Monoclonal antibody binding to human caveolin-1 and its application

Abstract

The present application relates to the technical field of human microcystin-1, and specifically to monoclonal antibodies binding to human microcystin-1 and their applications. The embodiment successfully developed a rabbit monoclonal antibody with high affinity against human microcystin-1 through single B cell screening and culture technology, which has high affinity and specificity for human microcystin-1, and the monoclonal antibody can be used to prepare immunoblotting kits and immune combination kits for detecting human microcystin-1. The monoclonal antibody also has important clinical value for the diagnosis and treatment of human microcystin-1 and related diseases.

Description

Technical Field

[0001] The present application relates to the technical field of human microcystin-1, and in particular to a monoclonal antibody binding to human microcystin-1 and its application. Background Art

[0002] Caveolin-1 is a transmembrane scaffold protein and the main structural component of the plasma membrane microvesicles. It participates in various complex cellular physiological processes and is interconnected with a variety of membrane structures, including the endoplasmic reticulum and the Golgi apparatus. After synthesis, caveolin-1 is inserted into the endoplasmic reticulum and transported to the Golgi apparatus and the cell surface to form pits. Caveolin-1 can be recovered from the plasma membrane in the opposite order. Human caveolin-1 (Homo sapiens caveolin-1) consists of 178 amino acids, with an oligomerization region and a CSD region (Caveolin scaffolding region). The conserved region between amino acids 60-70 is necessary for caveolin-1 to leave the endoplasmic reticulum, and the amino acids between 71-80 regulate the caveolin-1 oligomer to the Golgi apparatus. Phosphorylation at serine 80 converts caveolin-1 into a soluble secretory protein, which directly affects and regulates the activity of signal molecules through CSD.

[0003] As a scaffold protein, Caveolin-1 can increase or inhibit the activity of plasma membrane microvesicle-related proteins, inhibit cell migration or promote tumor formation, regulate cell apoptosis signaling pathways, affect growth and proliferation during the cell cycle, negatively or positively regulate angiogenesis in different tissues and under different conditions, and has certain anti-inflammatory activity. Therefore, Caveolin-1, as a member of the caveolin family closely related to tumors, has attracted widespread attention. Its function and its relationship with cancer are current research hotspots. Although scientists have different opinions on the role and function of Caveolin-1 in different cancers and at different stages of cancer, they all agree that it is crucial to the malignant phenotype transformation of cancer and is a target protein for cancer treatment.

[0004] Therefore, developing an antibody against human Caveolin-1 is of great significance for detecting human Caveolin-1 protein. Summary of the invention

[0005] The present application provides a class of rabbit monoclonal antibodies with high affinity to human microcystin-1 to solve one of the above technical problems to a certain extent. The embodiment successfully developed a rabbit monoclonal antibody with high affinity against human microcystin-1 through single B cell screening and culture technology, which has high affinity and specificity to human microcystin-1. Therefore, the monoclonal antibody can be used to prepare a kit for detecting the level of human microcystin-1. The monoclonal antibody provided in the present application has important clinical value for the diagnosis and treatment of human microcystin-1 and related diseases.

[0006] To this end, this application discloses at least the following technical solutions:

[0007] In a first aspect, the embodiment discloses a monoclonal antibody, which specifically binds to human microcystin-1, and the monoclonal antibody comprises:

[0008] 3 complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1 as set forth in SEQ ID NO:5, VL CDR2 as set forth in SEQ ID NO:6, and VL CDR3 as set forth in SEQ ID NO:7;

[0009] and / or

[0010] Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO:8, VH CDR2 as shown in SEQ ID NO:9, and VH CDR3 as shown in SEQ ID NO:10.

[0011] In a second aspect, the embodiments disclose a monoclonal antibody that specifically binds to human microcystin-1, wherein the monoclonal antibody comprises: a light chain variable region (VL) having a sequence as shown in SEQ ID NO:2 and a heavy chain variable region (VH) having a sequence as shown in SEQ ID NO:7.

[0012] In a third aspect, the embodiments disclose a monoclonal antibody that specifically binds to human microcystin-1, wherein the monoclonal antibody comprises: a light chain having a sequence as shown in SEQ ID NO:1 and a heavy chain having a sequence as shown in SEQ ID NO:6.

[0013] In a fourth aspect, the embodiments disclose an immunoblotting detection kit for detecting human microcystin-1, the kit comprising: the monoclonal antibody described in any one of the first to third aspects.

[0014] In the fifth aspect, the embodiment discloses an immunohistochemical detection kit for detecting human microcystin-1, the kit comprising: the monoclonal antibody described in any one of the first to third aspects.

[0015] In the sixth aspect, the embodiments disclose the use of the monoclonal antibody described in any one of the first to third aspects in the preparation of a reagent or a kit for detecting human microcystin-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The purification diagram of Caveolin-1 protein His tag detected by SDS-PAGE gel provided in the embodiment; Lane 1 and Lane 2 are 0.4 mg / mL and 0.2 mg / mL BSA respectively; Lane 3 is a marker; Lane 4 is supernatant ① (abbreviated as "Upper 1"); Lane 5 is the flow-through during the purification process (abbreviated as "FT"); Lane 6 is a 2-fold dilution of the collected Caveolin-1 protein (abbreviated as "*2"); Lane 7 is a 10-fold dilution of the collected Caveolin-1 protein (abbreviated as "*10").

[0017] Figure 2 Schematic diagram of the structure of the expression vector containing the heavy chain constant region of the rabbit monoclonal antibody constructed in the embodiment. In the figure, pBR322 origin and f1origin are replication promoters in Escherichia coli (E.Coli), Ampcillin is a plasmid resistance gene, CMV immearly promoter is a promoter in eukaryotes, SV40 PAterminator is a tailing signal, and Heavy chain constant is the nucleotide sequence of the heavy chain constant region of the rabbit monoclonal antibody.

[0018] Figure 3 Schematic diagram of the structure of the expression vector containing the rabbit monoclonal antibody light chain constant region constructed in the embodiment. In the figure, pBR322 origin and f1origin are replication promoters in Escherichia coli (E.Coli), Ampcillin is a plasmid resistance gene, CMV immearly promoter is a promoter in eukaryotes, SV40 PAterminator is a tailing signal, and Light chain constant is the nucleotide sequence of the rabbit monoclonal antibody light chain constant region.

[0019] Figure 4 The results of the immunoblotting kit constructed with the rabbit monoclonal antibody provided in the example for detecting Caveolin-1 in the cell sample HeLa.

[0020] Figure 5The detection results of Caveolin-1 in mouse lung tissue and rabbit lung tissue by the immunoblotting kit constructed with the rabbit monoclonal antibody provided in the example; "Mouse lung" is a mouse lung cell sample with high expression of Caveolin-1 from 6-8 week old c57bl / 6 mice; "Rat lung" is a mouse lung cell sample with high expression of Caveolin-1 from SD rats.

[0021] Figure 6 This is a staining localization diagram of Caveolin-1 in human lung samples detected by the immunohistochemistry kit constructed with the rabbit monoclonal antibody provided in the examples of the present application.

[0022] Figure 7 This is a staining localization diagram of Caveolin-1 in human colon samples using an immunohistochemistry kit constructed using the rabbit monoclonal antibody provided in the examples of the present application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0024] Explanation of terms

[0025] In this application, the term "antibody" should be interpreted in the broadest sense, including various antibody structures, including but not limited to Y-shaped antibodies, so-called full-length antibodies, antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications thereof. Among them, "antigen-binding portion" refers to one or more parts or fragments of a Y-shaped antibody that can retain the ability of the antibody to specifically bind to human microcystin-1.

[0026] In the present application, the term "monoclonal antibody" (mAb) includes a highly homogeneous antibody population with substantially identical antigenic determinants. That is, in this antibody population, individual antibodies are substantially identical, except for a small amount of mutations that may occur naturally. Monoclonal antibodies can show a single binding specificity and affinity for a specific epitope on an antigen. Compared with polyclonal antibodies that usually contain antibodies for different epitopes, each monoclonal antibody can be directed to the same or substantially the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and cannot be interpreted as an antibody that needs to be made by any specific method. The antibody can be prepared by a variety of methods, including but limited to hybridoma method, recombinant DNA method, phage antibody library, B cell screening and cultivation and similar methods.

[0027] In the present application, the modifier "rabbit" in the terms "rabbit antibody", "anti-human microcystin-1 rabbit monoclonal antibody", "rabbit monoclonal antibody to human microcystin-1" or similar terms indicates that the complementary determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody to human microcystin-1 may comprise the CDR and framework region (FR) of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit antibody or rabbit monoclonal antibody to human microcystin-1 may comprise the CDR of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody to human microcystin-1 may be an antibody whose CDR region is derived from a rabbit immunoglobulin sequence and whose FR is derived from the germline immunoglobulin sequence of other mammals (such as mouse or human). The term "rabbit monoclonal antibody to human microcystin-1" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, mutations introduced by random mutations or site-specific mutations in vitro, or by somatic mutations in vivo.

[0028] In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (L). Antibody light chains can be classified as κ (kappa) and λ (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 sequences of about 110 amino acids near the N-terminus of the heavy and light chains vary greatly, while the sequences of the other parts are relatively constant. Therefore, the regions of the light and heavy chains where the amino acid sequences near the N-terminus vary greatly are called variable regions (V), accounting for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the regions where the amino acid sequences near the C-terminus are relatively stable are called constant regions (C), accounting for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0029] The V region of the heavy chain and the V region of the light chain are called VH and VL, respectively. VH and VL each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions (HVR) or complementarity determining regions (CDR), including HVR1 (CDR1), HVR2 (CDR2) and HVR3 (CDR3), among which HVR3 (CDR3) has a higher degree of variation. Generally, VH includes three complementary determining regions VH CDR1, VH CDR2 and VH CDR3, and VL includes three complementary determining regions VL CDR1, VL CDR2 and VL CDR3. The three complementary determining regions of VH and VL constitute the antigen-binding site of the antibody alone or together, 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 region outside the CDR are relatively conservative, called the framework region (FR). VH or VL each has four framework regions, represented by FR1, FR2, FR3 and FR4. By replacing or changing the framework regions, multiple antibodies with the same specificity can be produced while ensuring that the site of antibody recognition and antigen binding remains unchanged.

[0030] The C region of the heavy chain and light chain is called CH and CL respectively. The length of CL of different types (κ or λ) of Ig is basically the same, but the length of CH of different classes of Ig is different. For example, IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4.

[0031] In the present application, the term "framework region" or "framework region" residues refers to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0032] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a specific 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 the antigen.

[0033] Antibody

[0034] The embodiments of the present application disclose an antibody that specifically binds to human microcystin-1.

[0035] In some embodiments, the antibody comprises complementary determining regions (CDRs) of the light chain variable region (VL) defined according to the three Kabat numbering systems: VL CDR1, VL CDR2, and VL CDR3. VL CDR1 is shown in SEQ ID NO:5, VL CDR2 is shown in SEQ ID NO:6, and VL CDR3 is shown in SEQ ID NO:7.

[0036] In some embodiments, the antibody comprises three complementary determining regions (CDRs) of the heavy chain variable region (HL) defined according to the Kabat numbering system: VH CDR1, VH CDR2 and VH CDR3. VL CDR1 is shown in SEQ ID NO:8, VL CDR2 is shown in SEQ ID NO:9, and VL CDR3 is shown in SEQ ID NO:10.

[0037] In some embodiments, the antibody comprises complementary determining regions (CDRs) of the light chain variable region (VL) defined according to the three Kabat numbering systems: VL CDR1, VL CDR2 and VL CDR3, and complementary determining regions (CDRs) of the heavy chain variable region (HL) defined according to the three Kabat numbering systems: VH CDR1, VH CDR2 and VH CDR3. VL CDR1 is shown in SEQ ID NO: 5, VLCDR2 is shown in SEQ ID NO: 6, and VL CDR3 is shown in SEQ ID NO: 7. VL CDR1 is shown in SEQ ID NO: 8, VLCDR2 is shown in SEQ ID NO: 9, and VL CDR3 is shown in SEQ ID NO: 10.

[0038] In certain embodiments, the anti-human microcystin-1 antibody may have a Y-shaped molecular structure. In one embodiment, the anti-human microcystin-1 antibody may include a pair of heavy chains and a pair of light chains. The heavy chain may include a heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally include five types of heavy chains: γ, δ, α, μ and ε, and the corresponding antibodies are called IgG, IgD, IgA, IgM and IgE. The light chain may be a polypeptide subunit that is smaller than the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is usually the N-terminal portion of the light chain, which shows higher variability in the amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, both the heavy chain variable region VH and the light chain variable region VL can be used to recognize and bind to human microcystin-1.

[0039] In some embodiments, the CDRs in the VL and VH regions may be separated from each other by FRs. FRs are conserved regions in the sequence structure. FRs can generally serve as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., human microcystin-1). The three-dimensional structure of FRs can be conserved in different antibodies.

[0040] Antibody preparation

[0041] The monoclonal antibodies provided in the embodiments of the present application can be prepared by a variety of methods, including but not limited to the hybridoma method, recombinant DNA method, phage antibody library, single B cell screening and culture, and similar methods.

[0042] In some embodiments, the preparation process of the monoclonal antibody includes: immunizing New Zealand white rabbits with recombinantly expressed human microcystin-1 as an immunogen; sorting antigen-specific B lymphocytes from spleen cells of the white rabbits; screening positive B lymphocytes that can secrete specific antibodies after antibody function verification; amplifying to obtain antibody heavy chain and light chain genes of positive B lymphocytes, and then recombinantly expressing them to obtain rabbit monoclonal antibodies against human MSH2.

[0043] In some embodiments, the method for preparing human microcystin-1 comprises:

[0044] (1) Expression plasmid construction

[0045] A plasmid containing the human Caveolin-1 CDS sequence (Thermo Fisher Scientific cDNA / ORFLibrary-Plate Map(s)) was used as a template, and PCR amplification was performed using primers Caveolin-1-Primer-F (shown in SEQ ID NO.11) and Caveolin-1-Primer-F (shown in SEQ ID NO.12) to express recombinant human Caveolin-1 (shown in SEQ ID NO.13) consisting of amino acids 1 to 80 at the N-terminus of the human Caveolin-1 protein.

[0046] Among them, the PCR reaction system: 1μL template, 1μL forward primer (10mM), 1μL reverse primer (10mM), 25μL 2×Gloria Hi-Fi PCR Master Mix with GC Buffer (Wuhan Aibotek Biotechnology Co., Ltd.), 22μL N.F H2O. PCR amplification program: 98℃ pre-denaturation for 30s, followed by 30 cycles of 98℃ for 10s, 64℃ for 30s, and 72℃ for 30s, and finally kept at 72℃ for 5min, and the obtained reaction solution was stored at 4℃. After the amplified PCR product was purified, it was loaded on the expression vector by homologous recombination. The prokaryotic expression vector used was pGEX-4T-AB1 (commercial transformation vector). After verification by sequencing (Jinkairui Biotechnology Co., Ltd.), the plasmid was preserved to obtain the expression plasmid for expressing human Caveolin-1.

[0047] (2) Recombinant expression of proteins

[0048] The expression plasmid for expressing human Caveolin-1 was transformed into E. coli Rosetta strain and incubated on LB agar plates (containing 75 μg / mL ampicillin) at 37° C. overnight to obtain several single colony transformants.

[0049] Inoculate a single colony transformant into 2 mL LB culture medium (75 μg / mL ampicillin) and culture at 37°C and 220 rpm until the OD600nm of the culture solution is 0.4-0.6. Next, transfer 2 mL of the culture of each strain to 400 mL LB expression medium and culture again at 37°C and 220 rpm until the OD600nm of the culture solution reaches about 0.45-0.55, add 0.8 mM IPTG, continue to induce at 37°C for 3-4 hours, centrifuge at 4000 rpm for 10 minutes, and take the precipitate as the bacterial body.

[0050] (3) Purification of recombinant protein

[0051] Resuspend the cells with 30 mL of lysis solution (50 mM Tris-300 mM NaCl) and fix in an ice box. Select a variable amplitude rod and place it in the lysis chamber. Set the power to 350 W, lysis time to 3 s, interval time to 3 s, count down to 5 min, then place in an ice-water mixture to cool for 5 min, and repeat the above steps to lysis for 5 min. After lysis, centrifuge at 9000 rpm for 10 min to obtain supernatant ① and precipitate.

[0052] Recombinant Caveolin-1 was extracted from the supernatant ① by affinity chromatography using Ni affinity chromatography resin. The purification results are shown in Figure 1As shown, recombinant Caveolin-1 with a purity of 80% was obtained, which contained Caveolin-1 protein fragment and glutathione S-transferase (GST) tag.

[0053] In some embodiments, the method for preparing a monoclonal antibody against human caveolin-1 comprises:

[0054] (1) Animal immunization

[0055] The recombinant human Caveolin-1 protein prepared in the above embodiment was used as an immunogen, and each New Zealand white rabbit was immunized with 200 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, and multiple injections were made subcutaneously in the rabbit's abdomen and back. After the first immunization, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier every 3 weeks, and multiple injections were made subcutaneously in the rabbit's abdomen and back, and booster immunization was performed twice. After three immunizations, rabbit serum samples were collected, and the titer against human Caveolin-1 protein was determined by ELISA. The serum was diluted 1:243000 and the titer was measured by ELISA. Rabbits with OD450nm exceeding 0.2 were taken, and 200 μg of the immunogen was injected subcutaneously at multiple points for booster immunization once, and the spleen was taken 3 days later.

[0056] (2) Isolation of splenocytes

[0057] Under a sterile environment, the rabbit spleen obtained above was washed with basal culture medium, excess connective tissue and fat were cut off, and the cells were ground in a cell screen. The grinding liquid (i.e., the intramembrane cells) was collected, centrifuged at 400g for 5 minutes at room temperature, the supernatant was removed, the cells were retained, 13mL of normal temperature RBC lysing solution (purchased from BioGems) was added, and the erythrocytes were lysed for 1 minute. 37mL of basal culture medium was added, and the erythrocytes were lysed. Centrifuged at 400g for 5 minutes at room temperature, the supernatant was removed, 40mL of normal temperature basal culture medium was added, and the cell clusters were gently blown away with a pipette to resuspend the cells. Centrifuged at 400g for 5 minutes at room temperature again, the supernatant was removed, the cells were retained, 20mL of normal temperature basal culture medium was added, and the cell clusters were gently blown away with a pipette to resuspend the cells; the resuspended cells were filtered again through a cell screen to remove the clumping cells, and then the cells were counted.

[0058] (3) B lymphocyte sorting and culture

[0059] B lymphocyte sorting and culture: see the patent "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A)" and the patent "A B lymphocyte in vitro culture system and application (Publication No.: CN111518765A)".

[0060] (4) Cloning of the gene encoding rabbit monoclonal antibody

[0061] The supernatant of cultured B cells was used to identify positive clones by antigen-coated ELISA. After the cells of the positive clones were collected and lysed, RNA was extracted using the Quick-RNATM MicroPrep kit (purchased from ZYMO) and reverse transcribed into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of the corresponding positive clones by PCR, and several clones were selected for sequencing.

[0062] The PCR reaction system was as follows: 4 μL cDNA, 1 μL, 10 mM forward primer, 1 μL, 10 mM reverse primer, 12.5 μL 2×Gloria HiFi (provided by Wuhan Abotek Biotechnology Co., Ltd.), 6.5 μL N.F H2O. PCR amplification procedure: 98°C pre-denaturation for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally kept at 72°C for 5 min. The resulting reaction solution was stored at 4°C.

[0063] Among them, the light chain variable region primer pair is: VL-Primer-F: SEQ ID NO: 14; VL-Primer-R: SEQ ID NO: 15.

[0064] The heavy chain variable region primer pair is: VH-Primer-F: SEQ ID NO: 16; VH-Primer-R: SEQ ID NO: 17.

[0065] (5) Construction of heavy chain and light chain expression vectors

[0066] The heavy chain genes and light chain genes of several rabbit monoclonal antibodies selected in the above steps were loaded onto expression vectors respectively. The mammalian expression vector pBR322 used was shown in Figure 2 and Figure 3 Among them, the selected light chain coding gene is shown in SEQ ID NO.18, and the selected light chain variable region coding gene is shown in SEQ ID NO.19. The selected heavy chain coding gene is shown in SEQ ID NO.20, and the selected heavy chain variable region coding gene is shown in SEQ ID NO.21.

[0067] (6) Monoclonal antibody preparation and purification

[0068] The constant region of the heavy chain of rabbit monoclonal antibody ( Figure 2 ) and the light chain constant region ( Figure 3) mammalian cell (such as CHO) expression vectors were conventionally linearized with NheI, XbaI and XbaI, BamHI restriction endonucleases respectively.

[0069] After purifying the PCR product amplified in the above step, the heavy chain variable region gene and the light chain variable region gene are constructed into the corresponding mammalian expression vector by homologous recombination; after sequencing verification, the expression vector containing the corresponding rabbit monoclonal antibody light chain gene and heavy chain gene are transfected into 293F cells together, and the transfection is carried out for 72 to 96 hours. The obtained transformants are continuously cultured, and the culture fluid is collected. The rabbit monoclonal antibody that recognizes human Caveolin-1 is purified and harvested from the supernatant by centrifugation using protein A affinity gel resin, and the purity of the antibody is verified by 12% SDS-PAGE gel electrophoresis. The purified rabbit monoclonal antibody of human Caveolin-1 protein is subjected to amino acid sequencing, and the purified rabbit monoclonal antibody is stored at -20°C for future use.

[0070] As a result, the amino acid sequence of the light chain of the obtained rabbit monoclonal antibody is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain is shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, the amino acid sequence of the light chain complementary determining region VL CDR1 is shown in SEQ ID NO.5, the amino acid sequence of the light chain complementary determining region VL CDR2 is shown in SEQ ID NO.6, the amino acid sequence of the light chain complementary determining region VL CDR3 is shown in SEQ ID NO.7, the amino acid sequence of the heavy chain complementary determining region VH CDR1 is shown in SEQ ID NO.8, the amino acid sequence of the heavy chain complementary determining region VH CDR2 is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain complementary determining region VH CDR3 is shown in SEQ ID NO.10. In addition, the light chain constant region of the rabbit monoclonal antibody is a κ chain, and the heavy chain constant region is an IgG1 type.

[0071] In some detection examples, Hela cells (cell strain of the Cell Bank of the Chinese Academy of Sciences) that highly express Caveolin-1 were used to detect the specificity of the rabbit monoclonal antibody against the human Caveolin-1 protein prepared in the above examples. Hela cells (cell strain of the Cell Bank of the Chinese Academy of Sciences) that highly express Caveolin-1 were taken, and the recognition specificity of the monoclonal antibody was detected by immunoblotting, and 7% polyacrylamide gel electrophoresis was performed. The gel protein band was transferred to a PVDF membrane in an electrotransfer system according to conventional methods. The membrane was placed in a TBST blocking solution containing 3% skimmed milk powder and incubated at room temperature for 1 hour, and the Caveolin-1 rabbit monoclonal antibody provided in the example (1:10000 dilution) was added, and incubated at 4°C overnight. After washing the membrane with TBST, a 1:10000 diluted goat anti-rabbit secondary antibody (purchased from Jackson ImmunoResearch, catalog number 111-035-045) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST again, and ECL color developing solution was added for development, with an exposure time of 90 seconds. See the results. Figure 4 The full length of Caveolin-1 is 178aa, and the theoretical molecular weight is 22kd. The screened rabbit monoclonal antibody against human Caveolin-1 protein has good specificity and detects specific target bands.

[0072] In some detection examples, mouse lung cell samples (6-8 week old c57bl / 6 mice and SD rats) that highly express Caveolin-1 were used, and the recognition specificity of the monoclonal antibody was detected by immunoblotting, and 7% polyacrylamide gel electrophoresis was performed. The gel protein band was transferred to a PVDF membrane in an electrotransfer system according to conventional methods. The membrane was placed in a TBST blocking solution containing 3% skimmed milk powder and incubated at room temperature for 1 hour, and the Caveolin-1 rabbit monoclonal antibody provided in the example (1:10000 dilution) was added, and incubated at 4°C overnight. After washing the membrane with TBST, a 1:10000 diluted goat anti-rabbit secondary antibody (purchased from Jackson ImmunoResearch, catalog number 111-035-045) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST again, and ECL color developing solution was added for development, with an exposure time of 10 seconds. See the results. Figure 5 The full length of Caveolin-1 is 178aa, and the theoretical molecular weight is 20kd. The screened rabbit monoclonal antibody against human Caveolin-1 protein has good specificity, and specific target bands were detected (the two bands are two splice forms of Caveolin-1, and the post-translational modifications after phosphorylation and lipidation are 24kD and 21kD).

[0073] In some detection cases, rabbit monoclonal antibodies against human caveolin-1 protein were used as primary antibodies, and immunohistochemical staining was used to qualitatively detect caveolin-1 in human lung samples and human colon samples. The specific process includes:

[0074] (1) Chip selection:

[0075] Immunohistochemistry chip, catalog number TMA000-42, Bios Corporation.

[0076] (2) IHC staining and analysis

[0077] Baking slices: Place the paraffin slices (human lung samples and human colon samples) on the slice rack in the same direction, and bake them in a 56°C constant temperature box for 30 minutes; at the same time, put the dewaxing liquid 1 cylinder into the 56°C constant temperature box; Dewaxing to water: Put the paraffin slices together with the slice rack into the dewaxing liquid 1 cylinder, and then take them out of the constant temperature box and place them at room temperature. After 5 minutes, take out the slices and immerse them in the room temperature dewaxing liquid 2 cylinder, and put the paraffin slices into the cylinders in the order of dewaxing liquid 2, dewaxing liquid 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. Each cylinder of the dewaxing liquid reagent cylinder is 5 minutes, and each cylinder of the anhydrous ethanol reagent cylinder is 3 minutes; wash the slices with running water for 3 minutes.

[0078] Antigen retrieval: 0.01 M sodium citrate retrieval solution (pH 6.0) with high pressure heat retrieval.

[0079] Inactivation of endogenous peroxidase: Wash with PBS buffer three times, 1 min each time, to remove the buffer on the slices; immerse the slices completely in 3% hydrogen peroxide solution, incubate at room temperature for 10 min.

[0080] Blocking: Wash with PBS buffer 3 times, 3 minutes each time, and remove the buffer on the slice; circle the tissue area to be tested on the slide with an immunohistochemistry pen, and add blocking solution - PBS blocking solution in the circled area; place the slice horizontally in an incubation wet box with water at the bottom, incubate at room temperature for 30 minutes, and start timing from the addition of the blocking solution.

[0081] Primary antibody incubation: remove the blocking solution, add the primary antibody diluted with PBS buffer (antibody dilution ratio is 1:150) on the tissue section, place it horizontally in an incubation wet box, and incubate it at room temperature for 60 minutes; remove the antibody working solution, rinse quickly once with PBS buffer, and soak and wash three times with buffer PBS, each time for 3 minutes; during the soaking and washing period, it is necessary to repeatedly pull up and down several times (primary antibody dilution ratio: 1:150).

[0082] Secondary antibody incubation: Add ready-to-use secondary antibody working solution (Dako REAL EnVision Detection System, Peroxidase / DAB, Rabbit / Mouse, HRP; Dako) to the tissue sections and place them horizontally in an incubation humidified box for incubation at room temperature for 25 minutes; remove the reagents on the sections, quickly rinse once with buffer PBS, and soak and wash three times with buffer PBS, each time for 3 minutes; during the soaking and washing period, lift up and down repeatedly

[0083] Color development: Add color developing solution to the tissue section, observe the color change closely under the microscope, and after obtaining the appropriate staining intensity, immerse the section in a large amount of distilled water to stop the color development. After the color development is stopped, wash the section in running water for 10 minutes.

[0084] Counterstaining: Immerse the slightly drained sections in Mayer's hematoxylin for counterstaining for 1 minute. After counterstaining, wash with running water for 3 minutes.

[0085] Bluing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, and then rinse with running water for 3 minutes;

[0086] Dehydration: Soak the washed slices in anhydrous ethanol once, lift them up and down several times during the soaking period, and take them out after 10 seconds; place them in a constant temperature air drying oven at high temperature (54-58°C) to completely dry;

[0087] Sealing: Add an appropriate amount of neutral gum to the center of the slice and cover it with a coverslip. The amount of glue added should be appropriate. After adding the coverslip, the tissue must be completely covered and no glue should overflow from the slice for scanning.

[0088] It should be noted that immunohistochemical staining results are divided into: positive and negative. Positive expression must be in specific antigenic sites of cells and tissues to be considered positive. Immunohistochemical staining was performed using the anti-human Caveolin-1 rabbit monoclonal antibody prepared in the above example as the primary antibody. The results are shown in FIG. Figure 6 , Figure 7 As shown. The staining is clear without nonspecific staining and the background is clean. Combined with the results of immunoblotting, it shows that the anti-human Caveolin-1 rabbit monoclonal antibody specifically recognizes human Caveolin-1 protein without nonspecific staining, effectively avoiding false positive results.

[0089] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A monoclonal antibody that specifically binds to human microcystin-1, comprising: three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1 as set forth in SEQ ID NO:5, VL CDR2 as set forth in SEQ ID NO:6, and VL CDR3 as set forth in SEQ ID NO:7; and Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO:8, VH CDR2 as shown in SEQ ID NO:9, and VH CDR3 as shown in SEQ ID NO:

10.

2. A monoclonal antibody that specifically binds to human microcystin-1, the monoclonal antibody comprising: The light chain variable region (VL) of the sequence shown in SEQ ID NO: 3; and The heavy chain variable region (VH) has the sequence shown in SEQ ID NO:

4.

3. A monoclonal antibody that specifically binds to human microcystin-1, the monoclonal antibody comprising: The light chain of the sequence shown in SEQ ID NO: 1; and The heavy chain has the sequence shown in SEQ ID NO:

2.

4. A nucleic acid molecule carrying a gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. A host cell carrying the nucleic acid molecule according to claim 4 and capable of autonomously expressing the monoclonal antibody according to any one of claims 1 to 3.

6. A kit for detecting human microcystin-1, comprising: The monoclonal antibody according to any one of claims 1 to 3.

7. The kit according to claim 6, wherein the kit is selected from the group consisting of an immunohistochemistry kit, an immunoblotting kit, and an immunoprecipitation kit.

8. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent or kit for detecting human microcystin-1.

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