CN118561999A - Anti-human granulocyte-macrophage colony stimulating factor antibody, antibody pair and application thereof - Google Patents

Abstract

The invention belongs to the technical field of antibodies, and particularly relates to an anti-human granulocyte-macrophage colony stimulating factor antibody, an antibody pair and application thereof. The antibody is a first antibody or a second antibody, the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are respectively shown in SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown in SEQ ID NO. 8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are shown as SEQ ID NO.13-15 respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NO.18-20 respectively. The invention provides the antibody with good specificity and high affinity to human GM-CSF, can effectively improve the accuracy, reliability and sensitivity of an immune detection result, and provides a more accurate and reliable antibody tool for detecting the GM-CSF protein level.

Description

Anti-human granulocyte-macrophage colony stimulating factor antibody, antibody pair and application thereof

Technical Field

The present invention relates to the field of antibody technology, and in particular to anti-human granulocyte-macrophage colony stimulating factor antibodies, antibody pairs and applications thereof.

Background Art

Granulocyte-macrophage colony stimulating factor (GM-CSF), also known as colony stimulating factor 2 (CSF-2), is a monomeric glycoprotein with pleiotropic cytokine functions, with a molecular weight of 30 kDa. It is secreted by various activated immune, mesenchymal and epithelial cell types and circulates in the form of monomers with variable glycosylation. Under physiological homeostasis, GM-CSF induces myeloid stem cells to produce granulocytes (neutrophils, eosinophils and basophils) and monocytes. Monocytes leave the circulation and migrate into tissues to mature into macrophages and dendritic cells. GM-CSF can also act as a chemoattractant for neutrophils and dendritic cells, stimulate the activation of mature granulocytes and macrophages, enhance the ability to phagocytize and kill bacteria, and thus enhance the body's immune defense function. In addition, it can promote the proliferation and differentiation of hematopoietic stem cells, increase the number of blood cells (including white blood cells, red blood cells and platelets), improve the survival and function of related cells, and play a role in tissue repair, angiogenesis and regulation of adaptive immune responses.

In many pathological conditions, the expression and activity of GM-CSF are abnormally elevated, which may lead to or aggravate inflammatory responses and autoimmune diseases. During the inflammatory response, GM-CSF is mainly produced by pathogenic T cells (mainly Th1). T-cell-derived GM-CSF mediates inflammation by stimulating antigen-presenting cells such as macrophages and dendritic cells to secrete IL-1 and IL-23, which in turn promote T cells to further secrete GM-CSF, thus forming a signal reinforcement loop. In addition, GM-CSF produced by T cells can also act as a "messenger" to promote macrophage polarization to the pro-inflammatory M1 phenotype to enhance the inflammatory response. Studies have shown that the pro-inflammatory effect of GM-CSF is involved in a series of inflammatory diseases. For example, in patients with rheumatoid arthritis (RA), the level of GM-CSF in the synovial fluid is significantly increased. GM-CSF promotes the differentiation of monocytes in synovial tissue into inflammatory dendritic cells, and induces Th1 cells to produce pro-inflammatory factors such as IL-17, promoting disease progression, and GM-CSF levels are related to the activity and severity of the disease. Therefore, evaluating GM-CSF concentration is of core value for understanding the development of inflammatory diseases, performing accurate diagnosis, evaluating the condition, and predicting patient prognosis.

In the serum of healthy individuals, the natural content of GM-CSF is extremely low, usually measured in pg/mL. This tiny baseline level increases the difficulty of measuring this indicator in biological samples. Therefore, it is extremely urgent and important to develop a method that can detect the content of GM-CSF in human serum with high sensitivity. At present, enzyme-linked immunosorbent assay, immunofluorescence assay, etc. are mostly used to determine the level of GM-CSF. Different methods require specific monoclonal antibodies against GM-CSF. Rabbit monoclonal antibodies have significant advantages in affinity, specificity, antigen epitope coverage, and cross-reactivity with human antigens, which successfully make up for the limitations of mouse monoclonal antibodies in immunoassay reagents. However, there are currently no high-performance antibodies against GM-CSF.

Summary of the invention

In view of the problems existing in the prior art, the present invention provides a rabbit monoclonal antibody with good specificity and high affinity for human GM-CSF protein, which can effectively improve the accuracy, reliability and sensitivity of the immunoassay results when used for immunoassay, and provides a more accurate and reliable antibody tool for scientific research, clinical diagnosis and treatment monitoring of GM-CSF protein levels. The present invention also provides the use of the aforementioned antibodies and antibody pairs in the preparation of human GM-CSF detection reagents or kits, and provides detection reagents or kits containing the aforementioned antibodies and antibody pairs. The present invention is achieved through the following technical solutions:

The first aspect of the present invention provides an anti-human granulocyte-macrophage colony stimulating factor antibody, selected from a first antibody or a second antibody, wherein: the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are respectively shown as SEQ ID NO.13-15, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.18-20.

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

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

Further, 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 .

The second aspect of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody or the second antibody as described above.

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

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

The third aspect of the present invention provides an anti-human granulocyte-macrophage colony stimulating factor antibody pair, which consists of the first antibody and the second antibody as described above.

The fourth aspect of the present invention provides use of the anti-human granulocyte-macrophage colony stimulating factor antibody or antibody pair as described above in the preparation of a human granulocyte-macrophage colony stimulating factor detection reagent or kit.

The fifth aspect of the present invention provides a human granulocyte-macrophage colony stimulating factor detection reagent or kit, comprising the anti-human granulocyte-macrophage colony stimulating factor antibody or antibody pair as described above.

Furthermore, the detection reagent or kit is a double antibody sandwich enzyme-linked immunosorbent assay reagent or kit, comprising a first antibody and a second antibody, wherein the first antibody serves as a capture antibody, and the second antibody modified with a detection marker serves as a detection antibody.

Compared with the prior art, the advantages and positive effects of the present invention are:

1. The first antibody and the second antibody of the present invention have ultra-high affinity, good specificity, and high specificity for human granulocyte-macrophage colony-stimulating factor (GM-CSF), and can effectively avoid false positive or false negative results when used in immunoassays, thereby improving the accuracy, reliability, and sensitivity of immunoassay results, and providing a more accurate and reliable antibody tool for scientific research, clinical diagnosis, and treatment monitoring of GM-CSF protein levels.

2. The first antibody and the second antibody of the present invention can recognize different antigenic epitopes of human GM-CSF protein. The double antibody sandwich enzyme-linked immunosorbent assay system is established by pairing the two, and the detection limit of human GM-CSF is as low as 0.01 pg/mL, which can achieve high-precision, high-specificity and high-sensitivity detection of human GM-CSF protein in the sample to be tested, which is of great significance in clinical diagnosis and scientific research applications and has good application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a vector map used to construct a rabbit monoclonal antibody expression vector in Example 1 of the present invention, which includes, from left to right, pBR322 vector maps carrying a light chain constant region and a heavy chain constant region;

FIG2 is a graph showing the affinity of monoclonal antibody 1G1 binding to human GM-CSF protein according to Example 2 of the present invention;

FIG3 is a graph showing the affinity of monoclonal antibody 8H9 binding to human GM-CSF protein in Example 2 of the present invention;

FIG4 is a standard curve for detecting human GM-CSF protein using a double antibody sandwich ELISA system established based on monoclonal antibodies 1G1 and 8H9 in Example 3 of the present invention;

FIG5 is a graph showing the specificity determination results of the double antibody sandwich ELISA system established based on rabbit monoclonal antibodies 1G1 and 8H9 in Example 4 of the present invention;

6 is a graph showing the results of thermal stability determination of the double antibody sandwich ELISA system established based on rabbit monoclonal antibodies 1G1 and 8H9 in Example 5 of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is 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.

Based on the information contained in the present invention, it is easy for those skilled in the art to 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 the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are all within the scope of the appended claims.

In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values used in the present invention to express the amount, percentage, etc. should be understood as 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 the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

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

The meaning of the terms "including", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the results can be added. The term "and/or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is regarded as including the following situations: (i) A, (ii) B and (iii) A and B. The terms "first", "second", etc. are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that such usage can be interchangeable under appropriate circumstances.

The terms "monoclonal antibody", "antibody" and "monoclonal antibody" and other similar words have the same meaning and can be used interchangeably. Unless otherwise specified, in the present invention, they all refer to rabbit antibodies that specifically bind to human granulocyte-macrophage colony stimulating factor (GM-CSF). The modifier "rabbit" indicates that the complementary determining region of the antibody is derived from a rabbit immunoglobulin sequence. The terms "human granulocyte-macrophage colony stimulating factor", "Human GM-CSF" and other similar words have the same meaning and can be used interchangeably.

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

A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, namely κ chain and λ chain; heavy chains can be classified into five types, namely μ, δ, γ, α and ε chains, and antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions near the N-terminus of the light and heavy chains where the amino acid sequences vary greatly are called variable regions (V), and the regions near the C-terminus where the amino acid sequences are relatively stable are called constant regions (C). The heavy chain variable region (VH) and the light chain variable region (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity determining regions (CDRs), which are ring structures. The heavy chain CDRs and light chain CDRs are closely together and cooperate with each other through the FR region, forming a surface that can complement the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. The FR region is the more conservative part of VH and VL. They are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop. Each VH and VL is usually composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the accumulation of both 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 known in the art. As used herein, the Kabat numbering system is used to define.

The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of antibodies. The length of CL of different types of Ig (κ or λ) is basically the same, but the length of CH of different classes of Ig is different, such as 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 heavy chain and light chain constant regions of antibodies are well known in the art.

A full-length antibody is the most complete antibody molecular structure and has 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.

An antibody fragment is one or more parts or fragments of a full-length antibody, which substantially retains the same biological function or activity as the full-length form. Specifically, an antibody fragment at least includes the same CDR region as the full-length antibody, and more preferably has the same variable region, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially 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.

(i) Fab: Antigen-binding fragment (Fab) is a monovalent fragment consisting 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 proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab') ₂ , and Fab' can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab') ₂ fragment and one pFc' fragment. The F(ab') ₂ fragment is further reduced to form two Fab' fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

(ii) F(ab) ₂ : A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

(iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). 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 to antigens, although the affinity is lower than that of the intact antibody.

(iv) (Fv) ₂ : It is composed of two covalently linked Fv fragments.

(v) scFv: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (generally composed of 10-25 amino acids), which retains the binding specificity of the original antibody to the antigen. The linker in the present invention is not particularly limited as long as it does not hinder the expression of the antibody variable regions connected to its two ends. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side effects.

(vi) sc(Fv) ₂ fragment is composed of two heavy chain variable regions and two light chain variable regions connected via a linker or the like.

The terms "monoclonal antibody" or "single antibody" and the like are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a small amount of mutations and/or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitopes on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population 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 hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

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

In order to make the above-mentioned objects and advantages of the present invention more obvious and understandable, the specific implementation methods of the present invention are described in detail below.

One embodiment of the present invention provides an anti-human GM-CSF antibody, selected from a first antibody or a second antibody, wherein the first antibody and the second antibody both comprise a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region both comprise three complementary determining regions (CDRs), respectively named CDR1, CDR2 and CDR3; 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, and 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 in 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 in the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.

The rabbit-derived antibody provided by the present invention has ultra-high affinity, good specificity, and high specificity for human granulocyte-macrophage colony stimulating factor (GM-CSF), can efficiently bind to human GM-CSF protein, and provides a more accurate and reliable antibody tool for scientific research, clinical diagnosis and treatment monitoring of GM-CSF protein. The use of the antibody of the present invention for immunodetection of GM-CSF protein in biological samples can significantly improve the accuracy, sensitivity, stability and reliability of the detection results, and reduce false negative or false positive results. Moreover, the two antibodies provided by the present invention recognize different antigenic epitopes on the surface of human GM-CSF protein, and a double antibody sandwich enzyme-linked immunosorbent (ELISA) detection system is constructed with the first antibody as the capture antibody and the second antibody as the detection antibody. The detection limit of human GM-CSF is as low as 0.01pg/mL, which can achieve high-precision, high-specificity and high-sensitivity detection of trace amounts of human GM-CSF protein in the sample to be tested, which is of great significance in clinical diagnosis and scientific research applications and has good application prospects.

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

Optionally, the first antibody and the second antibody further include a light chain constant region (CL) and a heavy chain constant region (CH), and each antibody CL and VL constitute a light chain (FL), and CH and VH constitute a heavy chain (FH). The constant region of an antibody can usually be obtained through public inquiries, such as: searching for rabbit IgG gamma C reign to obtain CH through the IMGT online database (www.imgt.org), and searching for rabbit IgG Kappa Creign to obtain CL. Specifically, 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.

Optionally, the first antibody and/or the second antibody is a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially maintains the same biological function or activity as the full-length form of the antibody. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab) ₂ , Fab', F(ab') ₂ , Fv, (Fv) ₂ , scFv and sc(Fv) _2. These antigen-binding regions can be obtained by conventional techniques in the art.

Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody and/or the second antibody as described above.

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

The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. The obtained nucleic acid molecule is inserted into an expression vector, then introduced into a host cell, and cultured under specific conditions to express the antibody.

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

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

The original vector for constructing the recombinant vector is various vectors conventional in the art, as long as it can hold the nucleic acid molecule. Typical vectors include plasmids (e.g., pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (e.g., 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 to a host and multiply in a 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 a 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.

The nucleic acid molecules encoding the antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into a vector, and each nucleic acid sequence is connected to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to different promoters, or the nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to a single promoter, so 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.

The recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth phase and treated with CaCl ₂ or MgCl ₂ ; they can also be transfected by microinjection, electroporation or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging or particle bombardment to achieve gene introduction.

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

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

Another embodiment of the present invention provides an anti-human GM-CS antibody pair, which consists of the first antibody and the second antibody as described above.

The first antibody and the second antibody of the present invention bind to different antigenic epitopes of human GM-CSF and are used to develop a double antibody sandwich enzyme-linked immunosorbent assay system, which has the advantages of high specificity, high detection sensitivity, strong anti-interference ability, and the like.

Another embodiment of the present invention provides use of the anti-human GM-CSF antibody or antibody pair described above in preparing a human GM-CSF detection reagent or kit.

The advantages of using the anti-human GM-CSF antibody or antibody pair in preparing a human GM-CSF detection reagent or kit are the same as the advantages of the anti-human GM-CSF antibody or antibody pair described above over the prior art, and will not be repeated here.

Based on the same inventive concept as above, an embodiment of the present invention further provides a human GM-CSF detection reagent or kit, wherein the detection reagent or kit comprises the first antibody and/or the second antibody as described above.

It should be emphasized that the first antibody and the second antibody can be used separately, together, or in pairs. When detecting, if used separately or together, the first antibody and/or the second antibody is used as a primary antibody or a capture antibody, the sample to be tested is contacted with the antibody of the present invention, and then the antibody is detected. In some embodiments, the first antibody and/or the second antibody can be coupled to a detection label, and the qualitative or quantitative detection of GM-CSF is achieved by analyzing the change in the identifiable signal generated by the detection label. In other embodiments, the anti-human GM-CSF antibody (as a primary antibody or a capture antibody) is not labeled, and the detection label is coupled to a secondary antibody (as a detection antibody) or other molecules that can bind to the primary antibody. For example, if the anti-human GM-CSF antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, thereby generating a change in the identifiable signal by coupling the detection label to the secondary antibody. If used in pairs, one of the first antibody and the second antibody is used as a primary antibody or a capture antibody, and the other is used as a secondary antibody or a detection antibody.

The above-mentioned detection method adopts common immunological methods, including but not limited to: enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB) and flow cytometry (FC). The detection object includes recombinantly expressed GM-CSF protein, cell-secreted GM-CSF protein or GM-CSF protein in human serum. The detection sample includes but is not limited to serum, plasma, urine or cell culture fluid.

Preferably, the detection reagent or kit is a double antibody sandwich enzyme-linked immunosorbent assay reagent or kit, comprising a first antibody and a second antibody, wherein the first antibody serves as a capture antibody (or primary antibody), and the second antibody modified with a detection marker serves as a detection antibody (or secondary antibody).

The above-mentioned detection labels for generating identifiable 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.

In a preferred embodiment, the detection label is biotin.

The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

Example 1 Preparation of anti-human GM-CSF rabbit monoclonal antibody

Human granulocyte-macrophage colony stimulating factor (GM-CSF) (from ABclonal, catalog number RP00094) was used as an antigen to immunize New Zealand white rabbits, and single antigen-specific B lymphocytes were sorted out. The antibody encoding gene in the cell was then amplified based on PCR technology, and recombinant expression was performed to obtain candidate antibodies. Finally, the target antibody 1G1 and 8H9 strains were screened by affinity testing and antigen epitope identification. The antibody sequencing work was completed by Jinkairui Biotechnology Co., Ltd. The amino acid (AA) and nucleotide (DNA) sequences of the antibodies are shown in Tables 1-2, respectively. In the table, LCDR1-3 represents the light chain complementary determining region CDR1-3, and HCDR1-3 represents the heavy chain complementary determining region CDR1-3.

Table 1 Sequence information of rabbit monoclonal antibody 1G1 in this example

Table 2 Sequence information of rabbit monoclonal antibody 8H9 in this example

The preparation method of rabbit monoclonal antibodies 1G1 and 8H9 specifically comprises the following steps:

1. Animal immunization: Each rabbit was immunized with 200 μg GM-CSF protein. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to make an emulsifier, which was injected subcutaneously at multiple points on the rabbit's abdomen and back; after the first immunization, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to make an emulsifier every 3 weeks, which was injected subcutaneously at multiple points on the rabbit's abdomen and back for two booster immunizations; after three immunizations, rabbit serum samples were collected, and the serum titer was determined by ELISA method. Rabbits with high titer were selected and booster immunized once with 200 μg of the immunogen at multiple points subcutaneously. Three days later, the animals were sacrificed and the spleen was taken.

2. Isolate spleen cells: Take out a culture dish in a safety cabinet under aseptic operation, add 30-40 mL of basal culture medium (RPMI Medium 1640 basic + 1% Pen Strep; RPMI 1640 purchased from Gibco, item number C11875500BT; PenStrep purchased from Gibco, item number 15140-163), put a cell sieve, take out the spleen and put it in the cell sieve, cut off the excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into pieces and put it into the cell sieve for grinding, take a clean grinding rod, and use the end of the pressing part to crush the tissue. The cells in the grinding membrane will slowly free themselves, pass through the cell sieve, and suspend 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 400g for 5min at room temperature, remove the supernatant, retain the cells, add 13mL of RBC lysis solution at room temperature (purchased from BioGems), gently blow away the cell clusters with a pipette and then time for 1min to lyse the red blood cells, add 37mL of basal culture medium and mix well to terminate the red blood cell lysis, centrifuge at 400g for 5min at room temperature, remove the supernatant, retain the cells, add 40mL of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, resuspend the cells, complete the first wash, centrifuge at 400g for 5min at room temperature, remove the supernatant, retain the cells, add 20mL of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, and resuspend the cells; filter the resuspended cells again through a cell sieve to remove the clumping cells, and then count the cells.

3. Isolate B lymphocytes from the spleen and sort B lymphocytes: Use conventional methods to isolate B lymphocytes from the spleen. For related methods, please refer to the patent "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication number: CN110016462A, publication date: 2019-07-16)" and the patent "A B lymphocyte in vitro culture system and application (publication number: CN111518765A, publication date: 2020-08-11)".

4. Cloning of the gene encoding rabbit monoclonal antibody: The cultured B lymphocyte supernatant was identified by GM-CSF-coated ELISA to obtain antigen-specific B lymphocytes; total RNA of the cells was extracted according to the instructions of the Quick-RNA ^TM Micro Prep Kit (purchased from ZYMO, catalog number R1051), and reverse transcribed into cDNA; using cDNA as a template, the naturally paired light chain variable region (VL) and heavy chain variable region (VH) of the rabbit monoclonal antibody were amplified from the cDNA of the corresponding positive clone by PCR and sequenced. The PCR reaction system included: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from ABclonal, catalog number RK20717) and 6.5 μL H ₂ O; the PCR amplification program included: 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min, and the resulting reaction solution was stored at 4°C. The primer sequences for amplifying VL and VH genes are shown in Table 3, where F and R represent forward primers and reverse primers, respectively.

Table 3 Primer sequence information for amplifying rabbit monoclonal antibodies in this example

Primer name Sequence information VL-F 5’-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3’ (see SEQ ID NO. 29) VL-R 5’-cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG-3’ (see SEQ ID NO. 30) VH-F 5’-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3’ (see SEQ ID NO. 31) VH-R 5’-gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG-3’ (see SEQ ID NO. 32)

5. Large-scale production and purification of rabbit monoclonal antibodies: The mammalian expression vector pBR322 loaded with the light chain constant region (CL) and heavy chain constant region (CH) genes was linearized with XbaI and NheI restriction enzymes respectively. After the VL and VH genes containing the signal peptide amplified by the above PCR were purified, they were constructed into the above expression vectors by homologous recombination to obtain light chain gene and heavy chain gene expression vectors. The expression vectors were successfully constructed by sequencing verification. The expression map of the vector used is shown in Figure 1. pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMVimmearly promoter is a transcription promoter, SV40 PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of the light chain constant region (left figure), and Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right figure). CL and CH genes were obtained by searching the IMGT online database (www.imgt.org), searching rabbit IgG gamma Creign to obtain CH, and searching rabbit IgG Kappa C reign to obtain CL.

The signal peptide of this embodiment can adopt the antibody expression signal peptide sequence commonly used in the art, such as the patent "Rabbit monoclonal antibody against human interferon α2 and its application (publication number: CN116063487A, publication date: 2023-05-05)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)" The light chain variable region upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGATF", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

The expression vectors containing the light chain gene and the heavy chain gene verified by sequencing were transfected into 293F cells together, and cultured for 72-96 hours after transfection to obtain the culture supernatant containing the recombinant rabbit monoclonal antibody that recognizes human GM-CSF. The target antibody was purified using protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100) and stored at -20°C for future use.

Example 2 Affinity test of rabbit monoclonal antibodies 1G1 and 8H9

The affinity of antibodies 1G1 and 8H9 was identified using the Gator biomolecular interaction analyzer from Probe Life, and the probe used was the Pro1 probe. The 1G1 and 8H9 antibody strains to be tested were immobilized on the Pro1 probe at a concentration of 3 μg/mL; then the human GM-CSF protein at two concentrations of 150 nM and 70 nM was used to bind the antibodies to be tested, and the affinity curve was obtained, as shown in Figure 2-3, where the ordinate represents the change in the thickness of the conjugate after the probe binds to the antibody and 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 curve. The affinity constants calculated by curve fitting are shown in Table 4.

Table 4 Affinity-related parameter determination results of rabbit monoclonal antibodies

Antibody K _off (1/s) K _on (1/Ms) K _D (M) 1G1 5.58×10 ^-4 6.79×10 ⁵ 8.22× ^10-10 8H9 1.78×10 ^-3 1.02×10 ⁶ 1.75× ^10-9

The dissociation coefficient K _off is used to characterize the constant of the dissociation rate between the antibody and the antigen, the binding coefficient K _on is used to characterize the constant of the binding rate between the antibody and its target, and the affinity constant K _D is the ratio of K _off /K _on , which represents the equilibrium dissociation constant between the antibody and its antigen. The results showed that the dissociation coefficients of antibodies 1G1 and 8H9 for human GM-CSF were 5.58×10 ^-4 and 1.78×10 ^-3 , respectively, and the binding coefficients were 6.79×10 ⁵ and 1.02×10 ⁶ , respectively, and the affinity constants were 8.22×10 ^-10 (M) and 1.75×10 ^-9 (M), indicating that the antibodies have a high affinity for human GM-CSF protein.

Example 3 Establishment of double antibody sandwich ELISA based on antibodies 1G1 and 8H9 and its sensitivity test

Biotin labeling of antibody 8H9: Mix antibody 8H9 and biotin (purchased from Uni-Land, catalog number: B5064) at a mass ratio of 10:1, place in a 2-8°C refrigerator for reaction for 16-20 hours before use.

The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method was established using antibody 1G1 as the capture antibody and biotin-labeled antibody 8H9 as the detection antibody. The steps are as follows:

1) Coating capture antibody 1G1: dilute antibody 1G1 to 4 μg/mL with 1× PBS, mix on a vortexer, add 100 μL/well to a 96-well microplate, cover with a cover film, and incubate in a refrigerator at 2-8°C for 16-20 hours;

2) Washing: After incubation, discard the liquid in the wells, wash the plate once with 1×PBST (1×PBS containing 0.05% Tween-20), add 300 μL of sample, let it stand for 40 seconds, then discard the liquid in the wells and pat dry on flat paper;

3) Blocking: Add 200 μL/well of blocking solution (1×PBS containing 2% BSA, 5% sucrose, 0.05% Tween 20 and 0.1% proclin 300, pH 7.2) into the plate wells, cover with cover film, block at 37°C for 2 h, discard the blocking solution after blocking, pat the ELISA plate dry, place in a 37°C oven to dry for 0.5-2 h, and take out for use;

4) Adding antigen protein: Human GM-CSF protein (purchased from RD, catalog number: 215-GM-010) was serially diluted with diluent (1×PBS containing 2% BSA, 0.05% Tween 20, 0.1% proclin 300, pH 7.2), with dilution concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0 pg/mL, and then different concentrations were added to the ELISA plate in sequence at 100 μL/well, covered with a cover film, and incubated at 37°C for 2 h;

5) Wash the plate: same as step 2);

6) Add detection antibody 8H9: dilute the biotin-labeled antibody 8H9 (8H9-biotin) to 0.1 μg/mL with diluent, add 100 μL/well to the ELISA plate, cover with cover film, and incubate at 37°C for 1 hour;

7) Washing the plate: same as step 2);

8) Add SA-HRP: dilute 100×SA-HRP (horseradish peroxidase labeled streptavidin, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number SA00001-0) concentrate) with diluent 100 times, add 100 μL/well to the ELISA plate, cover with cover film, and incubate at 37°C for 0.5 h;

9) Washing the plate: same as step 2);

10) Add colorimetric solution: Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution (purchased from Tetramethylbenzidine, catalog number 4ATMB1000) to the ELISA plate at 100 μL/well, cover with cover film, and incubate at 37°C for 15 min;

11) Reading: After incubation, remove the ELISA plate, add 50 μL of stop solution (1 mol/L hydrochloric acid) to each well, and immediately read the plate at OD _{450 nm} using an ELISA reader.

The standard curve was obtained by plotting the concentration of human GM-CSF protein as the horizontal axis and the absorbance value as the vertical axis (see Figure 4). The absorbance signal value of the sensitivity was the sum of the average absorbance value of the 16 dilution blank wells and 2 times the standard deviation. The absorbance signal value was substituted into the standard curve, and the concentration was back-calculated to obtain a sensitivity of 0.01 pg/mL for the antibody pair (see Table 5).

Table 5 Sensitivity test data of double antibody sandwich ELISA based on antibodies 1G1 and 8H9

Example 4 Specificity test of double antibody sandwich ELISA system based on antibodies 1G1 and 8H9

The cross-reactive test proteins are: Human G-CSF (from ABclonal, Catalog No. RP01722), Human M-CSF (from ABclonal, Catalog No.: RP01221), Human IL-3 (from ABclonal, Catalog No. RP01903), Human IL-4 (purchased from RD, Catalog No. DY204), Mouse IL-4 (purchased from RD, Catalog No. DY404), Human IL-5 (purchased from RD, Catalog No.: 205-IL-005) and Mouse IL-5 (purchased from RD, Catalog No.: DY405), dilution concentration 1000pg/mL; human GM-CSF protein is diluted in series, dilution concentrations: 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0pg/mL. The detection method is the same as in Example 3, and the results are shown in Figure 5. The results showed that only human GM-CSF could cause an increase in absorbance values, indicating that the double antibody sandwich ELISA system established based on antibodies 1G1 and 8H9 had no obvious cross-reaction to other proteins except the target protein. Antibodies 1G1 and 8H9 had high antigen recognition specificity and specifically recognized and bound human GM-CSF protein.

Example 5 Thermal stability test of double antibody sandwich ELISA system based on antibodies 1G1 and 8H9

The ELISA plate coated with capture antibody 1G1, freeze-dried human GM-CSF protein, and 100× concentrated detection antibody 8H9 were sealed and stored at 2-8°C and 37°C for 7 days, respectively, and then taken out and the absorbance value was tested according to the method of Example 3. The thermal stability of the double antibody sandwich ELISA system was compared based on the standard curves established by comparing the antibody samples treated at different temperatures, and the results are shown in Figure 6. The results show that the coefficient of variation CV of the standard curve of the antibody of the present invention under heat destruction at 37°C for 7 days and low temperature storage is 6.45%, and the detection system has good thermal stability.

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

Claims (10)

1. An anti-human granulocyte-macrophage colony-stimulating factor antibody, which is a primary antibody or a secondary antibody, wherein:

The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10;

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.

2. The anti-human granulocyte-macrophage colony stimulating factor antibody according to claim 1, wherein the amino acid sequence of the first antibody light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7;

The amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17.

3. The anti-human granulocyte-macrophage colony stimulating factor antibody according to claim 2, wherein the amino acid sequence of the first antibody light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6;

The amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 16.

4. The anti-human granulocyte-macrophage colony-stimulating factor antibody according to claim 1, wherein the first antibody and/or the second antibody is a full-length antibody or is an antigen-binding region of the full-length antibody;

The antigen binding region is selected from the group consisting of Fab, F (ab) ₂、Fab'、F(ab')₂、Fv、(Fv)₂, scFv, or sc (Fv) ₂.

5. A nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-human granulocyte-macrophage colony stimulating factor antibody of any one of claims 1-4, wherein the antibody is a first antibody or a second antibody.

6. The nucleic acid molecule of claim 5, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleotide sequence of the first antibody light chain variable region 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 as SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 28.

7. The nucleic acid molecule, recombinant vector comprising said nucleic acid molecule or host cell comprising said nucleic acid molecule of claim 6, wherein the nucleotide sequence of the light chain of said 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 as SEQ ID NO.25, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 27.

8. An anti-human granulocyte-macrophage colony-stimulating factor antibody pair, consisting of the first antibody of any one of claims 1-4 and the second antibody.

9. A human granulocyte-macrophage colony-stimulating factor detection reagent or kit comprising the anti-human granulocyte-macrophage colony-stimulating factor antibody of any one of claims 1-4 or the anti-human granulocyte-macrophage colony-stimulating factor antibody pair of claim 8.

10. The human granulocyte-macrophage colony-stimulating factor detection reagent or kit according to claim 9, wherein the detection reagent or kit is a double-antibody sandwich-method enzyme-linked immunosorbent assay reagent or kit, comprising a first antibody and a second antibody, wherein the first antibody is used as a capture antibody, and wherein the second antibody modified with a detection label is used as a detection antibody.