Antibody for resisting human secretory gelsolin, antibody pair, kit and application

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an antibody for resisting human secretory gelsolin, an antibody pair, a kit and application. The antibody is a first antibody or a second antibody, amino acid sequences of light chains CDR1-3 of the first antibody are respectively shown as SEQ ID NO.3-5, and amino acid sequences of heavy chains CDR1-3 of the first antibody are respectively shown as SEQ ID NO.8-10; the amino acid sequences of light chains CDR1-3 of the second antibody are respectively as shown in SEQ ID NO.13-15, and the amino acid sequences of heavy chains CDR1-3 of the second antibody are respectively as shown in SEQ ID NO.18-20. The antibody disclosed by the invention has good specificity and high affinity to human secreted gelsolin, can effectively recognize low-concentration antigens and recognize different antigen epitopes, and provides an ideal antibody tool and a detection system for efficiently, specifically, accurately, sensitively, reliably and stably detecting extremely trace amount of gelsolin.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and particularly to antibodies, antibody pairs, kits and applications against human secreted gelsolin. Background Art

[0002] Gelsolin (GSN) is a core member of the Gelsolin superfamily and a multifunctional actin-binding protein. It can regulate the structure of actin in a calcium-dependent manner by cutting, capping actin microfilaments or nucleating actin aggregation, etc., and plays a key role in the mutual conversion between the cytoplasmic sol state and the gel state. The mutual conversion between the cytoplasmic sol state and the gel state is the basis of many cellular processes. Through this mode of action, GSN plays an important role in many cellular processes related to cytoskeleton rearrangement, and also participates in the regulation of cell movement, cell signal transduction and apoptosis. GSN has a cytoplasmic type and a secreted type (secreted gelsolin, sGSN) distributed in extracellular fluids, including plasma, interstitial fluid, etc. The two types are encoded by the same gene through transcription at different transcription start sites.

[0003] sGSN exists in plasma at a certain concentration and can maintain the balance of extracellular actin. Its concentration will change under different physiological and pathological conditions. In cases such as cell damage or inflammation, intracellular actin will leak into the extracellular space. sGSN can bind to extracellular actin and cut it into smaller fragments, which helps to maintain blood fluidity and prevent thrombosis, and is also of great significance for physiological processes such as inflammatory response and wound healing. In the tumor microenvironment, sGSN can affect the occurrence and migration processes of tumors by regulating intracellular signal transduction and cell motility, and its expression and deletion play a two-way role in different tumors. The overexpression of gelsolin has been widely reported to increase the motility and invasiveness of tumor cells, and its overexpression is associated with a significant increase in the incidence of early tumor recurrence and metastasis and a decrease in patient survival rate, such as hepatocellular carcinoma, colorectal cancer, prostate cancer, oral cancer and lung adenocarcinoma, etc. In the process of tumor immune escape, sGSN also plays a key role. sGSN blocks the binding of the receptor DNGR-1 on the surface of dendritic cells to F-actin, interferes with the ability of dendritic cells to recognize and process dead tumor cells, and inhibits the antigen presentation reaction derived from dead tumor cells, thereby helping tumor cells to escape the attack of the immune system. Other data show that gelsolin can inhibit tumorigenesis by remodeling the cytoskeletal actin microfilaments, and its level decreases in many cancers, including bladder cancer, colon cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast ductal carcinoma, etc., and the expression level decreases with tumor progression and reduced differentiation degree. sGSN in blood has been proven to have great potential as a cancer diagnostic marker.

[0004] Detection of blood GSN levels can assist in the early diagnosis of cancer. In inflammatory diseases, its levels may also be abnormal. Currently, immunological analysis methods based on the specific reaction of antigen and antibody are mostly used to detect the level of sGSN protein. However, the currently commercially available anti-human sGSN antibodies have problems such as poor affinity and / or low specificity, and the detection sensitivity and reliability for extremely low concentrations in pathological samples, such as those at the ng / mL level, are not high. Summary of the Invention

[0005] Aiming at the problems of poor affinity and / or low specificity of the existing anti-human secreted gelsolin (sGSN), resulting in low detection sensitivity and poor reliability, the present invention provides two high-affinity anti-human sGSN antibodies and an antibody pair composed of them, as well as a kit containing the aforementioned antibody or antibody pair and its application. The antibody pair of the present invention has good specificity and high affinity for human sGSN, can effectively recognize low-concentration antigens and recognize different antigen epitopes, and provides an ideal antibody tool and a double-antibody sandwich enzyme-linked immunosorbent assay system for detecting extremely trace levels of gelsolin efficiently, specifically, accurately, sensitively, reliably, and stably.

[0006] To achieve the above object, the present invention is specifically realized through the following technical solutions:

[0007] In the first aspect of the present invention, an antibody against human secreted gelsolin is provided, which is a first antibody or a second antibody, wherein: the amino acid sequences of complementarity-determining regions CDR1-3 in the light chain variable region of the first antibody are respectively as shown in SEQ ID NO.3-5, and the amino acid sequences of complementarity-determining regions CDR1-3 in the heavy chain variable region are respectively as shown in SEQ ID NO.8-10; the amino acid sequences of complementarity-determining regions CDR1-3 in the light chain variable region of the second antibody are respectively as shown in SEQ ID NO.13-15, and the amino acid sequences of complementarity-determining regions CDR1-3 in the heavy chain variable region are respectively as shown in SEQ ID NO.18-20.

[0008] Furthermore, the amino acid sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the second antibody is as shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.17.

[0009] Furthermore, the amino acid sequence of the light chain of the first antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.6; the amino acid sequence of the light chain of the second antibody is as shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.16.

[0010] Further, the first antibody or the second antibody is a full-length antibody or an antigen-binding region of the full-length antibody; the antigen-binding region is selected from Fab fragment, F(ab) 2 fragment, Fv fragment, (Fv) 2 fragment, scFv fragment, and sc(Fv) 2 fragment, and at least one of them.

[0011] The second aspect of the present invention provides a nucleic acid molecule encoding the first antibody or the second antibody as described above.

[0012] The third aspect of the present invention provides a recombinant vector containing the nucleic acid molecule as described above.

[0013] The fourth aspect of the present invention provides an antibody pair against human secreted gelsolin, which is composed of the first antibody and the second antibody as described above.

[0014] The fifth aspect of the present invention provides the use of the antibody or antibody pair against human secreted gelsolin as described above in the preparation of a kit for detecting human secreted gelsolin.

[0015] The sixth aspect of the present invention provides a kit for detecting human secreted gelsolin, which includes the antibody or antibody pair against human secreted gelsolin as described above.

[0016] Further, the kit is a double-antibody sandwich enzyme-linked immunosorbent assay kit, including a first antibody and a second antibody. The first antibody is used as a capture antibody, and the second antibody is used as a detection antibody and is conjugated with a detection label.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides 2 rabbit-derived antibodies against human secreted gelsolin (sGSN) with high affinity. The affinity constant with human sGSN is at the 0.1 nM level, and it can effectively recognize low-concentration antigens. Moreover, these 2 antibodies recognize different epitopes of human sGSN and can be used to develop a double-antibody sandwich enzyme-linked immunosorbent assay system, which has the advantages of high specificity and good sensitivity for detecting the target antigen, and the detection limit can be as low as 0.067 ng / mL. The present invention provides an antibody tool and a detection system that can efficiently, specifically, accurately, sensitively, reliably, and stably detect extremely trace levels of sGSN in blood, urine, or other body fluids as well as cells / tissues, and has good application potential in the clinical diagnosis, long-term monitoring, and prognosis evaluation of gelsolin expression abnormal diseases as well as scientific research. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0020] Figure 1 This is the vector map used to construct the monoclonal antibody expression vector in Example 1 of the present invention. From left to right, it is the vector map of pRB322 carrying the light chain constant region and the heavy chain constant region.

[0021] Figure 2 This is the affinity curve graph of monoclonal antibodies 6C7 and 6D1 in Example 1 of the present invention binding to human secreted gelsolin.

[0022] Figure 3 This is the epitope curve graph of monoclonal antibodies 6C7 and 6D1 in Example 1 of the present invention binding to human secreted gelsolin.

[0023] Figure 4 This is the standard curve for detecting human secreted gelsolin by the double - antibody sandwich enzyme - linked immunosorbent assay system established based on monoclonal antibodies 6C7 and 6D1 in Example 2 of the present invention. Detailed implementation manners

[0024] In order to make the purpose, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Based on the information included in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for illustrative purposes of specific aspects of the present invention. In fact, all various changes that those skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0026] In order to better understand the present invention rather than limit its scope, all numbers representing amounts, percentages and other values used in the present invention should be understood as being modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired properties. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by the conventional rounding method.

[0027] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, scientific and technical terms used shall have the meanings commonly understood by those of ordinary skill in the art.

[0028] The meanings of terms such as "comprising", "including", "containing", "having", etc. are non-restrictive, that is, other steps and other components can be added without affecting the result.

[0029] The term "and / or" shall be regarded as a specific disclosure of each of two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0030] Terms such as "first", "second", etc. are used to distinguish similar objects and do not necessarily have to be used to describe a specific order or sequence. It should be understood that such use can be interchanged under appropriate circumstances.

[0031] Terms such as "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody", and "rabbit mAb" have the same meaning. Unless otherwise specified, they all refer to rabbit-derived antibodies that specifically bind to human (Human) secreted gelsolin. The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. Terms such as "secreted gelsolin", "secreted gelsolin", "sGSN", etc. have the same meaning and can be used interchangeably.

[0032] An antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity. An antibody fragment can be one or more parts or fragments of a full-length antibody, retaining the ability of the antibody to specifically bind to the target antigen.

[0033] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). The light chains can be divided into two types, namely κ chains and λ chains; the heavy chains can be classified into five types, namely μ, δ, γ, α, and ε chains, and the antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions with relatively large amino acid sequence variations near the N-terminus in 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 the variable region of the 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 regions are also called complementarity-determining regions (CDR), which are loop structures. The CDRs of the heavy chain and the CDRs of the light chain are closely juxtaposed and cooperate with each other through the FR regions to jointly form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the specificity of the antibody and being the site where the antibody recognizes and binds to the antigen. The FR regions are the more conserved parts of VH and VL. They generally have a β-sheet configuration and are connected by three CDRs that form linker loops. Each VH and VL usually 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.

[0034] The CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the cumulative of the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition or any CDR determination method well-known in the art. As used in the present invention, it is defined by the Kabat numbering system.

[0035] The constant region of the light chain (CL) and the constant region of the heavy chain (CH) do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity. The lengths of the CLs of different Ig types (κ or λ) are basically the same, but the lengths of the CHs of different Ig classes are different. 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 constant regions of the heavy and light chains of the antibody are well-known in the art and can be obtained by querying the IMGT database.

[0036] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "intact antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0037] Antibody fragments are one or more parts or fragments of full-length antibodies, which basically maintain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR regions as full-length antibodies, and more preferably have the same variable regions, thereby retaining a complete antigen recognition and binding site, and being able to bind to the same antigen as full-length antibodies, especially binding to the same epitope. In a typical example, antibody fragments include: Fab, F(ab) 2 、Fab’、F(ab’) 2 、Fv、(Fv) 2 、scFv、sc(Fv) 2 , and these antibody fragments can be obtained by conventional techniques in the art.

[0038] (i) Fab: The antigen-binding fragment (Fab) is a monovalent fragment composed of a complete light chain (variable region and constant region) and a partial heavy chain (variable region and the first constant region). By protease digestion of full-length antibodies, fragments such as Fab, F(ab’) 2 、Fab’ etc. can be obtained. 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. Since Fab has an antigen-binding region and a partial constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration ability as scFv, etc., but also has a more stable structure.

[0039] (ii) F(ab) 2 : A bivalent fragment containing two Fabs linked by a hinge region disulfide bridge.

[0040] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, only contains the variable region, and is composed of the variable regions of one light chain and one heavy chain. It is a non-covalently bound dimer of a VH and a VL (VH-VL dimer). The 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, and have the ability to recognize and bind antigens, although the affinity is lower than that of full-length antibodies.

[0041] (iv) (Fv) 2: It consists of two Fv fragments covalently linked together.

[0042] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is formed by connecting a heavy-chain variable region (VH) and a light-chain variable region (VL) through a flexible linker (usually composed of 10 - 25 amino acids). It retains the binding specificity of the original antibody to the antigen. In the present invention, the linker only needs to not interfere with the expression of the antibody variable regions connected to its two ends, and there is no particular limitation. Compared with the full-length antibody, scFv has the characteristics of a small molecular weight, so it has higher penetrability and lower immune side reactions.

[0043] (vi) sc(Fv) 2 fragment, which is formed by connecting two heavy-chain variable regions and two light-chain variable regions through a linker or the like.

[0044] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include complementarity-determining regions (CDRs) and framework regions (FRs) from rabbit immunoglobulin sequences. In other embodiments, the antibody may contain amino acid residues encoded by non-rabbit immunoglobulin sequences, such as humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which a part is derived from a specific source or species, while the remaining part is derived from a different source or species. The term "humanized antibody" is a chimeric antibody with the CDR regions of a non-human antibody such as a rabbit antibody and the FR regions from a human. In some cases, the variable region of the non-human antibody is combined with the constant region of the human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR regions of the non-human antibody are combined with the FR regions and constant regions derived from human antibody sequences, that is, the CDR regions of the non-human antibody are grafted onto the human antibody framework (FR) sequences, and such framework sequences are derived from the FR sequences of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric antibody or humanized antibody are derived from rabbit CDR regions.

[0045] The terms "monoclonal antibody" or "mAb" and similar terms are used interchangeably and refer to a homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for minor mutations and / or post-translational modifications (such as isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially the same epitope on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as limiting the source or method of preparation of the antibody. The antibody can be prepared by a variety of methods, including but not limited to the hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening or single cell sequencing method.

[0046] The term "specific binding" is well known in the art and a molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a particular target antigen or epitope than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0047] To make the above objects and advantages of the present invention more obvious and understandable, the following provides a detailed description of the specific embodiments of the present invention.

[0048] An embodiment of the present invention provides an antibody against human secreted gelsolin (sGSN), which is a first antibody or a second antibody. The antibody includes a light chain variable region and a heavy chain variable region, and both the light chain variable region and the heavy chain variable region include 3 complementarity determining regions (CDRs), named CDR1, CDR2, and CDR3 respectively; wherein: the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the first antibody are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively, and 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; the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20 respectively.

[0049] The present invention develops rabbit monoclonal antibodies with high affinity against human secreted gelsolin (sGSN) using single B cell screening and culture techniques. The obtained antibodies have an affinity constant for binding to human sGSN at the 0.1 nM level and can effectively recognize low concentrations of antigens. Moreover, these two antibodies can recognize different epitopes of the human sGSN protein. By using them to develop a sandwich enzyme-linked immunosorbent assay (ELISA) detection system, with the first antibody as the capture antibody and the biotin-labeled second antibody as the detection antibody, quantitative detection of the human sGSN protein is carried out. Its standard curve has good linearity, a wide detection concentration range, high specificity for the target antigen to be detected, good sensitivity, and a detection limit as low as 0.067 ng / mL. The present invention provides an antibody tool and a detection system that can efficiently, specifically, accurately, sensitively, reliably, and stably detect extremely trace levels of sGSN in blood, urine, or other body fluids, as well as in cells / tissues, and has broad practical application value in the clinical diagnosis, monitoring, and prognosis evaluation of gelsolin expression abnormal diseases, as well as in scientific research.

[0050] Optionally, both the light chain variable region and the heavy chain variable region include 4 framework regions (FRs), and the 4 FRs and 3 CDRs are arranged alternately in sequence to form the variable region. The amino acid sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7. The amino acid sequence of the light chain variable region of the second antibody is as shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.17.

[0051] Optionally, the antibodies of the present invention further include a light chain constant region (CL) and a heavy chain constant region (CH). CL and VL form the light chain, and CH and VH form the heavy chain. The constant regions of the antibodies are usually obtained by querying the IMGT online database.

[0052] Specifically, the amino acid sequence of the light chain (FL) of the first antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH) is as shown in SEQ ID NO.6. The amino acid sequence of the light chain (FL) of the second antibody is as shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain (FH) is as shown in SEQ ID NO.16.

[0053] It should be noted that the antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an 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 of the antibody. Specifically, the antigen-binding region includes the CDR regions as described above, and more preferably has the variable regions as described above, thereby retaining a complete antigen recognition and binding site and being able to bind to the same antigen as the full-length antibody, especially binding to the same epitope. Optionally, the antigen-binding region is selected from Fab, F(ab) 2 ', Fab', F(ab') 2 , Fv, (Fv) 2 , scFv and sc(Fv) 2 and at least one of them. 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 aforementioned nucleic acid molecule, or a host cell containing the aforementioned nucleic acid molecule, and the nucleic acid molecule encodes the first antibody and / or the second antibody as described above.

[0055] The nucleic acid molecule can be in the form of DNA (such as cDNA, genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). The DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be obtained by conventional means such as codon encoding rules based on the amino acid sequence of the antibody.

[0056] The full-length sequence or fragment of the nucleic acid molecule can usually be obtained by PCR amplification, recombination or artificial synthesis methods.

[0057] The original vector for constructing the recombinant vector is various conventional vectors in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., used to transfer the nucleic acid molecule into a host and multiply it in the host cell) 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). Insert the nucleic acid molecule into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is well-known technology in the art.

[0058] The nucleic acid molecules encoding the antibodies FL and FH of the present invention can be inserted into two vectors respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under appropriate conditions to form an antibody. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into one vector, and each nucleic acid sequence is linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably linked to different promoters, or the nucleic acid sequences encoding the heavy chain and the light chain can be operably linked to a single promoter such that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0059] Transfection or transformation of the recombinant vector into the host cell is carried out by conventional techniques. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA are harvested after the exponential growth phase and treated with CaCl 2 method or MgCl 2 treatment; it can also be carried out by microinjection, electroporation or liposome packaging, etc. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, microinjection method, electroporation method, liposome packaging or particle bombardment to achieve gene introduction.

[0060] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (such as DH5α, JM109, BL21, W3110), Bacillus spp. (such as Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (such as 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 the host cell transfected or transformed with the recombinant vector as described above, it is cultured under suitable conditions, and then the antibody can be expressed and separated to obtain a purified antibody.

[0061] Transfection or transformation of the recombinant vector of the present invention into the host cell is carried out by conventional techniques. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl 2 method or MgCl 2Treatment. If necessary, methods such as microinjection, electroporation, liposome packaging, or gene gun can also be used. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, microinjection, electroporation, liposome packaging, or gene gun, etc.

[0062] In a typical embodiment, the method for preparing the antibody of the present invention includes ligating the light chain and heavy chain genes of the antibody in series with a signal peptide, respectively loading them on the expression vector pBR322, co-transfecting human renal epithelial cells (293F), culturing the 293F cells, collecting the cell culture supernatant, and purifying to obtain the target antibody strain. The selection of the signal peptide is designed according to the host cell, and the present invention has no special limitation in this regard.

[0063] Another embodiment of the present invention provides an antibody pair against human secreted gelsolin, which is composed of the first antibody and the second antibody as described above.

[0064] The first antibody and the second antibody provided by the present invention recognize and bind to different epitopes of human sGSN, and can be used to pair antibodies to develop a double-antibody sandwich ELISA system or kit. The detection limit for detecting human sGSN protein can reach 0.067 ng / mL, and it has the advantages of high specificity for detecting the target antigen, good sensitivity, and high reliability of the detection results.

[0065] Another embodiment of the present invention provides the use of the antibody or antibody pair against human secreted gelsolin as described above in the preparation of a kit for detecting human secreted gelsolin.

[0066] The advantages of the use of the antibody or antibody pair against human secreted gelsolin in the preparation of a kit for detecting human secreted gelsolin are the same as those of the antibody or antibody pair against human secreted gelsolin as described above, and will not be elaborated here.

[0067] Based on the same inventive concept as above, an embodiment of the present invention also provides a kit for detecting human secreted gelsolin, and the kit includes the first antibody and / or the second antibody as described above.

[0068] It should be emphasized that the first antibody and the second antibody can be used separately, jointly, or in pairs. During detection, when used separately or jointly, the first antibody and / or the second antibody is used as the primary antibody or capture antibody, and the sample to be detected is contacted with the capture antibody, and then the antibody is detected. In some embodiments, the capture antibody can be conjugated (covalently or non-covalently) with a detection label, and qualitative or quantitative detection of gelsolin is achieved by analyzing the signal change generated by the detection label. In other embodiments, the primary antibody against human sGSN protein is not labeled, but the detection label is conjugated to a secondary antibody (used as a detection antibody) or other molecules that can bind to the capture antibody. For example, if the antibody against human sGSN protein is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, and thus a signal change is generated by the secondary antibody conjugated with the detection label. When used in pairs, one of the two antibodies is used as the primary antibody or capture antibody, and the other is used as the secondary antibody or detection antibody.

[0069] The detection method uses common immunological methods, such as: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), flow cytometry (FC), etc. The detection targets include recombinantly expressed and cell / tissue-naturally secreted or expressed human sGSN protein, and the detection samples include but are not limited to serum, plasma, urine, cells or cell culture supernatants, tissues or tissue homogenates, etc.

[0070] Preferably, the kit is a double-antibody sandwich enzyme-linked immunosorbent assay kit, including a first antibody and a second antibody. The first antibody is used as the capture antibody (or primary antibody), and the second antibody is used as the detection antibody (or secondary antibody) and is conjugated with a detection label.

[0071] The above detection labels for generating recognizable signal changes include but are not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP, and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0072] The present invention will be further described below in conjunction with specific embodiments. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" published by Cold Spring Harbor Laboratory, or usually according to the conditions recommended by the manufacturer.

[0073] Example 1 Screening and Preparation of Rabbit-derived Antibodies 6C7 and 6D1 Against Human Secreted Gelsolin

[0074] This example is based on the monoclonal antibody development technology of screening and culturing single B lymphocytes. B cells that can recognize the target antigen are enriched and isolated from the spleen of rabbits immunized with human secreted gelsolin (sGSN). Then, the isolated B cells are cultured in single-cell form to obtain monoclonal antibodies 6C7 and 6D1. Finally, the coding genes of the aforementioned monoclonal antibodies are expressed through gene recombination expression technology to mass-produce the monoclonal antibodies. The antibody sequencing work was completed by Wuhan Kingcare Biotechnology Co., Ltd., and its amino acid (AA) and gene (DNA) sequences are shown in Tables 1-2 respectively. For convenience of description, light chain CDR1-3 are represented by LCDR1-3 respectively, and heavy chain CDR1-3 are represented by HCDR1-3 respectively.

[0075] Table 1 Sequence information of monoclonal antibody 6C7 in this example

[0076]

[0077]

[0078] Table 2 Sequence information of monoclonal antibody 6D1 in this example

[0079]

[0080] 1.1 Antigen preparation: The immunogen used to prepare rabbit monoclonal antibodies is human secreted gelsolin (sGSN). The amino acid sequence can be found in NCBI accession number NP_000168.1 or Uniprot number P06396, and the gene sequence can be found in NCBI accession number NM_000177.5. The complete domain of secreted gelsolin is the amino acid fragment from position 28 to position 782. The gene sequence corresponding to 28-782AA was constructed into the pET-28a-sumo vector and transfected into Escherichia coli Rosetta. The Human sGSN protein with a purity greater than 90% and biological activity was expressed using the prokaryotic expression system and used as an antigen to immunize animals.

[0081] 1.2 Animal immunization: Two New Zealand white rabbits were immunized with 200 μg / rabbit of sGSN protein. Before the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to make an emulsifier, which was injected subcutaneously at multiple points on the abdomen and back of the rabbit. After 3 weeks, 100 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant to make an emulsifier, which was injected subcutaneously at multiple points on the abdomen and back of the rabbit for two booster immunizations. After three immunizations, the animals were boosted once again with 200 μg of the immunogen. Three days later, the animals were sacrificed and the spleens were taken.

[0082] 1.3. Isolation of B cells in the spleen and sorting of antigen-specific B cells: For relevant methods, refer to the published patent "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: July 16, 2019)" and the patent "An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: August 11, 2020)".

[0083] 1.4. Cloning of genes encoding monoclonal antibodies: The supernatant of cultured rabbit B cells was identified for positive clones by antigen-coated ELISA. After collecting and lysing the positive cells, RNA was extracted using the Quick-RNA TM MicroPrep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using the cDNA as a template, the naturally paired antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified from the positive clones by PCR. PCR reaction system: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from ABclonal, catalog number RK20717) and 6.5 μL H 2 O; Amplification program: 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, 72°C for 30 s, and finally 72°C for 5 min. The reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying VL and VH genes are shown below, where F and R represent forward and reverse primers respectively:

[0084] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO.21);

[0085] VL-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO.22);

[0086] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO.23);

[0087] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO.24).

[0088] The amplified products were sequenced to obtain the variable regions of the antibodies and their encoding gene sequences; and the heavy chain constant region (CH) was obtained by searching the rabbit IgG gamma C reign in the IMGT online database (www.imgt.org), and the light chain constant region (CL) was obtained by searching the rabbit IgGKappa C reign, resulting in the antibody 6C7 with the complete light chain (FL) and heavy chain (FH) shown in SEQ ID NO.1 and SEQ ID NO.6, and the antibody 6D1 with the complete light chain (FL) and heavy chain (FH) shown in SEQ ID NO.11 and SEQ ID NO.16.

[0089] 1.5. Production and purification of monoclonal antibodies: The obtained heavy and light chain genes of the antibody were respectively loaded onto expression vectors. In this example, CL and the heavy chain CH were pre-inserted into the mammalian expression vector pBR322, and the vector map obtained is shown in Figure 1 , where pBR322 origin and f1 origin are replication promoters, Ampcillin is the resistance gene, CMV promoter is the transcription promoter, SV40 PA terminator is the polyadenylation signal, Light chain constant is the CL gene sequence (left figure), and Heavy chain constant is the CH gene sequence (right figure). Then, the amplified VL and VH genes were ligated to the expression vector pBR322 carrying the CL and CH genes, which were linearized with XbaI and NheI restriction endonucleases respectively, by homologous recombination to obtain the FL and FH gene expression vectors, and the vector construction was verified to be successful by sequencing.

[0090] To more conveniently purify the antibody, the secretion expression of the antibody was achieved by adding signal peptides upstream of VL and VH. The signal peptides commonly used in the art for antibody expression can be used, such as in the patent "Rabbit monoclonal antibody against human interferon α2 and its application (Publication No.: CN116063487A, Publication Date: May 5, 2023)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: March 21, 2023)", with the signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" upstream of VL, and the signal peptide "METGLRWLLLVAVLKGVQC" upstream of VH. Of course, those skilled in the art can also replace other signal peptides for expression after obtaining the antibody sequences of the present invention. Therefore, the signal peptide sequences are not shown in the antibody sequences in Table 1 of this example.

[0091] The successfully constructed expression vector containing the FL and FH genes was co-transfected into 293F cells. After transfection, the cells were cultured for 72 - 96 h, and the culture supernatant was collected. A recombinant rabbit antibody that recognizes human sGSN was purified from the culture supernatant using protein A affinity gel resin (purchased from Tiandi Renhe, product number SA023100). The concentration of antibody 6D1 was 6.54 μg / mL, and the purity was greater than 95%. The concentration of antibody 6C7 was 5.22 μg / mL, and the purity was greater than 95%.

[0092] 1.6 Detection of the performance of antibodies 6C7 and 6D1: The Gator biomolecular interaction analyzer from Probe Life was used to measure the antigen-antibody binding curve to identify the antibody affinity and antigen-binding epitope.

[0093] 1) Identification of antibody affinity: First, Human sGSN was immobilized on the Anti-HIgG FC (HFC) Probe (purchased from Gator, product number 160003) at a concentration of 3 μg / mL. The probes with immobilized antigen were placed in the antibody dilutions of 6.54 μg / mL 6D1 and 5.22 μg / mL 6C7 respectively to test the affinity of the antibodies binding to the antigen. The affinity curves of antibody 6D1 (upper figure) and antibody 6C7 (lower figure) are shown in Figure 2 , where the ordinate represents the change in the thickness of the conjugate after the probe binds to the antibody and the protein, the abscissa represents the binding time, the dark gray curve is the real-time binding value curve, and the light gray curve is the fitted average value curve. The affinity constants obtained by curve fitting and calculation are shown in Table 3, where the dissociation coefficient K off is a constant representing the dissociation rate of the antibody from the antigen, and the association coefficient K on is a constant representing the binding rate of the antibody to its target, and the affinity constant K D is K off / K on ratio, representing the dissociation equilibrium constant between the antibody and the antigen.

[0094] Table 3 Determination results of affinity-related parameters of monoclonal antibodies 6C7 and 6D1

[0095] Monoclonal antibody <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> 6C7 <![CDATA[5.60×10 -4 > <![CDATA[6.31×10 5 > <![CDATA[8.88×10 -10 > 6D1 <![CDATA[1.84×10 -4 > <![CDATA[5.34×10 5 > <![CDATA[3.44×10 -10 >

[0096] From Figure 2 and Table 3, it can be seen that the dissociation equilibrium constants KD of rabbit monoclonal antibodies 6C7 and 6D1 against human sGSN protein are 0.888 nM and 0.344 nM respectively, showing excellent affinity.

[0097] 2) Identification of antigen recognition epitopes: First, immobilize human sGSN protein on the HFC Probe at a concentration of 3 μg / mL. Then, place the probe with immobilized antigen in a 3 μg / mL antibody 6C7 solution to allow antibody 6C7 and the antigen to bind until saturation. Next, place the probe in a 3 μg / mL antibody 6D1 solution to allow antibody 6D1 and the antigen to bind until saturation. Identify the recognition epitopes of the antibodies by analyzing the binding characteristics of different antibodies to the antigen. The results are shown in Figure 3 , where the vertical axis represents the thickness change of the conjugate after the probe binds to the antibody and the protein, and the horizontal axis represents the binding time.

[0098] As can be seen from Figure 3 , the probe immobilized with human sGSN can significantly bind 6D1 after binding 6C7, confirming that the antigen epitopes bound by 6C7 and 6D1 on human sGSN are different.

[0099] Example 2 Establishment of a sandwich enzyme-linked immunosorbent assay system based on antibodies 6C7 and 6D1 and its analytical sensitivity

[0100] Biotin labeling of antibody 6D1: Prepare a 1 mg / mL antibody 6D1 solution and a 60 mg / mL NHS-LC-biotin solution; take 200 μL of the 1 mg / mL antibody 6D1 solution, add 10 μL of the 60 mg / mL NHS-LC-biotin solution, mix well and let stand at room temperature for 30 min; then add 50 μg of a 500 mM Tris solution (pH 9.0) to terminate the reaction; finally, add a large amount of 1×PBS buffer at pH 7.4 and centrifuge using a centrifugal column with an exclusion limit of 30 KD to remove excess biotin molecules and balance the buffer system, obtaining biotin-labeled antibody 6D1 (6D1-biotin).

[0101] Using antibody 6C7 as the capture antibody and antibody 6D1-biotin as the detection antibody, a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection system was established as follows: 1) Coating the capture antibody 6C7: Dilute antibody 6C7 with 1×PBS to 2 μg / mL, add 100 μL per well to a 96-well microplate, cover with a cover membrane, and incubate at 4°C for 16 - 20 h; 2) Washing the plate: After incubation, discard the liquid in the wells, wash the plate once with 300 μL of 1×PBST, let it stand for 40 s, and then discard the liquid in the wells; 3) Blocking: Add the blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween 20, and 0.1% proclin 300 in 1×PBS, pH 7.2) to the wells at 200 μL per well, cover with a cover membrane, block at 37°C for 2 h. After blocking, discard the blocking solution and place it in a 37°C oven to dry for 0.5 - 2 h; 4) Adding antigen protein: Dilute human sGSN protein with a dilution solution (containing 2% BSA, 0.05% Tween 20, and 0.1% proclin 300 in 1×PBS, pH 7.2) to concentrations of: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0 ng / mL, and then add 100 μL per well to the microplate in sequence, cover with a cover membrane, and incubate at 37°C for 2 h; 5) Washing the plate: The same as step 2); 6) Adding detection antibody 6D1: Dilute 6D1-biotin antibody to 0.125 μg / mL, add 100 μL per well to the microplate in sequence, cover with a cover membrane, and incubate at 37°C for 1 h; 7) Washing the plate: The same as step 2); 8) Adding SA-HRP: After diluting the 100×SA-HRP (horseradish peroxidase-labeled streptavidin, purchased from Wuhan Sanying Co., Ltd., product number SA00001-0) concentrate, add 100 μL per well to the microplate in sequence, cover with a cover membrane, and incubate at 37°C for 0.5 h; 9) Washing the plate: The same as step 2); 10) Adding TMB chromogenic solution: Add the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution (purchased from Sizhengbai, product number 4ATMB1000) to the wells at 100 μL per well in sequence, cover with a cover membrane, and incubate at 37°C for 15 min; 11) Reading: Take out the microplate, add 50 μL of stop solution (1 mol / L hydrochloric acid) to each well, and immediately read with an enzyme-linked immunosorbent assay reader.

[0102] Using the sGSN protein concentration as the abscissa and the corrected absorbance value ΔOD (ΔOD = OD 450nm - OD 630nm ) as the ordinate to plot a graph, and using the four-parameter fitting of the Logistic curve, the fitting curve (R 2 = 0.9999) is shown in Figure 4The results showed that antibodies 6C7 and 6D1, as paired antibodies, were used to detect human sGSN, showing good linearity, and the detection results had good accuracy and reliability. Taking three times the average absorbance value of 16 dilution blank wells as the absorbance signal value of the sensitivity, substituting it into the fitting curve to calculate the sensitivity of the detection system (see Table 4). The detection limit was 0.067 ng / mL, indicating high sensitivity.

[0103] Table 4 Sensitivity of the double-antibody sandwich enzyme-linked immunosorbent assay established based on antibodies 6C7 and 6D1

[0104]

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-human secretory gelsolin antibody, characterized in that Selected from the first antibody or the second antibody, wherein: The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown in SEQ ID NOs. 3-5, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NOs. 8-10, respectively; The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NOs.13-15, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NOs.18-20, respectively.

2. The anti-human secretory gelsolin antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the first antibody 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; The amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

17.

3. The anti-human secretory gelsolin antibody according to claim 2, characterized in that The amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; The amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

16.

4. The anti-human secretory gelsolin antibody according to claim 1, characterized in that The first antibody or the second antibody is a full-length antibody or an antigen-binding region of the full-length antibody; The antigen binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the first antibody or the second antibody as described in any one of claims 1-4.

6. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 5.

7. An antibody pair against human secretory gelsolin, characterized in that: The method comprises the first antibody and the second antibody as described in any one of claims 1 to 4.

8. Use of the anti-human secretory gelsolin antibody according to any one of claims 1 to 4 or the anti-human secretory gelsolin antibody pair according to claim 7 in preparing a kit for detecting human secretory gelsolin.

9. A kit for detecting human secretory gelsolin, characterized in that: The method comprises the anti-human secretory gelsolin antibody according to any one of claims 1 to 4 or the anti-human secretory gelsolin antibody pair according to claim 7.

10. The kit for detecting human secretory gelsolin according to claim 9, characterized in that: The kit is a double antibody sandwich ELISA kit, comprising a first antibody and a second antibody, wherein the first antibody is a capture antibody, the second antibody is a detection antibody and the second antibody is coupled with a detection label.

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