Anti-human SMMHC rabbit monoclonal antibody as well as antigen binding fragment, preparation method and application thereof

Abstract

The invention relates to the technical field of immune globulin, in particular to an anti-human SMMHC monoclonal antibody and an antigen binding fragment, a preparation method and application thereof, the anti-human SMMHC monoclonal antibody comprises a light chain variable region VL and a heavy chain variable region VH, and amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL are respectively shown as SEQ ID NO.3-5; the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 of the VH are as shown in SEQ ID NO.7 to SEQ ID NO.9. The anti-human SMMHC rabbit monoclonal antibody disclosed by the invention has good affinity, can be specifically combined with SMMHC protein, remarkably improves the specificity, sensitivity, accuracy and reliability of SMMHC protein immunodetection, is mainly used for identifying and marking smooth muscle cells, can be suitable for marking breast muscle epithelium, and has the advantages of high sensitivity and high sensitivity. The kit can be used for differential diagnosis of breast duct internal cancer and invasive cancer, and can also be used for diagnosis of smooth muscle-derived tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunoglobulin, in particular to an anti-human SMMHC rabbit monoclonal antibody and antigen-binding fragment thereof, a preparation method and application thereof. BACKGROUND

[0002] Myosin is a highly conserved protein and an important component of cytoskeleton. At present, 40 human myosin genes have been found, representing 12 super families (I-XII). Smooth muscle myosin belongs to class II myosin and is involved in muscle contraction and relaxation. It is a hexameric complex, including two heavy chains, two regulatory light chains and two essential light chains LC 17. Myosin heavy chain protein is encoded by MYH gene family and can be involved in myofilament connection and assembly through protease hydrolysis. Different MYH encodes different myosin heavy chains, for example, α-MyHC encoded by MYH6 gene is expressed in atrial muscle; β-MyHC encoded by MYH7 gene is expressed in ventricular muscle. Smooth muscle myosin heavy chain (SMMHC) encoded by MYH11 gene located at human chromosome 16p13.11 is specifically expressed in smooth muscle cells of all internal organs such as blood vessels, digestive tract, respiratory tract and urogenital tract, and is an important marker in the process of vascular smooth muscle cell contraction, playing an important role in muscle physiology and smooth muscle contraction function.

[0003] SMMHC is mainly expressed in smooth muscle cells of all internal organs such as blood vessels, digestive tract, respiratory tract and urogenital tract, and is one of the most reliable markers of mature smooth muscle cells. SMMHC can be used to label breast myoepithelial cells, for differential diagnosis of intraductal carcinoma and invasive carcinoma of the breast, and for diagnosis of smooth muscle-derived tumors.

[0004] However, different myosins encoded by MYH have high homology, so it is necessary to develop a monoclonal antibody specifically recognizing SMMHC to ensure that the antibody only binds to SMMHC and avoids cross-reaction with other proteins, thereby reducing false positive or false negative results and improving the accuracy of diagnosis. In addition, with the help of SMMHC monoclonal antibody, precise diagnosis of intraductal carcinoma and invasive carcinoma of the breast and smooth muscle-derived tumors can be made, providing reliable support for precise diagnosis, treatment and prognosis. SUMMARY

[0005] Therefore, the present application provides an anti-human SMMHC rabbit monoclonal antibody, a preparation method and application thereof, to provide a monoclonal antibody specifically binding to SMMHC, as well as a preparation method and application of the antibody, which significantly improves the specificity and sensitivity of SMMHC immunodetection and is suitable for labeling of smooth muscle.

[0006] In a first aspect, the present application provides an anti-human SMMHC rabbit monoclonal antibody or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region VH, The light chain variable region VL comprises a LCDR1, a LCDR2 and a LCDR3, The amino acid sequence of the LCDR1 is SEQ ID NO. 3 or a variant having one or two amino acid substitutions, deletions, or additions compared with SEQ ID NO. 3, The amino acid sequence of the LCDR2 is LVS or a variant having one or two amino acid substitutions, deletions, or additions compared with LVS, The amino acid sequence of the LCDR3 is SEQ ID NO. 5 or a variant having one or two amino acid substitutions, deletions, or additions compared with SEQ ID NO. 5; The heavy chain variable region VH comprises a HCDR1, a HCDR2 and a HCDR3, The amino acid sequence of the HCDR1 is SEQ ID NO. 7 or a variant having one or two amino acid substitutions, deletions, or additions compared with SEQ ID NO. 7, The amino acid sequence of the HCDR2 is SEQ ID NO. 8 or a variant having one or two amino acid substitutions, deletions, or additions compared with SEQ ID NO. 8, The amino acid sequence of the HCDR3 is SEQ ID NO. 9 or a variant having one or two amino acid substitutions, deletions, or additions compared with SEQ ID NO. 9; And, the anti-human SMMHC rabbit monoclonal antibody or antigen-binding fragment thereof can specifically bind to smooth muscle myosin heavy chain.

[0007] Further, the light chain variable region VL comprises an amino acid sequence having 95% or more homology compared with the amino acid sequence shown in SEQ ID NO. 2 obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; and the heavy chain variable region VH comprises an amino acid sequence having 95% or more homology compared with the amino acid sequence shown in SEQ ID NO. 6 obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.

[0008] Further, the amino acid sequence having 95%, 96%, 97%, 98% or 99% identity.

[0009] Further, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO. 2, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO. 6.

[0010] Further, the light chain variable region VL has a full length of 103 amino acids, the four domains of FR have amino acid numbers of 26, 17, 36 and 10 respectively, the three domains of LCDR have amino acid numbers of 6, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-93aa respectively, and the amino acid sequences are QSIYNN (SEQ ID NO. 3), LVS (SEQ ID NO. 4) and QAYSG (SEQ ID NO. 5) respectively.

[0011] Further, the heavy chain variable region VH has a full length of 111 amino acids, the four domains of FR have amino acid numbers of 25, 17, 39 and 11 respectively, the three domains of HCDR have amino acid numbers of 7, 7 and 5 respectively, the regions of HCDR1, HCDR2 and HCDR3 are 26aa-32aa, 50aa-56aa and 96aa-100aa respectively, and the amino acid sequences are GFSLSYT (SEQ ID NO. 7), INPSGNT (SEQ ID NO. 8) and SRRWG (SEQ ID NO. 9) respectively.

[0012] Further, the antigen binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv and bispecific antibody.

[0013] In a second aspect, the present application further provides a biological material selected from a polynucleotide, a vector or a cell, The polynucleotide encodes the anti-human SMMHC rabbit monoclonal antibody or the antigen binding fragment thereof according to the first aspect. The vector carries the polynucleotide. The cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-human SMMHC rabbit monoclonal antibody or the antigen binding fragment thereof according to the first aspect.

[0014] In a third aspect, the present application further provides a preparation method of the anti-human SMMHC rabbit monoclonal antibody or the antigen binding fragment thereof according to the first aspect, which comprises culturing the cell according to the second aspect, wherein the cell is prepared by transforming a polynucleotide encoding the anti-human SMMHC rabbit monoclonal antibody or the antigen binding fragment thereof into the cell, the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0015] Further, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

[0016] In a fourth aspect, the present application also provides the use of the anti-human SMMHC rabbit monoclonal antibody or antigen binding fragment thereof of the first aspect or the biological material of the second aspect, wherein the use is selected from one or more of the following, 1) detecting human SMMHC for non-diagnostic and therapeutic purposes; 2) preparing an immunoassay product for detecting human SMMHC; 3) detecting human intraductal carcinoma of the breast, invasive carcinoma and / or smooth muscle-derived tumor for non-diagnostic and therapeutic purposes; 4) preparing an immunoassay product for detecting human intraductal carcinoma of the breast, invasive carcinoma and / or smooth muscle-derived tumor.

[0017] In the present application, the use is for non-diagnostic and therapeutic purposes, which can be specifically researching the phenotypic transformation of smooth muscle cells in diseases such as atherosclerosis, hypertension and vascular restenosis, which can be using SMMHC as a key marker to verify the differentiation efficiency by immunostaining or flow cytometry when inducing pluripotent stem cells or mesenchymal stem cells to differentiate into smooth muscle cells, which can be evaluating the effect of drugs on smooth muscle cells by detecting the expression change of SMMHC when screening compounds for regulating smooth muscle function, and which can be exploring the potential of SMMHC as a disease-related biomarker by detecting the level of SMMHC in cell culture supernatant, tissue lysate or blood sample using antibodies.

[0018] In a fifth aspect, the present application also provides an immunoassay product of human SMMHC, wherein the immunoassay product comprises the anti-human SMMHC rabbit monoclonal antibody or antigen binding fragment thereof of the first aspect or the biological material of the second aspect.

[0019] Further, the immunoassay product is an enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM) and immunoprecipitation, etc. detection reagent or kit.

[0020] Further, the immunoassay product is an immunoassay kit, wherein the immunoassay kit comprises a primary antibody reagent, and the primary antibody reagent contains the anti-human SMMHC rabbit monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application.

[0021] Furthermore, the immunoassay kit also includes antigen retrieval solution, endogenous peroxidase inhibitor, hypersensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution; preferably, the hypersensitive secondary antibody reagent is a hypersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0022] Sixthly, this application also provides an SMMHC polypeptide immunogen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0023] In a seventh aspect, this application also provides the use of the SMMHC polypeptide immunogen described in the sixth aspect in the preparation of anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment.

[0024] Further, the application involves immunizing animals with purified SMMHC polypeptide immunogen, and isolating peripheral blood mononuclear cells (PBMCs) from the immunized animals; sorting the PBMCs using purified SMMHC polypeptide immunogen to obtain antigen-specific B cells; and culturing and screening the obtained B cells to obtain positive clones.

[0025] Compared with existing technologies, the anti-human SMMHC monoclonal antibody or its antigen-binding fragment provided in this application can specifically bind to human SMMHC protein, significantly improving the specificity, accuracy and reliability of SMMHC immunoassay. Secondly, using the anti-human SMMHC monoclonal antibody and kit of this application, the expression of SMMHC protein in smooth muscle cells can be clearly detected by IHC method, indicating that the anti-human SMMHC monoclonal antibody of this application can be well applied to the immunohistochemical detection of SMMHC protein in tissue cells. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of this application, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0027] Figure 1 The image shows the results of immunohistochemical detection of SMMHC protein expression in six normal tissues (liver, prostate, colon, placenta, pancreas, and kidney) as described in Example 5. The primary antibody used was the anti-human SMMHC rabbit monoclonal antibody OTIR5D12 described in this invention.

[0028] Figure 2 The image shows the results of immunohistochemical detection of SMMHC protein expression in breast and lung tissues as described in Example 5. The primary antibody used was the anti-human SMMHC rabbit monoclonal antibody OTIR5D12 described in this invention.

[0029] Figure 3The image shows the results of immunohistochemical detection of SMMHC protein expression in myocardium and skeletal muscle as described in Example 5. The primary antibody used was the anti-human SMMHC rabbit monoclonal antibody OTIR5D12 described in this invention.

[0030] Figure 4 The image shows the results of immunohistochemical detection of SMMHC protein expression in two cases of leiomyosarcoma as described in Example 5. The primary antibody used was the anti-human SMMHC rabbit monoclonal antibody OTIR5D12 described in this invention. Detailed Implementation

[0031] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Unless otherwise specified, the test reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods unless otherwise specified.

[0032] Example 1: Preparation of anti-human SMMHC rabbit monoclonal antibody Step 1: Preparation of Immunogen The sequence and structural characteristics of SMMHC were analyzed using UniProt and DNAStar sequence analysis software, including hydrophilicity / hydrophobicity, three-dimensional structural features, family sequence homology, and homology between human and rabbit sequences. The amino acid sequence of the synthesized SMMHC protein from position 1932 to 1951 (SEQ ID NO.1) was selected as the SMMHC polypeptide immunogen for the development of monoclonal antibodies. The immunogen SMMHC polypeptide was synthesized by Zhongtai Biochemical Co., Ltd.

[0033] Step 2, Animal Immunization The synthesized SMMHC peptide was used as an immunogen in immunized animals, with New Zealand white rabbits selected as experimental animals. The peptide (800 μg / rabbit) was thoroughly mixed with an adjuvant and injected into the animals. Two weeks later, a second immunization was performed at a dose of 400 μg / rabbit. A third immunization was then performed two weeks later at the same dose. Two weeks later, blood was collected from the ear vein, and serum titers were determined using serial dilutions with ELISA and IHC. The absorbance at 128,000 ELISA titers and the staining signal intensity in colon and kidney tissues detected by IHC were used as criteria to determine whether an immune response was generated in the immunized animals and whether the titer reached the level required for PBMC collection.

[0034] Step 3: PBMC isolation, specific B cell sorting, and clonal recombination Heart blood was collected from New Zealand white rabbits with serum titers meeting the standard using a 50mL syringe for the collection and separation of peripheral blood mononuclear cells (PBMCs). The specific procedure was performed according to the instructions for the lymphocyte separation reagent. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., the mononuclear cell layer), a separation solution layer, and a red blood cell layer. The mononuclear cell layer was aspirated and washed with PBS to remove platelets and lymphocyte separation solution, yielding rabbit PBMCs.

[0035] The SMMHC naked peptide synthesized in step 1 was coupled with magnetic beads, and the PBMCs obtained above were mixed with it. Antigen-specific B cells were sorted by magnetic rack and cultured. The synthesized SMMHC naked peptide was used to screen B cell culture supernatant by ELISA. The ELISA-positive B cell supernatant was tested by IHC in colon and kidney tissues. Clones that were positive in smooth muscle cells of colon and kidney tissues were selected. OTIR5D12 was the optimal clone after comparative analysis.

[0036] Cells corresponding to OTIR5D12 were lysed with lysis buffer, and the lysis products were collected. RNA was extracted from the lysate and reverse transcribed into cDNA. The full-length light and heavy chains of the naturally paired rabbit monoclonal antibody were amplified from the cDNA. The rabbit monoclonal antibody expression vector was constructed by clonal recombination.

[0037] Step 4: Preparation and purification of monoclonal antibodies The recombinant plasmid was constructed by inserting the heavy and light chain gene fragments encoding the rabbit monoclonal antibody OTIR5D12 into an expression vector using molecular cloning technology. The recombinant plasmid was then transfected into HEK293 cells using transfection reagents. Five to six days after transfection, the presence of the rabbit monoclonal antibody against human SMMHC in the culture supernatant was detected by ELISA and IHC (the specific procedure is the same as in step 3). The supernatant was collected by centrifugation and purified by affinity chromatography. A rabbit monoclonal antibody specifically recognizing human SMMHC was obtained. The concentration of the purified monoclonal antibody was determined by the BCA method, and the antibody concentration was found to be 5.4 mg / mL.

[0038] Example 2: Variable region gene and amino acid sequence analysis of anti-human SMMHC monoclonal antibody Using the recombinant plasmid of the SMMHC monoclonal antibody OTIR5D12 as a DNA template, the variable region light and heavy chain gene sequences of the antibody were amplified using 5' RACE (Rapid Amplification of cDNA Ends) technology. For detailed experimental procedures, please refer to the Takara Bio USA SMARTer® RACE 5' / 3' Kit user manual.

[0039] Based on the described antibody subtypes, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of their Ig and Kappa constant regions were designed. The primer sequences are as follows: pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 10) pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 11) The light and heavy chain gene fragments of the antibody were amplified by RACE and then inserted into the pUC119 cloning vector using an enzyme digestion-ligation method. After transformation, positive clones were preliminarily identified by blue-white screening. Subsequently, recombinant plasmids were extracted and sequenced using the Sanger method. An ABI 3730 sequencer was used, and the universal primers M13F and M13R were used for sequencing.

[0040] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO: 12) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 13).

[0041] The heavy and light chain nucleotide sequences of the monoclonal antibody obtained from the sequencing were submitted to the V-QUEST analysis tool on the IMGT website (http: / / www.imgt.org) for professional analysis. The VL amino acid sequence of the anti-human SMMHC monoclonal antibody is shown in SEQ ID NO.2, and the VH amino acid sequence is shown in SEQ ID NO.6.

[0042] The VL is 103 amino acids long. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89-93aa, respectively. Their amino acid sequences are QSIYNN (SEQ ID NO.3), LVS (SEQ ID NO.4) and QAYSG (SEQ ID NO.5), respectively.

[0043] VH is 111 amino acids long. The number of amino acids in the four domains of its FR are 25, 17, 39 and 11, respectively. The number of amino acids in the three domains of HCDR are 7, 7 and 5, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-32aa, 50aa-56aa and 96aa-100aa, respectively. Their amino acid sequences are: GFSLSYT (SEQ ID NO.7), INPSGNT (SEQ ID NO.8) and SRRWG (SEQ ID NO.9).

[0044] Example 3: Affinity Identification of Anti-human SMMHC Monoclonal Antibody The non-competitive ELISA method for determining the antibody affinity constant (Ka) is as follows: 1) Coating: The synthesized SMMHC naked peptide was diluted with carbonate buffer and the concentration gradient was set as follows: 0.625 μg / mL, 0.313 μg / mL, 0.156 μg / mL, 0.078 μg / mL. The solution was added to the coated plate in 100 μL wells and incubated overnight at 4°C.

[0045] 2) Blocking: After removing the coating solution, wash 3 times with PBS buffer, then incubate with 200 μL / well blocking buffer (5% skim milk powder) at 37°C for 1 h.

[0046] 3) Incubation of primary antibody: Serially dilute the SMMHC monoclonal antibody, with the concentration gradient set as follows: 9 × 10⁻⁶ -2 μg / mL, 3×10 -2 μg / mL, 1×10 -3 μg / mL, 3.33×10 -3 μg / mL, 1.11×10 -3 μg / mL, 3.7×10 -4 μg / mL, 1.23×10 -4 μg / mL was added to the coated plate at a rate of 100 μL / well and incubated at 37°C for 1 h.

[0047] 4) Incubation with secondary antibody: Remove primary antibody, wash 3 times with PBS buffer, dilute HRP-labeled goat anti-rabbit secondary antibody 1:20000, add 100 μL / well to the coated plate, incubate at 37℃ for 30 min.

[0048] 5) Color development and termination: Remove the secondary antibody, wash 3 times with PBS buffer, add 50 μL of color development solution to the coated plate at 37°C for 15 min, and then add 50 μL of stop solution (1 mol / L H2SO4) to terminate the reaction.

[0049] 6) Data reading: Set the parameter to 450nm in the microplate reader and read the absorbance data.

[0050] 7) Data Processing: An S-shaped curve was plotted with the logarithm of antibody concentration (mol / L) on the x-axis and the corresponding absorbance on the y-axis. The affinity constant was calculated, and the calculated affinity constant for the anti-human SMMHC monoclonal antibody was Ka = 2.47 × 10⁻⁶. 10 L / mol.

[0051] Example 4: Immunohistochemical detection kit containing anti-human SMMHC monoclonal antibody This kit is an immunohistochemical detection system based on anti-human SMMHC monoclonal antibody. The core detection antibody was prepared and purified in Example 1, and the concentration used is 0.27 μg / mL. The kit also provides a complete set of reagents, including: antigen retrieval solution (1 mM EDTA, 10 mM Tris Buffer, pH 8.0), endogenous peroxidase inhibitor (hydrogen peroxide), ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB substrate buffer for the DAB chromogenic system, DAB concentrated chromogenic solution, and hematoxylin staining solution.

[0052] Example 5: Analysis of SMMHC expression distribution in various human tissues using an anti-human SMMHC immunohistochemical kit. The immunohistochemical detection kit described in Example 4 was used to detect SMMHC protein in various tissues. The specific steps are as follows.

[0053] (1) Preparation of tissue sections To assess antibody expression in various tissues, representative paraffin-embedded samples from the lung, placenta, myocardium, and leiomyoma were selected. Serial sections were prepared using a Leica microtome, with a section thickness of 4 μm, and the sections were mounted on glass slides for subsequent immunohistochemical staining analysis.

[0054] (2) Tissue section pretreatment The prepared tissue sections were dewaxed three times in analytical grade xylene for 10 min each time. They were then subjected to a gradient ethanol hydration process: three times in anhydrous ethanol for 1 min each time, followed by 1 min each in 95%, 85%, and 75% ethanol. Finally, they were rinsed three times in deionized water for 2 min each time to complete the dewaxing and hydration process.

[0055] (3) Antigen thermal retrieval Antigen retrieval was performed using a high-temperature, high-pressure method. The slides were immersed in EDTA antigen retrieval solution at pH 8.0 and heated in an autoclave for 2.5 minutes. After retrieval, the slides were allowed to cool naturally to room temperature and then rinsed three times with deionized water for 2 minutes each time.

[0056] (4) Blocking endogenous enzyme activity To eliminate interference from endogenous peroxidase, a sufficient amount of 3% hydrogen peroxide solution was added to the reaction area of ​​the slice and incubated at room temperature in the dark for 15 minutes.

[0057] (5) Primary antibody incubation Add anti-human SMMHC monoclonal antibody (0.27 μg / mL), place the slide in a humidified chamber, and incubate at 37°C for 60 min. Wash three times with 0.1% PBST for 2 min each time.

[0058] (6) Secondary antibody incubation Add 100 μL of high-sensitivity HRP-labeled goat anti-mouse / rabbit IgG polymer to the reaction area of ​​a glass slide and incubate at 37°C for 30 min. Wash three times with 0.1% PBST for 2 min each time.

[0059] (7) DAB colorimetric reaction Add 120 μL of freshly prepared DAB colorimetric solution, react at room temperature for 5 min, then stop the color development and wash 3 times.

[0060] (8) Re-dyeing and blueing Counterstaining was performed using hematoxylin solution for 12 seconds, followed by three washes to stop the staining process. Differentiation was then performed in 1% hydrochloric acid ethanol solution, followed by three rinses with deionized water to stop the reaction. The solution was then placed in a pH 8.0 Tris-EDTA disodium solution to restore the blue color, followed by three rinses with deionized water.

[0061] (9) Dehydration sealing sheet The stained sections were dehydrated by sequentially passing them through 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, and 100% ethanol for 1 min three times, then treated with xylene for 1 min three times, and finally mounted with neutral resin.

[0062] (10) Microscopic examination Images were observed and acquired under an optical microscope to analyze the tissue localization and expression level of SMMHC protein.

[0063] To elucidate the expression status of smooth muscle myosin heavy chain in different physiological and pathological tissues, an optimized immunohistochemical detection protocol was established using the specific anti-human SMMHC monoclonal antibody prepared in this invention. Screening was performed on a diverse set of tissue microarrays, including various normal tissues and related tumor samples, to comprehensively validate the detection efficacy of the antibody and explore the expression patterns of SMMHC. Specific results are analyzed below:

[0064] Figure 1 Six different tissues were displayed: liver, prostate, colon, kidney, placenta, and pancreas. The results showed that all brown-positive cells were of smooth muscle origin, while other cell types of non-muscle origin were negative, such as…Figure 1 In the middle (c) colon, the smooth muscle bundles of the lamina propria, the smooth muscle layer of the blood vessel wall, and the muscle layer around the glands showed positive staining of cell cytoplasm, while non-smooth muscle cells such as colonic gland epithelial cells, goblet cells, lymphocytes, and plasma cells did not show positive staining. Figure 1 In the middle (f) pancreas, the smooth muscle layer of the blood vessel wall and the smooth muscle layer of the duct wall showed positive staining, while the pancreatic parenchymal cells (acini and islets) did not show positive staining.

[0065] Figure 2 The left image shows breast tissue, with results indicating positive staining in the smooth muscle layer of the blood vessel walls and myoepithelial cells, while glandular epithelial cells, adipocytes, and lymphocytes are negative. The right image shows lung tissue, with results indicating positive staining primarily in the smooth muscle layer of the blood vessel walls and bronchial smooth muscle layers, while alveolar epithelium and inflammatory cells are negative.

[0066] Figure 3 The left image shows the myocardium, and the right image shows the staining results of skeletal muscle. The results show that neither myocardial cells nor skeletal muscle cells showed brown positive expression, while the cytoplasm of smooth muscle cells in the smooth muscle layer of the blood vessel wall showed a brown positive signal.

[0067] Figure 4 The two leiomyosarcoma tissue samples were from different cases. Both samples showed positive expression of leiomyosarcoma cells and smooth muscle layer of blood vessel walls, while inflammatory cells and other substances were negative.

[0068] The results showed positive staining only in smooth muscle-derived cell types, while other cell types showed negative staining. This indicates that SMMHC protein expression is only present in myogenic cells, and not in other cell types. These results are consistent with the theoretical expression pattern of SMMHC. The monoclonal antibody used for the detection was at a concentration of 0.27 μg / mL. Staining results under these conditions meet clinical diagnostic requirements and did not produce background staining that could affect diagnosis. The anti-human SMMHC rabbit monoclonal antibody OTIR5D12 exhibits good specificity and high sensitivity.

[0069] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment, characterized in that, Includes the light chain variable region VL and the heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

3. The amino acid sequence of LCDR2 is LVS or a variant with one or two amino acid substitutions, deletions, or additions compared to LVS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.5; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.7 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

7. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.8 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

8. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.9 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.9; Furthermore, the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment can specifically bind to the smooth muscle myosin heavy chain.

2. The anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.2, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.6, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.

3. The anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.2, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.

6.

4. The anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

5. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4.

6. The method for preparing the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The method includes culturing the cells as described in claim 5, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a polynucleotide comprising the anti-human SMMHC rabbit monoclonal antibody or an antigen-binding fragment thereof, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

7. The application of the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment according to any one of claims 1-4, or the biomaterial according to claim 5, characterized in that, The application is selected from one or more of the following: 1) Testing for human SMMHC for non-diagnostic and non-therapeutic purposes; 2) Prepare immunoassay products for detecting human SMMHC; 3) Detection of human breast ductal carcinoma, invasive carcinoma, and / or smooth muscle tumors for non-diagnostic and non-therapeutic purposes; 4) Prepare immunoassay products for detecting ductal carcinoma, invasive carcinoma and / or smooth muscle-derived tumors in human breast.

8. An immunoassay product for human SMMHC, characterized in that, The immunoassay product comprises the anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, or the biological material as described in claim 5.

9. An SMMHC polypeptide immunogen, characterized in that, The amino acid sequence of the SMMHC polypeptide immunogen is shown in SEQ ID NO.

1.

10. The use of the SMMHC polypeptide immunogen of claim 9 in the preparation of anti-human SMMHC rabbit monoclonal antibody or its antigen-binding fragment.

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