CN117362431A - Anti-mouse interleukin 10 rabbit monoclonal antibody and application thereof - Google Patents
Anti-mouse interleukin 10 rabbit monoclonal antibody and application thereof Download PDFInfo
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- CN117362431A CN117362431A CN202311080455.4A CN202311080455A CN117362431A CN 117362431 A CN117362431 A CN 117362431A CN 202311080455 A CN202311080455 A CN 202311080455A CN 117362431 A CN117362431 A CN 117362431A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody for resisting mouse interleukin 10 and application thereof. The rabbit monoclonal antibody comprises 2H4 and/or 3F5, wherein the amino acid sequences of light chain CDR1-3 of the rabbit monoclonal antibody 2H4 are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of heavy chain CDR1-3 are respectively shown as SEQ ID NO. 8-10; the amino acid sequences of the light chain CDR1-3 of the rabbit monoclonal antibody 3F5 are shown as SEQ ID NO.13-15 respectively, and the amino acid sequences of the heavy chain CDR1-3 are shown as SEQ ID NO.18-20 respectively. The rabbit monoclonal antibody 2H4 and/or 3F5 has high affinity and good specificity to mouse IL-10, and has the advantages of high accuracy, good reliability, excellent anti-interference capability, excellent detection sensitivity and the like when being used for immunodetection.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody resisting mouse interleukin 10 and application thereof.
Background
Interleukin 10 (IL-10), also known as cytokine synthesis inhibitor (cytokine synthesis inhibitory factor, CSIF), is a pleiotropic cytokine that is secreted primarily by antigen presenting cells, such as activated T cells (including primarily Th2 cells), monocytes, B cells and macrophages. Both the mouse and human IL-10 genes are located on chromosome 1, the genome of which comprises 5 exons and 4 introns. Human and mouse IL-10 have 81% and 73% homology at the DNA and amino acid levels, respectively, human IL-10 can act on cells of murine origin, whereas mouse IL-10 has no cross-reaction with human cells. IL-10 has bidirectional immunoregulatory effect, can play a role in immunosuppression or immunostimulation in various cell types, on one hand, IL-10 can weaken the function of Antigen Presenting Cells (APC) by reducing the expression of dendritic cells and major histocompatibility antigen II (MHC 2) molecules on the surface of macrophages, can also down regulate the activity of T lymphocytes, and can inhibit the activation, migration and adhesion of inflammatory cells, thus having negative regulatory effect on immune response in tumor environment; on the other hand, IL-10 has stimulatory effects on T, B lymphocytes, and IL-10 can also exert stimulatory effects in the tumor environment. The bidirectional regulation of IL-10 not only affects the immune system, but can also affect many pathophysiological processes including angiogenesis, tumor formation and infection by modulating growth factors, cytokines, and can also establish roles in peripheral tolerance by inducing regulatory T cells.
IL-10 abnormal expression such as over-expression and deficiency has pathophysiological significance, tumor growth, systemic Lupus Erythematosus (SLE), lymphoma, skin cancer are diseases in which IL-10 is over-expressed, and crohn's disease, psoriasis, inflammatory bowel disease, rheumatoid arthritis, asthma, organ transplantation reactions are diseases in which IL-10 is under-expressed; therefore, detection of IL-10 levels is of great instructive significance in the diagnosis and assessment of the progression of related diseases and in prognostic assays.
The development of anti-IL-10 antibodies with good specificity is the basis for realizing accurate and sensitive detection of IL-10. Currently, patents have disclosed the development of immunodetection antibodies for IL-10 proteins using immunogens that are predominantly human, avian or fish derived IL-10 recombinant proteins and no monoclonal antibodies are developed against murine IL-10 proteins. However, a considerable number of animal disease models related to IL-10 research are modeled with mouse species, so developing a monoclonal antibody against mouse interleukin 10 with excellent performance has very important significance and clinical use value for intensive study of the biological function of the protein and for detection of the expression level of IL-10 antigen in related diseases.
Disclosure of Invention
In view of the above problems of the prior art, the present invention provides a rabbit monoclonal antibody against mouse interleukin 10 and its use, which aim to solve some of the problems of the prior art or at least to alleviate some of the problems of the prior art.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
in a first aspect the invention provides a rabbit monoclonal antibody against mouse interleukin 10, comprising rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5, said rabbit monoclonal antibody comprising a light chain variable region and a heavy chain variable region, said light chain variable region and said heavy chain variable region each comprising 3 Complementarity Determining Regions (CDRs); wherein: the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 2H4 are respectively shown in SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 8-10; the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 3F5 are respectively shown in SEQ ID NO.13-15, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 18-20.
Further, the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody 2H4 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 rabbit monoclonal antibody 3F5 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.
Further, the rabbit monoclonal antibody also comprises a light chain constant region and a heavy chain constant region, wherein the light chain constant region of the rabbit monoclonal antibody 2H4 and the light chain constant region of the rabbit monoclonal antibody 3F5 are both kappa chains, and the heavy chain constant region is of an IgG1 type.
Further, the amino acid sequence of the light chain of the rabbit monoclonal antibody 2H4 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 rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 16.
In a second aspect the invention provides a nucleic acid molecule for encoding the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
Further, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody 2H4 is shown as SEQ ID NO.21, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 22; the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.23, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 24.
In a third aspect the invention provides a recombinant vector comprising a nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
In a fourth aspect the invention provides a host cell comprising a recombinant vector as described above or having integrated in its genome a nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
In a fifth aspect the invention provides a kit for the immunodetection of mouse interleukin 10, said kit comprising the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
Further, the immunodetection method includes one or more of an enzyme immunoassay, an enzyme-linked immunosorbent assay, an immunohistochemical method, an immunofluorescence method, an immunoblotting method and a flow cytometry.
Further, the immunodetection method is a double-antibody sandwich enzyme-linked immunosorbent method, in which the capture antibody is the rabbit monoclonal antibody 2H4, and the detection antibody is the rabbit monoclonal antibody 3F5 marked by a detectable marker.
Further, the immunodetection sample is one or more of a tissue sample expressing IL-10 protein, a cell line sample expressing IL-10 protein, a serum sample secreting IL-10 protein, and a recombinantly expressed IL-10 protein.
The invention has the advantages and positive effects that:
1. the rabbit monoclonal antibody 2H4 and/or the rabbit monoclonal antibody 3F5 provided by the invention has high affinity and good specificity to the mouse interleukin 10, has high accuracy, good reliability and excellent anti-interference capability when being used for immunodetection of the mouse IL-10 protein, and provides an effective antibody raw material for immunodetection of the distribution, expression level or biological function of the mouse IL-10 protein.
2. The rabbit monoclonal antibodies 2H4 and 3F5 provided by the invention recognize and specifically bind different antigen epitopes on the surface of the mouse IL-10 protein, do not interfere with each other, can be used as a pairing antibody of a double-antibody sandwich ELISA method, and can be used as a capture antibody by taking the rabbit monoclonal antibody 2H4 as a capture antibody, and the detection limit of the detection of the mouse IL-10 protein is 1.965pg/mL by taking the rabbit monoclonal antibody 3F5 as a detection antibody.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of a vector used for constructing a rabbit monoclonal antibody expression vector according to example 1 of the present invention, from left to right, respectively pRB322 vector map carrying light chain constant regions and heavy chain constant regions in advance;
FIG. 2 is a graph showing the affinity of the rabbit monoclonal antibody 2H4 of example 1 of the present invention for binding to mouse IL-10 protein;
FIG. 3 is a graph showing the affinity of the rabbit monoclonal antibody 3F5 of example 1 of the present invention for binding to mouse IL-10 protein;
FIG. 4 is a graph showing the epitope recognition of rabbit monoclonal antibodies 2H4 and 3F5 of example 1 of the present invention;
FIG. 5 is a standard graph of the double antibody sandwich ELISA method established based on rabbit monoclonal antibodies 2H4 and 3F5 in example 2 of the present invention;
FIG. 6 is a graph showing the results of specific measurement of the double antibody sandwich ELISA method established based on rabbit monoclonal antibodies 2H4 and 3F5 in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In addition, the terms "comprising," "including," "containing," "having," and the like are intended to be non-limiting, as other steps and other ingredients may be added that do not affect the result.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" will be considered to encompass the following: (i) A, (ii) B, and (iii) A and B.
In the context of the present invention, the terms "rabbit monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to the mouse interleukin 10 (IL-10) protein. The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" shall be taken to meanThe broadest interpretation and includes different antibody structures including but not limited to so-called full length antibodies, antibody fragments and their genetic or chemical modifications, provided they exhibit the desired antigen binding activity. Where "antibody fragment" refers to one or more portions or fragments of a full-length antibody, in typical examples, the antibody fragment comprises: fab, fab', F (ab) 2 、F(ab’) 2 、Fv、(Fv) 2 、scFv、sc(Fv) 2 。
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, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate to form a surface complementary to the three-dimensional structure of the antigen or epitope of interest, which determines the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs and FR can be identified according to Kabat definition, chothia definition, a combination of both Kabat definition and Chothia definition, abM definition, contact definition, IMGT unique number definition and/or conformational definition, or any CDR determination method known in the art. As used herein, is defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. 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 antibody heavy and light chain constant regions are well known in the art.
The terms "monoclonal antibody" or "mab" and the like are used interchangeably and refer to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. "monoclonal antibodies" are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as limiting the source or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method. "Rabbit monoclonal antibody" indicates that the antibody was produced by a rabbit.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a rabbit monoclonal antibody resisting mouse interleukin 10, which comprises a rabbit monoclonal antibody 2H4 and/or a rabbit monoclonal antibody 3F5, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs); wherein: the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 2H4 are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10; the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 3F5 are respectively shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20.
The invention uses the mouse interleukin 10 (IL-10) protein as immunogen, and utilizes single B cell screening and culturing technology to successfully develop the rabbit monoclonal antibody 2H4 and 3F5 resisting the mouse IL-10. The biological function of the antibody is that the space conformation between the Complementarity Determining Region (CDR) of the antibody and the antigenic determinant is complementary, the affinity and specificity of the antibody to the potential antigen are mostly determined by the length of the CDR sequence and the amino acid composition, the rabbit monoclonal antibodies 2H4 and 3F5 with the CDR sequence can specifically recognize the recombinant expressed mouse IL-10 protein and the natural IL-10 protein secreted by mouse serum or expressed on the tissue and cell surface, the recombinant expressed mouse IL-10 protein is taken as a detection object, and the affinity constants (K) of the rabbit monoclonal antibodies 2H4 and 3F5 D ) 1.38X10 respectively -10 (M) and 1.24X10 -10 (M) has high affinity, further, through cross experiments, the two are only specifically identified and combined with the mouse IL-10 protein, have no cross reaction to other interleukin series proteins and human IL-10 proteins similar to the mouse IL-10 protein, have high specificity, prove that the antibody has high accuracy, good reliability and excellent anti-interference capability in immunodetection of the mouse IL-10 protein, and provide effective antibody raw materials for immunodetection of the distribution, expression level or biological function of the mouse IL-10 protein. In addition, rabbit monoclonal antibodies 2H4 and 3F5 can recognize different epitopes on the surface of the mouse IL-10 protein, and based on the different epitopes, the mouse IL-10 protein can be developed The double-antibody sandwich ELISA adsorption method uses the rabbit monoclonal antibody 2H4 as a capture antibody, uses the rabbit monoclonal antibody 3F5 as a detection antibody, and adopts the double-antibody sandwich ELISA method to detect mouse IL-10, wherein the detection limit is 1.964752pg/mL, thus the method can stably detect the mouse IL-10 with trace level in serum, cells and tissue samples, has the advantages of good specificity, high detection sensitivity, strong stability and the like, and has important significance in clinical diagnosis and scientific research application.
Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the rabbit monoclonal antibody 2H4 is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 7. The amino acid sequence of the light chain variable region (VL) of the rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 17.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region and a heavy chain constant region, CL and VL comprising the complete light chain, CH and VH comprising the complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: through IMGT online database (www.imgt.org), rabbit source IgG gamma C reign is searched for CH and rabbit source IgG Kappa C reign is searched for CL.
Illustratively, the light chain constant regions of the rabbit monoclonal antibody 2H4 and the rabbit monoclonal antibody 3F5 are both kappa chains and the heavy chain constant regions are both of the IgG1 type.
Correspondingly, the amino acid sequence of the light chain (L chain) of the rabbit monoclonal antibody 2H4 is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 6. The amino acid sequence of the light chain (L chain) of the rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 16.
Yet another embodiment of the invention provides a nucleic acid molecule for encoding the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody.
Illustratively, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody 2H4 is shown as SEQ ID NO.21, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 22. The nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.23, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 24. It will be appreciated by those skilled in the art that nucleic acid molecules other than those exemplified above may likewise encode rabbit monoclonal antibodies 2H4 and 3F5 due to the degeneracy of the genetic code, and therefore the nucleic acid molecules exemplified above should not be taken as limiting the scope of the invention.
The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. Furthermore, for ease of purification, the coding sequences for the heavy and light chains and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Another embodiment of the invention provides a recombinant vector comprising a nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 as described above.
The recombinant vector may be constructed by ligating the nucleic acid molecule provided by the present invention to various vectors by a method conventional in the art. The vector is one which is capable of carrying the nucleic acid molecule. Commonly used vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and in the context of the present invention, vectors are used interchangeably with plasmids.
The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The cloning vector may comprise a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector comprises regulatory elements (e.g., promoters, enhancers) for expression in the specified host cell. The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecules of the invention. This is well known in the art and will not be described in detail herein.
Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, nucleic acid molecules encoding the heavy and light chains of a rabbit monoclonal antibody of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or alternatively, the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter described later depends on the type of host cell used to produce the antibody.
Transformation of host cells with recombinant vectors can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 By a method or MgCl 2 The method can also be microinjection, electroporation or liposome packaging. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome encapsulation, and the like.
In a further embodiment the invention provides a host cell comprising a recombinant vector as described above or having integrated in its genome a nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or the rabbit monoclonal antibody 3F5 as described above.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomycete, salmonella typhimurium, fungal cells such as yeast, insect cells of drosophila S2 or Sf9, CHO, COS7, 293 series cells, and the like. After obtaining a host cell transformed with the expression vector as described above, the cell is cultured under appropriate conditions to express the monoclonal antibody, and then isolated 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).
The embodiment of the invention also provides application of the rabbit monoclonal antibody against the mouse interleukin 10, the nucleic acid molecule, the recombinant vector and the host cell in preparation of a kit for immunodetection of the mouse interleukin 10.
The advantages of the application in preparing the kit for immunodetection of mouse interleukin 10 are the same as those of the rabbit monoclonal antibody against mouse interleukin 10 described above with respect to the prior art, and are not described in detail herein.
The rabbit monoclonal antibodies 2H4 and 3F5 of the invention can be used individually, or in pairs, or can be conjugated or coupled to a detectable label (for generating a detection signal), respectively. Detectable labels for generating a detection signal include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.
In the case of immunodetection, if 2H4 or 3F5 is used separately, the antibody 2H4 or 3F5 of the present invention is used as an antigen-binding antibody (capture antibody) which specifically recognizes and binds to IL-10 protein in a sample to be detected, and then a recognizable signal change is generated by a detectable label attached thereto, or a detection antibody such as IgG conjugated with a detectable label specifically binds to the antibody of the present invention to generate a recognizable signal change, thereby achieving qualitative or quantitative detection of mouse IL-10 protein.
In preferred embodiments, the immunoassay method includes, but is not limited to: enzyme immunoassay (Enzyme immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunosorbent assay (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), flow Cytometry (FCM), and the like.
Optionally, the immunodetection sample includes, but is not limited to: a tissue sample expressing an IL-10 protein, a cell line sample expressing an IL-10 protein, a serum sample secreting an IL-10 protein, and a recombinantly expressed IL-10 protein, wherein the IL-10 protein may be present in a native state in serum, cells, or tissues.
Based on the different antigen epitope recognition of the rabbit monoclonal antibodies 2H4 and 3F5, the immunodetection method is preferably an enzyme-linked immunosorbent assay, especially a double-antibody sandwich enzyme-linked immunosorbent assay, wherein in the double-antibody sandwich enzyme-linked immunosorbent assay, the capture antibody is the rabbit monoclonal antibody 2H4, and the detection antibody is the rabbit monoclonal antibody 3F5 marked by a detectable marker (such as biotin). The mouse IL-10 is detected by the rabbit monoclonal antibody 2H4 and 3F5 in a pairing way, so that the sensitivity and the specificity are high, and the detection limit is as low as 1.965pg/mL.
Based on the same inventive concept, the embodiment of the invention also provides a kit for immunodetection of mouse interleukin 10, which comprises the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5.
The advantages of the kit for immunodetection of mouse interleukin 10 are the same as those of the rabbit monoclonal antibody against mouse interleukin 10 described above with respect to the prior art, and are not described in detail herein.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
EXAMPLE 1 preparation of Rabbit monoclonal antibodies against mouse IL-10 protein
The immunogen used for preparing the mouse interleukin 10 (IL-10) rabbit monoclonal antibody is a recombinant mouse IL-10 mature protein (purchased from ABclonal, cat. No. RP 01465) with biological activity, which is obtained by expressing the immunogen in HEK293 cells from a mammalian expression system, the IL-10 protein sequence is referred to NCBI accession No. NP-034678.1, the mature protein expression region is Ser19-Ser178 (19-178 aa) of the sequence, the C terminal band is 6 XHis tag, the mature protein is in a monomer form, the theoretical molecular weight is 17.6kDa, and the size is 20-24kDa in the subsequent practical detection due to glycosylation modification. The antibody preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture, and specifically comprises the following steps:
1. Immunization of animals
2 New Zealand white rabbits were immunized with recombinant mouse IL-10 mature protein (purchased from ABclonal, cat. No. RP 01465) as an immunogen; each white rabbit was immunized 200. Mu.g, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma Co.) to prepare an emulsion before the first immunization, and injected subcutaneously in the abdomen and back of the rabbits at multiple points. 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare an emulsifier, which was subcutaneously injected at the abdomen and back of rabbits at multiple points to boost the immunization twice. After three immunizations, rabbit serum samples were collected, serum was taken at 1: after 243000 times dilution, the titer of the sample against mouse IL-10 was measured by ELISA, and OD was obtained 450nm Rabbits exceeding 0.2 were boosted subcutaneously with 200 μg immunogen at multiple points and spleens were taken three days later.
2. Isolation of B lymphocytes from spleen
Taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by rolling the tail end of the pressed part. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company), gently blowing off cell clusters by using a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
3. B lymphocyte sorting and culturing
See patent "method for efficiently isolating individual antigen-specific B lymphocytes from spleen cells (publication No. CN 110016462A)" and patent "an in vitro B lymphocyte culture system and use (publication No. CN 111518765A)".
4. Cloning of genes encoding Rabbit monoclonal antibodies
Positive clones capable of binding to mouse IL-10 protein were identified from the cultured B lymphocyte supernatants by antigen-coated ELISA. Positive clones were collected and lysed and then treated with Quick-RNA TM Micro Prep kit instructions (available from ZYMO under accession number R1051) extract RNA and reverse transcribe into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of the naturally paired rabbit monoclonal antibodies from the cDNA of the corresponding positive clone, and the sequence is determined by sequencing of Jin Kairui biotechnology limited company. The PCR reaction system is as follows: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2 XGloriaHiFi (available from Wuhan Aibolag Biotechnology Co., ltd., cat., product No. RK 20717) and 6.5. Mu. L H 2 O; PCR amplificationThe program comprises the following steps: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.
The nucleic acid sequences (5 '-3') of the forward primer and the reverse primer described above are as follows:
VL-F (see SEQ ID NO. 25): tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC;
VL-R (see SEQ ID NO. 26): cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG;
VH-F (see SEQ ID No. 27): tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG;
VH-R (see SEQ ID No. 28): gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG.
5. Production and purification of Rabbit monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies recognizing mouse IL-10 protein, the heavy chain genes and the light chain genes (with signal peptides at the upstream) of the plurality of rabbit monoclonal antibodies selected above are respectively loaded on an expression vector pBR322, the expression vector pBR322 used carries light chain and heavy chain constant regions in advance, the expression patterns of which are shown in FIG. 1, wherein pBR322Ori and f1Ori are replication initiation sites in E.coli, ampcillin is a plasmid resistance gene, CMV immearly promotor is a transcription promoter, SV40 PAtermate is a tailing signal, light chain constant is a nucleic acid sequence of the light chain constant region (left graph), and Heavy chain constant is a nucleic acid sequence of the heavy chain constant region (right graph). The specific method comprises the following steps: carrying out conventional linearization treatment on a mammalian cell expression vector pBR322 containing rabbit monoclonal antibodies CH and CL by using NheI and XbaI restriction endonucleases respectively, purifying the amplified PCR product, respectively constructing VL genes and VH genes with signal peptide coding genes at the upstream into the expression vector by adopting a homologous recombination mode, and transfecting the expression vector containing light chain genes and heavy chain genes of the corresponding rabbit monoclonal antibodies into 293F cells together after sequencing and verifying that the expression vector is successfully constructed; culturing for 72-96h after transfection to obtain supernatant containing rabbit monoclonal antibody recognizing mouse IL-10. Purifying rabbit monoclonal antibody recognizing mouse IL-10 from culture supernatant by using protein A affinity gel resin, verifying antibody purity by using 12% SDS-PAGE gel electrophoresis, sub-packaging after verification, and preserving at-20deg.C for use. Affinity chromatography uses procedures conventional in the art and will not be described in detail herein.
The signal peptide upstream of the VL gene and the VH gene in this example may be expressed by using an antibody commonly used in the art, and specifically, see "rabbit monoclonal antibody against Human interferon alpha 2" and its application (publication No. CN116063487A, publication No. 2023-05-05) ", or" high affinity Human IL-5 rabbit monoclonal antibody and its application (publication No. CN115819578A, publication No. 2023-03-21) ", with a signal peptide" MDTRAPTQLLGLLLLWLPGATF "or" MDTRAPTQLLGLLLLWLPGARC "upstream of the light chain variable region, and a signal peptide" METGLRWLLLVAVLKGVQC "upstream of the heavy chain variable region.
6. Monoclonal antibody screening and identification
After a multi-strain mouse IL-10 rabbit monoclonal antibody is obtained, the rabbit monoclonal antibody is firstly subjected to preliminary identification and screening, including identification of antibody affinity and identification of antigen recognition epitopes, and the specific method is as follows:
1) Antibody affinity assay: the affinity of the obtained rabbit monoclonal antibody was preliminarily determined using a gate biomolecular interaction analyzer from Probe Life. The antigen material used is recombinant mouse IL-10 protein, the working concentration is 3 mug/mL, and the working concentration of the rabbit monoclonal antibody is 2 mug/mL; by comparing the affinities of the respective antibodies, the dissociation constant K is selected from D ≤1×10 -9 An antibody of (M).
2) Identification of antigen recognition epitopes: dissociation constant K using Gator biomolecular interaction Analyzer from Probe Life D ≤1×10 -9 Performing a pairing reaction on the rabbit monoclonal antibodies of (1) to test the epitope determinants recognized by the rabbit monoclonal antibodies; wherein the antigen is recombinant mouse IL-10 protein, the working concentration is 3 mug/mL, and the working concentration of the first antibody and the working concentration of the second antibody are both 3 mug/mL. Two antibodies recognizing different epitopes were selected from the two antibodies, designated rabbit monoclonal antibodies 2H4 and 3F5, respectively, by analyzing the pairing data between the two antibodies.
FIGS. 2-3 show affinity curves for binding of rabbit monoclonal antibodies 2H4 and 3F5 to mouse IL-10 protein, respectively, wherein the abscissa indicates binding time, the ordinate indicates thickness variation of the conjugate after probe binding to antibody and protein, rabbit monoclonal antibody 2H4 working concentration of 5.94. Mu.g/mL, rabbit monoclonal antibody 3F5 working concentration of 8.61. Mu.g/mL; the dark gray curve is a real-time combined numerical curve, and the light gray curve is a fitted average curve. Affinity-related parameters of the antibodies were obtained by curve fitting and calculation (see Table 1), dissociation coefficient K off Constant for characterizing the dissociation rate of antibodies from antigens, binding coefficient K on Constant for characterizing the binding rate of an antibody to its target, dissociation constant K D For K off /K on Represents the equilibrium dissociation constant between an antibody and its antigen. As can be seen from FIGS. 2-3 and Table 1, the affinity constants K of the rabbit monoclonal antibodies 2H4 and 3F5 with recombinant mouse IL-10 D 1.38X10 respectively -10 (M) and 1.24X10 -10 (M), all below the nanoscale, indicating high affinity for IL-10 protein.
TABLE 1 determination of affinity-related parameters for Rabbit monoclonal antibodies 2H4 and 3F5
| Rabbit monoclonal antibodies | K off (1/s) | K on (1/Ms) | K D (M) |
| 2H4 | 8.33×10 -5 | 6.04×10 5 | 1.38×10 -10 |
| 3F5 | 6.59×10 -5 | 5.33×10 5 | 1.24×10 -10 |
FIG. 4 shows the results of epitope recognition by rabbit monoclonal antibodies 2H4 and 3F5, wherein the ordinate indicates the thickness change of the conjugate after binding of the probe to the antibody and the protein, and the abscissa indicates the binding time, and 2H4 indicates rabbit monoclonal antibody 2H4,3F5 indicates rabbit monoclonal antibody 3F5. From the figure, after the rabbit monoclonal antibody 2H4 and the recombinant mouse IL-10 protein are combined, the rabbit monoclonal antibody 3F5 can still be combined with the IL-10 protein, and the fact that the rabbit monoclonal antibody 2H4 and the rabbit monoclonal antibody 3F5 are combined at different positions on the surface of the IL-10 protein, recognize different antigen epitopes and do not interfere with each other is proved, and based on the result, the two can be used as a paired antibody for double-antibody sandwich method ELISA.
The amino acid and nucleotide sequences of the rabbit monoclonal antibodies 2H4 and 3F5 obtained in this example are shown in tables 2-3, respectively, and the light chain variable region VL and heavy chain variable region VH sequence identity of the rabbit monoclonal antibodies 2H4 and 3F5 are 75.45% and 66.38%, respectively. For convenience of description, light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively, AA represents the amino terminal sequence, and DNA represents the nucleotide sequence.
TABLE 2 summary of sequence information relating to monoclonal antibody 2H4 of this example
TABLE 3 summary of sequence information relating to monoclonal antibody 3F5 of this example
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Example 2 method for establishing double antibody sandwich ELISA based on rabbit monoclonal antibodies 2H4 and 3F5
The double-antibody sandwich method ELISA method comprises the following steps:
2.1, capture antibody coating: the rabbit monoclonal antibody 2H4 is diluted to 2 mug/mL by 1 XPBS, and after being uniformly mixed by a vortex meter, 100 mug/hole is added into a 96-well ELISA plate, a cover plate film is covered, and the mixture is placed in a refrigerator at 4 ℃ for incubation for 16-20H.
2.2, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 350. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.3, sealing: adding blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween 20 and 0.1%proclin300,pH 7.2) into plate holes at a ratio of 200 μl/hole, covering with cover plate film, sealing at 37deg.C for 2 hr, discarding blocking solution after sealing, drying ELISA plate, baking at 37deg.C for 0.5-2 hr, and taking out.
2.4, adding protein: mouse IL-10 protein (available from R & D under the trade designation 417-ML) was diluted with phosphate buffer pH 7.2 containing 2% bovine serum albumin, 0.05% Tween-20 and 0.1% proclin300 at the following concentrations: 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.6pg/mL, 7.8pg/mL, and then added sequentially to the ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated for 2h at 37 ℃.
2.5, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.6, adding detection antibody: after dilution of the biotin-labeled rabbit monoclonal antibody 3F5 to 0.05. Mu.g/mL, the mixture was sequentially added to the ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 1 hour. The preparation method of the biotin-labeled rabbit monoclonal antibody 3F5 comprises the following steps: 1mg/mL of rabbit monoclonal antibody 3F5 was prepared, N-succinimidyl 6-biotin aminocaproic acid (NHS-LC-biotin, available from Thermo company) was prepared as a solution with a concentration of 60mg/mL, 200. Mu.L of 1mg/mL of rabbit monoclonal antibody 3F5 solution was added to 10. Mu.L of 60mg/mL of NHS-LC-biotin, and after mixing, the mixture was left at room temperature for 3min, 50. Mu.L of 500mM Tris-HCl solution (pH 9.0) was added to terminate the reaction; finally, 4mL of 1 XPBS solution (pH 7.4) is added, and the mixture is centrifuged by a centrifugal column with the exclusion limit of 30kDa, so as to remove redundant biotin molecules and balance a buffer system, thus obtaining the rabbit monoclonal antibody 3F5-biotin.
2.7, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.8, adding SA-HRP: 100 XSA-HRP (horseradish peroxidase labeled streptavidin, available from Wuhan Sanying Biotechnology Co., ltd., product No. SA 00001-0) concentrate was diluted 100 times, and then added to an ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.
2.9, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.10, adding TMB color development liquid: the 3,3', 5' -Tetramethylbenzidine (TMB) color development solution was added to the ELISA plate at 100. Mu.L/well, covered with a cover plate film, and incubated at 37℃for 15min.
After incubation was completed, the microplate was removed, 50. Mu.L of stop solution (1 mol/L hydrochloric acid) was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
The standard curve drawn according to the detection result is shown in fig. 5. The result shows that the established double-antibody sandwich enzyme-linked immunosorbent assay method for detecting the mouse IL-10 protein by using the rabbit monoclonal antibody 2H4 as a capture antibody and the rabbit monoclonal antibody 3F5 as a detection antibody has the advantages of high brightness and good reliability, and the detection sensitivity can be as low as 1.965pg/mL through standard curve calculation.
EXAMPLE 3 specific detection of Rabbit monoclonal antibodies 2H4 and 3F5
Based on the double-antibody sandwich ELISA method established in example 2, six proteins similar to the mouse IL-10 protein are used for detecting the specificity of the rabbit monoclonal antibodies 2H4 and 3F5, and the concentration of each standard protein is 10ng/mL. Six proteins were each mouse interferon gamma (IFN-gamma, manufacturer R & D, manufacturer No. 485-MI-100/CF), mouse interferon beta (IFN-beta, manufacturer R & D, manufacturer No. 8234-MB-010), mouse interleukin 20 (IL-20, manufacturer R & D, manufacturer No. 1204-ML-025/CF), mouse interleukin 22 (IL-22, manufacturer R & D, manufacturer No. 582-ML-010/CF), mouse interleukin 24 (IL-24, manufacturer R & D, manufacturer No. 7807-ML-010/CF), human interleukin 10 (IL-10, manufacturer ABclonal, manufacturer RP 00093), all of which were commercially available, and the results of specific assays are shown in FIG. 6.
When the rabbit monoclonal antibody is combined with antigen, an optical signal is generated, and only mouse IL-10 protein can cause the change of absorbance value, which indicates that the double-antibody sandwich ELISA detection method based on the rabbit monoclonal antibodies 2H4 and 3F5 is only combined with the mouse IL-10 protein and does not generate any cross reaction with other six proteins, and indicates that the rabbit monoclonal antibodies 2H4 and 3F5 prepared by the method have high specificity to the mouse IL-10 and are suitable for detecting the mouse interleukin 10 with high specificity and high sensitivity.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A rabbit monoclonal antibody against mouse interleukin 10, comprising rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5, said rabbit monoclonal antibody comprising a light chain variable region and a heavy chain variable region, said light chain variable region and said heavy chain variable region each comprising 3 complementarity determining regions; wherein:
the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 2H4 are respectively shown in SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 8-10;
the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the rabbit monoclonal antibody 3F5 are respectively shown in SEQ ID NO.13-15, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown in SEQ ID NO. 18-20.
2. The rabbit monoclonal antibody against mouse interleukin 10 according to claim 1, wherein the amino acid sequence of the light chain variable region of said rabbit monoclonal antibody 2H4 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 rabbit monoclonal antibody 3F5 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 rabbit monoclonal antibody against mouse interleukin 10 according to claim 1, further comprising a light chain constant region and a heavy chain constant region, wherein the light chain constant regions of both the rabbit monoclonal antibody 2H4 and the rabbit monoclonal antibody 3F5 are kappa chains, and wherein the heavy chain constant regions are of the IgG1 type.
4. The rabbit monoclonal antibody against mouse interleukin 10 according to claim 1, wherein the amino acid sequence of the light chain of said rabbit monoclonal antibody 2H4 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 rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 16.
5. A nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or the rabbit monoclonal antibody 3F5 according to any one of claims 1-4.
6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the light chain variable region of said rabbit monoclonal antibody 2H4 is shown in SEQ ID NO.21 and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 22;
The nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody 3F5 is shown as SEQ ID NO.23, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 24.
7. A recombinant vector comprising a nucleic acid molecule encoding the rabbit monoclonal antibody 2H4 and/or the rabbit monoclonal antibody 3F5 according to any one of claims 1-4.
8. A host cell comprising the recombinant vector of claim 7 or having integrated into its genome a nucleic acid molecule encoding the nucleic acid of claim 5.
9. A kit for immunodetection of mouse interleukin 10, comprising the rabbit monoclonal antibody 2H4 and/or rabbit monoclonal antibody 3F5 of any one of claims 1-4;
the immunodetection method is one or more of enzyme immunoassay, enzyme-linked immunosorbent assay, immunohistochemical method, immunofluorescence method, immunoblotting method and flow cytometry;
the immunodetection sample is one or more of a tissue sample expressing IL-10 protein, a cell line sample expressing IL-10 protein, a serum sample secreting IL-10 protein, and a recombinantly expressed IL-10 protein.
10. The kit for immunodetection of mouse interleukin 10 according to claim 9, wherein the immunodetection method is a double antibody sandwich enzyme-linked immunosorbent method, wherein in the double antibody sandwich enzyme-linked immunosorbent method, the capture antibody is the rabbit monoclonal antibody 2H4, and the detection antibody is the rabbit monoclonal antibody 3F5 labeled with a detectable label.
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