Monoclonal antibody of anti-human LAMP-2A protein and application of monoclonal antibody in immunodetection

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human LAMP-2A protein monoclonal antibody and application of the anti-human LAMP-2A protein monoclonal antibody in immunodetection. Amino acid sequences of CDR1-3 on a light chain variable region of the antibody are respectively shown as SEQ ID NO.3-5, and amino acid sequences of CDR1-3 on a heavy chain variable region of the antibody are respectively shown as SEQ ID NO.8-10. The antibody disclosed by the invention is good in performance, strong in specificity to human LAMP-2A protein, high in recognition sensitivity and good in binding affinity, not only can accurately locate target protein, but also can effectively resist interference of non-target protein, is free of non-specific reaction when being used for detecting complex samples such as cells, tissues and the like, and can be used for detecting the human LAMP-2A protein. Therefore, the accuracy, the sensitivity, the specificity and the reliability of immunodetection of the LAMP-2A protein can be improved, the coincidence degree of the antibody detection result and the actual expression condition of the LAMP-2A protein is high, and the occurrence rate of false positive or false negative results is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, and in particular to monoclonal antibodies against human LAMP-2A protein and their application in immunoassay. Background Technology

[0002] Lysosome-associated membrane protein 2 (LAMP-2), also known as LAMPB, LAMP2, CD107b, CD107-B, DND, LGP-96, or LGP110, is a highly glycosylated type I transmembrane protein expressed in the lysosomal membranes of almost all cells. It is a crucial protein for maintaining lysosomal membrane integrity and plays a vital role in lysosomal biogenesis, lysosomal pH regulation, and lysosomal protein degradation in response to starvation. The structure of LAMP-2 consists of a large N-terminal lysosomal intraluminal domain, a hydrophobic transmembrane domain, and a short C-terminal cytoplasmic tail. Alternative splicing of its mRNA can produce three different isoforms: LAMP-2A, LAMP-2B, and LAMP-2C. These are identical in their intraluminal lysosomal domain but differ in their transmembrane domain and C-terminal tail. LAMP-2A plays a key role in chaperone-mediated autophagy (CMA), serving as a core receptor and protein channel for CMA. During CMA, degradation substrates carrying KFERQ-like motifs are recognized and bound by the heat shock protein HSC70. The HSC70-substrate complex binds to the lysosomal membrane protein LAMP-2A to form a transmembrane translocation complex, after which the substrate is transported into the lysosome for degradation. LAMP-2A-mediated CMA is highly selective and helps to clear damaged, misfolded, or excess proteins from the cell, maintain intracellular protein homeostasis and normal function, and respond to various stress responses.

[0003] Under normal physiological conditions, LAMP-2A expression levels are tightly regulated to ensure a balance in intracellular protein degradation. CMA dysfunction is closely associated with the development and progression of neurodegenerative diseases, cardiovascular diseases, and some cancers. In neurodegenerative diseases such as Huntington's disease (HD) and Parkinson's disease (PD), abnormal and misfolded proteins accumulate in the form of toxic multimeric complexes, which are fundamental to the development of these diseases. LAMP-2A deficiency or dysfunction can lead to the abnormal aggregation of these pathogenic proteins, thereby exacerbating disease manifestations. In cardiovascular diseases such as atherosclerosis, macrophage CMA dysfunction leads to impaired lipid metabolism and increased inflammatory responses, thus accelerating the progression of atherosclerosis. Increasing research also demonstrates that LAMP-2A is often highly expressed in cancerous tissues, and knocking out LAMP-2A expression can effectively inhibit the proliferation of various tumor cells, suggesting that targeting LAMP-2A to inhibit CMA activity may be a potential cancer treatment.

[0004] LAMP-2A, a key protein in CMA (cell metabolites), can be used to assess CMA activity by detecting its levels. This also helps in exploring the molecular mechanisms and regulatory processes of autophagy, revealing the regulatory networks of protein metabolism under different physiological and pathological states. This is of great significance for disease diagnosis, prognostic assessment, and drug development in related fields. Utilizing the specific binding properties of antigens and antibodies to achieve quantitative and / or qualitative immunoassays is currently the main method for detecting antigen proteins, including Western blotting, immunofluorescence, immunohistochemistry, and flow cytometry. The core material in these methods is the antibody, and the quality of the antibody directly determines the accuracy and reliability of the detection results. Currently, highly sensitive and reliable immunoassay antibodies against human LAMP-2A protein have not been effectively researched and developed. Related antibodies are few in number, and existing antibodies suffer from weak affinity, low specificity, and low detection sensitivity. Therefore, developing a novel, high-performance anti-human LAMP-2A monoclonal antibody has become an urgent problem to be solved. Summary of the Invention

[0005] To address the lack of monoclonal antibodies against human LAMP-2A protein with high affinity, specificity, and / or high detection sensitivity in existing technologies, this invention provides a monoclonal antibody against human LAMP-2A protein, and further provides the application of this antibody or its antibody conjugate in the preparation of a human LAMP-2A protein immunoassay kit, as well as related immunoassay kits. To achieve the aforementioned objectives, this invention specifically utilizes the following technical solutions:

[0006] The first aspect of the present invention provides a monoclonal antibody against human LAMP-2A protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO. 3-5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO. 8-10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

[0010] A second aspect of the present invention provides a nucleic acid molecule or a recombinant vector comprising said nucleic acid molecule, said nucleic acid molecule encoding a monoclonal antibody against human LAMP-2A protein as described above.

[0011] Furthermore, the nucleic acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.12 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a complementary sequence thereto.

[0012] Furthermore, the nucleic acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.11 or is complementary to it, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or is complementary to it.

[0013] A third aspect of the present invention provides the use of the monoclonal antibody against human LAMP-2A protein as described above or an antibody-conjugate thereof in the preparation of a human LAMP-2A protein immunoassay kit, wherein the antibody-conjugate is formed by conjugating the monoclonal antibody with a detection label.

[0014] Furthermore, the immunoassay kit is selected from immunoblotting kits, immunofluorescence kits, immunohistochemical kits, or flow cytometry kits.

[0015] A fourth aspect of the present invention provides a human LAMP-2A protein immunoassay kit, the immunoassay kit comprising a monoclonal antibody against human LAMP-2A protein as described above or an antibody-conjugate thereof, the antibody-conjugate being formed by conjugating the monoclonal antibody with a detection label.

[0016] Furthermore, the immunoassay kit also includes a fluorescein-conjugated anti-rabbit IgG secondary antibody.

[0017] The advantages and positive effects of this invention are as follows:

[0018] The antibody against human lysosome-associated membrane glycoprotein 2A (LAMP-2A) screened by this invention exhibits excellent performance, with high specificity, high recognition sensitivity, and good binding affinity for human LAMP-2A protein. It can not only accurately locate the target protein but also effectively resist interference from non-target proteins. It shows no non-specific reaction when used for detection in complex samples such as cells and tissues. This is beneficial to improving the accuracy, sensitivity, specificity, and reliability of immunoassay for LAMP-2A protein. The antibody detection results are highly consistent with the actual expression of LAMP-2A protein, greatly reducing the incidence of false positive or false negative results. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the results of serum titer detection in rabbits immunized with human LAMP-2A protein in Example 1 of the present invention.

[0021] Figure 2 This is a map of the monoclonal antibody expression vector used to construct anti-human LAMP-2A protein in Example 1 of the present invention. From left to right, it shows the pBR322 vector pre-loaded with the antibody light chain constant region and heavy chain constant region.

[0022] Figure 3 This is a Western blot image showing the results of immunoblotting detection of human A549 cells using a monoclonal antibody against human LAMP-2A protein in Example 2 of the present invention.

[0023] Figure 4 This is an image showing the immunofluorescence detection results of human HeLa cells using a monoclonal antibody against human LAMP-2A protein in Example 2 of the present invention.

[0024] Figure 5This is an image showing the immunohistochemical detection results of a human kidney tissue section sample using a monoclonal antibody against human LAMP-2A protein in Example 2 of the present invention.

[0025] Figure 6 This is a flow cytometry result of HeLa cells before and after homozygous knockout of the LAMP2 gene, using a monoclonal antibody against human LAMP-2A protein in Example 2 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0027] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0028] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0029] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0030] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0031] The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is considered to include (i) A, (ii) B, and (iii) A and B.

[0032] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and "rabbit monoclonal antibody," etc., have the same meaning. Unless otherwise specified, they all refer to antibodies that specifically bind to human lysosome-associated membrane glycoprotein 2A. The terms "lysosome-associated membrane glycoprotein 2A," "LAMP-2A," "LAMP2A," "LAMP-2A protein," "CD107b," "LAMPB," and "CD107-B," etc., have the same meaning. The modifier "rabbit" indicates that the antibody's complementarity-determining region (CDR) is derived from a rabbit immunoglobulin sequence.

[0033] An antibody is an immunoglobulin molecule that specifically binds to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In this invention, the term "antibody" is to be interpreted in the broadest sense and includes various antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, provided they exhibit the desired antigen-binding activity. An antibody fragment may be one or more portions or fragments of a full-length antibody, retaining the antibody's ability to specifically bind to a target antigen.

[0034] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be classified into two types: κ chains and λ chains; heavy chains can be classified into five types: μ, δ, γ, α, and ε chains, with antibodies defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of both the heavy and light chains vary considerably, while the amino acid sequences of other parts are relatively constant. The regions with significant amino acid sequence variation near the N-terminus in both the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable regions of the heavy chain (VH) and light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable region, also known as the complementarity-determining region (CDR), is a ring structure. Heavy chain CDRs and light chain CDRs are tightly joined together by the FR region and cooperate to form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the antibody's specificity and serving as the site for antibody recognition and antigen binding. The FR regions are the more conserved parts of the VH and VL, generally exhibiting a β-sheet configuration, linked by three CDRs forming a connecting loop. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0035] CDRs and FRs can be identified according to Kabat definitions, Chothia definitions, the sum of Kabat and Chothia definitions, AbM definitions, contact definitions, IMGT unique numbering definitions and / or conformational definitions, or any CDR determination method known in the art. As used in this invention, they are defined by the Kabat numbering system.

[0036] The light chain constant region (CL) and heavy chain constant region (CH) do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The CL lengths of different Ig types (κ or λ) are generally consistent, but the CH lengths differ among Ig classes. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well-known in the art and can be obtained by searching the IMGT database.

[0037] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-type molecular structure. Therefore, in the context of this invention, "full-length antibody," "complete antibody," and "Y-type antibody" have the same meaning and can be used interchangeably.

[0038] An antibody fragment is one or more portions or segments of a full-length antibody that substantially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR region as the full-length antibody, and more preferably the same variable region, thereby retaining complete antigen recognition and binding sites, enabling it to bind to the same antigens, especially the same epitopes, as the full-length antibody. Typical examples of antibody fragments include Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2, which can be obtained using conventional techniques in the art.

[0039] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of a heavy chain (variable and first constant region). Fragments such as Fab, F(ab')2, and Fab' can be obtained by protease cleavage of a full-length antibody. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab possesses an antigen-binding region and a portion of a constant region, it not only has antibody-antigen affinity and excellent tissue penetration like scFv, but also has a more stable structure.

[0040] (ii)F(ab)2: Contains a bivalent segment consisting of two Fabs connected by a disulfide bridge in the hinge region.

[0041] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment. It contains only the variable region and consists of a variable region of one light chain and one heavy chain. It is a non-covalently bound dimer of VH and VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0042] (iv)(Fv)2: Consists of two Fv segments covalently linked together.

[0043] (v)scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a flexible linker (typically composed of 10-25 amino acids). It retains the original antibody's specificity for binding to the antigen. The linker in this invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked to its two ends. Compared to full-length antibodies, scFv has a smaller molecular weight, thus exhibiting higher penetration and lower immune side effects.

[0044] The (vi)sc(Fv)2 segment is formed by connecting two heavy chain variable regions and two light chain variable regions through a joint, etc.

[0045] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a CDR region and a FR region derived from a rabbit immunoglobulin sequence. In other embodiments, the antibody may contain amino acid residues encoded by a non-rabbit immunoglobulin sequence, such as humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection response while maintaining the desired specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a specific source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody containing the CDR region of a non-human antibody, such as a rabbit antibody, and a FR region derived from a human antibody. In some cases, the variable region of the non-human antibody binds to the constant region of a human antibody, such as in human-rabbit chimeric antibodies; in other cases, the CDR region of the non-human antibody binds to both the FR region and the constant region derived from a human antibody sequence, i.e., grafting the CDR region of the non-human antibody onto a human antibody frame (FR) sequence derived from the FR sequence of one or more other human antibody variable regions. In this invention, the CDR region in the chimeric antibody or humanized antibody is derived from the rabbit CDR region.

[0046] The terms "monoclonal antibody" or similar terms are used interchangeably and refer to a homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for a small number of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). A "monoclonal antibody" is highly specific, exhibiting a single binding specificity and affinity for the same or substantially identical epitopes on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as limiting the source or method of preparation of the antibody. This antibody can be prepared by a variety of methods, including but not limited to hybridoma, phage display, yeast display, recombinant DNA, single-cell screening, or single-cell sequencing.

[0047] The term "specific binding" is a well-known term in the art. A molecule exhibits "specific binding" if its reaction with a specific target antigen or epitope is more frequent, faster, longer-lasting, and / or has a greater affinity than its reaction with other target antigens or epitopes. "Specific binding," also known as "preferential binding," does not necessarily require (although may include) exclusive binding. To make the above-described objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the invention are described in detail below with reference to the accompanying drawings.

[0048] This invention provides a monoclonal antibody against human LAMP-2A protein, comprising a light chain variable region and a heavy chain variable region. Both the light chain and heavy chain variable regions include three complementarity-determining regions (CDRs), named CDR1, CDR2, and CDR3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively.

[0049] The antibody against human lysosome-associated membrane glycoprotein 2A (LAMP-2A) screened in this invention exhibits excellent performance, high specificity, high recognition sensitivity, and good binding affinity for human LAMP-2A protein. It not only accurately locates the target protein but also effectively resists interference from non-target proteins. It shows no non-specific reactions when used in the detection of complex samples such as cells and tissues. This improves the accuracy, sensitivity, specificity, and reliability of immunoassay for LAMP-2A protein. It demonstrates good applicability in various immunoassay systems, especially immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry. The antibody detection results show a high degree of agreement with the actual expression of LAMP-2A protein, significantly reducing the incidence of false positives or false negatives. This invention provides an effective antibody tool and detection method for the high-specificity, high-accuracy, and high-sensitivity detection of human LAMP-2A protein, and has broad market prospects in clinical diagnosis and scientific research.

[0050] Optionally, both the light chain variable region and the heavy chain variable region include four frame regions (FRs), which are arranged in an alternating sequence with three core parameters (CDRs) to form the variable region. The amino acid sequence of the light chain variable region (VL) of the antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.7.

[0051] Optionally, the antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH), wherein CL and VL constitute a complete light chain (FL), and CH and VH constitute a complete heavy chain (FH). The constant regions of the antibody can usually be obtained through public searches, such as searching for rabbit-derived IgG gamma C reign to obtain CH and searching for rabbit-derived IgG Kappa C reign to obtain CL through the IMGT online database (www.imgt.org).

[0052] Specifically, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0053] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or the antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining an intact antigen recognition and binding site, capable of binding to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0054] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule, wherein the nucleic acid molecule encodes a monoclonal antibody against human LAMP-2A protein as described above.

[0055] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA, or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0056] The sequence of a nucleic acid molecule can be derived from the antibody AA sequence using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or its fragments can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0057] For example, the nucleic acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.12 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a complementary sequence thereto.

[0058] For example, the nucleic acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.11 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or a complementary sequence thereto.

[0059] Those skilled in the art will understand that, due to the degeneracy of the genetic code, nucleic acid molecules other than those in the above examples can also encode the antibodies of the present invention. Therefore, the nucleic acid molecules in the above examples should not be regarded as limiting the scope of protection of the present invention.

[0060] The original vector used to construct the recombinant vector can be any vector conventional in the art, as long as it can contain the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1, and M13), viscera, and mini-chromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules into a host and multiply them in host cells) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, which is then introduced into a host cell and cultured under specific conditions to express and obtain an antibody. This is a well-known technique in the art and will not be described in detail here.

[0061] The nucleic acid molecules encoding the antibodies FL and FH of this invention can be inserted into two vectors, which can be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules encoding antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence ligated downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy and light chains can be operatively ligated to different promoters, or the nucleic acid sequences encoding the heavy and light chains can be operatively ligated to a single promoter, such that both the heavy and light chains can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibodies.

[0062] Recombinant vector transfection or transformation into host cells is performed using conventional techniques. When the host is a prokaryote such as *E. coli*, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2; alternatively, methods such as microinjection, electroporation, liposome packaging, or gene gun can be used. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or gene gun.

[0063] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in this invention include, but are not limited to, *Escherichia coli* (e.g., DH5α, JM109, BL21, W3110), *Bacillus* spp. (e.g., *Bacillus subtilis*, *Bacillus thuringiensis*), *Enterobacterium* strains (e.g., *Salmonella typhimurium*, *Serratia marcescens*), and *Pseudomonas* spp. Eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining host cells transfected or transformed with the recombinant vector described above, they can be cultured under suitable conditions to express antibodies, which can then be isolated to obtain purified antibodies.

[0064] In a preferred embodiment, the recombinant vector described above is the mammalian expression vector pBR322, and the host cell is human renal epithelial cells (293F cells).

[0065] In a preferred embodiment, the method for preparing the monoclonal antibody includes: tandemly loading the heavy chain gene, light chain gene, and signal peptide of the antibody onto the expression vector pBR322, co-transfecting 293F cells, culturing and collecting the cell culture supernatant, and purifying to obtain the target antibody. The selection of the signal peptide is designed based on the host cell, and this invention does not have any particular limitations in this regard.

[0066] In another embodiment of the present invention, the application of the monoclonal antibody against human LAMP-2A protein as described above or its antibody-drug conjugate thereof in the preparation of a human LAMP-2A protein immunoassay kit is provided, wherein the antibody-drug conjugate comprises the monoclonal antibody against human LAMP-2A protein as described above and a detection marker linked to the monoclonal antibody.

[0067] The advantages of using the monoclonal antibody against human LAMP-2A protein or its antibody conjugate in the preparation of a human LAMP-2A protein immunoassay kit are the same as the advantages of the monoclonal antibody against human LAMP-2A protein over the prior art as described above, and will not be repeated here.

[0068] It is important to emphasize that the antibodies of this invention can be used alone or linked (covalently or non-covalently) with detection markers to form antibody-conjugates. In some embodiments, the antibodies of this invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to the LAMP-2A protein in the sample to be tested. LAMP-2A is then qualitatively or quantitatively detected by analyzing the signal of the detection marker linked to it. In other embodiments, the antibody against the LAMP-2A protein is not labeled (as a primary antibody or capture antibody), but the detection marker is linked to a secondary antibody (as a detection antibody) or other molecules. For example, if the anti-LAMP-2A antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. Thus, the qualitative or quantitative detection of LAMP-2A is achieved by analyzing the change in the detection marker signal generated after the secondary antibody specifically binds to the antibody of this invention, as exemplified by the multiple detection systems established in Example 2 of this invention below.

[0069] The detection markers are used to generate identifiable signal changes to identify the antibodies or secondary antibodies of the present invention based on these signal changes, thereby identifying the expression of LAMP-2A protein in the sample to be tested through a specific antigen-antibody reaction. Detection markers include, but are not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride), fluorescent proteins (such as isophycocyanin, phycoerythrin, PerCP, and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, and avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0070] The aforementioned immunoassay methods include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB), immunoprecipitation (IP), and flow cytometry (FC).

[0071] Based on the same inventive concept, embodiments of the present invention also provide a human LAMP-2A protein immunoassay kit, the kit comprising a monoclonal antibody against human LAMP-2A protein or an antibody-drug conjugate thereof as described above.

[0072] Optionally, the immunoassay kit is an enzyme-linked immunosorbent assay (ELISA) kit, an ELISA kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.

[0073] Preferably, the immunoassay kit is an immunoblotting kit, an immunofluorescence kit, an immunohistochemistry kit, or a flow cytometry kit, and the immunoassay kit further includes a fluorescein-conjugated anti-rabbit IgG secondary antibody.

[0074] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0075] Example 1: Preparation of rabbit monoclonal antibody against human LAMP-2A protein

[0076] 1.1 Antigen Preparation: In this embodiment, recombinant human LAMP-2A protein was used as the antigen to immunize animals. Based on single B cell labeling and sorting technology, B cells capable of recognizing the target antigen were enriched and sorted from the animal spleen. These cells were cultured in single-cell form to obtain monoclonal antibodies secreted by the B cells. The amino acid sequence of the LAMP-2 protein can be found in Uniprot ID: P13473 or NCBI accession number: NP_002285.1, and the gene sequence can be found in NCBI accession number: NM_002294.3. The gene sequence corresponding to amino acid fragments 29-375 of human LAMP-2 was constructed into the pBR322 vector, and high-quality mature recombinant human LAMP-2 protein with biological activity was expressed in 293F cells as the antigen. The specific amino acid (AA) fragment of human LAMP-2 from amino acid 29-375 is shown below, belonging to the LAMP-2A subtype:

[0077] LELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTT VPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQDCSADDDNF (see SEQ ID NO.15).

[0078] 1.2 Animal Immunization: Four New Zealand white rabbits were immunized with 200 μg / rabbit of human LAMP-2A protein. Before the first immunization, the antigen was mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits. Every three weeks after the first immunization, 100 μg of immunogen was mixed with an equal volume of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits for two booster immunizations. After the three immunizations, rabbit serum was collected, and the titer of targeting LAMP-2A protein was determined by antigen-coated enzyme-linked immunosorbent assay (ELISA). Rabbits with an OD450nm greater than 0.2 were given a booster immunization with 200 μg of immunogen injected subcutaneously at multiple sites. Spleens were harvested three to four days later.

[0079] Results of ELISA determination of immune serum titers are shown below. Figure 1 The antigen was recombinant human LAMP-2A protein, with a final coating concentration of 1 μg / mL. The test serum served as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (purchased from Jackson, catalog number 111-035-045) served as the secondary antibody. The secondary antibody dilution ratio was 1:5000. Pre-immunization rabbit serum was used as a negative control, and the detection system without immunized serum served as a blank control (NC). WA-63982D is the project number, and N17298-N17299 are the rabbit numbers. Figure 1 It can be seen that a strong immune response was generated in the rabbits after the third and fourth booster immunizations, with high titers of neutralizing antibodies. The serum titer of the fourth immunization was the best and can be used to isolate antibodies.

[0080] 1.3 Isolation of B cells and sorting of antigen-specific B cells in the spleen: For relevant methods, please refer to the published patents “Method for efficient isolation of single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)” and “An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: 2020-08-11)”.

[0081] 1.4 Cloning of the gene encoding a rabbit-derived monoclonal antibody: The supernatant from cultured B cells was used to identify antigen-specific B cells using an antigen-coated ELISA. Lysed cells were collected and analyzed using Quick-RNA... TM RNA was extracted using the Micro Prep kit (ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR and sequenced. The PCR system consisted of: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (ABclonal, catalog number RK20717), and 6.5 μL H2O. The PCR program consisted of: 98℃ for 30 s, followed by 40 cycles of 98℃ for 10 s, 64℃ for 30 s, and 72℃ for 30 s, with a final temperature of 72℃ for 5 min. The reaction solution was stored at 4℃. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward and reverse primers, respectively.

[0082] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 16);

[0083] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 17);

[0084] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 18);

[0085] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 19).

[0086] The amplified DNA product was sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7; then the sequence of the constant region was obtained by querying the IMGT online database (www.imgt.org), resulting in antibody 1F12 with the complete light chain (FL) shown in SEQ ID NO.1 and the complete heavy chain (FH) shown in SEQ ID NO.6.

[0087] Antibody sequencing was performed by Kinkai Biotechnology Co., Ltd. The amino acid (AA) and nucleotide (DNA) sequences of the antibodies are shown in Table 1. In the table, LCDR1-3 represent complementarity-determining regions CDR1-3 on the light chain variable region, and HCDR1-3 represent complementarity-determining regions CDR1-3 on the heavy chain variable region. The variable region numbering system is the Kabat numbering system.

[0088] Table 1. Sequence information of rabbit-derived monoclonal antibody 1F12 in this embodiment.

[0089]

[0090]

[0091] 1.5 Expression and Large-Scale Production of Antibody 1F12: Using recombinant expression technology, the amplified VL and VH genes were inserted in tandem with the light chain constant region (CL) and heavy chain constant region (CH) into an expression vector. Monoclonal antibody 1F12 was then mass-produced through recombinant expression of the antibody genes. In this embodiment, the CL and CH genes were pre-inserted into the mammalian expression vector pBR322, and its expression profile is shown below. Figure 2 In the figure, pBR322 origin and f1 origin are replication promoters, Ampcillin is the resistance gene, CMV promoter is the transcription promoter, SV40 PA terminator is the tailing signal, Light chain constant is the nucleotide sequence of CL (left figure), and Heavy chain constant is the nucleotide sequence of CH (right figure). Then, the VL and VH genes were ligated to the expression vector pBR322 carrying the CL and CH genes, linearized with XbaI (955bp) and NheI (949bp) restriction endonucleases respectively, via homologous recombination to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors. Sequencing confirmed the successful construction of the vectors.

[0092] To facilitate antibody purification, a signal peptide is added upstream of the VL and VH genes to achieve secretory expression of the antibody. The signal peptide can be a commonly used antibody expression signal peptide in the field, such as the patent "Rabbit Monoclonal Antibody against Human Interferon α2 and its Application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and the patent "High Affinity Human..." The IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21) has a signal peptide "MDTRAPTQLLGLLLLWLPGATF (encoding gene in this embodiment is atggacacgagggcccccactcagctgctggggctcctgcttttatggctgccaggtgctaccttt)" upstream of VL and "METGLRWLLLVAVLKGVQC (encoding gene in this embodiment is atggagactgggctgcgctggcttctcctggtggctgtcctcaaaggggtccaatgc)" upstream of VH. Of course, those skilled in the art can replace the signal peptide with other ones after obtaining the antibody sequence of this invention for antibody expression; therefore, the signal peptide sequence is not shown in Table 1 of this embodiment.

[0093] The successfully constructed FL and FH expression vectors were co-transfected into 293F cells. After transfection and culture for 72-96 hours, the culture supernatant contained a recombinant rabbit monoclonal antibody recognizing human LAMP-2A protein. The target antibody was purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100). The antibody purity was verified to be ≥95% by 12% polyacrylamide gel electrophoresis (SDS-PAGE). The purified antibody was aliquoted and stored at -20°C for later use.

[0094] Example 2: Establishment and efficacy evaluation of an immunoassay method based on anti-human LAMP-2A protein antibody 1F12.

[0095] This embodiment establishes an immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry analysis system for rabbit monoclonal antibody 1F12 to evaluate the antibody's application performance.

[0096] 2.1 Establishment of an immunoblotting (WB) detection system

[0097] The WB experimental analysis procedure is as follows: (1) Cell sample preparation: Human non-small cell lung cancer cells (A549) with high expression of Human LAMP-2A are passaged and cultured. When the cell confluence is 70-80%, the culture supernatant is aspirated, and the cells are carefully washed twice with PBS solution. The cells are then lysed to obtain protein lysis buffer; (2) Electrophoresis: 6% separating gel is selected for polyacrylamide gel electrophoresis (SDS-PAGE); (3) Transfer: The gel protein band is transferred to a PVDF membrane using the conventional method in the electrotransfer system; (4) Blocking: The PVDF membrane is placed in TBST buffer containing 3% skim milk powder. (5) Blocking at room temperature for 1 hour; (6) Primary antibody incubation: Add antibody 1F12 prepared in Example 1 (working concentration of primary antibody is 0.01 μg / mL), incubate at 4°C overnight; (7) Secondary antibody incubation: Wash the membrane 3-4 times with TBST, add goat anti-rabbit IgG secondary antibody (from Abclonal, catalog number AS014, secondary antibody dilution ratio 1:100000), incubate at room temperature for 1 hour; (8) Color development: Wash the membrane 3-4 times with TBST, add ECL ultrasensitive color development solution (from Abclonal, catalog number RM00020), and develop.

[0098] Human LAMP-2A has a full length of 410 aa and a theoretical molecular weight of 100-130 kDa. Figure 3 It can be seen that the rabbit monoclonal antibody 1F12 against human LAMP-2A protein obtained by screening was detected as a single target band with obvious band signal in A549 cell lysate, with no non-specific bands. The actual detected molecular weight was consistent with the expectation, indicating that the antibody has good specificity, high affinity for human LAMP-2A protein, and good antigen recognition sensitivity and anti-interference ability of cell components.

[0099] 2.2 Establishment of Immunofluorescence (IF) Detection System

[0100] The IF detection method is as follows: (1) Sample processing: Take human cervical cancer cells (HeLa) that highly express LAMP-2A protein, add 4% neutral formaldehyde fixative, fix at room temperature for 15 min, then centrifuge to collect cell pellet, wash and resuspend with PBS buffer, aspirate the cell suspension and spread it on a glass slide to prepare a cell smear; (2) Blocking: Completely cover the sample with TBS buffer containing 5% goat serum, place in a 37℃ constant temperature and humidity incubator for 30 min, and remove the blocking solution; (3) Primary antibody incubation: Add antibody 1F12 solution prepared in Example 1 (primary antibody working concentration is 2.75 μg / mL), incubate at 4℃ overnight; take out and warm to room temperature for 15 min, remove the primary antibody working solution, and then wash with TBST; (4) Secondary antibody incubation: Add Cy3 fluorescent secondary antibody solution (Cy3-conjugated Goat anti-Rabbit) IgG (H+L), from ABclonal, catalog number AS007, secondary antibody dilution ratio 1:500), incubated at 37℃ in the dark for 1 h; remove the secondary antibody working solution, then wash with TBST; (5) staining the nucleus: add 1 μg / mL DAPI nuclear dye working solution, stain at room temperature for 30 min, remove the DAPI working solution, wash once with TBST, then wash twice with buffer TBS, 5 min each time; (6) color development: after adding anti-fluorescence attenuation mounting medium, observe and acquire images under a fluorescence microscope. α-Tubulin cytoskeletal protein was used as an intracytoplasmic reference, its primary antibody was α-Tubulin Mouse mAb (from ABclonal, catalog number AC012, dilution ratio 1:400), and the secondary antibody was... 488-conjugated Goat Anti-Mouse IgG(H+L)Ab (from ABclonal, catalog number AS076, dilution ratio 1:500).

[0101] IF test results are shown below Figure 4The top image, from left to right, shows fluorescence imaging of human HeLa cell line samples bound with antibody 1F12 and anti-α-Tubulin antibody. After antibody 1F12 binds to LAMP-2A protein in HeLa cells, it is labeled red with fluorescent secondary antibody, and after anti-α-Tubulin antibody binds to α-Tubulin protein in HeLa cells, it is labeled green with fluorescent secondary antibody. The bottom image, from left to right, shows fluorescence imaging of cell samples counterstained with DAPI and a comparison image showing the red, blue, and green merging. The cell nucleus is blue after DAPI counterstaining. LAMP-2A is mainly expressed on the lysosomal membrane, and is also expressed in the cell membrane, endosomal membrane, cytoplasmic vesicles, autophagosome membrane, exosomes, extracellular space, lysosomal lumen, perinuclear region of the cytoplasm, and trans-Golgi network, appearing as diffusely distributed granular staining in the cytoplasm. As can be seen from the figure, the antibody of the present invention can specifically bind to the LAMP-2A protein in HeLa cell samples. The detection results are consistent with its theoretical localization, further proving that antibody 1F12 has good recognition specificity and binding sensitivity to human LAMP-2A.

[0102] 2.3 Construction of Immunohistochemistry (IHC) Detection System

[0103] The IHC detection steps are as follows: (1) Baking: The paraffin sections of human kidney tissue baked at 56℃ for 30 min are immersed in dewaxing solution 1. After 5 min, the sections are taken out and immersed in dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3 in sequence. When placed in dewaxing solution, they are placed for 5 min and when placed in anhydrous ethanol, they are placed for 3 min. Then, the sections are rinsed with running water for 3 min. Dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; (2) Antigen retrieval: 0.01M Tris-EDTA retrieval solution (pH 9.0) High-pressure heat repair; (3) Endogenous peroxidase inactivation: Wash with PBS buffer 3 times, 3 min each time, and remove the buffer from the slide; then immerse the slide in 3% hydrogen peroxide solution and incubate at room temperature for 10 min; (4) Blocking: Wash with PBS buffer 3 times, 3 min each time, then remove the buffer, delineate the area to be tested on the slide, add PBS blocking solution to the area to be tested, and incubate at room temperature for 30 min; (5) Primary antibody incubation: Remove the blocking solution, add antibody 1F12 dilution solution (final primary antibody concentration is 0.2 μg / mL), and incubate at room temperature for 60 min; remove the antibody working solution, rinse quickly with PBS buffer once, and soak and wash 3 times, 3 min each time; (6) Secondary antibody incubation: Add ready-to-use secondary antibody working solution (Dako REAL EnVision Detection) System, Peroxidase / DAB, Rabbit / Mouse, HRP; purchased from Dako, catalog number K5007), incubated at room temperature for 25 min; remove the secondary antibody working solution, rinse quickly once with PBS buffer, and soak and wash three times, each time for 3 min; (7) staining: add staining working solution, closely observe the color change under a microscope, and after obtaining a suitable staining intensity, immerse the section in a large amount of distilled water to stop staining, and then rinse with running water for 10 min; (8) counterstaining: drain the slightly dried tissue sections (9) Blueing: Immerse the slightly drained sections in a saturated lithium carbonate solution for 3 seconds and rinse with running water for 3 minutes; (10) Dehydration: Immerse the sections in anhydrous ethanol twice, lifting them up and down several times during the immersion process, and remove them after 10 seconds; dry the sections at high temperature (54-58℃); (11) Mounting: Add an appropriate amount of neutral resin to the center of the section and cover it with a coverslip. The amount of resin should be appropriate. After covering with a coverslip, the tissue should be completely covered and no resin should overflow. Finally, scan the sections.

[0104] IHC staining results of human kidney tissue samples as follows Figure 5As shown, LAMP-2A protein is highly expressed in peroxisomes, lysosomes, etc. in human kidney tissue samples. The results show that human kidney tissue has specific brown staining, and positive staining granules are distributed in small spots in the cytoplasm. The staining location is consistent with the actual expression of the positive marker LAMP-2A protein. Moreover, the background color is light or unstained, showing a clear positive signal. This indicates that antibody 1F12 has clear and accurate localization, high specificity and sensitivity in recognizing LAMP-2A protein, and its binding to LAMP-2A protein is not affected by the complexity of cells / tissues.

[0105] 2.4 Establishment of a Flow Cytometry (FC) Detection System

[0106] The specificity of FC detection in immune serum includes the following steps: (1) Disinfect the ultra-clean workbench with ultraviolet irradiation for 15-20 min, turn on the fan for 5 min, and prepare for aseptic work; (2) Collect and wash cells, determine the total number of cells, and check that the cell viability is 90%-95%; the cell samples to be tested include HeLa cells and HeLa cells with homozygous knockout of the LAMP2 gene (LAMP2Knockout HeLa Cell Line); (3) Resuspend the cells in PBS buffer to about 3×10 6 -5×10 6 (3) Distribute cells at 100 μL / well into 96-well V-plates and wash once with 1×PBS; (4) Dilute the L / D staining solution (Live / Dead staining solution) of Zombie NIR Fixable Viability Kit (purchased from Biolegend, catalog number 423105) at a ratio of 1:1500 and distribute the diluted staining solution at 100 μL / well into the wells, resuspend the cells in the wells, and react at room temperature for 15 min; (5) Centrifuge at 400g for 5 min, discard the supernatant, and wash twice with FACS buffer (formulation: 1000 mL 1×PBS + 40 mL FBS + 1 mL 0.5 MEDTA); (6) Distribute 0.5% BSA / PBS (formulation: 1 L 1×PBS + 5 g) at 100 μL / well into 96-well V-plates and wash once with 1×PBS. (7) Dispense the diluted BSA-containing immune serum (as primary antibody) into the wells of the plate, resuspend the cells in each well, and incubate at room temperature for 30 min; (8) Centrifuge at 400g for 5 min, discard the supernatant, and wash twice with 0.5% BSA / PBS; (9) Add 100 μL / well of the fluorescent secondary antibody (Alexa) diluted with 0.5% BSA / PBS (dilution ratio 1:600). 647 Affini Pure F(ab')2 Fragment Goat Anti-Rabbit IgG (purchased from Jackson, catalog number 111-096-046) was dispensed into wells, and the cells were resuspended in the wells and reacted at room temperature for 30 min; (9) the cells were washed twice with 0.5% BSA / PBS, and then resuspended in each well with 200 μL of 0.5% BSA / PBS and stored in the dark; (10) the analysis was performed according to the Beckmancy toflex flow cytometer operation and maintenance SOP-105-AND-CA-008. Rabbit IgG isotype control (from ABclonal, catalog number AC042) was used as isotype control.

[0107] The FC detection results of HeLa cell samples before and after homozygous knockout of the LAMP2 gene are as follows: Figure 6 As shown, from left to right, HeLa and LAMP2 Knockout HeLa cells are represented. The horizontal axis represents the relative intensity of the fluorescence signal / scattered light signal of the channel, and the vertical axis represents the corresponding cell number. The red curve is the blank control without any antibody, the blue curve is the isotype control, and the yellow curve is the antibody 1F12 against human LAMP2 protein. It can be seen that antibody 1F12 shows a significant fluorescence transition signal after binding to HeLa cells (positive cells), but no fluorescence transition signal after binding to the LAMP2 Knockout HeLa cell line. Furthermore, the blank control and isotype control show no significant signal transition, confirming that anti-rabbit monoclonal antibody 1F12 has good specificity and high affinity for FC detection, and does not specifically recognize non-target proteins other than LAMP-2A protein.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody against human LAMP-2A protein, characterized in that, It includes a light chain variable region and a heavy chain variable region. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The monoclonal antibody against human LAMP-2A protein according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against human LAMP-2A protein according to claim 2, characterized in that, The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against human LAMP-2A protein according to claim 1, characterized in that, The monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and sc(Fv)2 fragment.

5. A nucleic acid molecule or recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule, which encodes a monoclonal antibody against human LAMP-2A protein as described in any one of claims 1-4.

6. The nucleic acid molecule or recombinant vector according to claim 5, characterized in that, The nucleic acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.12 or is complementary to it, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO.14 or is complementary to it.

7. The nucleic acid molecule or recombinant vector according to claim 6, characterized in that, The nucleic acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.11 or is complementary to it, and the nucleic acid sequence of the heavy chain is shown in SEQ ID NO.13 or is complementary to it.

8. The use of the monoclonal antibody against human LAMP-2A protein or its antibody-conjugate as described in any one of claims 1-4 in the preparation of a human LAMP-2A protein immunoassay kit, characterized in that, The antibody conjugate is formed by linking the monoclonal antibody to a detection marker.

9. A human LAMP-2A protein immunoassay kit, characterized in that, Includes a monoclonal antibody against human LAMP-2A protein as described in any one of claims 1-4, or an antibody-conjugate thereof, wherein the antibody-conjugate is formed by conjugating the monoclonal antibody with a detection label.

10. The human LAMP-2A protein immunoassay kit according to claim 9, characterized in that, The immunoassay kit is selected from immunoblotting kits, immunofluorescence kits, immunohistochemistry kits, or flow cytometry kits, and the immunoassay kit also includes a fluorescein-conjugated anti-rabbit IgG secondary antibody.

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