Anti-human calcium binding protein S100A1 antibody, antibody pair and detection kit

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human calcium binding protein S100A1 antibody, an antibody pair and a detection kit. 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 two antibodies provided by the invention have good specificity, strong affinity and high antigen recognition sensitivity to the human S100A1 protein, and can be used for pairing the antibodies to construct a double-antibody sandwich enzyme-linked immunosorbent assay system so as to realize efficient detection of extremely trace level S100A1 protein.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and particularly to antibodies and antibody pairs against human calcium-binding protein S100A1 and a detection kit. Background Art

[0002] S100A1 (S100 calcium binding protein A1) is a small calcium-binding protein belonging to the S100 protein family. It has two typical EF-hand type domains within its molecule, which can respond to changes in intracellular and extracellular calcium concentrations and expose a hydrophobic surface by changing its own conformation, and then interact with target proteins to exert various biological functions. As a calcium-binding protein, S100A1 plays an important role in maintaining intracellular calcium homeostasis; by binding to and releasing calcium ions, it can regulate the concentration and distribution of intracellular calcium ions. In cardiomyocytes, S100A1 regulates the calcium ion balance in cardiomyocytes by interacting with proteins such as the sarcoplasmic reticulum calcium release channel (ryanodine receptor 2, RyR2) and the sarcoplasmic reticulum calcium pump (SERCA2a). S100A1 can also affect and regulate myocardial mitochondrial function, bind to the adenine nucleotide translocator (ANT), mediate the phosphorylation pathway, enhance the expression of nuclear transcription factors, and improve the mitochondrial energy metabolism of cardiomyocytes. The above-mentioned mechanisms help to regulate myocardial calcium transport, inhibit ventricular remodeling, reduce cardiomyocyte apoptosis, and restore myocardial energy supply, thereby improving myocardial contraction and diastolic function. Therefore, the S100A1 protein plays an important role in improving cardiac function after heart failure and maintaining blood pressure stability. Increasing the expression of S100A1 can reverse the progression of heart failure, providing a new idea for the clinical treatment of cardiovascular diseases. S100A1 is also involved in regulating the calcium signal transduction process related to cell growth, proliferation, and apoptosis.

[0003] In recent years, the application of S100A1 in laboratory medicine has received extensive attention. The expression of S100A1 in the myocardial tissue of heart failure patients is significantly reduced, and the serum S100A1 level may also change. Therefore, S100A1 can be used as an auxiliary index for diagnosing heart failure and evaluating its severity. After myocardial ischemia injury, S100A1 is released from damaged cells into the blood. Combined detection with traditional myocardial injury markers such as troponin helps to improve the accuracy of myocardial infarction diagnosis. In addition, S100A1 is abnormally expressed in a variety of tumor tissues and can affect the invasion, metastasis of tumors. For example, the expression of S100A1 is enhanced in endometrial cancer and atypical endometrial hyperplasia tissues, promoting the proliferation of endometrial cancer cells. S100A1 is also an important indicator for evaluating the prognosis of patients with clear cell renal cell carcinoma (CCRCC). S100A1-high-expressing cells have increased euchromatin and enhanced transcriptional activity, which has a driving effect on the high-grade transformation of CCRCC. Detecting the expression level of S100A1 can be used as an auxiliary index for the diagnosis and prognosis evaluation of these diseases.

[0004] At present, immunological methods such as enzyme-linked immunosorbent assay, immunoturbidimetry, immunoprecipitation, and immunofluorescence for detecting S100A1 have become the mainstream direction because of their simple operation and easy result determination. In particular, enzyme-linked immunosorbent assay (ELISA) is widely used for the detection of biological samples such as serum, plasma, urine, tissue lysates, and cell culture supernatants. Different detection methods are all based on the principle of specific binding of antibodies to S100A1. However, the monoclonal antibodies used in traditional clinical diagnosis are all murine-derived, and their affinity and specificity are generally low, resulting in problems such as low detection sensitivity and / or insufficient reliability, and unable to effectively monitor the low-concentration S100A1 antigen in pathological samples. Therefore, developing a new type of antibody with high specificity and / or high affinity is of great significance for the highly sensitive detection of human S100A1. Summary of the Invention

[0005] Aiming at the problems of insufficient affinity and / or specificity of the existing anti-human calcium-binding protein S100A1 antibodies, the present invention provides 2 strains of monoclonal antibodies with excellent performance and the antibody pair composed thereof, as well as a kit containing the aforementioned antibodies or antibody pairs. The antibodies of the present invention have good specificity, strong affinity, and high antigen recognition sensitivity for human S100A1 protein, and can be used to construct a double-antibody sandwich enzyme-linked immunosorbent assay system for paired antibodies to achieve the efficient detection of extremely trace levels of S100A1 protein.

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

[0007] In the first aspect of the present invention, an antibody against human calcium-binding protein S100A1 is provided, which is a first antibody or a second antibody, wherein: the amino acid sequences of the complementarity-determining regions CDR1-3 on the light chain variable region of the first antibody are respectively shown in SEQ ID NO.3-5, and the amino acid sequences of the complementarity-determining regions CDR1-3 on the heavy chain variable region are respectively shown in SEQ ID NO.8-10; the amino acid sequences of the complementarity-determining regions CDR1-3 on the light chain variable region of the second antibody are respectively shown in SEQ ID NO.13-15, and the amino acid sequences of the complementarity-determining regions CDR1-3 on the heavy chain variable region are respectively shown in SEQ ID NO.18-20.

[0008] Further, 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.

[0009] Further, the light chain of the first antibody and / or the second antibody is a κ chain, and the heavy chain is of IgG1 type.

[0010] Further, 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.

[0011] Further, the first antibody and / or the second antibody is a full-length antibody or the antigen-binding region of the full-length antibody; the antigen-binding region is selected from at least one of Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and sc(Fv)2 fragment.

[0012] In the second aspect of the present invention, a nucleic acid molecule or a recombinant vector is provided, the recombinant vector contains the nucleic acid molecule, and the nucleic acid molecule encodes the first antibody or the second antibody as described above.

[0013] In the third aspect of the present invention, an antibody pair against human calcium-binding protein S100A1 is provided, which is composed of the first antibody and the second antibody as described above.

[0014] In the fourth aspect of the present invention, the application of the antibody or antibody pair against human calcium-binding protein S100A1 as described above in the preparation of a detection kit for human calcium-binding protein S100A1 is provided.

[0015] The fifth aspect of the present invention provides a kit for detecting human calbindin S100A1, and the detection kit includes the antibody or antibody pair against human calbindin S100A1 as described above.

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

[0017] Further, the double-antibody sandwich enzyme-linked immunosorbent assay kit further includes a diluent, a blocking solution, a horseradish peroxidase-labeled streptavidin solution, and a 3,3',5,5'-tetramethylbenzidine chromogenic solution.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides 2 monoclonal antibodies against human calbindin S100A1 with high affinity. The affinity constants of the antibodies binding to human S100A1 protein are 9.44×10 -9 and 1.29×10 -8 M. Moreover, these two antibodies can be used to pair with antibodies to construct a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection system. The quantitative detection of human S100A1 protein has the characteristics of good specificity, strong affinity, high antigen recognition sensitivity, etc., and the detection sensitivity is as low as 0.41 ng / mL. It provides excellent antibodies, antibody pairs and detection systems for specifically, sensitively, accurately and reliably detecting the content of S100A1 protein in biological samples such as blood, urine, cell / tissue culture medium or homogenate, and has good application prospects in the fields of clinical diagnosis and treatment of related diseases and basic medical research. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 This is the vector map used for constructing 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;

[0022] Figure 2 This is the affinity curve graph of the monoclonal antibody binding to human calbindin S100A1 in Example 2 of the present invention;

[0023] Figure 3This is the standard curve for detecting human calbindin S100A1 by the double - antibody sandwich enzyme - linked immunosorbent assay system established based on monoclonal antibodies 1D12 and 2F7 in Example 3 of the present invention. Detailed implementation manners

[0024] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to 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 schematically illustrating specific aspects of the present invention. In fact, various changes that can be made by those skilled in the relevant art to the embodiments of the present invention are all covered within the scope of the appended claims.

[0026] To better understand the present invention rather than limit its scope, all numbers representing amounts, percentages and other numerical values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and appended claims are approximate values, which may vary according to the different desired properties to be obtained. Each numerical parameter should at least be regarded as obtained according to 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 should have the meanings commonly understood by those of ordinary skill in the art.

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

[0029] The term "and / or" should be regarded 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 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 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] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody", "rabbit mAb" and similar terms have the same meaning and, unless otherwise specified, all refer to rabbit-derived antibodies that specifically bind to human S100A1 protein. The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. The terms "calcium-binding protein S100A1", "S100 calcium binding protein A1", "S100A1" and similar terms 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 that retain 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 of 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 heavy chain variable region (VH) and the light chain variable region (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 (HVRs) and framework regions (FRs). The hypervariable regions are also called complementarity-determining regions (CDRs) and are loop structures. The heavy chain CDRs and the light chain CDRs 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 forming connecting 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, they are defined by the Kabat numbering system.

[0035] The light chain constant region (CL) and the heavy chain constant region (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-mediated cytotoxicity (ADCC). The CL lengths of different Ig isotypes (κ or λ) are basically the same, but the CH lengths 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 antibody heavy chain and light chain 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, having a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "complete 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 a full-length antibody, which basically retain the same biological function or activity as the full-length form. Specifically, the antibody fragment at least includes the same CDR regions as the full-length antibody, and more preferably has the same variable region, 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. 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 the full-length antibody, fragments such as Fab, F(ab’)2, and Fab’ 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, but also has a more stable structure.

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

[0040] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment and contains only the variable regions. It consists of a variable region of a light chain and a variable region of a heavy chain, and is a non-covalently bound dimer of a VH and a 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 full-length antibody.

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

[0042] (v) scFv: The single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is formed by linking a variable region of a heavy chain (VH) and a variable region of a light chain (VL) through a flexible linker (usually composed of 10-25 amino acids). It retains the antigen-binding specificity of the original antibody. The linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions connected at both ends. Compared with the full-length antibody, scFv has the characteristics of a small molecular weight, so it has higher penetration and lower immune side effects.

[0043] (vi) The sc(Fv)2 fragment is formed by connecting two variable regions of heavy chains and two variable regions of light chains 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 comprise complementarity-determining regions (CDRs) and framework regions (FRs) from rabbit immunoglobulin sequences. In other embodiments, the antibody may comprise amino acid residues encoded by non-rabbit immunoglobulin sequences, such as in the types of humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the desired specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a particular source or species, while the remainder 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, and in some cases, the variable region of the non-human antibody is combined with the constant region of a 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 framework (FR) sequences of a human antibody, and this framework sequence is derived from the FR sequences of one or more 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., each antibody constituting the population is identical except for minor mutations and / or post-translational modifications (such as isomerization, amidation) that may occur naturally. A "monoclonal antibody" is highly specific and exhibits 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 "specifically binds" is a well-known term in the art, and a molecule exhibits "specifically binds" 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. "Specifically binds" or "preferentially binds" 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 calcium-binding protein S100A1, 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 developed 2 high-affinity antibodies against human calcium-binding protein S100A1 (hereinafter referred to as S100A1 for short) using single B cell screening and culture technology. The affinity constants of the antibodies binding to human S100A1 protein are 9.44×10 -9 and 1.29×10 -8 M. These two antibodies can be used to pair antibodies to construct a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection system. Using one antibody as the capture antibody and the other antibody labeled with biotin as the detection antibody, the human S100A1 protein can be quantitatively detected. It has the characteristics of good specificity, strong affinity, and high antigen recognition sensitivity. The detection sensitivity is as low as 0.41 ng / mL, and the efficient detection of extremely trace levels of S100A1 protein can be achieved. The present invention provides antibodies, antibody pairs, and detection systems with excellent performance for specifically, sensitively, accurately, and reliably detecting the content of S100A1 protein in biological samples such as blood, urine, cell / tissue culture medium, or homogenate. It has important clinical application value in the early diagnosis, condition monitoring, and prognosis evaluation of cardiovascular diseases such as heart failure and other diseases targeting S100A1, and has broad application prospects in basic medical research such as exploring the biological function of S100A1 protein and its regulatory role in cell metabolism and signal pathways.

[0050] Optionally, both the light chain variable region and the heavy chain variable region include four framework regions (FRs), and the four FRs and three 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 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.

[0051] Optionally, the antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH), and CL and VL form the light chain, and CH and VH form the heavy chain. The constant region of the antibody is usually obtained through the IMGT online database.

[0052] In a preferred embodiment, the light chain of the first antibody and / or the second antibody is a κ chain, and the heavy chain is of the IgG1 type.

[0053] Specifically, the amino acid sequence of the light chain (FL) of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.6. The amino acid sequence of the light chain (FL) of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.16.

[0054] It should be noted that the antibody of the present invention can be a full-length antibody (with 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 region as described above, and more preferably has the variable region as described above, thereby retaining the 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 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.

[0055] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the foregoing nucleic acid molecule, or a host cell containing the foregoing nucleic acid molecule, and the nucleic acid molecule encodes the first antibody and / or the second antibody as described above.

[0056] 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 the coding strand or the non-coding strand. The sequence of the nucleic acid molecule can be derived by conventional means such as codon coding rules based on the amino acid sequence of the antibody.

[0057] The full-length sequence or fragment of the nucleic acid molecule can usually be obtained by PCR amplification, recombination or artificial synthesis methods.

[0058] The original vectors for constructing the recombinant vectors are various conventional vectors in the art, as long as they can accommodate the said 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). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or an expression vector carrying the said nucleic acid molecule. This is a well-known technique in the art.

[0059] 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 suitable conditions to form the antibody. In some other embodiments, the nucleic acid molecules of the antibodies FL and FH can also be cloned into one vector, and each nucleic acid sequence is ligated 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 the expression vector / promoter depends on the type of host cell used for producing the antibody.

[0060] The 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, the competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with the CaCl2 method or MgCl2 method; 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, etc.

[0061] 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), 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 cells transfected or transformed with the recombinant vector as described above, culturing them under suitable conditions can express the antibody, and then separating it to obtain the purified antibody.

[0062] In a typical embodiment, the method for preparing the antibody of the present invention includes, after tandemly connecting the light chain and heavy chain genes of the antibody 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 sequence of the signal peptide is designed according to the host cell, and the present invention has no special limitation on this. The signal peptide coding gene is located upstream (5' end) of the antibody gene.

[0063] Another embodiment of the present invention provides an antibody pair against human calcium-binding protein S100A1, which is composed of the first antibody and the second antibody as described above.

[0064] The first antibody and the second antibody of the present invention can be used as paired antibodies in a double antibody sandwich. Using this antibody pair to construct a double antibody sandwich ELISA method can specifically and sensitively detect human S100A1 protein, providing a powerful tool for efficiently and accurately detecting low-concentration S100A1 protein.

[0065] Another embodiment of the present invention provides the use of the antibody or antibody pair against human calcium-binding protein S100A1 as described above in the preparation of a human calcium-binding protein S100A1 detection kit.

[0066] The advantages of the use of the antibody or antibody pair against human calcium-binding protein S100A1 in the preparation of a human calcium-binding protein S100A1 detection kit are the same as those of the antibody or antibody pair against human calcium-binding protein S100A1 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 human calcium-binding protein S100A1 detection kit, which 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 the 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 S100A1 can be achieved by analyzing the signal change generated by the detection label. In other embodiments, the primary antibody against human S100A1 protein is not labeled, but the detection label is conjugated with the secondary antibody against the capture antibody (as the detection antibody) or other molecules. For example, if the antibody against human S100A1 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 the capture antibody, and the other is used as the secondary antibody or the 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 naturally secreted or expressed human S100A1 protein in cells / tissues, 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] Optionally, the double-antibody sandwich enzyme-linked immunosorbent assay kit further includes a diluent, a blocking solution, a horseradish peroxidase-labeled streptavidin solution, and a 3,3',5,5'-tetramethylbenzidine chromogenic solution.

[0073] The present invention will be further described below in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are generally 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 generally according to the conditions recommended by the manufacturer.

[0074] Example 1 Screening and Preparation of Rabbit-derived Antibodies against Human Calcium-binding Protein S100A1

[0075] Based on the monoclonal antibody development technology of screening and culturing single B lymphocytes, this example enriches and isolates B lymphocytes that can recognize the target antigen from the spleen of rabbits immunized with human calcium-binding protein S100A1, and then cultures them in the form of single cells to obtain monoclonal antibodies 1D12 and 2F7 secreted by B lymphocytes. Finally, the coding genes of the aforementioned monoclonal antibodies are expressed by gene recombination expression technology for mass production. The antibody sequencing work was completed by Wuhan Kingcare Biotechnology Co., Ltd. The amino acid sequences of antibodies 1D12 and 2F7 are shown in Table 1-2 respectively. For convenience of description, the light chain CDR1-3 are represented by LCDR1-3 respectively, and the heavy chain CDR1-3 are represented by HCDR1-3 respectively.

[0076] Table 1 Sequence Information of Monoclonal Antibody 1D12 in this Example

[0077]

[0078]

[0079] Table 2 Sequence Information of Monoclonal Antibody 2F7 in this Example

[0080]

[0081] 1.1 Animal Immunization

[0082] Two New Zealand white rabbits were immunized with 200 μg / rabbit of commercially available recombinant Human S100A1 protein (from Abclonal, catalog number 10179-HNAE, protein sequence see NCBI accession number: NP_006262.1). Before the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to make an emulsifier, and 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, and injected subcutaneously at multiple points on the abdomen and back of the rabbit for two booster immunizations. After three immunizations, the animals were boosted again with 200 μg of the immunogen, and sacrificed three days later to obtain the spleen.

[0083] 1.2 Isolation of B Lymphocytes in the Spleen and Sorting of Antigen-specific B Lymphocytes

[0084] For related methods, please 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)".

[0085] 1.3 Cloning of Genes Encoding Monoclonal Antibodies

[0086] The supernatant of cultured B cells was taken and used for antigen-coated ELISA to identify positive clones. After collecting and lysing the positive cells, RNA was extracted using the Quick-RNA TM MicroPrep Kit (purchased from ZYMO, product number R1051) and reverse transcribed into cDNA. The reverse transcription system was as follows: 1 μL of Oligo(dT)12-18 primer, 1 μL of dNTPs (10 mM), and 1 μL of RNA. After reacting at 65°C for 5 min, 4 μL of 5×FS Buffer, 1 μL of DTT (10 mM), 1 μL of RNase OUT (40 U / μL), and 1 μL of ABScriptⅡ RT (200 U / μL, from ABconal) were added to the above products. After mixing, the reaction was carried out at 42°C for 1 h and 85°C for 5 min to obtain cDNA. Using cDNA as a template, the naturally paired antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified by PCR. The PCR reaction system was: 4 μL of cDNA, 1 μL of forward primer (10 mM), 1 μL of reverse primer (10 mM), 12.5 μL of 2×Gloria HiFi (from ABclonal, product number RK20717), and 6.5 μL of H2O. The amplification program was: 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 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. F and R represent forward and reverse primers respectively:

[0087] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO.21);

[0088] VL-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO.22);

[0089] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO.23);

[0090] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO.24).

[0091] The amplified products were sequenced to obtain the variable regions of the antibodies against human Human S100A1 protein and their encoding gene sequences; and the heavy chain constant region (CH) was obtained by searching the rabbit-derived 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-derived IgG Kappa C reign, resulting in the antibody 1D12 with the complete light chain (FL) and heavy chain (FH) as shown in SEQ ID NO.1 and SEQ ID NO.6, and the antibody 2F7 with the complete light chain (FL) and heavy chain (FH) as shown in SEQ ID NO.11 and SEQ ID NO.16.

[0092] 1.4. Large-scale production of monoclonal antibodies 1D12 and 2F7

[0093] The obtained antibody heavy chain and light chain genes were respectively loaded onto the expression vector pBR322. In this example, CL and the heavy chain CH were previously inserted into pBR322, and the vector map obtained is shown in Figure 1 , where pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PA terminator is a 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 that had been linearly processed with XbaI and NheI restriction endonucleases by homologous recombination to obtain the FL and FH gene expression vectors, and the successful construction of the vectors was verified by sequencing.

[0094] To purify the antibody more conveniently, the antibody was secreted and expressed by adding a signal peptide upstream of VL and VH. The signal peptide can be a commonly used antibody expression signal peptide in the art, such as the signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" upstream of VL 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)", 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 sequence of the present invention. Therefore, the signal peptide sequence is not shown in the antibody sequences in Table 1-2 of this example.

[0095] 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. The recombinant rabbit-derived antibody that recognizes human S100A1 was purified from the culture supernatant using protein A affinity gel resin (purchased from Tiandi Renhe, product number SA023100). After detection, the concentration of antibody 1D12 was 1 mg / mL, and the concentration of antibody 2F7 was 1 mg / mL, and the purity of both was greater than 95%.

[0096] Example 2 Detection of the affinity of antibodies 1D12 and 2F7 for S100A1 protein

[0097] The Gator Prime biomolecular interaction analyzer from Probe Life was used to determine the antigen-antibody binding curve to identify the antibody affinity. First, the antibody to be tested was immobilized on a Pro A probe (purchased from Gator Bio, product number 160003). The immobilization concentration of antibodies 1D12 and 2F7 was 2 μg / mL. Then, the probes immobilized with the antibody were placed in 131.58 nM and 263.16 nM S100A1 protein dilutions respectively to test the affinity of the antibody for binding to antigen S100A1 under different molar concentration conditions.

[0098] As Figure 2 shown, the upper and lower figures are the affinity curves of antibody 2F7 and antibody 1D12 binding to human S100A1 protein respectively. The vertical axis is the change in the thickness of the conjugate after the probe binds to the antibody and the protein, and the horizontal axis is 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. The dissociation coefficient K off A constant characterizing the dissociation rate of the antibody from the antigen. The association coefficient Kon The constant characterizing the binding rate of an antibody to its target, the affinity constant K D is K off / K on The ratio of, which characterizes the dissociation equilibrium constant between the antibody and the antigen.

[0099] Table 3 Results of the determination of affinity-related parameters of monoclonal antibodies 1D12 and 2F7

[0100] antibody <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> 1D12 <![CDATA[6.38×10 -4 > <![CDATA[6.77×10 4 > <![CDATA[9.44×10 -9 > 2F7 <![CDATA[3.11×10 -3 > <![CDATA[2.42×10 5 > <![CDATA[1.29×10 -8 >

[0101] As can be seen from Table 3, the affinity constants K of rabbit monoclonal antibodies 1D12 and 2F7 with Human S100A1 protein D are 9.44×10 -9 and 1.29×10 -8 (M) respectively, indicating that the antibody affinity is relatively high.

[0102] Example 3 Establishment of a sandwich enzyme-linked immunosorbent assay based on antibodies 1D12 and 2F7 and its sensitivity analysis

[0103] Biotin labeling of detection antibody 2F7: Take 100 μg of 2F7 antibody, add 10 μg of biotin (purchased from Biosai Biotechnology, product number B5064), mix well after addition, and incubate overnight at 4°C to obtain biotin-labeled antibody 2F7 (2F7-biotin).

[0104] Using antibody 1D12 as the capture antibody and antibody 2F7-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 1D12: Prepare a 2 μg / mL antibody 1D12 solution with 1×PBS, add 100 μL / well to a 96-well microplate, 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 E013 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 / well, block at 37°C for 2 h. After blocking, discard the blocking solution and dry it in an oven at 37°C for 0.5 - 2 h; 4) Adding antigen protein: Gradient dilute the HumanS100A1 protein (purchased from SinoBiological, catalog number 10179-H01H) with the E003 diluent (containing 2% BSA, 0.05% Tween 20, and 0.1% proclin 300 in 1×PBS, pH 7.2) to concentrations of: 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 ng / mL, and add 100 μL / well to the microplate respectively, and incubate at 37°C for 2 h; 5) Washing the plate: The same as step 2); 6) Adding the detection antibody 2F7: Prepare a 0.01 μg / mL antibody 2F7-biotin solution, add 100 μL / well to the microplate, and incubate at 37°C for 1 h; 7) Washing the plate: The same as step 2); 8) Adding SA-HRP: After diluting the concentrated solution of 100× streptavidin-horseradish peroxidase solution (SA-HRP, purchased from Wuhan Sanying, catalog number SA00001-0), add 100 μL / well to the microplate and incubate at 37°C for 0.5 h; 9) Washing the plate: The same as step 2); 10) Adding the chromogenic solution: Add the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution (purchased from Sizhengbai, catalog number 4ATMB1000) to the wells at 100 μL / well and incubate at 37°C for 15 min; 11) Reading: Take out the microplate, add 50 μL of 1 mol / L hydrochloric acid to each well to terminate the reaction, and immediately read with an enzyme-linked immunosorbent assay reader.

[0105] Using the concentration of human S100A1 protein as the abscissa and the corrected absorbance value ΔOD (ΔOD = OD 450nm - OD 630nm ) as the ordinate to plot a graph, and use the four-parameter fitting of the Logistic curve. The fitted standard curve (R 2 = 0.9999) is as shown in Figure 3As shown in the figure. The average absorbance value of 16 diluent blank wells greater than three times the average absorbance value of the blank control was used as the signal value of the sensitivity, which was substituted into the standard curve to calculate the sensitivity of the detection system (see Table 4), and its detection limit was 0.41 ng / mL.

[0106] Table 4 Sensitivity of the double-antibody sandwich enzyme-linked immunosorbent assay established based on antibodies 1D12 and 2F7

[0107]

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, 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 antibody against human calcium-binding protein S100A1, characterized in that, Selected from the first antibody or the second antibody, wherein: The amino acid sequences of complementarity-determining regions CDR1, CDR2, and CDR3 in the light chain variable region of the first antibody are shown in SEQ ID NO.3-5 respectively, and the amino acid sequences of complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8-10 respectively; The amino acid sequences of complementarity-determining regions CDR1, CDR2, and CDR3 in the light chain variable region of the second antibody are shown in SEQ ID NO.13-15 respectively, and the amino acid sequences of complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.18-20 respectively.

2. The antibody against human calcium-binding protein S100A1 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 antibody against human calcium-binding protein S100A1 according to claim 1, wherein The light chain of the first antibody and / or the second antibody is a κ chain, and the heavy chain is of IgG1 type.

4. The antibody against human calcium-binding protein S100A1 according to claim 1, 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.

5. The antibody against human calcium-binding protein S100A1 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 Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

6. A nucleic acid molecule or recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule encoding the first antibody or the second antibody as described in any one of claims 1-5.

7. An antibody pair against human calcium-binding protein S100A1, characterized in that, Composed of the first antibody and the second antibody as described in any one of claims 1-5.

8. Use of the antibody against human calbindin S100A1 as described in any one of claims 1-5 or the antibody pair against human calbindin S100A1 as described in claim 7 in the preparation of a kit for detecting human calbindin S100A1.

9. A kit for detecting human calcium-binding protein S100A1, characterized in that, Comprising the antibody against human calbindin S100A1 as described in any one of claims 1-5 or the antibody pair against human calbindin S100A1 as described in claim 7.

10. The human calcium-binding protein S100A1 detection kit according to claim 9, characterized in that, The detection kit is a double-antibody sandwich enzyme-linked immunosorbent assay 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 conjugated with a detection label.

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