Anti-AchR (alpha) 1 subunit antibody as well as preparation method and application thereof

Abstract

The invention relates to an anti-AchR (alpha) 1 subunit antibody and a preparation method thereof. The anti-AchR (alpha) 1 subunit antibody comprises a heavy chain variable region and a light chain variable region, the antibody is not only suitable for various experimental methods such as cellular immunofluorescence (IF) and tissue immunofluorescence (IHC), but also can specifically recognize the alpha 1 subunit of an AChR receptor, and especially shows efficient binding capacity to an AChR receptor protein complex. Due to the characteristics, the antibody can accurately reflect the real condition of combination of an in-vivo autoimmune antibody and an antigen, so that the antibody is very suitable for being used as a positive standard substance of an acetylcholine receptor autoantibody CBA detection kit.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an anti-AchRɑ1 subunit antibody and a preparation method and application thereof. Background Art

[0002] Myasthenia gravis is an autoimmune disease mediated by AChR antibodies. AChR antibodies disrupt signaling at the neuromuscular junction, leading to muscle weakness and fatigability. Symptoms often worsen with activity and improve with rest. Common manifestations include: eye muscle weakness, drooping eyelids (ptosis), and diplopia; limb weakness, with fatigue in the arms and legs; bulbar muscle weakness, with difficulty swallowing, dysarthria, and chewing; and respiratory muscle weakness, which can lead to respiratory failure (myasthenic crisis) in severe cases.

[0003] The AChR complex is a key structure located on the postsynaptic membrane of the neuromuscular junction, responsible for transmitting nerve signals to muscle cells and triggering muscle contraction. It is a pentameric protein composed of five subunits: two α subunits (α1), one β subunit (β1), one δ subunit, and one γ subunit (in the fetus) or ε subunit (in adults). Acetylcholine receptor (AChR) antibodies are the main autoantibodies in myasthenia gravis, and are detected in approximately 85% of patients with generalized myasthenia gravis and 50% of patients with ocular myasthenia gravis. AChR antibodies target acetylcholine receptors (AChRs) at the neuromuscular junction. Their mechanisms of action are as follows: first, they block acetylcholine binding, inhibiting nerve signal transmission or accelerating receptor degradation; second, they reduce the number of AChRs through cross-linking and internalization; and third, they activate complement, leading to damage to the postsynaptic membrane and further disrupting neuromuscular transmission.

[0004] Detection of AChR antibodies is crucial for diagnosis and disease assessment. A positive AChR antibody is a crucial indicator for the diagnosis of myasthenia gravis. Currently, detection of anti-AChR antibodies often relies on laboratory testing. Three common techniques exist for detecting anti-AChR antibodies: cell-based immunofluorescence assay (CBA), ELISA, and radioimmunoassay. CBA is generally considered the gold standard.

[0005] Positive standards are a core component of IVD diagnostic kits. However, in the field of diagnostic kits for anti-AChR receptor antibodies, a standard human positive standard is currently unavailable on the market. Although commercial AChR receptor antibodies are available, most are rabbit or mouse derived and cannot be directly used in human autoantibody detection kits because different secondary antibody systems are required. To address this technical bottleneck, we screened a specific AChR receptor antibody using single-cell immune repertoire sequencing technology and systematically validated its function and application.

[0006] In summary, there is an urgent need to develop a new technical solution to solve the problems existing in the existing technology and meet the development needs of the current market. Summary of the Invention

[0007] Based on this, the present invention discloses an anti-AchRɑ1 subunit antibody and its preparation method. Verified by cellular immunofluorescence (IF) analysis, the anti-AchRɑ1 subunit monoclonal recombinant antibody obtained in the present invention can specifically recognize the native conformation of the AchR complex and can be used as a positive standard in acetylcholine receptor autoantibody CBA detection kits, improving the accuracy and stability of the test.

[0008] One object of the present invention is to provide an anti-AchRɑ1 subunit antibody, wherein the anti-AchRɑ1 subunit antibody comprises an α1 subunit, a β1 subunit, a δ subunit, a γ subunit, and an ε subunit;

[0009] The method for preparing the anti-AchRɑ1 subunit antibody comprises the following steps:

[0010] S1. Culture of recombinant cell vectors;

[0011] S2. The recombinant cell vector is mixed with α1 subunit-based plasmid, β1 subunit-based plasmid, δ subunit-based plasmid, γ subunit-based plasmid, ε subunit-based plasmid and a transfection reagent, and then transfected to obtain an anti-AchRɑ1 subunit antibody.

[0012] Furthermore, in step S2, the mass ratio of the α1 subtype based plasmid, the β1 subtype based plasmid, the δ subtype based plasmid, the γ subtype based plasmid, and the ε subtype based plasmid is 2-4:1:1:1:1:1.

[0013] Furthermore, in step S1, the culture density of the recombinant cell vector is 40-50%.

[0014] The present invention also provides a DNA molecule comprising the anti-AchRɑ1 subunit antibody or a fragment thereof.

[0015] Furthermore, the recombinant vector is selected from prokaryotic cells or eukaryotic cells.

[0016] Furthermore, the recombinant vector is selected from one or more of HEK293T cells, Hela cells, and Hep2 cells.

[0017] The present invention also provides use of the composition in preparing a CBA detection kit.

[0018] Furthermore, the anti-AchRɑ1 subunit antibody is used as a positive control.

[0019] The beneficial effects of the present invention are:

[0020] The antibody of the present invention is not only suitable for various experimental methods such as cellular immunofluorescence (IF) and tissue immunofluorescence (IHC), but also can specifically identify anti-AChR receptor antibodies in serum, especially showing efficient binding ability to AChR receptor protein complexes. These characteristics enable the antibody to accurately reflect the actual binding of autoimmune antibodies to antigens in the body, making it very suitable as a positive standard for acetylcholine receptor autoantibody CBA detection kits. At the same time, the antibody also has important application value in the field of AChR receptor function research and has broad commercial prospects, bringing significant technological breakthroughs and innovative value to related scientific research and diagnostic fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Screening fluorescence images for anti-AchR antibody-positive samples. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0023] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0024] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0025] Cell line: HEK293T cells were purchased from ATCC, catalog number: crl-3216;

[0026] Plasmid: α subunit plasmid (α1) (Sino Biological, Cat. No. HG18361-ACG);

[0027] β-subunit matrix plasmid (β1) (Sino Biological, Cat. No.: HG20046-CF);

[0028] Delta subunit matrix plasmid (Sino Biological, Cat. No.: HG19458-UT);

[0029] γ subunit matrix granules (in the fetal stage) (Sino Biological, Cat. No.: HG15656-CM);

[0030] ε subunit matrix granules (in adult stage) (Sino Biological, Cat. No.: HG18373-UT);

[0031] PBS was purchased from VivaCell, catalog number: C3580-0500;

[0032] 10% FBS + DMEM cell culture medium was purchased from Procell, catalog number: PM150210B;

[0033] Transfection reagent PEI MAX 40K, purchased from Polysciences, catalog number: 24765-100;

[0034] AlexaFluor 546 Anti-human IgG was purchased from ThermoFisher, catalog number: A-21089;

[0035] Example 1

[0036] Preparation of anti-AchRɑ1 subunit antibody:

[0037] Patient enrollment: Myasthenia gravis (MG) patients with positive anti-AchR receptor antibodies were enrolled and serum samples were obtained. The patients signed the clinical informed consent.

[0038] The patient's serum is first subjected to an immune reaction between antigen and antibody to verify that it does contain anti-AchR receptor antibodies. Currently, the detection of anti-AchR receptor antibodies is mainly done by cellular immunofluorescence (IF).

[0039] Preparation of cell matrix for anti-AchR receptor antibody detection:

[0040] HEK293T cells were plated in 48-well plates and cultured in DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator for 72 h.

[0041] When the cell density reaches 40%, α1 subunit matrix plasmid, β1 subunit matrix plasmid, δ subunit matrix plasmid, γ subunit matrix plasmid, and ε subunit matrix plasmid are mixed with HEK293T cells in a 48-well plate using 3 μg PEI MAX 40K transfection reagent and co-transfected. After 6 hours of transfection, fresh medium is replaced to remove the transfection reagent. After 48 hours of transfection, the cell density reaches 80%.

[0042] The mass ratio of the α1 subunit matrix, the β1 subunit matrix, the δ subunit matrix, the γ subunit matrix, and the ε subunit matrix is ​​2:1:1:1:1;

[0043] Cell fixation: Fix the cells with 2 wt % paraformaldehyde at room temperature for 30 min, wash twice with 200 μL of PBS, and prepare the cell product for acetylcholine receptor autoantibody CBA detection.

[0044] Immunofluorescence experiments:

[0045] Approximately 20 μl of patient serum was diluted with 20 μl of PBS and added to a 48-well plate transfected with AchR receptor antigen. The plate was incubated at 37°C for 1 hour in the dark, washed five times with PBS, and then 100 μl of secondary antibody conjugated to Alexa Fluor 546 Anti-human IgG was added and incubated for 1 hour. The plate was washed five times with PBS, and the plate was observed under a 20X fluorescence microscope and the fluorescence image was captured.

[0046] The fluorescence image is as follows Figure 1 As shown, it can be seen that since the patient's serum contains anti-AchR receptor antibodies, the sample initially diluted 10 times produces an obvious red fluorescent signal after reacting with the transfected AchR receptor cells, and a positive signal can still be seen when the serum sample is diluted 1000 times.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0048] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An anti-AchRɑ1 subunit antibody, characterized in that: The anti-AchRɑ1 subunit antibody includes α1 subunit, β1 subunit, δ subunit, γ subunit, and ε subunit; The method for preparing the anti-AchRɑ1 subunit antibody comprises the following steps: S1. Culture of recombinant cell vectors; S2. The recombinant cell vector is mixed with α1 subunit-based plasmid, β1 subunit-based plasmid, δ subunit-based plasmid, γ subunit-based plasmid, ε subunit-based plasmid and a transfection reagent, and then transfected to obtain an anti-AchRɑ1 subunit antibody.

2. The anti-AchRɑ1 subunit antibody according to claim 1, wherein in step S2, the mass ratio of the α1 subunit-based plasmid, the β1 subunit-based plasmid, the δ subunit-based plasmid, the γ subunit-based plasmid, and the ε subunit-based plasmid is 2-4:1:1:1:1:

1. 3 . The anti-AchRɑ1 subunit antibody according to claim 1 , wherein in step S1 , the recombinant cell vector is cultured at a density of 40-50%.

4. A DNA molecule, characterized in that The invention comprises the anti-AchRɑ1 subunit antibody or a fragment thereof according to any one of claims 1 to 3.

5. The method for preparing the anti-AchRɑ1 subunit antibody according to claim 1, characterized in that: The recombinant vector is selected from prokaryotic cells or eukaryotic cells.

6. The method for preparing the anti-AchRɑ1 subunit antibody according to claim 1, characterized in that: The recombinant cell vector is selected from one or more of HEK293T cells, Hela cells, and Hep2 cells.

7. Use of the composition according to any one of claims 1 to 3 in preparing a CBA detection kit.

8. Use according to claim 7, characterized in that The anti-AchRɑ1 subunit antibody is used as a positive control substance of the kit.

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