and replace the buffer with at least 10 times the volume of PBS to obtain 0.5 mL of purified human LRP4 antibody (LR-5).

[0099] Take 2 μg of purified human LRP4 antibody, perform electrophoresis according to the method in the above example, and stain with Coomassie brilliant blue to observe the protein band of human LRP4 antibody.

[0100] The results are as follows Figure 4 As shown.

[0101] Figure 4 This is a Coomassie Brilliant Blue staining image of the purified human LRP4 antibody, where M represents the protein marker (20-250 kDa). Lanes are labeled as follows: 1 is flow-through buffer; 2 is wash buffer I; 3 is wash buffer II; and 4 is elution buffer. Flow-through buffer is the supernatant obtained after overnight incubation at 4°C with agarose beads. Wash buffer I is 10 times its volume, wash buffer II is 20 times its volume, and elution buffer is 20 times its volume.

[0102] Approximately 0.5 mL of human LRP4 antibody was obtained. Its protein concentration was measured using a nanodrop A280 and found to be 0.4 mg / mL. Therefore, it can be deduced that 45 mL of transfected HEK 293T cells can express approximately 0.4 mg of human LRP4 antibody, and the expression level of human LRP4 antibody in transfected HEK 293T cells is approximately 8.8 mg / L.

[0103] Example 3 In this embodiment, the human LRP4 antibody obtained in the above embodiment was used for cell immunofluorescence detection (CBA) to verify its effectiveness.

[0104] In the prior art, the detection of anti-LRP4 antibodies has been widely used in the detection of myasthenia gravis, and the detection method used is mainly cell immunofluorescence assay (CBA). Therefore, the inventors used the same method to evaluate and analyze the titer of the anti-LRP4 antibody (LR-5) in this invention in recognizing the LRP4 protein.

[0105] Anti-LRP4 antibody was prepared according to the method described in the above embodiments to detect the cell matrix. The efficacy of the human anti-LRP4 antibody obtained in the above embodiments was verified by immunofluorescence (IF). Specifically, the purified human anti-LRP4 antibody was diluted with PBS at volume ratios of 1:100, 1:500, and 1:1000, and added to 48-well plates containing anti-LRP4 antibody-loaded cell matrix. The plates were then incubated with HEK 293T cells expressing LRP4 at 37°C for 1.5 h in the dark. The liquid in the wells was discarded, and 200 μL of 0.25% Triton X-100 solution was added to each well. After standing for 5 min, the solution was discarded. This process was repeated 3-5 times. Secondary antibody Alexa Fluor 488 Anti-human IgG (ThermoFisher, catalog number: A-11013, volume ratio: 1:1000) diluted with 0.5% Triton X-100 solution was added, and the plates were incubated for 1.5 h. Discard the liquid in each well, add 200 μL of 0.25% Triton X-100 solution to each well, let stand for 5 min, then discard. Repeat 3-5 times. Add 100 μL of PBS to each well to prevent drying. Observe and photograph the fluorescence under a 20× objective lens using a fluorescence microscope.

[0106] The results are as follows Figure 5 As shown.

[0107] It was observed that both LR-5 and LRP4-transfected cells at different dilution gradients (1:100-1000) produced significant red fluorescent signals after reaction. The positive signal gradually weakened with increasing LR-5 dilution, but remained visible at a 1:1000 dilution. These results demonstrate that the human LRP4 antibody in this embodiment can be used for immunofluorescence experiments on transfected cells and can serve as a specific antibody against LRP4.

[0108] Example 4 In this embodiment, the efficacy of the human LRP4 antibody prepared in the above embodiment was further verified using the IHC method. Specifically, in this embodiment, mouse gastrocnemius muscle tissue was used to verify the recognition effect of the human LRP4 antibody on LRP4 protein in mouse muscle tissue.

[0109] In this embodiment, the mouse gastrocnemius muscle tissue was conventionally frozen mouse gastrocnemius muscle tissue preserved in the inventor's laboratory. After thawing the frozen mouse gastrocnemius muscle tissue, it was washed with PBS for 5 minutes. Myofilaments were extracted from the mouse gastrocnemius muscle tissue, fixed with 4% paraformaldehyde for 20 minutes, and then washed three times with PBS. 0.5% Triton X-100 solution was added to completely immerse the myofilaments, and permeation was performed at room temperature for 15 minutes. After permeation, it was washed three times with PBS. Then, it was blocked with 5% BSA solution at 37°C for 1 hour, ensuring that the myofilaments were completely immersed in the solution. After blocking, it was washed three times with PBS. Rhodamine-labeled α-BTX (α-B-x)-Alexa Fluor 594 was diluted with 5% BSA at a ratio of 1:200. The diluted α-BTX-Alexa Fluor 594 was added to the myofilaments, incubated overnight at 4°C, and then washed five times with PBS for 5 minutes each time. Then, the purified human LRP4 antibody prepared in the above examples was used to thoroughly soak the myofilaments, and the mixture was incubated at 37°C for 1.5 h in the dark. After washing with PBS, the mixture was allowed to stand for 5 min, and the solution was discarded. This washing process was repeated 3-5 times. Alexa Fluor 488 Anti-human IgG (ThermoFisher, catalog number: A-11013, dilution ratio 1:1000) diluted with 0.5% Triton X-100 solution was added, and the mixture was incubated for 1.5 h. After washing with PBS, the mixture was allowed to stand for 5 min and then discarded. This process was repeated 3-5 times. The myofilaments were then immersed in PBS, and the results were observed and recorded using a Nikon Eclipse Ti2 fluorescence microscope.

[0110] Test results as follows Figure 6 As shown.

[0111] Figure 6Immunofluorescence images of mouse muscle tissue neuronal synapses identified using the human anti-LRP4 antibody from this embodiment of the invention. Staining results of mouse gastrocnemius muscle incubated with the human anti-LRP4 antibody from this embodiment of the invention as the primary antibody showed a significant positive signal in mouse muscle tissue, indicating that it can recognize the LRP4 protein in mouse gastrocnemius muscle.

[0112] Example 5 In this embodiment, the human anti-LRP4 antibody from the above embodiments is used for ELISA detection.

[0113] Specifically, LRP4-ECD (extracellular domain protein) was diluted to 10 μg / mL with 1× carbonate buffer and used for coating (50 μL per well in a 96-well plate), and incubated overnight at 4 °C. The coating solution was discarded, and the plate was washed with PBST for 5 min each time, 3 times. The plate was blotted dry on absorbent paper, and 150 μL of 5% BSA solution was added to each well. The plate was sealed with a sealing membrane and incubated at 37 °C for 2 h or at 4 °C overnight. The blocking solution was discarded, and the plate was washed with PBST for 5 min each time, 3 times, blotted dry on absorbent paper, and dried at 25 °C and 10% humidity for 4 h. 50 μL of the human anti-LRP4 antibody from the above examples was added to each well, and the plate was incubated at 37 °C for 1.5 h, washed 5 times with PBST, and blotted dry on absorbent paper. Add 100 μL of HRP-labeled goat anti-human IgG (1:6000 dilution) to each well, seal with sealing film, incubate at 37℃ for 1 h, wash 5 times with washing buffer, and pat dry on absorbent paper. Discard the enzyme-labeled secondary antibody, wash 3-5 times with PBST, add 50 μL / well of chromogenic buffer (Solution A:Solution B = 1:1), and incubate at 37℃ in the dark for 10-15 min. Stop the reaction by adding 50 μL of 2 M H2SO4 stop solution to each well, and measure the OD value at 450 nm using a microplate reader. A negative control (NC) is set up without any antibodies.

[0114]

[0115] One commercially available LRP4 monoclonal antibody was also used as a positive control; it was an Anti-LRP4 antibody (purchased from abcam, ab174637).

[0116] Test results as follows Figure 7 As shown.

[0117] It can be observed that the OD value of the human anti-LRP4 antibody (LR-5) in the embodiments of the present invention is 2.6±0.12, the OD value of the commercial LRP4 monoclonal antibody is 0.33±0.05, and the OD value of NC is 0.12±0.01. Therefore, the human anti-LRP4 antibody used in the embodiments of the present invention can be used for LRP4 detection, and can also serve as a positive standard in LRP4 autoantibody detection kits. Furthermore, it can share anti-human secondary antibodies with human samples, offering the advantage of not requiring additional reagent components.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An anti-LRP4 antibody, characterized in that, The anti-LRP4 antibody includes: The heavy chains CDR1-3 are shown in sequence as SEQ ID NO: 2-4 and the light chains CDR1-3 are shown in sequence as SEQ ID NO: 6-8.

2. The anti-LRP4 antibody according to claim 1, characterized in that, The anti-LRP4 antibody includes: (1) The heavy chain variable region as shown in SEQ ID NO: 3 and the light chain variable region as shown in SEQ ID NO: 5; or (2) A heavy chain variable region and a light chain variable region having at least 80% sequence identity with at least one of SEQ ID NO: 3 and 5, and still retaining the anti-LRP4 antibody variants of heavy chain CDR1-3 and light chain CDR1-3 as described in claim 1.

3. The anti-LRP4 antibody according to claim 1 or 2, characterized in that, The anti-LRP4 antibody includes: (1) The heavy chain sequence as shown in SEQ ID NO: 9 and the light chain sequence as shown in SEQ ID NO: 10; or (2) A heavy chain sequence and a light chain sequence having at least 80% sequence identity with at least one of SEQ ID NO: 9 and 10, and still retaining the anti-LRP4 antibody variants of heavy chain CDR1-3 and light chain CDR1-3 as described in claim 1.

4. A nucleic acid molecule encoding the anti-LRP4 antibody according to any one of claims 1-3.

5. An expression cassette containing the nucleic acid molecule as described in claim 4.

6. A transformant containing the nucleic acid molecule as described in claim 4 and / or the expression cassette as described in claim 5.

7. A coupling, characterized in that, The conjugate contains the anti-LRP4 antibody as described in any one of claims 1-3.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the anti-LRP4 antibody according to any one of claims 1-3, and / or the conjugate according to claim 7.

9. Use of at least one of the anti-LRP4 antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression cassette according to claim 5, the transformant according to claim 6, and the conjugate according to claim 7 in LRP4 detection; in, The intended use does not involve the diagnosis of diseases.

10. Use of at least one of the anti-LRP4 antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression cassette of claim 5, the transformant of claim 6, the conjugate of claim 7, and the pharmaceutical composition of claim 8 in the preparation of products for the diagnosis, prevention, and / or treatment of LRP4-related diseases; in, The LRP4-related diseases include: myasthenia gravis, amyotrophic lateral sclerosis (ALS), temporal lobe epilepsy, Alzheimer's disease, and osteoporosis.

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