CN116355094A - Monoclonal antibody against interleukin 12 of mouse and preparation method - Google Patents
Monoclonal antibody against interleukin 12 of mouse and preparation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The application discloses a monoclonal antibody against mouse interleukin 12, which can specifically bind to mouse interleukin 12, so that the monoclonal antibody can be applied to preparing a kit for detecting the level of mouse interleukin 12. In addition, as the monoclonal antibody has high affinity to the mouse interleukin 12, the protein interaction nonstandard technology verifies that the monoclonal antibody is combined with different antigenic determinants on the surface of the mouse interleukin 12, can be used for establishing a double-antibody sandwich enzyme-linked immunosorbent assay method for resisting the mouse interleukin 12 protein, and has important significance in clinical diagnosis and scientific research application.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a monoclonal antibody for resisting mouse interleukin 12 and a preparation method thereof.
Background
Interleukin 12 is a multi-active cytokine whose target cells are T cells, NK cells and myeloid progenitor cells. Interleukin 12 promotes proliferation of activated T cells and NK cells, enhances cytotoxic activity of T cells and NK cells, and induces the same to produce gamma interferon (IFN-gamma), beta tumor necrosis factor (tumor necrosis factor beta, TNF-beta), etc. Interleukin 12 plays an important role in the balance of Th1/Th2 responses, and Interleukin 12 can induce Tn cells to differentiate into Th1 cells and stimulate the development and proliferation of Th1 cells, thereby playing an important role in cell-mediated immunity.
Interleukin 12 is a multi-biologically active cytokine whose important role in anti-tumor immunity is to establish a link between natural resistance mediated by macrophages, NK cells, and acquired immunity mediated by Th1 cells, NK1.1+tcrint cells, CTL cells. Interleukin 12 regulates the immune response of tumor patients as a new, promising strategy for tumor therapy. Therefore, the development of a high-sensitivity interleukin 12 protein detection methodology has very important clinical significance.
In order to improve the specificity and sensitivity of ELISA detection methods, it is currently common in the industry to employ monoclonal antibody pairs against interleukin 12 to develop corresponding ELISA detection kits. However, the ELISA detection kit for interleukin 12 in the market at present adopts mouse anti-interleukin 12 monoclonal antibodies, and the affinity and the specificity of the ELISA detection kit are generally lower than those of rabbit monoclonal antibodies. The used mouse monoclonal antibody is developed and produced by a traditional hybridoma method, and the preparation process is more complex than that of the recombinant monoclonal antibody, and has larger batch-to-batch difference. Therefore, ELISA detection kits developed by using mouse monoclonal antibodies have the challenges of low sensitivity, difficult control of batch-to-batch differences and the like.
Disclosure of Invention
To solve or alleviate the above technical problems, the present application provides a rabbit monoclonal antibody against mouse interleukin 12 instead of the conventional mouse monoclonal antibody, the present application successfully develops monoclonal antibodies A and B against mouse interleukin 12 with high affinity and specificity to mouse interleukin 12 by single B cell screening and culturing technique, the binding rate constant of the antibody A and recombinant expressed mouse interleukin 12 is 7.85×10 4 Antibody B and recombinant expression mouse interleukin 12 binding rate constant of 1.40X10 5 . Therefore, the monoclonal antibody can be used for preparing a kit for detecting the interleukin 12 level of mice. The monoclonal antibody provided by the application has important clinical value for diagnosis and treatment of the mouse interleukin 12 and related diseases.
Therefore, the application at least discloses the following technical scheme:
an antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said light chain variable region having: VL CDR1 consisting of the sequence shown in SEQ ID NO. 3 or 13, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; VL CDR2 consisting of the sequence shown in SEQ ID NO. 4 or 14 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and VL CDR3 consisting of the sequence shown in SEQ ID NO. 5 or 15, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence;
And/or
A heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said heavy chain variable region having: a VH CDR1 consisting of the sequence shown in SEQ ID NO. 8 or 18 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; a VH CDR2 consisting of the sequence shown in SEQ ID NO 9 or 19 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; and a VH CDR3 consisting of the sequence shown in SEQ ID NO 10 or 20, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; preferably, the substitutions are conservative substitutions.
An antibody that specifically binds to mouse interleukin 12, the antibody comprising: a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 2; heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 7.
An antibody that specifically binds to mouse interleukin 12, the antibody comprising: a light chain consisting of the sequence shown in SEQ ID NO. 1; and a heavy chain consisting of the sequence shown in SEQ ID NO. 6.
An antibody that specifically binds to mouse interleukin 12, the antibody comprising: a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 12; heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 17.
An antibody that specifically binds to mouse interleukin 12, the antibody comprising: a light chain consisting of the sequence shown in SEQ ID NO. 11; and a heavy chain consisting of the sequence shown in SEQ ID NO. 16.
A conjugate comprising said antibody, and a detectable label attached to said antibody.
A kit for detecting mouse interleukin 12, the kit comprising: said antibody or said conjugate.
The application also provides the application of the high-affinity rabbit monoclonal antibody against mouse interleukin 12 in the establishment of an enzyme-linked immunosorbent assay method for mouse interleukin 12 of non-diagnostic purposes.
Further, the enzyme-linked immunosorbent assay method is a double-antibody sandwich method enzyme-linked immunosorbent assay method.
In the double-antibody sandwich ELISA detection method, the capture antibody is a rabbit monoclonal antibody A, and the detection antibody is a biotin-labeled rabbit monoclonal antibody B.
The present application also provides a kit comprising an antibody or conjugate as described previously.
The application also provides application of the kit in detection of mouse interleukin 2.
Further, the mouse interleukin 12 includes recombinant expressed mouse interleukin 12 or mouse interleukin 12 protein secreted by cells.
To sum up, the application has the advantages and positive effects that:
1. the application successfully develops a pair of high-affinity anti-mouse interleukin 12 protein rabbit monoclonal antibodies, and simultaneously develops a double-antibody sandwich method ELISA detection method aiming at the mouse interleukin 12 protein with high sensitivity and specificity, thereby having important significance in clinical diagnosis and scientific research application.
2. The rabbit monoclonal antibodies A and B of the anti-mouse interleukin 12 protein prepared in the application prove that the rabbit monoclonal antibodies A and B are combined with different antigenic determinants on the surface of the mouse interleukin 12 through the verification of a protein interaction nonstandard technology, and can be used for establishing a double-antibody sandwich enzyme-linked immunosorbent assay method aiming at the mouse interleukin 12 protein.
3. The invention provides a mouse interleukin 12 double-antibody sandwich ELISA detection method established based on the mouse interleukin 12 protein rabbit monoclonal antibody, wherein a capture antibody is the mouse interleukin 12 rabbit monoclonal antibody A, a detection antibody is the mouse interleukin 12 rabbit monoclonal antibody B marked by biotin, a standard sample is human mouse interleukin 12 protein expressed in vitro in a recombination way, the detection sensitivity is 500pg/ml (500 ng/L), and the established method can be used for high-sensitivity detection of the mouse interleukin 12 protein.
Drawings
FIG. 1 is a block diagram of an antibody light chain expression plasmid provided in the examples of the present application.
FIG. 2 is a block diagram of an antibody heavy chain expression plasmid provided in the examples of the present application.
FIG. 3 is a graph showing the affinity of the anti-mouse interleukin 12 antibody A provided in the examples of the present application.
FIG. 4 is a graph showing the affinity of the anti-mouse interleukin 12 antibody B provided in the examples of the present application.
FIG. 5 is the results of an EP experiment for anti-mouse interleukin 12 antibodies A and B provided in the examples of the present application.
FIG. 6 is a graph showing ELISA detection results of the mouse interleukin 12 by the antibody A and the antibody B provided in the example of the present application.
FIG. 7 is a graph showing the cross-reaction results of antibody A and antibody B provided in the examples of the present application.
Fig. 8 is a graph showing the results of the stability experiments of the antibodies a and B provided in the examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
Interpretation of the terms
In the present application, the term "antibody" is to be interpreted in the broadest sense, having a variety of antibody structures, including but not limited to, Y-type antibodies, so-called full length antibodies, antigen binding portions of Y-type antibodies, and genetic or chemical modifications thereof. Wherein "antigen binding portion" refers to one or more portions or fragments of a Y-type antibody that retains the ability of the antibody to specifically bind to mouse interleukin 12.
In this application, the term "monoclonal antibody" (mAb) includes a population of highly homogeneous antibodies having substantially identical antigenic determinants. That is, the individual antibodies are essentially identical in the population, except for the small number of mutations that may occur naturally. Monoclonal antibodies may exhibit a single binding specificity and affinity for a particular epitope on an antigen. Each monoclonal antibody may be directed against the same or substantially the same epitope on the antigen, as compared to a polyclonal antibody which typically comprises antibodies directed against different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring preparation by any particular method. The antibodies can be prepared by a variety of methods including, but not limited to, hybridoma methods, recombinant DNA methods, phage antibody libraries, and the like.
In this application, the modifier "rabbit" in the term "rabbit antibody" or "anti-mouse interleukin 12 monoclonal antibody" or similar terms means that the Complementarity Determining Regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. In one embodiment, the anti-mouse interleukin 12 rabbit monoclonal antibody may comprise the CDRs and Framework Regions (FR) of the antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit antibody or rabbit monoclonal antibody against mouse interleukin 12 may comprise CDRs from an antibody of rabbit immunoglobulin sequence. In one embodiment, the anti-mouse interleukin 12 rabbit monoclonal antibody may be one in which the CDR regions are derived from rabbit immunoglobulin sequences, while the FR is derived from other mammalian germline immunoglobulin sequences (e.g., mouse or human). The term "anti-mouse interleukin 12 rabbit monoclonal antibody" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g., mutations introduced by random or point-specific mutations in vitro, or by somatic mutations in vivo. However, the term "anti-mouse interleukin 12 rabbit monoclonal antibody" does not include antibodies in which the CDR regions are derived from the germline of other mammals (e.g., mice).
In this application, the term "antibody" refers to an immunoglobulin molecule consisting of four heterologous polypeptide chains, of which the two chains with the larger molecular weight are referred to as heavy chains (H) and the two chains with the smaller molecular weight are referred to as Light chains (L). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The heavy and light chains vary widely in about 110 amino acid sequences near the N-terminus, with the other portions of the amino acid sequences being relatively constant. Thus, the regions of the light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V) and account for 1/4 and 1/2 of the heavy and light chains, respectively; the region of relatively stable amino acid sequence near the C-terminus is called constant region (C) and occupies 3/4 and 1/2 of the heavy and light chains, respectively.
The V chains of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains a region of highly variable 3 amino acid composition and arrangement sequence, termed hypervariable region (hypervariable region, HVR) or complementarity determining region (complementarity determining region, CDR), including HVRl (CDRl), HVR2 (CDR 2) and HVR3 (CDR 3), wherein HVR3 (CDR 3) varies to a greater extent. The 3 CDRs of VH and VL together form the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the regions outside the CDRs are relatively conserved, called Framework Regions (FR). VH or VL has four framework regions, denoted FR1, FR2, FR3 and FR4, respectively.
The C chains of the heavy and light chains are referred to as CH and CL, respectively. CL lengths of different classes (kappa or lambda) of Ig are substantially identical, but CH lengths of different classes of Ig are different, e.g., igG, igA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CHl, CH2, CH3, and CH4.
In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In this application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.
In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar (of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue.
Antibodies to
An antibody disclosed in the examples of the present application that specifically binds to mouse interleukin 12, the antibody comprising a light chain variable region (VL) defined according to the Kabat numbering system, the light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the sequence shown in SEQ ID NO. 3 or 13, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; VL CDR2 consisting of the sequence shown in SEQ ID NO. 4 or 14 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and VL CDR3 consisting of the sequence shown in SEQ ID NO. 5 or 15, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
In certain embodiments, VL CDR1, consisting of the sequence shown in SEQ ID NO. 3, or a sequence having 1 to 3 amino acid substitutions, deletions or additions compared thereto; VL CDR2 consisting of the sequence shown in SEQ ID NO. 4 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and VL CDR3 consisting of the sequence shown in SEQ ID NO. 5 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
In certain embodiments, VL CDR1, consisting of the sequence shown in SEQ ID NO. 13, or a sequence having 1 to 3 amino acid substitutions, deletions or additions compared thereto; VL CDR2 consisting of the sequence shown in SEQ ID NO. 14 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and VL CDR3 consisting of the sequence shown in SEQ ID NO. 15 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
Also disclosed in the examples herein is an antibody that specifically binds to mouse interleukin 12 comprising the following 3 heavy chain variable regions (VH) of Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said heavy chain variable regions having: a VH CDR1 consisting of the sequence shown in SEQ ID NO. 8 or 18 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; a VH CDR2 consisting of the sequence shown in SEQ ID NO 9 or 19 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; and a VH CDR3 consisting of the sequence shown in SEQ ID NO 10 or 20, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
In certain embodiments, a VH CDR1 consisting of the sequence shown in SEQ ID NO. 8 or a sequence having 1 to 3 amino acid substitutions, deletions or additions compared thereto; a VH CDR2 consisting of the sequence shown in SEQ ID NO. 9 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and a VH CDR3 consisting of the sequence shown in SEQ ID NO. 10 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
In certain embodiments, a VH CDR1 consisting of the sequence shown in SEQ ID NO. 18 or a sequence having 1 to 3 amino acid substitutions, deletions or additions compared thereto; a VH CDR2 consisting of the sequence shown in SEQ ID NO. 19 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and a VH CDR3 consisting of the sequence shown in SEQ ID NO. 20 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution.
In certain embodiments, the antibody comprises a light chain variable region (VL CDR1, VL CDR2, and VL CDR 3) and a heavy chain variable region (VH CDR1, VH CDR2, and VH CDR 3) of Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system. VL CDR1 consists of the sequence shown in SEQ ID NO. 3 or 13 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it. VL CDR2 consists of the sequence shown in SEQ ID NO. 4 or 14 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it. VL CDR3 consists of the sequence shown in SEQ ID NO. 5 or 15, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; wherein the substitution is a conservative substitution. VH CDR1 consists of the sequence shown in SEQ ID NO. 8 or 18 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. The VH CDR2 consists of the sequence shown in SEQ ID NO 9 or 19 or has a substitution, deletion or addition of 1 to 3 amino acids as compared with the sequence. The VH CDR3 consists of the sequence shown in SEQ ID NO 10 or 20 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. Wherein the substitution is a conservative substitution.
In certain examples of this embodiment, VL CDR1 consists of the sequence shown in SEQ ID NO. 3 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared to it. VL CDR2 consists of the sequence shown in SEQ ID NO. 4 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it. VL CDR3 consists of the sequence shown in SEQ ID No. 5 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it. Wherein the substitution is a conservative substitution. VH CDR1 consists of the sequence shown in SEQ ID NO. 8 or has a substitution, deletion or addition of 1 to 3 amino acids compared with it. The VH CDR2 consists of the sequence shown in SEQ ID NO 9 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. The VH CDR3 consists of the sequence shown in SEQ ID NO. 10 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. Wherein the substitution is a conservative substitution.
In certain examples of this embodiment, the VH CDR1 consists of the sequence shown in SEQ ID NO. 13 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared to it. The VH CDR2 consists of the sequence shown in SEQ ID NO. 14 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. The VH CDR3 consists of the sequence shown in SEQ ID NO. 15 or has a substitution, deletion or addition of 1 to 3 amino acids as compared with the sequence. Wherein the substitution is a conservative substitution. VH CDR1 consists of the sequence shown in SEQ ID NO. 18 or has a substitution, deletion or addition of 1 to 3 amino acids compared with it. The VH CDR2 consists of the sequence shown in SEQ ID NO. 19 or has a substitution, deletion or addition of 1 to 3 amino acids as compared with the sequence. The VH CDR3 consists of the sequence shown in SEQ ID NO. 20 or has a substitution, deletion or addition of 1 to 3 amino acids compared with the sequence. Wherein the substitution is a conservative substitution.
In certain embodiments, the antibody comprises: a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 2; heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 7.
In certain embodiments, the antibody comprises: a light chain consisting of the sequence shown in SEQ ID NO. 1; and a heavy chain consisting of the sequence shown in SEQ ID NO. 6.
In certain embodiments, the antibody comprises: a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 12; heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 17.
In certain embodiments, the antibody comprises: a light chain consisting of the sequence shown in SEQ ID NO. 11; and a heavy chain consisting of the sequence shown in SEQ ID NO. 16.
In certain embodiments, the humanized antibody against mouse interleukin 12 may have a Y-type molecular structure. In one embodiment, the humanized antibody against mouse interleukin 12 may comprise a pair of heavy chains and a pair of light chains. The heavy chain may include one heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally comprise five types of heavy chains: antibodies of corresponding composition are termed IgG, igD, igA, igM and IgE five antibodies. The light chain may be a smaller polypeptide subunit relative to the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is typically the N-terminal part of the light chain, exhibiting higher variability in amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, the heavy chain variable region VH and the light chain variable region VL can both be used to recognize and bind to mouse interleukin 12. In one embodiment, the light chain constant region of the antibody is a kappa chain and the heavy chain constant region of the antibody is of the IgG1 type.
In certain embodiments, the CDRs located in the VL and VH regions can be separated from each other by a FR. FR is a conserved region in the sequence structure. FR can generally act as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., mouse interleukin 12). The three-dimensional structure of FR may be conserved among different antibodies. CDRs can be grafted between the FRs of another antibody from another species while retaining its ability to bind to mouse interleukin 12, forming a fusion antibody. In one embodiment, the CDRs of the Y-type rabbit monoclonal antibody are grafted between the FRs of the human antibody to form a humanized antibody against mouse interleukin 12. In certain embodiments, the antibody comprises an FR region derived from a human immunoglobulin, optionally comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 back mutations from a human residue to a corresponding rabbit residue.
In certain embodiments, the CDRs located in the VH and VL regions can be separated from each other by a FR. FR is a conserved region in the sequence structure. FR can generally act as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., mouse interleukin 12). The three-dimensional structure of FR may be conserved among different antibodies. CDRs of a rabbit monoclonal antibody can be grafted between the FRs of another antibody from another species while retaining its ability to bind to mouse interleukin 12, forming a fusion antibody. In one embodiment, the CDRs of the Y-type rabbit monoclonal antibody are grafted between the FRs of the human antibody to form a humanized antibody against mouse interleukin 12.
The anti-mouse interleukin 12 rabbit monoclonal antibodies provided in the embodiments of the present application may also include antigen binding portions thereof derived from the structures provided in the embodiments described above or obtained by genetic modification. In certain embodiments, the anti-mouse interleukin 12 rabbit monoclonal antibody can have a different transgenic antibody structure, including, but not limited to, humanized antibodies and chimeric antibodies. In one embodiment, the anti-mouse interleukin 12 rabbit monoclonal antibody may be a humanized antibody having a protein sequence with a high degree of homology to that of a naturally variant antibody adapted to the human body. Wherein the protein sequence of the "humanized antibody" may be substantially identical to the protein sequence of the human variant antibody while maintaining the binding capacity of the rabbit CDR regions to mouse interleukin 12. In one embodiment, the "humanized antibody" may be created by inserting CDR regions of a non-humanized antibody, e.g., a CDR region of a rabbit antibody into a humanized antibody scaffold to produce a humanized antibody. In one embodiment, the anti-mouse interleukin 12 rabbit monoclonal antibody may be a chimeric antibody. In one embodiment, the chimeric antibody may be an antibody produced by grafting variable regions of heavy and light chains of Y-type antibodies of different sources to constant regions of another animal (e.g., human). In one embodiment, the anti-mouse interleukin 12 rabbit monoclonal antibody may be a single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by two separate genes, the two separate encoding genes can be joined by recombinant methods to form one linker, thereby encoding an expressed scFv. In one of the embodiments, the relevant genetic modification and transgenic manipulation can be performed according to methods well known to those skilled in the art, and the transgenic antibody structure can be screened in the same manner as full length antibodies are screened.
Preparation of humanized antibodies against mouse interleukin 12
The present application discloses methods for preparing the above antibodies, and the monoclonal antibodies of the present application may be prepared by various methods known in the art, such as by genetic engineering recombinant techniques; DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, then the host cells are transfected, and the transfected host cells are cultured under specific conditions and the antibodies of the present application are expressed.
In certain embodiments, the method of preparation is a monoclonal antibody development technique based on single B lymphocyte selection and culture, the method of preparation comprising: taking recombinant mouse interleukin 12 protein as an immunogen to immunize New Zealand white rabbits; b lymphocytes are sorted from spleen cells of the large white blood cell line and cultured; extracting RNA in B lymphocytes, and reversely transcribing the RNA into cDNA; the cDNA is amplified by PCR to obtain a natural paired rabbit monoclonal antibody; the heavy chain variable region (VH) gene and the light chain variable region (VL) gene of the rabbit monoclonal antibody which are paired are respectively loaded on an expression vector, the vector is transfected into a host cell, the host cell is cultured, and the monoclonal antibody is obtained by separating and purifying the culture solution of the host cell.
In some examples of the foregoing embodiments, the specific steps include:
animal immunization: in order to obtain the rabbit monoclonal antibody for recognizing the mouse interleukin 12 protein, the invention takes the self-produced recombinant mouse interleukin 12 protein as an immunogen to immunize New Zealand white rabbits; immunizing 200 mug of each white rabbit, firstly immunizing, mixing an immunogen with an equivalent amount of complete Freund adjuvant to prepare an emulsifying agent, subcutaneously injecting the mixture into the abdomen and the back at multiple points, taking 100 mug of the immunogen and an equivalent amount of incomplete Freund adjuvant at intervals of 3 weeks, subcutaneously injecting the mixture into the abdomen and the back at multiple points, boosting the mixture twice, measuring serum titer by an ELISA method after three times of immunization, taking a rabbit with high serum titer, boosting the mixture once by 200 mug of the immunogen by subcutaneous multiple points, and taking spleen after three days;
spleen cell separation;
b lymphocyte sorting: see patent 201910125091.4, methods for efficient isolation of individual antigen-specific B lymphocytes from spleen cells;
cloning of the gene for encoding the rabbit monoclonal antibody; the cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clone using PCR methods, and the primer pairs for amplifying the light chain variable region (VL) were as follows:
Forward primer: the nucleotide sequence is shown as SEQ ID NO. 21;
reverse primer: the nucleotide sequence is shown as SEQ ID NO. 22;
primer pairs for amplifying the heavy chain variable region (VH) are as follows:
forward primer: the nucleotide sequence is shown as SEQ ID NO. 23;
reverse primer: the nucleotide sequence is shown as SEQ ID NO. 24; sequencing the amplification result to determine a sequence;
production and purification of monoclonal antibodies; in order to obtain a plurality of rabbit monoclonal antibodies for recognizing the mouse interleukin 12 protein, the invention loads the heavy chain and the light chain genes of the rabbit monoclonal antibodies on expression vectors respectively, wherein the construction structure diagram of the expression vectors of the light chain is shown in figure 1; the expression vector of the heavy chain is shown in FIG. 2; transfecting the plasmid into 293F cells; the rabbit monoclonal antibody which is used for recognizing the mouse interleukin 12 protein and is recombined in the culture supernatant is obtained after 72-96 hours of transfection. Purifying recombinant rabbit monoclonal antibody recognizing mouse interleukin 12 protein from transfected culture medium supernatant by using protein A affinity gel resin, split charging after antibody identification, and preserving at-20 ℃ for standby.
Results: in the embodiment of the application, two rabbit monoclonal antibodies A and B which recognize mouse interleukin 12 are screened; wherein the light chain amino acid sequence of the antibody A is SEQ ID NO. 1; the heavy chain amino acid sequence is SEQ ID NO. 6; the amino acid sequence of the light chain variable region is SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region is SEQ ID NO. 7; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively.
The light chain amino acid sequence of the antibody B is SEQ ID NO. 11, and the heavy chain amino acid sequence of the antibody B is SEQ ID NO. 16; the amino acid sequence of the light chain variable region is SEQ ID NO. 12; the amino acid sequence of the heavy chain variable region is SEQ ID NO. 17; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, respectively; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, respectively.
Monoclonal antibody and mouse interleukin 12 affinity detection
In the embodiment of the application, after a plurality of anti-mouse interleukin 12 rabbit monoclonal antibodies are obtained, the antibodies are firstly subjected to preliminary identification and screening, including identification of antibody affinity and identification of antigen recognition epitopes; the method comprises the following specific steps of carrying out preliminary identification of antibody affinity on the obtained anti-mouse interleukin 12 rabbit monoclonal antibody, carrying out the preliminary identification by using Gator Prime equipment, and using a probe Protein A probe, wherein the specific steps comprise:
pre-wet: before use, vibrating and wetting the Pro A probe in a matched buffer at 1000rpm for 300sec; baseline1: the Pro A probe is placed in a buffer solution for initial point calibration, so that the probe is ensured to be in a stable state at 1000rpm for 60sec; loading: the antibody ARC54512 to be tested is solidified on the Pro A probe, and the solidifying concentration is 1 mug/mL, 1000rpm,120sec; antibody ARC54512 to be tested was immobilized on Pro A probe at a concentration of 3 μg/mL,1000rpm,120sec; baseline2: placing the probe after the antibody is solidified in a buffer solution for vibration washing at 1000rpm for 60sec; association: the probes after the antibody is solidified are respectively placed in 178.57nM and 357.14nM antigen dilutions, and the affinity of the antibodies for binding the antigen under different molar concentrations is tested, 1000rpm,180sec; dissociation: after antigen-antibody binding reached saturation, the probe was transferred to a dissociation system to complete the dissociation process at 1000rpm for 480sec.
In the embodiment of the application, the Anti-mouse interleukin 12 rabbit monoclonal antibody and the mouse interleukin 12 protein antigen recognition epitope obtained in the invention are identified, the identification is carried out through a Gator Prime device, and the obtained antibody is subjected to pairing reaction by using a probe as an Anti-HisProbe to test the recognized epitope determinant; the method specifically comprises the following steps:
pre-wet: before use, the Anti-His probe is vibrated and wetted in a matched buffer at 1000rpm for 300sec; baseline1: placing the Anti-His probe in a buffer solution for initial point calibration, and ensuring that the probe is initially in a stable state at 1000rpm for 60sec; loading: solidifying target protein on the Anti-His probe, wherein the solidifying concentration is 5 mug/mL, 1000rpm,47sec; baseline2: placing the probe after the protein solidification in a buffer solution, and vibrating the probe in a first antibody diluent of 5 mug/mL to enable the first antibody and the target protein to be combined to saturation, 1000rpm and 480sec; association2: the probe after immobilization of the target protein and the first antibody was placed in a dilution of the second antibody so that the second antibody bound to the target protein at 1000rpm,240sec on an epitope different from the first antibody.
Results: the results of the affinity assays for mouse interleukin 12 for antibodies a and B are shown in table 1:
TABLE 1
| Name of the name | Binding constant (1/Ms) |
| Antibody A | 7.85E+004 |
| Antibody B | 1.40E+005 |
Results: as can be seen from the table, the a and B antibodies have higher affinity for mouse interleukin 12, and as shown in fig. 3-4, the a and B antibodies bind to mouse interleukin 12 to reach a stable value at 120sec, and the binding rate is also faster; the above results show that the screened A and B antibodies have specific binding capacity to the mouse interleukin 12 protein; as shown in fig. 5, antibody a as a capture antibody and antibody B as a detection antibody were each stably bound to a different epitope of mouse interleukin 12, and thus, antibody a and antibody B can be used for detection of mouse interleukin 12.
Antibody A and B established double-antibody sandwich method ELISA detection
In the embodiment of the application, a double-antibody sandwich ELISA experiment is performed by taking an antibody A as a capture antibody and an antibody B as a detection antibody, and the specific experimental steps comprise:
coating: diluting the rabbit antibody A into 4 mug/ml by using 1X PBS, uniformly mixing by a vortex instrument, adding into a 96-well micro-pore plate by 100 mug/well, covering a cover plate film, and placing into a refrigerator at 4 ℃ for incubation for 16-20 h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed once with 1 x pbst, 300 μl of sample was added, and after 40s of standing, the liquid in the wells was discarded, and the liquid in the wells was dried on a piece of flat paper; closing: adding E013 sealing liquid into a plate hole at 200 μl/well, covering a cover plate film, sealing for 2h at 37 ℃, discarding the sealing liquid after sealing, drying an ELISA plate, drying in a baking oven at 37 ℃ for 0.5-2 h, and taking out for later use; protein adding: the Mouse IL-12p70 protein was diluted with a diluent at a concentration of: 500,250,125,62.5,31.25,15.625,7.8125,0pg/mL, then sequentially adding 100. Mu.l/well to the ELISA plate, covering the cover plate membrane, and incubating at 37 ℃ for 2h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding a detection antibody: diluting antibody B-biotin to 0.1 mu g/ml, sequentially adding the diluted antibody B-biotin into an ELISA plate at 100 mu l/well, covering a cover plate film, and incubating at 37 ℃ for 1h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding SA-HRP: 100 times of SA-HRP concentrated solution is diluted by 100 times, 100 mu l/well of the diluted concentrated solution is added into an ELISA plate in sequence, a cover plate film is covered, and the incubation is carried out for 0.5h at 37 ℃; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding TMB color development liquid: adding TMB color development liquid into an ELISA plate in sequence at a rate of 100 mu l/well, covering a cover plate film, and incubating for 15min at 37 ℃; after the incubation was completed, the microplate was removed, 50. Mu.l of stop solution was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
The biotin labeling treatment method comprises the following steps: preparing an anti-mouse interleukin 12 rabbit monoclonal antibody B into a solution with the concentration of 1mg/ml, and preparing NHS-LC-biotin into a solution with the concentration of 10mg/ml by using DMSO; 200 μl of 1mg/ml anti-mouse interleukin 12 rabbit monoclonal antibody B solution is taken, and 10 μl of 10mg/ml NHS-LC-biotin solution is added; after mixing, the mixture was left at room temperature for 30 minutes, and then 50. Mu.g of 500mM PH9.0 Tris solution was added to stop the reaction; finally, a large amount of 1 XPBS buffer, pH7.4, was added and centrifuged with a 30KD exclusion limit for removing excess biotin molecules and performing equilibration of the buffer system.
As shown in FIG. 6, the result shows that the antibody A is used as a capture antibody, the antibody B is used as a detection antibody, and ELISA detection is performed on the mouse interleukin 12, so that good linearity is realized, and the detection has good sensitivity and accuracy.
Cross-reaction test of antibodies A and B
In the examples of the present application, the specific steps of the cross-reaction test of anti-mouse interleukin 12 protein rabbit monoclonal antibodies A and B include:
coating: diluting the rabbit antibody A into 4 mug/ml by using 1X PBS, uniformly mixing by a vortex instrument, adding into a 96-well micro-pore plate by 100 mug/well, covering a cover plate film, and placing into a refrigerator at 4 ℃ for incubation for 16-20 h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed once with 1x pbst, 300 μl of sample was added, and after 40s of standing, the liquid in the wells was discarded, and the liquid in the wells was dried on a piece of flat paper; closing: adding E013 sealing liquid into a plate hole at 200 μl/well, covering a cover plate film, sealing for 2h at 37 ℃, discarding the sealing liquid after sealing, drying an ELISA plate, drying in a baking oven at 37 ℃ for 0.5-2 h, and taking out for later use; protein adding: diluting the Mouse IL-12p70 protein to 500pg/mL by using a diluent, diluting other proteins needing to be tested for cross reaction to 10ng/mL, sequentially adding 100 mu l/well into an ELISA plate, covering a cover plate film, and incubating for 2h at 37 ℃; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding a detection antibody: diluting antibody B-biotin to 0.1 mu g/ml, sequentially adding the diluted antibody B-biotin into an ELISA plate at 100 mu l/well, covering a cover plate film, and incubating at 37 ℃ for 1h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding SA-HRP: 100 times of SA-HRP concentrated solution is diluted by 100 times, 100 mu l/well of the diluted concentrated solution is added into an ELISA plate in sequence, a cover plate film is covered, and the incubation is carried out for 0.5h at 37 ℃; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding TMB color development liquid: adding TMB color development liquid into an ELISA plate in sequence at a rate of 100 mu l/well, covering a cover plate film, and incubating for 15min at 37 ℃; after the incubation was completed, the microplate was removed, 50. Mu.l of stop solution was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
As shown in FIG. 7, the antibodies A and B are used for detecting the mouse interleukin 12, and have good specificity.
Antibody A and antibody B stability experiments
In the embodiment of the application, the specific coating: diluting the rabbit antibody A into 4 mug/ml by using 1X PBS, uniformly mixing by a vortex instrument, adding into a 96-well micro-pore plate by 100 mug/well, covering a cover plate film, and placing into a refrigerator at 4 ℃ for incubation for 16-20 h; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed once with 1 x pbst, 300 μl of sample was added, and after 40s of standing, the liquid in the wells was discarded, and the liquid in the wells was dried on a piece of flat paper; closing: adding E013 sealing liquid into a plate hole at 200 μl/well, covering a cover plate film, sealing for 2h at 37 ℃, discarding the sealing liquid after sealing, beating the ELISA plate, and drying in a baking oven at 37 ℃ for 0.5-2 h; thermal destruction: dividing the coated ELISA plate, lyophilized protein and 100X concentrated detection antibody into three parts, respectively placing at-20deg.C, 4deg.C and 37deg.C, sealing and preserving for 7 days, and taking out for testing; protein adding: diluting the Mouse IL-12p70 protein stored under different conditions into 500pg/mL by using a diluent, sequentially adding 100 mu l/well into an ELISA plate stored at a corresponding temperature, covering a cover plate film, and incubating at 37 ℃ for 2 hours; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding a detection antibody: after the antibody B-biotin preserved under different conditions is diluted by 100 times, sequentially adding 100 mu l/well into an ELISA plate preserved at a corresponding temperature, covering a cover plate film, and incubating for 1h at 37 ℃; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding SA-HRP: 100 times of SA-HRP concentrated solution is diluted by 100 times, 100 mu l/well of the diluted concentrated solution is added into an ELISA plate in sequence, a cover plate film is covered, and the incubation is carried out for 0.5h at 37 ℃; washing the plate: after incubation, the liquid in the wells was discarded, the plate was washed three times with 1 x pbst, 300 μl was added, the liquid in the wells was discarded after 40s of standing, and the liquid in the wells was dried on a piece of flat paper; adding TMB color development liquid: adding TMB color development liquid into an ELISA plate in sequence at a rate of 100 mu l/well, covering a cover plate film, and incubating for 15min at 37 ℃; after the incubation was completed, the microplate was removed, 50. Mu.l of stop solution was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
Results: as shown in FIG. 8, when the antibody subjected to 7-day thermal destruction treatment is used for detecting interleukin 12 in mice, the dominant performance can still be maintained, and further, the rabbit monoclonal antibodies A and B provided by the application have good stability.
Application of antibody in preparation of kit
The application also discloses a kit for detecting mouse interleukin 12, the kit includes: said antibody or said conjugate.
In certain embodiments, the monoclonal antibodies of the present application are capable of specifically binding to mouse interleukin 12, and thus can be used to detect the level of mouse interleukin 12.
In certain embodiments, the antibody or antigen binding fragment thereof does not comprise a detectable label.
In certain embodiments, the kit comprises a second antibody that specifically recognizes a monoclonal antibody described herein; wherein the secondary antibody comprises a detectable label such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.
In certain embodiments, the second antibody is specific for an antibody from a species (e.g., rabbit or human) from which the constant regions comprised by the monoclonal antibodies described herein are derived.
In certain embodiments, the second antibody is an anti-immunoglobulin (e.g., human or rabbit immunoglobulin) antibody, such as an anti-IgG antibody.
In certain embodiments, the second antibody is an anti-rabbit IgG antibody or an anti-human IgG antibody.
In certain embodiments, the kits of the present application comprise reagents for causing the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethyl benzidine (TMB), ABTS, or luminol for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. The kit may further comprise a pre-excitation and/or excitation liquid for chemiluminescence, such as when the detectable label is a chemiluminescent reagent, such as an acridine ester compound.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (10)
1. An antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said light chain variable region having:
VL CDR1 consisting of the sequence shown in SEQ ID NO. 3 or 13, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it;
VL CDR2 consisting of the sequence shown in SEQ ID NO. 4 or 14 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it; and
VL CDR3 consisting of the sequence shown in SEQ ID NO. 5 or 15, or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with it;
and/or
A heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said heavy chain variable region having:
a VH CDR1 consisting of the sequence shown in SEQ ID NO. 8 or 18 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence;
a VH CDR2 consisting of the sequence shown in SEQ ID NO 9 or 19 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence; and
a VH CDR3 consisting of the sequence shown in SEQ ID NO 10 or 20 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the sequence;
Preferably, the substitutions are conservative substitutions.
2. The antibody of claim 1, wherein the antibody comprises:
a light chain variable region (VL) comprising 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said light chain variable region having a VL CDR1 of the sequence shown in SEQ ID No. 3, a VL CDR2 of the sequence shown in SEQ ID No. 4 and a VL CDR3 of the sequence shown in SEQ ID No. 5;
and/or
A heavy chain variable region (VH) comprising 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said heavy chain variable region having a VL CDR1 of the sequence shown in SEQ ID No. 13, a VL CDR2 of the sequence shown in SEQ ID No. 14 and a VL CDR3 of the sequence shown in SEQ ID No. 15.
3. The antibody of claim 1, wherein the antibody comprises:
a light chain variable region (VL) comprising 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said light chain variable region having a VL CDR1 of the sequence shown in SEQ ID No. 8, a VL CDR2 of the sequence shown in SEQ ID No. 9 and a VL CDR3 of the sequence shown in SEQ ID No. 10;
and/or
A heavy chain variable region (VH) comprising 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system, said heavy chain variable region having a VL CDR1 of the sequence shown in SEQ ID No. 18, a VL CDR2 of the sequence shown in SEQ ID No. 19 and a VL CDR3 of the sequence shown in SEQ ID No. 20.
4. An antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 2; a kind of electronic device with high-pressure air-conditioning system
Heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 7.
5. An antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain consisting of the sequence shown in SEQ ID NO. 1; a kind of electronic device with high-pressure air-conditioning system
And a heavy chain consisting of the sequence shown in SEQ ID NO. 6.
6. An antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain variable region (VL); the light chain variable region consists of a sequence shown in SEQ ID NO. 12; a kind of electronic device with high-pressure air-conditioning system
Heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO. 17.
7. An antibody that specifically binds to mouse interleukin 12, the antibody comprising:
a light chain consisting of the sequence shown in SEQ ID NO. 11; a kind of electronic device with high-pressure air-conditioning system
A heavy chain consisting of the sequence shown in SEQ ID NO. 16.
8. The antibody of any one of claims 1 to 7, wherein the antibody is a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof, which substantially retains its biological function derived from the aforementioned monoclonal antibody.
9. A conjugate comprising the antibody of any one of claims 1-8, and a detectable label attached to the antibody.
10. A kit for detecting mouse interleukin 12, the kit comprising: an antibody according to any one of claims 1 to 8 or a conjugate according to claim 9.
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