Anti-human intercellular adhesion molecule-1 antibody, antibody pair and their applications

Abstract

The present invention belongs to the technical field of antibodies, and particularly relates to anti-human intercellular adhesion molecule-1 antibodies, antibody pairs and their applications. The antibody is a first antibody or a second antibody. The amino acid sequences of CDR1-3 in the light chain variable region of the first antibody are shown in SEQ ID NOs. 3-5 respectively, and the amino acid sequences of CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs. 8-10 respectively; the amino acid sequences of CDR1-3 in the light chain variable region of the second antibody are shown in SEQ ID NOs. 13-15 respectively, and the amino acid sequences of CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs. 18-20 respectively. The present invention provides an antibody and an antibody pair that can specifically recognize human ICAM-1, and are used to develop an immunoassay system for detecting human ICAM-1, which has the advantages of high affinity and specificity, strong anti-interference ability, high detection sensitivity and good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly to anti-human intercellular adhesion molecule-1 antibodies, antibody pairs and their applications. Background Art

[0002] Intercellular cell adhesion molecule-1 (ICAM-1), also known as CD54, with a relative molecular weight of 70-110 kD, is a single-chain glycoprotein and belongs to the members of the immunoglobulin superfamily (IGSF) among adhesion molecules. It is an important adhesion molecule mediating cell adhesion reactions. It participates in important physiological processes such as cell signal transduction and activation, immune response, and inflammatory response. ICAM-1 is constitutively expressed at a low level in various types of cells, such as endothelial cells, immune cells, and some epithelial cells, etc. Under the stimulation of various inflammatory factors such as TNF-α, IFN-α, IFN-γ, IL-1β, and IL-6, its expression is up-regulated, but there is a certain degree of specificity among different types of cells. The expression of ICAM-1 in endothelial cells is induced by TNF-α, the expression of ICAM-1 in intestinal epithelial cells is induced by IFN-α, and the expression of ICAM-1 in macrophages is mainly induced by IFN-γ and polysaccharides. The expression of ICAM-1 is also regulated by the activity of microRNA. ICAM-1 mainly participates in leukocyte trafficking and immune cell recruitment. It mediates the vascular adhesion and paracellular migration of leukocytes expressing activated LFA-1 (CD11a / CD18) and Mac-1 (CD11b / CD18), and plays an important role in the processes of immune surveillance, inflammatory response, and immune response. It also binds several non-integrin ligands, including CD43 / sialophorin, fibrinogen, hyaluronic acid, rhinovirus, and red blood cells infected with Plasmodium falciparum, and participates in inflammatory allergic reactions and transplant rejection reactions, etc. ICAM-1 also participates in immune cell interactions, such as T cell activation, antigen presentation, and the formation of immune synapses, etc.

[0003] Abnormal expression of ICAM-1 is related to various pathological processes, especially the pathological processes related to inflammatory responses and immune-mediated diseases. During the inflammatory process, the up-regulation of ICAM-1 in endothelial cells promotes leukocyte adhesion and infiltration into tissues, and is related to the pathogenesis of diseases such as atherosclerosis, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. In addition, studies have shown that ICAM-1 can promote the migration of cancer cells; ICAM-1 is up-regulated in triple-negative breast cancer (TNBC) and colorectal cancer (CRC), and is closely related to tumor invasion, metastasis, and poor prognosis. Targeting ICAM-1 can reduce the metastasis and angiogenesis of CRC. Therefore, ICAM-1 is expected to become a diagnostic and therapeutic target for TNBC and CRC, and detecting this index has important clinical significance.

[0004] Currently, for the determination of ICAM-1 levels, immunological methods such as enzyme-linked immunosorbent assay, immunoturbidimetry, immunoprecipitation, and immunofluorescence are mostly used. Different methods require specific monoclonal antibodies against ICAM-1. Monoclonal antibodies derived from mice are used in traditional clinical diagnosis, and their affinity and specificity are generally lower than those of monoclonal antibodies derived from rabbits. Moreover, the mouse monoclonal antibodies used rely on traditional hybridoma methods for development and production. The preparation process is more complex than that of recombinant monoclonal antibodies and there are also significant batch-to-batch differences. Therefore, immunodetection reagents developed based on mouse monoclonal antibodies face challenges such as poor stability and low sensitivity. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an antibody and an antibody pair that can specifically recognize human ICAM-1. When used to develop an immunodetection system for detecting ICAM-1, they have the advantages of high affinity and specificity, strong anti-interference ability, high detection sensitivity, and good stability, providing an effective solution for the high-precision detection of trace levels of human ICAM-1 in biological samples. Accordingly, the present invention also provides the application of the aforementioned antibody and antibody pair in the preparation of human ICAM- detection reagents or kits, and provides a detection reagent or kit containing the aforementioned antibody and antibody pair. The present invention is achieved through the following technical solutions:

[0006] In the first aspect of the present invention, there is provided an anti-human intercellular adhesion molecule-1 antibody, selected from a first antibody or a second antibody, wherein: the amino acid sequences of 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 CDR1-3 in the heavy chain variable region are respectively as shown in SEQ ID NO.8-10; the amino acid sequences of 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 CDR1-3 in the heavy chain variable region are respectively as shown in SEQ ID NO.18-20.

[0007] 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.

[0008] 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.

[0009] Furthermore, the first antibody and / 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, F(ab)2, Fab’, F(ab’)2, Fv, (Fv)2, scFv or sc(Fv)2.

[0010] In the second aspect of the present invention, there is provided a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule or a host cell containing the nucleic acid molecule, and the nucleic acid molecule encodes the first antibody or the second antibody as described above.

[0011] Furthermore, the nucleotide sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO.22, and the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO.24; the nucleotide sequence of the light chain variable region of the second antibody is as shown in SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO.28.

[0012] Furthermore, the nucleotide sequence of the light chain of the first antibody is as shown in SEQ ID NO.21, and the nucleotide sequence of the heavy chain is as shown in SEQ ID NO.23; the nucleotide sequence of the light chain of the second antibody is as shown in SEQ ID NO.25, and the nucleotide sequence of the heavy chain is as shown in SEQ ID NO.27. [[ID=!2]]

[0013] In the third aspect of the present invention, there is provided an anti-human intercellular adhesion molecule-1 antibody pair, which is composed of the first antibody and the second antibody as described above.

[0014] In the fourth aspect of the present invention, there is provided the use of the anti-human intercellular adhesion molecule-1 antibody or antibody pair as described above in the preparation of a human intercellular adhesion molecule-1 detection reagent or kit.

[0015] In the fifth aspect of the present invention, there is provided a human intercellular adhesion molecule-1 detection reagent or kit, and the detection reagent or kit is a double antibody sandwich enzyme-linked immunosorbent assay reagent or kit, including a first antibody and a second antibody, and the first antibody serves as a capture antibody, and the second antibody modified with a detection label serves as a detection antibody.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0017] 1. The antibody provided by the present invention can specifically recognize human ICAM-1 protein, and has advantages such as good binding activity, high affinity and strong specificity, and has low cross-reactivity with similar proteins, providing a reliable antibody raw material for immunodetection of ICAM-1 protein, and having a broad market prospect and good economic benefits in the fields of clinical diagnosis and scientific research.

[0018] 2. The rabbit monoclonal antibody against human intercellular adhesion molecule-1 of the present invention binds to different antigenic epitopes on the surface of human intercellular adhesion molecule-1. The double-antibody sandwich enzyme-linked immunosorbent assay system developed using this antibody pair has the advantages of high specificity, strong anti-interference ability, high detection sensitivity and good stability, providing an effective solution for the high-precision detection of extremely trace levels of human ICAM-1 protein in biological samples.

[0019] 3. The present invention is based on B cell labeling and sorting technology, which directly amplifies the antibody gene sequence from immunized rabbit B lymphocytes and performs recombinant expression. The antibody preparation method is simple and can achieve large-scale antibody preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0021] Figure 1 A map of the vector used to construct the rabbit monoclonal antibody expression vector in Example 1 of the present invention, including, from left to right, the pBR322 vector carrying the light chain constant region and the heavy chain constant region;

[0022] Figure 2 This is an affinity curve of the monoclonal antibody 2B10 binding to human ICAM-1 protein in Example 2 of the present invention;

[0023] Figure 3 This is an affinity curve of the monoclonal antibody 1F10 binding to human ICAM-1 protein in Example 2 of the present invention;

[0024] Figure 4 This is a graph showing the recognition of human ICAM-1 epitopes by monoclonal antibodies 2B10 and 1F10 according to Example 2 of the present invention;

[0025] Figure 5 This is the standard curve for detecting human ICAM-1 protein using a double antibody sandwich enzyme-linked immunosorbent assay system established based on monoclonal antibodies 2B10 and 1F10 in Example 3 of the present invention;

[0026] Figure 6 This is a graph showing the specificity determination results of the double-antibody sandwich enzyme-linked immunosorbent assay system established based on rabbit monoclonal antibodies 2B10 and 1F10 in Example 4 of the present invention;

[0027] Figure 7 This is a graph showing the thermal stability test results of a double-antibody sandwich enzyme-linked immunosorbent assay system established based on rabbit monoclonal antibodies 2B10 and 1F10 in Example 5 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Based on the information contained 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, various changes that can be made by those skilled in the art in the relevant field to the embodiments of the present invention are all covered within the scope of the appended claims.

[0030] In order 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 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 be regarded as obtained at least according to the reported significant figures and by the conventional rounding method.

[0031] In addition, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of the present invention should have the meanings commonly understood by those of ordinary skill in the art.

[0032] The meanings of terms such as "comprising", "including", "containing", "having" and the like are non-restrictive, that is, other steps and other components can be added without affecting the result. The term "and / or" should 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. 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.

[0033] Terms such as "monoclonal antibody", "antibody" and "mAb" and the like have the same meaning and can be used interchangeably. Unless otherwise specified, in the present invention, they all refer to rabbit-derived antibodies that specifically bind to human intercellular adhesion molecule-1 (ICAM-1). The modifier "rabbit" indicates that the complementarity-determining regions of the antibody are derived from rabbit immunoglobulin sequences. Terms such as "human intercellular adhesion molecule-1", "Human ICAM-1" and the like have the same meaning and can be used interchangeably.

[0034] 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, provided that 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.

[0035] 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, and 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 region 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 the antigen. The FR regions are the more conserved parts of VH and VL. They are generally in a β-sheet conformation 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.

[0036] 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.

[0037] The constant regions of the light chain (CL) and the heavy chain (CH) do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity. The lengths of the CL of different Ig types (κ or λ) are basically the same, but the lengths of the CH 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 and light chains are well known in the art.

[0038] 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", "complete antibody", and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0039] Antibody fragments are one or more parts or fragments of full-length antibodies, which basically maintain the same biological functions or activities 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 the complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, especially binding to the same epitope. In typical examples, 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.

[0040] (i) Fab: The antigen-binding fragment (Fab) is a monovalent fragment composed of a complete light chain (variable region and constant region) and a part of the heavy chain (variable region and the first constant region). By protease digestion of full-length antibodies, 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 part of the 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.

[0041] (ii) F(ab)2: A bivalent fragment containing two Fabs linked by a hinge region disulfide bridge.

[0042] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable regions, and consists of the variable regions of one light chain and one heavy chain. It is a dimer (VH-VL dimer) formed by non-covalent binding of one VH and one VL. The three CDRs of each variable region interact with each other to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0043] (iv) (Fv)2: Composed of two Fv fragments covalently linked together.

[0044] (v) scFv: The single-chain antibody (Single-chain variable fragment, scFv) is an Fv fragment composed of a single polypeptide chain, which is formed by connecting one heavy-chain variable region (VH) and one light-chain variable region (VL) through a flexible linker (linker, generally composed of 10-25 amino acids). It retains the binding specificity of the original antibody to the antigen. The linker in the present invention only needs to not interfere with the expression of the antibody variable regions connected to both ends thereof, and there is no special limitation. Compared with the full-length antibody, scFv has the characteristics of a small molecular weight, so it has higher penetration and lower immune side reactions.

[0045] (vi) The sc(Fv)2 fragment is formed by connecting two heavy-chain variable regions and two light-chain variable regions through a linker or the like.

[0046] Terms such as "monoclonal antibody" or "mAb" can be used interchangeably and refer to a homogeneous group of antibodies, that is, each antibody constituting the group is the same except for a small number of possible natural mutations and / or post-translational modifications (such as isomerization, amidation). "Monoclonal antibody" is highly specific and shows a single binding specificity and affinity for the same or substantially the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed as limiting the source or preparation method 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.

[0047] The term "specific binding" is a well-known term in the art. If a molecule reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a specific target antigen or epitope than with other target antigens or epitopes, it exhibits "specific binding". "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0048] To make the above objects and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given.

[0049] An embodiment of the present invention provides an anti-human intercellular adhesion molecule-1 antibody, selected from the first antibody or the second antibody. Both the first antibody and the second antibody include a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region both include 3 complementarity-determining regions (CDRs), named CDR1, CDR2, and CDR3 respectively. Among them:

[0050] The amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the first antibody are shown as 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 as SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 respectively;

[0051] The amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the second antibody are shown as 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 as SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20 respectively.

[0052] Based on B cell labeling and sorting technology, the present invention directly enriches and isolates B lymphocytes capable of recognizing human intercellular adhesion molecule-1 protein from the immunized rabbit spleen, greatly improving the efficiency of screening antigen-specific B lymphocytes. Then, by PCR amplification and recombinant expression of specific antibodies in B lymphocytes, the cumbersome steps of multiple subcloning in the hybridoma technology are omitted, and the large-scale preparation of antibodies can be achieved.

[0053] The antibody containing the above CDR sequences prepared by the present invention has high affinity, good specificity, and excellent anti-interference ability for human intercellular adhesion molecule-1 (ICAM-1), can specifically bind to human ICAM-1 protein, has low cross-reactivity with similar proteins such as human VCAM-1, and is used for immunodetection of ICAM-1 protein in biological samples, which is beneficial to significantly improving the accuracy, sensitivity, stability, and reliability of detection results and reducing false negative or false positive results. Moreover, the two antibodies of the present invention recognize different antigen epitopes on the surface of human ICAM-1 protein. Using the first antibody as the capture antibody and the second antibody as the detection antibody to construct a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection system, the detection limit of human ICAM-1 is as low as 5.54 pg / mL, providing an effective solution for the high-precision detection of extremely trace levels of human ICAM-1 protein in biological samples, and having broad market prospects and good economic benefits in the fields of clinical diagnosis and scientific research.

[0054] Optionally, both the light chain variable region and the heavy chain variable region each 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 (VL) of the first antibody is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is as shown in SEQ ID NO.7. The amino acid sequence of the light chain variable region (VL) of the second antibody is as shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region (VH) is as shown in SEQ ID NO.17.

[0055] Optionally, the first antibody and the second antibody further include a light chain constant region (CL) and a heavy chain constant region (CH). For each antibody, CL and VL form the light chain (FL), and CH and VH form the heavy chain (FH). The constant regions of antibodies can generally be obtained through public queries. For example, through the IMGT online database (www.imgt.org), the CH can be obtained by searching for rabbit IgG gamma C reign, and the CL can be obtained by searching for rabbit IgG Kappa C reign. Specifically, 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. Among them, the light chain constant regions of the first antibody and the second antibody are κ chains, and the heavy chain constant regions are of the IgG1 type.

[0056] Optionally, the first antibody and / or the second antibody is a full-length antibody (having a typical Y-shaped molecular structure) or the antigen-binding region of the full-length antibody; this antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form of the rabbit monoclonal 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.

[0057] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell containing the nucleic acid molecule, and the nucleic acid molecule encodes the first antibody and / or the second antibody as described above.

[0058] 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 non-coding strand.

[0059] The sequence of the nucleic acid molecule can be obtained by derivation through conventional means such as codon coding rules based on the antibody AA sequence. The full-length sequence or fragments of the nucleic acid molecule can usually be obtained by PCR amplification, recombination or artificial synthesis methods. The obtained nucleic acid molecule is inserted into an expression vector, then introduced into a host cell, and cultured under specific conditions to express and obtain the antibody.

[0060] Exemplarily, the nucleotide sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO.22, and the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO.24; the nucleotide sequence of the light chain variable region of the second antibody is as shown in SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO.28.

[0061] Exemplarily, the nucleotide sequence of the light chain of the first antibody is as shown in SEQ ID NO.21, and the nucleotide sequence of the heavy chain is as shown in SEQ ID NO.23; the nucleotide sequence of the light chain of the second antibody is as shown in SEQ ID NO.25, and the nucleotide sequence of the heavy chain is as shown in SEQ ID NO.27.

[0062] The original vectors for constructing the recombinant vector are various conventional vectors in the art, as long as they 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). The nucleic acid molecule of the present invention can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule.

[0063] 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 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 expression vector / promoter depends on the type of host cell used to produce the antibody.

[0064] 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, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with the CaCl2 method or MgCl2; 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. to achieve gene introduction.

[0065] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include Escherichia coli (such as DH5α, JM109, BL21, W3110), Bacillus (such as Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (such as Salmonella typhimurium, Serratia marcescens) and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include yeast, insect cells and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHODG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, HEK293 cell lines, etc., but are not limited thereto. 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.

[0066] In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human renal epithelial cell (293F cell).

[0067] Another embodiment of the present invention provides an anti-human intercellular adhesion molecule-1 antibody pair, which consists of the first antibody and the second antibody as described above.

[0068] The first antibody and the second antibody of the present invention bind to different antigenic epitopes of human ICAM-1, and can be used to develop a sandwich enzyme-linked immunosorbent assay system, which has the advantages of high specificity, wide linear range, high detection sensitivity, and strong anti-interference ability.

[0069] Another embodiment of the present invention provides the use of the anti-human intercellular adhesion molecule-1 antibody or antibody pair as described above in the preparation of a human intercellular adhesion molecule-1 detection reagent or kit.

[0070] The advantages of the use of the anti-human ICAM-1 antibody or antibody pair in the preparation of a human ICAM-1 detection reagent or kit are the same as the advantages of the anti-human ICAM-1 antibody or antibody pair over the prior art, and will not be elaborated herein.

[0071] Based on the same inventive concept, an embodiment of the present invention also provides a human intercellular adhesion molecule-1 detection reagent or kit, and the detection reagent or kit includes the first antibody and / or the second antibody as described above.

[0072] 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 test sample is contacted with the first antibody and / or the second antibody, and then the antibody is detected. In some embodiments, the first antibody and / or the second antibody can be conjugated with a detectable label, and qualitative or quantitative detection of ICAM-1 can be achieved by analyzing the change in the recognizable signal generated by the detectable label. In other embodiments, the antibody against human ICAM-1 (used as the primary antibody or capture antibody) is not labeled, and the detectable label is conjugated with a secondary antibody (used as the detection antibody) or other molecules that can bind to the primary antibody. For example, if the antibody against human ICAM-1 is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, and thus a change in the recognizable signal is generated by the secondary antibody conjugated with the detectable label. When used in pairs, one of the first antibody and the second antibody is used as the primary antibody or capture antibody, and the other is used as the secondary antibody or detection antibody.

[0073] The detection methods described above use common immunological methods, including but not limited to: enzyme immunoassay (EIA), 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 ICAM-1 protein, ICAM-1 protein secreted by cells, or ICAM-1 protein in human serum. The detection samples include but are not limited to serum, plasma, urine, and cell culture medium.

[0074] Preferably, the detection reagent or kit is a double antibody sandwich enzyme-linked immunosorbent assay reagent or kit, including a first antibody and a second antibody. The first antibody serves as a capture antibody (or primary antibody), and the second antibody modified with a detection label serves as a detection antibody (or secondary antibody).

[0075] 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.

[0076] In a preferred embodiment, the detection label is biotin.

[0077] 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 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.

[0078] Example 1 Preparation of Rabbit Monoclonal Antibody Against Human ICAM-1

[0079] New Zealand white rabbits were immunized with human intercellular adhesion molecule-1 (ICAM-1) (from ABclonal, catalog number RP00272) as the antigen. Antigen-specific B lymphocytes were isolated from the immunized spleen cells and cultured. The genes of the heavy chain variable region (VH) and light chain variable region (VL) corresponding to the antibody were extracted by primers, constructed into an expression vector containing the heavy chain constant region and light chain constant region, and transfected into host cells. The host cells were cultured to obtain the supernatant containing the rabbit-derived monoclonal antibody, which was purified to obtain the candidate antibody. Subsequently, screening was carried out by methods such as affinity testing and antigen epitope identification, and antibody strains 2B10 and 1F10 were obtained. The selected antibodies were sequenced, and the sequencing work was completed by Wuhan Genecreate Biological Engineering Co., Ltd. The amino acid (AA) and nucleotide (DNA) sequences of the antibodies are shown in Tables 1-2 respectively. For the convenience of description, the light chain complementarity-determining regions CDR1, CDR2, and CDR3 are represented by LCDR1, LCDR2, and LCDR3 respectively, and the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 are represented by HCDR1, HCDR2, and HCDR3 respectively.

[0080] Table 1 Sequence Information of Rabbit Monoclonal Antibody 2B10 in This Example

[0081]

[0082]

[0083] Table 2 Sequence information of rabbit monoclonal antibody 1F10 in this embodiment

[0084]

[0085] The preparation methods of rabbit monoclonal antibodies 2B10 and 1F10 specifically include the following steps:

[0086] 1. Animal immunization: Each white rabbit is immunized with 200 μg of antigen (from ABclonal, catalog number RP00272). Before the first immunization, the immunogen is mixed with an equal amount of complete Freund's adjuvant to make an emulsifier, and it is injected subcutaneously at multiple points on the abdomen and back of the rabbit; every 3 weeks after the first immunization, 100 μg of the immunogen is mixed with an equal amount of incomplete Freund's adjuvant to make an emulsifier, and it is injected subcutaneously at multiple points on the abdomen and back of the rabbit for two booster immunizations; after three immunizations, rabbit serum samples are collected, and the titer against human ICAM-1 is measured by the ELISA method. The rabbit with a high serum titer is selected and boosted once by subcutaneous multi-point injection with 200 μg of the immunogen. The animal is sacrificed three days later and the spleen is taken.

[0087] 2. Isolate splenocytes: Under aseptic conditions in a safety cabinet, take out a culture dish and add 30 - 40 mL of basic medium (RPMI Medium 1640 basic + 1% Pen Strep; RPMI 1640 is purchased from Gibco, product number C11875500BT; Pen Strep is purchased from Gibco, product number 15140 - 163). Place a cell sieve, take out the spleen and place it in the cell sieve. Cut off the excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into small pieces and grind it in the cell sieve. Take a clean grinding rod and use the end of the pressing part to crush and grind the tissue. The cells in the membrane will slowly dissociate. After passing through the cell sieve, they will be suspended in the culture dish solution. Wash the cell sieve with 10 mL of basic medium and collect the basic medium outside the cell sieve. Centrifuge at 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 13 mL of room temperature RBC lysis buffer (purchased from BioGems), gently blow and disperse the cell clumps with a pipette and start timing for 1 min for RBC lysis. Add 37 mL of basic medium and mix well to terminate RBC lysis. Centrifuge at 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 40 mL of room temperature basic medium, gently blow and disperse the cell clumps with a pipette to resuspend the cells, and complete the first wash. Centrifuge at 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 20 mL of room temperature basic medium, gently blow and disperse the cell clumps with a pipette to resuspend the cells. Filter the resuspended cells through the cell sieve again to remove clumped cells, and then count the cells.

[0088] 3. Isolate B lymphocytes in the spleen and perform B lymphocyte sorting: Isolate B lymphocytes in the spleen by conventional methods. For relevant methods, refer to the patents "Method for efficiently isolating single antigen - specific B lymphocytes from spleen cells (Publication number: CN110016462A, Publication date: July 16, 2019)" and "An in vitro culture system for B lymphocytes and its application (Publication number: CN111518765A, Publication date: August 11, 2020)".

[0089] 4. Clone the gene encoding rabbit monoclonal antibody: Identify positive clones by ELISA coated with ICAM - 1 antigen using the supernatant of cultured B lymphocytes to obtain antigen - specific B lymphocytes. After collecting and lysing the positive clone cells, follow Quick - RNA TMExtract RNA using the Micro Prep Kit Instruction Manual (purchased from ZYMO, product number R1051), and reverse transcribe it into cDNA. Using the aforementioned cDNA as a template, adopt the PCR method to amplify the variable light chain (VL) and variable heavy chain (VH) of the natural paired rabbit monoclonal antibody from the cDNA of the corresponding positive clone and conduct sequencing. The sequencing work was completed by GeneCreate Biological Engineering Co., Ltd. The PCR reaction system includes: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×GloriaHiFi (from ABclonal, product number RK20717), and 6.5 μL H2O; the PCR amplification program includes: pre-denaturation at 98 °C for 30 s, and then perform 40 cycles according to the conditions of 98 °C for 10 s, 64 °C for 30 s, and 72 °C for 30 s, and finally hold at 72 °C for 5 min. The obtained reaction solution is stored at 4 °C. Among them, the primer sequences (5'-3') for amplifying the VL and VH genes are shown in Table 3, where F and R represent the forward primer and the reverse primer respectively.

[0090] Table 3 Primer sequence information for amplifying the rabbit monoclonal antibody in this example

[0091]

[0092]

[0093] 5. Production and purification of rabbit monoclonal antibody: The mammalian expression vector pBR322 carrying the light chain constant region (CL) and heavy chain constant region (CH) genes was linearly processed by conventional digestion with XbaI and NheI restriction enzymes. After purifying the VL and VH genes containing the signal peptide amplified by the above PCR, they were constructed into the aforementioned expression vector by homologous recombination to obtain the light chain gene and heavy chain gene expression vectors. The successful construction of the expression vector was verified by sequencing. The vector expression map is shown in Figure 1 , 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 nucleic acid sequence of the light chain constant region (left figure), Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right figure). The CL and CH genes were obtained by querying the IMGT online database (www.imgt.org), searching for rabbit-derived IgG gamma C reign to obtain CH, and searching for rabbit-derived IgGKappa C reign to obtain CL.

[0094] The signal peptide of this embodiment can adopt the signal peptide sequences commonly used in the art for antibody expression. For example, 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 Rabbit Monoclonal Antibody Against Human IL-5 and Its Application (Publication No.: CN115819578A, Publication Date: March 21, 2023)", there is a signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" upstream of the light chain variable region, and a signal peptide "METGLRWLLLVAVLKGVQC" upstream of the heavy chain variable region.

[0095] The expression vectors containing the light chain gene and the heavy chain gene that have been verified correctly by sequencing are co-transfected into 293F cells. After transfection, the cells are cultured for 72 - 96 h to obtain a recombinant rabbit monoclonal antibody against human ICAM-1 in the culture supernatant. The target antibody is purified from the culture supernatant using protein A affinity gel resin (purchased from Tiandi Renhe, product number SA023100). After sub-packaging the purified antibody, it is stored at -20 °C for future use.

[0096] Example 2 Performance Testing of Rabbit Monoclonal Antibodies 2B10 and 1F10

[0097] 1) Affinity testing: The affinities of the obtained antibodies 2B10 and 1F10 are identified using the Gator biomolecular interaction analyzer (Gator Prime device) from Probe Life, and the probe used is ProteinA Probe. The specific method is as follows: 1) Pre-wet: Shake and wet the probe in the supporting buffer at 1000 rpm for 300 s before use; 2) Baseline1: Place the probe in the buffer for initial point calibration to ensure that the probe is initially in a stable state, and process it at 1000 rpm for 60 s; 3) Loading: Immobilize the test antibodies 2B10 / 1F10 on the probe, and the immobilization concentrations are 2.49 μg / mL and 5.76 μg / mL respectively, and process it at 1000 rpm for 35 s; 4) Baseline2: Place the probe with immobilized antigen in the buffer for shaking and washing, and process it at 1000 rpm for 60 s; 5) Association: Place the probe with immobilized antibody in 75 nM and 150 nM antigen dilution solutions respectively, and process it at 1000 rpm for 240 s to test the affinity of the antibody binding to the antigen under different molar concentration conditions; 6) Dissociation: When the antigen-antibody binding reaches the saturation state, transfer the probe to the dissociation system to complete the dissociation process, and process it at 1000 rpm for 240 s. The affinity curves of antibodies 2B10 and 1F10 binding to human ICAM-1 are shown respectively inFigures 2 - 3 , 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 calculated by curve fitting are shown in Table 4.

[0098] Table 4 Determination results of affinity-related parameters of rabbit monoclonal antibodies

[0099] antibody <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> 2B10 <![CDATA[3.48×10 -4 > <![CDATA[9.39×10 5 > <![CDATA[3.7×10 -10 > 1F10 <![CDATA[2.26×10 -4 > <![CDATA[2.40×10 5 > <![CDATA[9.4×10 -10 >

[0100] The results show that the dissociation rate constants of antibodies 2B10 and 1F10 against human intercellular adhesion molecule-1 are 3.48×10 -4 and 2.26×10 -4 respectively; the association rate constants are 9.39×10 5 and 2.40×10 5 respectively; the dissociation equilibrium constants are 3.7×10 -10 and 9.4×10 -10 respectively, indicating that the antibodies have a high affinity for human intercellular adhesion molecule-1.

[0101] Figure 4

[0102]

[0103] 2) Identification of antigen recognition epitopes: The operation is basically the same as that for affinity determination, except that: the probe is Anti-His / FC Probe. First, the antigen protein is immobilized on the probe at a concentration of 10 μg / mL. Then, the test antibodies 2B10 / 1F10 are used to bind to the antigen on the probe at a concentration of 3 μg / mL. By analyzing the binding of the two antibodies to the antigen, the antigen epitopes recognized by them are determined. The antigen determinant recognition results of the two antibodies are shown in Figure 4 , where the ordinate represents the change in the thickness of the conjugate after the probe binds to the antibody and the protein, and the abscissa represents the binding time of the antibody and the antigen protein.

[0102] It can be seen from the figure that antibodies 2B10 and 1F10 recognize different antigen epitopes of human intercellular adhesion molecule-1 respectively, and the binding sites do not interfere with each other. Therefore, the two can be used as paired antibodies for sandwich enzyme-linked immunosorbent assay.

[0103] Example 3 Establishment of a sandwich enzyme-linked immunosorbent assay based on antibodies 2B10 and 1F10 and its sensitivity test

[0104] Biotinylation of detection antibody - antibody 1F10: Prepare a 1 mg / mL solution of antibody 1F10, and prepare a 10 mg / mL solution of biotin (purchased from Youyilan Di, product number: B5064) with DMSO; take 200 μL of the 1 mg / mL antibody 1F10 solution and add 2 μL of the 10 mg / mL biotin solution; after mixing, place it in a refrigerator at 2 - 8 °C and react for 16 - 20 h before use.

[0105] Using antibody 2B10 as the capture antibody and biotin-labeled antibody 1F10 as the detection antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was established, and the steps were as follows: 1) Coating the capture antibody 2B10: Dilute antibody 2B10 with 1×PBS to 2 μg / mL, mix well with a vortex mixer, and then add 100 μL / well to a 96-well microplate. Cover with a cover film and incubate in a 4°C refrigerator for 16 - 20 h; 2) Washing the plate: After incubation, discard the liquid in the wells, wash the plate once with 1×PBST, add 300 μL of sample, let stand for 40 s, then discard the liquid in the wells, and pat dry the liquid in the wells on a flat piece of paper; 3) Blocking: Add the E013 blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween, and 0.1% proclin 300 in 1×PBS, pH 7.2) to the wells at 200 μL / well, cover with a cover film, block at 37°C for 2 h. After blocking, discard the blocking solution, pat dry the microplate, and then dry it in a 37°C oven for 0.5 - 2 h, and take it out for standby; 4) Adding antigen protein: Gradient dilute human ICAM-1 protein (purchased from RD, catalog number ADP4-050) with the E003 blocking solution (containing 2% BSA, 0.05% Tween 20, and 0.1% proclin 300 in 1×PBS, pH 7.2). The dilution concentrations are: 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL. Then add different concentrations to the microplate at 100 μL / well in sequence, cover with a cover film, and incubate at 37°C for 2 h; 5) Washing the plate: After incubation, discard the liquid in the wells, wash the plate three times with 1×PBST, add 300 μL of sample, let stand for 40 s, then discard the liquid in the wells, and pat dry the liquid in the wells on a flat piece of paper; 6) Adding the detection antibody 1F10: Dilute the biotin-labeled antibody 1F10 (B-biotin) to 0.02 μg / mL, and then add it to the microplate at 100 μL / well in sequence, cover with a cover film, and incubate at 37°C for 1 h; 7) Washing the plate: The same as step 5); 8) Adding SA-HRP: Dilute the 100×SA-HRP (horseradish peroxidase-labeled streptavidin, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number SA00001-0) concentrate by 100 times, and then add it to the microplate at 100 μL / well in sequence, cover with a cover film, and incubate at 37°C for 0.5 h; 9) Washing the plate: The same as step 5); 10) Adding the chromogenic solution: Add the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution (purchased from Sizhengbai, catalog number 4ATMB1000) to the microplate at 100 μL / well, cover with a cover film, and incubate at 37°C for 15 min; 11) Reading: After incubation, take out the microplate, add 50 μL of the termination solution (1 mol / L hydrochloric acid) to each well, and immediately read with an enzyme reader. Using the human ICAM-1 protein concentration as the abscissa and the corrected absorbance value Y (=OD 450nm-OD 630nm ) Plot the absorbance values on the vertical axis to obtain the standard curve (see Figure 5 ). The absorbance value for sensitivity is the sum of the average absorbance value of 16 blank wells (replacing the antibody solution with E003 diluent) and twice the standard deviation. Substitute the absorbance value into the standard curve, and the resulting concentration is the sensitivity of the sandwich ELISA method for human intercellular adhesion molecule-1. The results are shown in Table 5.

[0106] Table 5 Sensitivity test data of the sandwich ELISA established based on antibodies 2B10 and 1F10

[0107]

[0108] The above results show that the sandwich ELISA system established with antibody 2B10 as the capture antibody and antibody 1F10 as the detection antibody for the detection of human intercellular adhesion molecule-1 exhibits good linearity. The detection sensitivity (sensitivity) is as low as 5.54 pg / mL, with good sensitivity and accuracy.

[0109] Example 4 Specificity test of the sandwich ELISA system established based on antibodies 2B10 and 1F10

[0110] Use Human Galectin 1 (from Abclonal, catalog number RP00007), Human sE-selectin (from Abclonal, catalog number RP00293), Human P-selectin (purchased from RD, catalog number 841156), Human VCAM-1 (from Abclonal, catalog number RP00972), Mouse VCAM-1 (from Abclonal, catalog number RP01176) as detection targets, and conduct cross-experiments with human ICAM-1 (purchased from RD, catalog number ADP4-050). Dilute the human ICAM-1 protein to 2000 pg / mL with E003 diluent, and the other proteins to 40 ng / mL. The detection method is the same as in Example 3. The results are shown in Figure 6 , where H. represents Human and M. represents Mouse in the figure.

[0111] When the antibody binds to the antigen, a light signal will be generated. Figure 6 It shows that only human ICAM-1 can cause an increase in the absorbance value, indicating that antibodies 2B10 and 1F10 have no obvious cross-reaction with other similar proteins except ICAM-1 protein, proving that the antibodies of the present invention have high specificity for recognizing and binding human ICAM-1.

[0112] Example 5 Thermal Stability Test of a Sandwich ELISA System Based on Antibodies 2B10 and 1F10

[0113] The ELISA plate coated with the capture antibody 2B10, the lyophilized human ICAM-1 protein, and the 100× concentrated detection antibody 1F10 were separately stored sealed at -20°C and 37°C for 7 days and then taken out. The Y value was measured according to the method of Example 3. The thermal stabilities of the rabbit monoclonal antibodies 2B10 and 1F10 against human intercellular adhesion molecule-1 were compared based on the standard curves established by comparing the antibody samples treated at different temperatures. The results are shown in Table 6 and Figure 7 .

[0114] Table 6 Stability Test Data of the Sandwich ELISA Method Based on Antibodies 2B10 and 1F10

[0115]

[0116] The results showed that the CV values of the ELISA plate coated with the capture antibody 2B10, the biotin-labeled detection antibody, and the antigen protein after being damaged at 37°C for 7 days compared with the control group were 6.88%, 1.67%, and 3.39% respectively. The CV value of all three aforementioned components after being damaged at 37°C for 7 days compared with the control group was 6.88%. It can be seen that the detection system of the present invention has good stability.

[0117] 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 intercellular adhesion molecule-1 antibody, characterized in that Selected from the first antibody or the second antibody, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in 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; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; 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.

2. The anti-human intercellular adhesion molecule-1 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 intercellular adhesion molecule-1 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 intercellular adhesion molecule-1 antibody according to claim 1, characterized in that The first antibody and / 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, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv or sc(Fv)2.

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 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleotide sequence of the light chain variable region of the first antibody is shown in SEQ ID NO. 22, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 24; The nucleotide sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

28.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleotide sequence of the light chain of the first antibody is shown in SEQ ID NO.21, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.23; The nucleotide sequence of the light chain of the second antibody is shown in SEQ ID NO. 25, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.

27.

8. An anti-human intercellular adhesion molecule-1 antibody pair, characterized in that: The method comprises the first antibody and the second antibody according to any one of claims 1 to 4.

9. Use of the anti-human intercellular adhesion molecule-1 antibody according to any one of claims 1 to 4 or the anti-human intercellular adhesion molecule-1 antibody pair according to claim 8 in the preparation of a human intercellular adhesion molecule-1 detection reagent or kit.

10. A human intercellular adhesion molecule-1 detection reagent or kit, characterized in that: The detection reagent or kit is a double antibody sandwich enzyme-linked immunosorbent assay reagent or kit, comprising the anti-human intercellular adhesion molecule-1 antibody pair as claimed in claim 8, with the first antibody serving as the capture antibody and the second antibody modified with a detection marker serving as the detection antibody.

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