An anti-human CD68 monoclonal antibody, its preparation method and application

Abstract

This invention relates to an anti-human CD68 monoclonal antibody, its preparation method, and its applications. The amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3) of the anti-human CD68 monoclonal antibody include the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the light chain complementarity-determining regions (CDR1, CDR2, and CDR3) include the sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. This invention develops and prepares a highly specific and affinity-based anti-human CD68 monoclonal antibody with good anti-interference ability, wide applicability, and is suitable for high-specificity, high-accuracy, and high-sensitivity detection of CD68, especially for immunohistochemical detection.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and relates to an anti-human CD68 monoclonal antibody, its preparation method, and its application. Background Technology

[0002] CD68, also known as macrophage sialic acid protein, GP110, LAMP4, or SCARD1, is a 110 kDa transmembrane glycoprotein widely expressed in monocytes such as macrophages, microglia, and osteoclasts. It is a member of the lysosome / endosome-associated membrane glycoprotein (LAMP) family. This protein is primarily located in lysosomes and endosomes, with a small fraction circulating to the cell surface. It is a type I integrated membrane protein with a highly glycosylated extracellular domain that can bind to tissue- and organ-specific lectins or selectins. Expressed in many tumor cell lines, it can attach to selectins on vascular endothelium, thereby promoting its spread to secondary sites.

[0003] CD68 plays a crucial role in various physiological and pathological processes, including inflammation and autoimmunity, promoting bone resorption, and tumor progression. Bone marrow-derived macrophages are the most common type of tumor-infiltrating immune cells in the tumor microenvironment (TME) and are important mediators of anti-tumor immune responses. Recent studies have found that CD68 is overexpressed in tumor-associated macrophages (TAMs) and tumor cells. High levels of CD68 are associated with higher tumor grade, larger tumor size, Ki67 positivity, and other malignant characteristics, indicating tumor progression and invasiveness. TAMSs identified by CD68 expression can be divided into two subtypes: classically activated type 1 (M1) macrophages and selectively activated type 2 (M2) macrophages. M1 macrophages have pro-inflammatory properties and express high levels of free radicals and major histocompatibility complex molecules, contributing to anti-tumor activity. In contrast, M2 macrophages, which release a variety of anti-inflammatory cytokines and chemokines, promote tumor growth and metastasis.

[0004] Increasing evidence suggests that CD68 is a promising biomarker for the diagnosis and prognosis of tumor-associated cancers. CD68, alone or in combination with other cellular markers of tumor-associated macrophages, has shown good predictive value as a prognostic biomarker for cancer patient survival. For example, CN118655317A discloses the application of GPER1 and CD68 as prognostic markers in the preparation of a hepatocellular carcinoma (HCC) prognostic prediction kit. The kit predicts HCC prognosis by detecting GPER1 and CD68 expression levels, using a CD68 antibody to detect CD68 expression levels and a GPER1 antibody to detect GPER1 expression levels.

[0005] In conclusion, the development of monoclonal antibodies against human CD68 can expand the detection tools for CD68 and is of great significance for tumor-related diagnosis and prognosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-human CD68 monoclonal antibody, its preparation method, and its application. It develops a novel monoclonal antibody against human CD68, expands the detection tools for CD68, and has broad applicability, suitable for high-specificity, high-accuracy, and high-sensitivity detection of CD68.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an anti-human CD68 monoclonal antibody, wherein the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2 and CDR3 of the anti-human CD68 monoclonal antibody include the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2 and CDR3 include the sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0009] In this invention, amino acid fragments at positions 124 to 135 and 146 to 158 of the C-terminus of human CD68 protein are selected as antigens. High-purity polypeptides are synthesized, and the CD68 polypeptide conjugated with thiol KLH is used as an immunogen to immunize New Zealand white rabbits. Based on monoclonal antibody development technology using single B lymphocyte screening and culture, rabbit monoclonal antibodies against CD68 protein, along with their heavy and light chain sequences, are obtained. The prepared rabbit monoclonal antibody exhibits high specificity, affinity, and good anti-interference ability, making it widely applicable and suitable for high-specificity, high-accuracy, and high-sensitivity detection of CD68, particularly for immunohistochemical detection.

[0010] It is understood that, based on the anti-human CD68 monoclonal antibody designed in this invention, any functionally similar nanobodies obtained by modifying the amino acid sequence using genetic engineering techniques in the art through substitution, deletion, or addition should be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the modified amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In the art, when amino acids with similar or comparable properties are conservatively substituted, the function of the protein is usually not changed. For example, when amino acids with similar properties are substituted in the CDR region and / or FR region, the amino acid residues that can be conservatively substituted are known in the art, and such substituted amino acid residues may or may not be encoded by the genetic code. Therefore, monoclonal antibodies obtained by conservative substitution of amino acids with similar or comparable properties are also within the scope of protection of this invention.

[0011] Preferably, the amino acid sequence of the heavy chain variable region of the anti-human CD68 monoclonal antibody includes the sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8.

[0012] Preferably, the rabbit monoclonal antibody of the present invention further includes a light chain constant region and a heavy chain constant region, where CL and VL constitute the complete light chain, and CH and VH constitute the complete heavy chain. The constant regions of the antibody can usually be obtained through public searches, such as searching for rabbit-derived IgG gamma C reign to obtain CH, and searching for rabbit-derived IgG Kappa Creign to obtain CL, using the IMGT online database (www.imgt.org).

[0013] Preferably, the light chain constant region of the anti-human CD68 monoclonal antibody may be a κ chain, and the heavy chain may be of the IgG type.

[0014] Preferably, the amino acid sequence of the heavy chain of the anti-human CD68 monoclonal antibody includes the sequence shown in SEQ ID NO.9, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.10.

[0015] It should be noted that the rabbit monoclonal antibody of the present invention can be a full-length antibody (with a typical Y-shaped molecular structure) or an antibody fragment. The antibody fragment 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 antibody fragment includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining complete antigen recognition and binding sites, and is able to bind the same antigen with the full-length antibody, especially the same epitope. The antibody fragments include, but are not limited to: (i) Fab fragments, monovalent fragments consisting of variable regions and a first constant region of each heavy and light chain; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fv fragments, consisting of a heavy chain variable region and a light chain variable region of the antibody; (iv) (Fv)2 fragments, consisting of two Fv fragments covalently linked together; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain, formed by connecting a heavy chain variable region and a light chain variable region via a linker; and (vi) sc(Fv)2 fragments, which are formed by linking two heavy chain variable regions and two light chain variable regions via a linker, etc. These antibody fragments can be obtained using conventional techniques in the art.

[0016] In a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-human CD68 monoclonal antibody described in the first aspect.

[0017] In this invention, nucleic acid molecules can be in the form of DNA (e.g., cDNA, genomic DNA, or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be derived from the amino acid sequence of the antibody using conventional methods such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragments can typically be obtained using PCR amplification, recombination, or artificial synthesis.

[0018] Preferably, the nucleic acid sequence of the light chain of the anti-human CD68 monoclonal antibody includes the sequence shown in SEQ ID NO.11.

[0019] Preferably, the nucleic acid sequence of the variable region of the light chain of the anti-human CD68 monoclonal antibody includes the sequence shown in SEQ ID NO.12.

[0020] Preferably, the nucleic acid sequence of the anti-human CD68 monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO.13.

[0021] Preferably, the nucleic acid sequence of the variable region of the heavy chain of the anti-human CD68 monoclonal antibody includes the sequence shown in SEQ ID NO.14.

[0022] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0023] In this invention, the starting vector for the recombinant vector only needs to be capable of carrying the nucleic acid molecule, and various vectors conventional in the art can be used. Typical vectors include plasmids, viral vectors, bacteriophages, granules, and minichromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules into a host and multiply them in host cells) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in the host cell). Therefore, a cloning vector may contain selection markers and a replication origin matching the cell type specified by the cloning vector, while an expression vector contains regulatory elements (such as promoters and enhancers) for expression in a specified host cell. The nucleic acid molecules of this invention can be inserted into suitable vectors to form cloning vectors or expression vectors. This is well-known in the art and will not be described in detail here.

[0024] The nucleic acid molecules encoding the heavy and light chains of the antibody of the present invention can be constructed onto two separate vectors, which can be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to form an antibody. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the rabbit monoclonal antibody of the present invention can also be cloned into a single vector, with each nucleic acid sequence ligated downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy and light chains can be operatively ligated to different promoters, or the nucleic acid sequences encoding the heavy and light chains can be operatively ligated to a single promoter, such that both the heavy and light chains can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0025] Fourthly, the present invention provides a host cell expressing the anti-human CD68 monoclonal antibody described in the first aspect.

[0026] In this invention, the host cell contains the recombinant vector or the nucleic acid molecule is integrated into the genome.

[0027] In this invention, the recombinant vector is transfected or transformed into host cells using conventional techniques. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2 or MgCl2. If necessary, microinjection, electroporation, or liposome packaging can also be used. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, etc.

[0028] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: *Escherichia coli*, *Streptomyces*, *Salmonella typhimurium*, yeast, *Drosophila* S2 or Sf9 cells, and CHO, COS7, and 293 series cells. After obtaining host cells transfected or transformed with the vectors described above, they can be cultured under suitable conditions to express monoclonal antibodies, which can then be isolated to obtain purified antibodies.

[0029] In one specific embodiment of the present invention, the starting vector of the recombinant vector is the mammalian expression vector pBR322, and the host cell is human kidney epithelial cells (293F cells).

[0030] Fifthly, the present invention provides a method for preparing the anti-human CD68 monoclonal antibody described in the first aspect, the method comprising:

[0031] The nucleic acid molecule encoding the anti-human CD68 monoclonal antibody is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and purified to obtain the anti-human CD68 monoclonal antibody.

[0032] It is understood that the sequence of the anti-human CD68 monoclonal antibody developed based on the present invention can be used for recombinant expression vector construction, cell culture and antibody isolation and purification using common techniques in the field, without any special restrictions.

[0033] In a sixth aspect, the present invention provides the use of the anti-human CD68 monoclonal antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the host cell described in the fourth aspect in the preparation of formulations targeting human CD68.

[0034] The anti-human CD68 monoclonal antibody developed in this invention has high specificity and affinity, as well as good anti-interference ability, and can be used to prepare formulations targeting human CD68, including immunoassay agents, drugs, etc.

[0035] In a seventh aspect, the present invention provides an antibody conjugate comprising the anti-human CD68 monoclonal antibody described in the first aspect and a conjugate thereof.

[0036] Preferably, the coupling substance includes at least one of cytotoxin, radioactive isotope, luminescent material, chromogenic substance or enzyme.

[0037] Eighthly, the present invention provides a human CD68 immunoassay kit, the kit comprising the anti-human CD68 monoclonal antibody described in the first aspect and / or the antibody conjugate described in the seventh aspect.

[0038] In immunoassay, the sample to be tested is contacted with an anti-human CD68 monoclonal antibody, which is then detected. In some embodiments, the anti-human CD68 monoclonal antibody can be conjugated to a detectable label, and qualitative or quantitative detection of CD68 is achieved by analyzing changes in a recognizable signal generated by the detectable label. In other embodiments, the anti-human CD68 monoclonal antibody (primary antibody or capture antibody) is not labeled, but a detectable label is conjugated to a secondary antibody (detection antibody) or other molecules that can bind to the monoclonal antibody. For example, if the anti-human CD68 monoclonal antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby generating a change in a recognizable signal by specifically binding the primary antibody to the secondary antibody conjugated with the detectable label.

[0039] The detectable markers used to generate identifiable signal changes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, chemifluorescent groups, fluorescent proteins, enzymes (such as horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0040] Optionally, the immunoassay methods include, but are not limited to: enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB), flow cytometry (FCM), etc. Therefore, the kit can be an enzyme immunoassay kit, an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit. In a preferred embodiment, the kit is an immunoblotting kit or an immunohistochemistry kit.

[0041] Preferably, the immune test sample includes serum, cells, or tissues, wherein the tissues include human pituitary tissue, human liver tissue, or mouse pituitary tissue.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] This invention uses amino acid fragments from positions 124 to 135 and 146 to 158 of the C-terminus of human CD68 protein as antigens. High-purity polypeptides are synthesized and used as immunogens for immunization. Subsequently, based on monoclonal antibody development technology using single B lymphocyte screening and culture, a rabbit monoclonal antibody against human CD68 is obtained. This antibody exhibits excellent performance, accurately distinguishing all positive and negative tissue samples by immunohistochemistry. It stains accurately in positive tissue samples and shows no non-specific staining in either negative or positive tissue samples, demonstrating high detection sensitivity and specificity. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the structure of a monoclonal antibody expression vector;

[0045] Figure 2 The image shows the results of detecting the recognition specificity of rabbit monoclonal antibodies against CD68 in cell samples using the Western blotting method.

[0046] Figure 3 This image shows the immunohistochemical detection results of rabbit monoclonal antibodies against human CD68 and tissue section samples. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0049] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0050] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0051] The terms “comprising,” “including,” “containing,” “having,” and similar words are non-restrictive and can include other steps and components that do not affect the result. The term “and / or” is considered a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is considered to include (i) A, (ii) B, and (iii) A and B.

[0052] The terms "monoclonal antibody," "monoclonal antibody," and "antibody" have the same meaning and can be used interchangeably, all referring to rabbit monoclonal antibodies that specifically bind to CD68. The modifier "rabbit" indicates that the antibody's complementarity-determining region (CDR) is derived from the rabbit monoclonal antibody sequence.

[0053] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be classified into two types: κ chains and λ chains; heavy chains can be classified into five types: μ, δ, γ, α, and ε chains, with antibodies defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of both the heavy and light chains vary considerably, while the amino acid sequences of other parts are relatively constant. The regions with significant amino acid sequence variation near the N-terminus in both the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable regions of the heavy chain (VH) and light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable region, also known as the complementarity-determining region (CDR), is a ring structure. Heavy chain CDRs and light chain CDRs are tightly joined together by the FR region and cooperate to form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the antibody's specificity and serving as the site for antibody recognition and antigen binding. The FR regions are the more conserved parts of the VH and VL, generally exhibiting a β-sheet configuration, linked by three CDRs forming a connecting loop. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0054] CDRs can be identified according to Kabat definitions, Chothia definitions, the sum of Kabat and Chothia definitions, AbM definitions, contact definitions, IMGT unique numbering definitions and / or conformational definitions, or any CDR determination method well known in the art. As used in this invention, CDRs are defined by the Kabat numbering system.

[0055] The term "antigen-binding fragment (Fab)" refers to the region of an antibody molecule that can bind to an antigen. It consists of a complete light chain (variable and constant regions) and a portion of a heavy chain (a variable region and a constant region fragment). Fragments such as Fab, F(ab')2, and Fab' can be obtained by protease cleavage of a full-length antibody. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab contains both an antigen-binding region and a portion of a constant region, it not only possesses the same antibody-antigen affinity and excellent tissue penetration as single-chain antibodies (scFv), but also has a more stable structure, making it widely used in clinical diagnosis and treatment.

[0056] The term "variable fragment (Fv)" is located at the N-terminus of the antibody Fab fragment. It contains only the variable region and consists of a variable region of one light chain and one heavy chain. It is a non-covalently bound dimer of VH and VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0057] The term "single-chain variable fragment (scFv)" refers to the smallest antibody fragment formed by the heavy chain variable region (VH) and the light chain variable region (VL) linked by a flexible linker (typically composed of 10-25 amino acids). It retains the original antibody's specificity for binding to the antigen. In this invention, the linker is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked to its two ends. Compared to full-length antibodies, scFvs have a smaller molecular weight, thus exhibiting higher penetration and lower immune side effects.

[0058] The full-length sequence of the antibody or antibody fragment of the present invention can be derived from a single species, such as rabbit, or it can be a chimeric or humanized antibody to reduce the body's rejection response while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a specific source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody containing the CDR region of a non-human antibody, such as a rabbit antibody, and a FR region derived from a human antibody. In some cases, the variable region of the non-human antibody binds to the constant region of a human antibody, such as in human-rabbit chimeric antibodies; in other cases, the CDR region of the non-human antibody binds to both the FR region and the constant region derived from a human antibody sequence, i.e., grafting the CDR region of the non-human antibody onto a human antibody frame (FR) sequence derived from one or more other human antibody variable region frame sequences. In the present invention, the CDR region in the chimeric or humanized antibody is derived from a rabbit CDR region.

[0059] The term "monoclonal antibody" refers to a homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for the possibility of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigen or epitope. The term "monoclonal" indicates that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as limiting the antibody's structure, origin, or method of preparation. In some embodiments, monoclonal antibodies are prepared using hybridoma, phage display, yeast display, recombinant DNA, single-cell screening, or single-cell sequencing methods.

[0060] The term “specific binding” is a well-known term in the art. A molecule exhibits “specific binding” if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity for a particular target antigen or epitope than it reacts with other target antigens or epitopes. “Specific binding”, or “preferred binding”, does not necessarily require (although may include) exclusive binding.

[0061] Example 1

[0062] This embodiment describes the preparation of rabbit polyclonal antibodies.

[0063] The amino acid sequences of the human CD68 protein synthesized by Sangon Biotech from positions 124 to 135 and from positions 146 to 158 are: TSNSTATSPGFT-C; SPSPSPTSKETI-C.

[0064] The two CD68 peptides were conjugated to keyhole hemocyanin (KLH) with thiol groups. The specific steps are as follows:

[0065] (1) Weigh 6 mg of KLH (purchased from Merck) on an analytical balance, dissolve it in 1×PBS, maintain the concentration at 3 mg / mL, and shake well; weigh 5 mg of SMCC (purchased from Thermo) on an analytical balance, dissolve it in 1×PBS in a 55°C water bath, maintain the concentration at 5 mg / mL, and shake well.

[0066] (2) Take 180 μL of SMCC and add it to the dissolved KLH. At room temperature, place it on a rotating incubator, mix and shake well for 1 h. White flocculent matter will appear.

[0067] (3) Dialyze with 1×PBS at pH 7.4 at a volume ratio of V(PBS):V(KLH) = 15:1 for 1 h to remove free SMCC.

[0068] (4) Take 12 mg of the peptide, dissolve it in 1×PBS to maintain a concentration of 10 mg / mL, and slowly add it to the activated KLH (dialyzed) in several portions. Place it in a small rotary culture device, at the lowest speed, and incubate overnight at 4°C.

[0069] (5) Animal Immunization: Four New Zealand white rabbits were immunized with a human CD68 peptide conjugated with thiol-KLH as the immunogen. Each rabbit was immunized with 700 μg of immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits. After the first immunization, every 3 weeks, 350 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits for four booster immunizations. After five immunizations, rabbit serum samples were collected, and the titer against human CD68 protein was determined by ELISA. The serum was diluted 1:243000 and the titer was determined by ELISA. 450nm For rabbits with a growth rate exceeding 0.2, administer a booster immunization via subcutaneous injection of 400 μg of immunogen at multiple sites, and harvest the spleen three days later.

[0070] Example 2

[0071] This embodiment describes the preparation of rabbit monoclonal antibodies.

[0072] (1) Isolation of B lymphocytes from rabbit spleen and sorting of B lymphocytes

[0073] Isolation of spleen cells: Under aseptic conditions in a biosafety cabinet, remove a culture dish, add 35 mL of basal culture medium, place a cell sieve inside, remove the spleen and place it in the cell sieve. Remove excess connective tissue and fat from the rabbit spleen tissue, mince the spleen tissue, and grind it in the cell sieve. Take a clean grinding stick and use the pressing end to grind the tissue. The intracellular cells will slowly disperse and, after passing through the cell sieve, will be suspended in the culture dish solution. Wash the cell sieve with 10 mL of basal culture medium and collect the basal culture medium outside the cell sieve. Centrifuge at 400×g for 5 min at room temperature, discard the supernatant, retain the cells, add 13 mL of room temperature RBC lysis buffer (purchased from BioGems, catalog number 64010-00-100), gently disperse the cell clumps with a pipette and time for 1 min to perform erythrocyte lysis, add 37 mL of basal culture medium, mix well, stop erythrocyte lysis, centrifuge at 400×g for 5 min at room temperature, discard the supernatant, retain the cells, add 40 mL of room temperature basal culture medium, gently disperse the cell clumps with a pipette to resuspend the cells, complete the first wash, centrifuge at 400×g for 5 min at room temperature, discard the supernatant, retain the cells, add 20 mL of B cell complete culture medium, gently disperse the cell clumps with a pipette to resuspend the cells; filter the resuspended cells again through a cell sieve to remove clumped cells, and then count the cells.

[0074] B lymphocytes were isolated from the spleen using conventional methods, and then antigen-specific B lymphocytes were obtained by sorting. The relevant methods can be found in "A method for efficient isolation of single antigen-specific B lymphocytes from spleen cells (CN110016462A)" and "An in vitro culture system for B lymphocytes and its application (CN111518765A)".

[0075] (2) Cloning of the gene encoding rabbit monoclonal antibody

[0076] Positive clones were identified in the supernatant of cultured B cells using an antigen-coated ELISA. Cells from positive clones were collected, lysed, and RNA was extracted using the Quick-RNA™ MicroPrep kit (ZYMO, catalog number R1100-250) and reverse transcribed into cDNA. Using cDNA as a template, PCR was employed to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of naturally paired rabbit monoclonal antibodies from the corresponding positive clones' cDNA. The PCR reaction mixture consisted of: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×GloriaHiFi (ABclonal, catalog number RK20717), and 6.5 μL H2O.

[0077] The primer pair for the light chain variable region is as follows:

[0078] VL-Primer-F:

[0079] 5'-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3' (SEQ ID NO: 15);

[0080] VL-Primer-R:

[0081] 5'-cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG-3' (SEQ ID NO: 16).

[0082] The heavy chain variable region primer pair is:

[0083] VH-Primer-F:

[0084] 5'-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3' (SEQ ID NO: 17);

[0085] VH-Primer-R:

[0086] 5'-gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG-3' (SEQ ID NO: 18).

[0087] PCR amplification program: pre-denaturation at 98℃ for 30s, followed by 40 cycles at 98℃ for 10s, 64℃ for 30s, and 72℃ for 30s, and finally holding at 72℃ for 5min. The resulting reaction solution was stored at 4℃.

[0088] (3) Production and purification of rabbit monoclonal antibodies

[0089] After purifying the heavy and light chain variable region genes of the obtained rabbit monoclonal antibody, they were loaded into the expression vector pBR322, with the heavy and light chain expression vectors being pBR322 OSLIC Hc and pBR322 OSLIC Lc, respectively (see schematic diagram of plasmid structure). Figure 1 As shown in the figure, pBR322Ori and f1Ori are the replication start sites in E. coli, Ampcillin is the plasmid resistance gene, CMV Promoter is the transcription promoter, SV40 PAterminator is the tailing signal, Heavy chain constant is the nucleic acid sequence of the heavy chain constant region, and Light chain constant is the nucleic acid sequence of the light chain constant region. The constant region sequences were obtained by querying the IMGT online database (www.imgt.org).

[0090] The construction process of rabbit monoclonal antibody expression vectors carrying light and heavy chain genes is as follows: The mammalian cell expression vector pBR322 containing rabbit monoclonal antibodies CH and CL was linearized using NheI and XbaI restriction endonucleases, respectively. Homologous recombination was then used to construct the VL and VH genes, which encode signal peptides, into the NheI (949 bp) and XbaI (955 bp) sites of the pBR322 expression vector, respectively. After sequencing verification, the expression vectors containing the corresponding rabbit monoclonal antibody light and heavy chain genes were transfected into 293F cells. After transfection and culturing for 80 h, a supernatant containing the recombinant rabbit monoclonal antibody recognizing human CD68 protein was obtained. The rabbit monoclonal antibody recognizing human CD68 was then purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100). The purification protocol was followed according to the Protein A affinity gel resin instruction manual and will not be described further here. The antibody purity was verified to be ≥95% by 12% SDS-PAGE gel electrophoresis, with a concentration of 1 mg / mL. The purified antibody was aliquoted and stored at -20°C for later use.

[0091] The signal peptide in this embodiment can be an antibody expression signal peptide sequence commonly used in the art, such as "rabbit monoclonal antibody against human interferon α2 and its application (CN116063487A)", with the signal peptide "MDTRAPTQLLGLLLLWLPGATF" upstream of the light chain variable region and the signal peptide "METGLRWLLLVAVLKGVQC" upstream of the heavy chain variable region.

[0092] The amino acid sequence of the rabbit monoclonal antibody against human CD68 obtained in this embodiment is shown below. For ease 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.

[0093] The selected light chain gene sequence of the rabbit monoclonal antibody is as follows:

[0094] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGTTTACTCTTGTTATGGCTTCCAGGAGCCACCTTTGCCCAGGTGTTGACACAAACGCCATCCCCCGTCTCTGCCCCAGTTGGGGGCACTGTGACCATTAATTGTCAAGCTAGCCAGAGTGTCTATGTGAACAAGAACCTGGCCTGGTTCCAACAAAAGCCCGGCCAGCCTCCCAAGCTCCTCATCTACACAGCCTCTACACTGGCGTCAGGCGTCAGCTCTCGCTTTCAGGGTAGTGGATCTGGGACAGAGTTTACCCTCACCATCTCTGACGTCCAGTGTGACGATGCCGCAACATACTATTGCCTGGGTGAATACTGTTGGGATAATGACTGTAACGTCTTCGGCGGAGGTACCGAGGTCGTTGTGAAGGGAGACCCAGTTGCCCCCACAGTGCTCATCTTCCCCCCCGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG(SEQ ID NO.11).

[0095] The variable region gene sequence of the light chain of the selected rabbit monoclonal antibody is as follows:

[0096] GCCCAGGTGTTGACACAAACGCCATCCCCCGTCTCTGCCCCAGTTGGGGGCACTGTGACCATTAATTGTCAAGCTAGCCAGAGTGTCTATGTGAACAAGAACCTGGCCTGGTTCCAACAAAAGCCCGGCCAGCCTCCCAAGCTCCTCATCTACACAGCCTCTACACTGGCGTCAGGCGTCAGCTCTCGCTTTCAGGGTAGTGGATCTGGGACAGAGTTTACCCTCACCATCTCTGACGTCCAGTGTGACGATGCCGCAACATACTATTGCCTGGGTGAATACTGTTGGGATAATGACTGTAACGTCTTCGGCGGAGGTACCGAGGTCGTTGTGAAG(SEQ ID NO.12).

[0097] The heavy chain gene sequence of the selected rabbit monoclonal antibody is:

[0098]

[0099] The selected heavy chain variable region gene sequence of the rabbit monoclonal antibody is as follows:

[0100] CAGTCTGTAGAAGAAAGCGGGGGGCGGCTGGTGACACCCGGCACCCCTCTTACCCTCACGTGTACTGTGAGCGGCTTCTCCCTCAGCGATTACTACATGTCCTGGGTTCGCCAGGCGCCAGGCGAGGGCCTGAAATGGATCGGAGTTATTACATATCATGGCAAAACATCCTAC GCCCGCTGGACTAGAGGACGGTTTACAATCAGTAAGACTAGCACAACAGTAGATCTCAAAATGACCGGCCTCACAACAGAGGATACCGCTACCTACTTTTGTGTGGCGTGACGTACATAAGTCTGGGGCTGCTGACCCATGGGGCCCAGGCACCCTCGTGACAGTGAGTAGT(SEQ ID NO.14).

[0101] The sequences of the selected rabbit monoclonal antibodies are as follows:

[0102] Light chain amino acid sequence:

[0103] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAPVGGTVTINCQASQSVYVNKNLAWFQQKPGQPPKLLIYTASTLASGVSSRFQGSGSGTEFTLTISDVQCDDAATYYCLGEYCWDND CNVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.10).

[0104] Heavy chain amino acid sequence:

[0105] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSDYYMSWVRQAPGEGLKWIGVITYHGKTSYARWTRGRFTISKTSTTVDLKMTGLTTEDTATYFCVRDV HKSGAADPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVA PSTCSKPMCPPPELPGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKT ISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.9).

[0106] The amino acid sequence of the light chain variable region:

[0107] AQVLTQTPSPVSAPVGGTVTINCQASQSVYVNKNLAWFQQKPGQPPKLLIYTASTLAS GVSSRFQGSGSGTEFTLTISDVQCDDAATYYCLGEYCWDNDCNVFGGGTEVVVK (SEQ ID NO. 8).

[0108] The amino acid sequence of the heavy chain variable region:

[0109] QSVEESGGRLVTPGTPLTLTCTVSGFSLSDYYMSWVRQAPGEGLKWIGVITYHGKTSY ARWTRGRFTISKTSTTVDLKMTGLTTEDTATYFCVRDVHKSGAADPWGPGTLVTVSS (SEQ ID NO. 7).

[0110] The amino acid sequence of the light chain complementarity-determining region LCDR1:

[0111] QSVYVNKNLAW (SEQ ID NO.4).

[0112] The amino acid sequence of the light chain complementarity-determining region LCDR2:

[0113] LIYTASTLASGV (SEQ ID NO.5).

[0114] The amino acid sequence of the light chain complementarity-determining region LCDR3:

[0115] LGEYCWDNDCNVF(SEQ ID NO.6).

[0116] The amino acid sequence of the heavy chain complementarity-determining region HCDR1:

[0117] FSLSDYYMS (SEQ ID NO.1).

[0118] The amino acid sequence of the heavy chain complementarity-determining region HCDR2:

[0119] WIGVITYHGKTSYARWTR (SEQ ID NO. 2).

[0120] The amino acid sequence of the heavy chain complementarity-determining region HCDR3:

[0121] YFCVRDVHKSGAADP (SEQ ID NO. 3).

[0122] The rabbit monoclonal antibody described above has a light chain constant region of the κ chain and a heavy chain constant region of the IgG1 type.

[0123] Example 3

[0124] This embodiment studies the reaction specificity and application of rabbit monoclonal antibodies.

[0125] (1) Immunoblot analysis of rabbit monoclonal antibodies (Western blot, WB)

[0126] Cell samples with high CD68 expression, including THP-1, U-397, Mouse spleen, and Rat lung, were collected. The recognition specificity of monoclonal antibodies was detected by immunoblotting and followed by 12% polyacrylamide gel electrophoresis.

[0127] (1-1) Electrophoresis: First, prepare the sample and then load it. The sample verification loading volume is 15 μL, and the internal control loading volume is 5 μL (concentration of 3) or 3 μL (concentration of 5). Next, prepare the Mona PE60201 MiniMP4 mini vertical electrophoresis tank, use SWE rapid high-resolution electrophoresis buffer (Servicebio) at a constant voltage of 200V, use Tris-Glycine-SDS-Buffer conventional electrophoresis solution (Monad), set the separating gel to 120V and the stacking gel to 80V, and start electrophoresis. When the bromophenol blue electrophoresis reaches the bottom of the gel, stop the electrophoresis and turn off the power of the electrophoresis apparatus.

[0128] (1-2) Transfer: Cut the NC membrane to a size of 4cm×7.5cm and transfer the gel protein bands onto the PVDF membrane using the conventional method in the electrotransfer system.

[0129] (1-3) Ponceau S staining: Immerse the marked membrane in Ponceau S staining solution and shake for 4 minutes or longer until clear bands appear; place the membrane on white filter paper and take pictures of each membrane in turn. The pictures should show clear bands and occupy 3 / 4 of the entire picture and be centered; rinse with TBST 3 times for 4 minutes each time to remove Ponceau S.

[0130] (1-4) Blocking: Using TBST as a buffer system, prepare skim milk with a mass-volume ratio of 3%, place it on a magnetic stirrer and stir for 30 minutes before use; place the membrane in a suitable antibody incubation tank, add blocking incubation solution and incubate at room temperature for 1 hour.

[0131] (1-5) Primary antibody incubation: Using TBST as a buffer system, prepare skim milk with a mass-volume ratio of 3%, place it on a magnetic stirrer and stir for 30 minutes before use; place the membrane in a suitable antibody incubation tank, add blocking incubation solution and incubate at room temperature for 1 hour; rinse 3 times with TBST.

[0132] (1-6) Secondary antibody incubation: After determining the species source of the primary antibody, select the appropriate secondary antibody for dilution at a ratio of 1:5000. The diluent is TBST. Add the secondary antibody working solution to the incubation tank, place it on a shaker, and incubate at 20 rpm for 1 hour at room temperature; rinse 4 times with TBST.

[0133] (1-7) Exposure: Add ECL developer, develop, and see results below. Figure 2 .

[0134] The theoretical molecular weight of CD68 is 32-37 kDa. However, due to the high degree of glycosylation modification in the CD68 protein, the actual detected band size is between 110-150 kDa, which differs from the theoretical value of 32-37 kDa. Figure 2 It can be seen that the CD68 rabbit monoclonal antibody obtained through screening has good specificity, and a single target band was detected.

[0135] (2) Immunohistochemical analysis of rabbit monoclonal antibodies (IHC)

[0136] (2-1) Sample preparation and slide baking: Place the paraffin slides on the slide rack with the same orientation and bake them in a 56°C constant temperature oven for 30 minutes; at the same time, put the dewaxing solution tank 1 into the 56°C constant temperature oven, put the paraffin slides and slide rack into the dewaxing solution tank 1, and then take them out of the constant temperature oven and place them at room temperature for 5 minutes. After that, take out the slides and immerse them in the room temperature dewaxing solution tank 2. Then, put the paraffin slides into the tanks in the following order: dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, anhydrous ethanol 3. Each dewaxing solution tank is used for 5 minutes, and each anhydrous ethanol tank is used for 3 minutes. Rinse the slides with running water for 3 minutes. The aforementioned dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.

[0137] (2-2) Antigen retrieval: High-pressure thermal retrieval using 0.01M Tris-EDTA retrieval solution (pH 9.0);

[0138] (2-3) Inactivation of endogenous peroxidase: Wash the slices three times with PBS buffer for 1 min each time to remove the buffer; immerse the slices completely in 3% hydrogen peroxide solution and incubate at room temperature for 10 min;

[0139] (2-4) Blocking: Wash the slides three times with PBS buffer for 3 minutes each time to remove the buffer solution; use an immunohistochemistry pen to circle the area of ​​tissue to be tested on the slide, and add PBS blocking solution to the circled area; place the slides horizontally in an incubation humidifier with water at the bottom, and incubate at room temperature for 30 minutes, starting from the time when the blocking solution is added;

[0140] (2-5) Primary antibody incubation: Remove the blocking solution, drop rabbit monoclonal antibody diluted with antibody dilution buffer-PBS working solution (primary antibody dilution ratio 1:900) onto the tissue section, place it horizontally in an incubation humidifier, and incubate at room temperature for 60 min; remove the antibody working solution, rinse once quickly with PBS buffer, and soak and wash three times with PBS buffer for 3 min each time; during soaking and washing, repeatedly lift up and down several times;

[0141] (2-6) Secondary antibody incubation: After adding ready-to-use secondary antibody working solution (purchased from DAKO, catalog number K5007) to the tissue section, place it horizontally in an incubation humidifier and incubate at room temperature for 25 min; remove the secondary antibody working solution from the section, rinse quickly once with PBS buffer, and soak and wash three times with PBS buffer for 3 min each time; during soaking and washing, repeatedly lift up and down several times;

[0142] (2-7) Color development: Add the color development working solution to the slide and closely observe the color change under the microscope. After obtaining the appropriate staining intensity, immerse the slide in a large amount of distilled water to stop the color development. After stopping the color development, rinse the slide in running water for 10 minutes.

[0143] (2-8) Counterstaining: Immerse the slightly drained tissue sections in Mayer's hematoxylin for 1 min, then rinse with running water for 3 min.

[0144] (2-9) Blueing: Immerse the slightly drained slices in a saturated lithium carbonate solution for 3 seconds to blue them, and then rinse with running water for 3 minutes.

[0145] (2-10) Dehydration: Immerse the cleaned slices in anhydrous ethanol once, lifting them up and down several times during the immersion period, and remove them after 10 seconds; place them in a constant temperature drying oven at a high temperature (56℃) for complete drying.

[0146] (2-11) Mounting: Add an appropriate amount of neutral resin to the center of the slide and cover it with a coverslip. The amount of resin should be appropriate. After covering with a coverslip, the tissue should be completely covered and there should be no resin overflow.

[0147] (2-12) Slice scanning.

[0148] Immunohistochemical staining results are divided into positive and negative. A positive signal requires brown staining in specific tissues and cells with a low background. That is, cells or tissues that should theoretically show brown staining should have moderate staining, and cells and tissues that should not theoretically be stained should not be stained to be considered positive.

[0149] from Figure 3 As can be seen from the results, the anti-human CD68 rabbit monoclonal antibody prepared in this invention has accurate localization in human tonsil tissue, clear staining without non-specific staining, and presents a positive signal. It also presents a positive signal in human colon tissue, which is consistent with the theoretical localization and expectations. This further illustrates that the anti-human CD68 rabbit monoclonal antibody has high specificity and strong anti-interference ability of cellular components, which is conducive to improving the accuracy and reliability of detection and effectively avoiding false positive results.

[0150] Example 4

[0151] Based on the above tests, the rabbit monoclonal antibody against human CD68 of this invention exhibits excellent performance. Immunohistochemical detection accurately distinguishes between all positive and negative tissue samples, showing accurate staining in positive tissue samples and no non-specific staining in either negative or positive samples, demonstrating high detection sensitivity and specificity. Further development of human CD68 immunoassay kits and other products is possible.

[0152] In summary, this invention uses amino acid fragments from positions 124 to 135 and 146 to 158 of the C-terminus of the human CD68 protein as antigens. A highly pure polypeptide is synthesized and used as an immunogen for immunization. Subsequently, based on monoclonal antibody development technology using single B lymphocyte screening and culture, a rabbit monoclonal antibody against human CD68 is obtained. This antibody exhibits excellent performance, accurately distinguishing all positive and negative tissue samples by immunohistochemistry. It stains accurately in positive tissue samples and exhibits no non-specific staining in either negative or positive tissue samples. It demonstrates high sensitivity and specificity, and wide applicability, making it suitable for high-specificity, high-accuracy, and high-sensitivity detection of CD68, particularly by immunohistochemistry.

[0153] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-human CD68 monoclonal antibody, characterized in that, The amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the anti-human CD68 monoclonal antibody are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.

6.

2. The anti-human CD68 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human CD68 monoclonal antibody comprises the sequence shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region comprises the sequence shown as SEQ ID NO.

8.

3. The anti-human CD68 monoclonal antibody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain of the anti-human CD68 monoclonal antibody comprises the sequence shown as SEQ ID NO. 9, and the amino acid sequence of the light chain comprises the sequence shown as SEQ ID NO.

10.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human CD68 monoclonal antibody according to any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that, The host cell expresses the anti-human CD68 monoclonal antibody according to any one of claims 1-3.

7. A method for producing the anti-human CD68 monoclonal antibody according to any one of claims 1 to 3, characterized by, The preparation method comprises: The nucleic acid molecule encoding the anti-human CD68 monoclonal antibody is inserted into an expression vector to obtain a recombinant vector, the recombinant vector is introduced into a host cell, and the host cell is cultured and separated and purified to obtain the anti-human CD68 monoclonal antibody.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the anti-human CD68 monoclonal antibody according to any one of claims 1-3 and a conjugate substance coupled thereto. The conjugate substance comprises a luminescent substance or a chromogenic substance.

9. A human CD68 immunoassay kit characterized in that, The kit comprises the anti-human CD68 monoclonal antibody according to any one of claims 1-3 and / or the antibody conjugate according to claim 8.

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