Anti-human mucin 6 (MUC6) rabbit monoclonal antibodies and uses thereof

Abstract

The present application relates to the field of biotechnology, and discloses a rabbit anti-human mucin 6 (MUC6) rabbit monoclonal antibody OTIR4C11, the amino acid sequences of the antibody light chain variable region (VL) and the heavy chain variable region (VH) are shown as SEQ ID NO. 2 and SEQ ID NO. 3. The present application also provides a preparation method of the anti-MUC6 rabbit monoclonal antibody, the antibody is screened from specific B cells after immunizing New Zealand white rabbits with MUC6 polypeptides, and is produced by molecular cloning and recombination. The anti-MUC6 rabbit monoclonal antibody of the present application is used in an immunodetection tool for detecting MUC6 protein, and is used in an immunohistochemical detection kit and a labeled tumor kit. The monoclonal antibody disclosed in the present application can be specifically combined with MUC6 protein, and the specificity and reliability of the immunodetection of MUC6 protein are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method and application for the specific binding of anti-human mucoprotein 6 (MUC6) rabbit monoclonal antibody to mucoprotein 6 for immunoassay. Background Technology

[0002] Mucins are a class of high-molecular-weight (>200kD) glycosylated proteins secreted by various epithelial cells in the gastrointestinal tract, ovary, and other organs. Their synthesis and secretion are characteristic of glandular epithelium. More than 20 types have been identified, which can be divided into two main categories: secretory mucins and transmembrane mucins. Secretory mucins are mainly secreted by goblet cells and constitute the main component of mucus, playing a protective and lubricating role for the mucosal epithelium, especially in the gastrointestinal tract, respiratory tract, and reproductive tract. These include MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC9. Transmembrane mucins are mainly highly expressed in the gastrointestinal tract, particularly in the large intestine, and include MUC1, MUC3, MUC4, MUC11, MUC12, and MUC13.

[0003] MUC6, located at 11p15.5, is a mucin gene cloned by Kim et al. in 1993 from a human gastric cDNA library. This gene consists of randomly numbered repeat units (VNTRs), each containing 169 amino acids. MUC6 is expressed in normal gastric mucosa (partially expressed in the mucosal neck cells of the gastric body and antral glands, and in the basal gland cells of the gastric body and antrum), seminal vesicles, gallbladder, pancreas, and duodenum. Muc-5AC and Muc-6 are expressed in normal gastric mucosa, also known as gastric mucus. Under physiological conditions, Muc-2 is only expressed in normal intestinal mucosa, also known as intestinal mucus. The combined use of these three genes is significant in the differential diagnosis of gastrointestinal tumors.

[0004] Immunohistochemical pathology, based on the protein level, can further determine the tissue origin, primary site, pathological type, and residual marginal cancer cells of a tumor. Besides its diagnostic role, it also guides prognosis. Immunohistochemical techniques have been applied to pathological diagnosis, significantly impacting tumor diagnosis, tumor classification, and prognosis assessment. They have also expanded our understanding of various diseases and tumor formation processes, improving the level of pathological diagnosis and research. At its core, immunohistochemical pathology requires highly specific and sensitive antibodies, especially monoclonal antibodies. With the deepening research on diseases and the MUC6 protein, the application scope of anti-MUC6 monoclonal antibodies continues to expand. Simultaneously, with the widespread use of automated instruments, differences in detection conditions across various testing platforms, and the rapid development of pathological AI, the requirements for antibody quality are becoming increasingly stringent. Therefore, the development of highly specific and sensitive anti-MUC6 monoclonal antibodies suitable for different detection platforms is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a rabbit monoclonal antibody that specifically binds to the MUC6 protein, and its application in the preparation of an immunoassay tool for detecting the MUC6 protein.

[0006] The present invention provides a rabbit monoclonal antibody against MUC6, OTIR4C11. The antibody light chain variable region (VL) contains 112 amino acids, the sequence of which is shown in SEQ ID NO.2; the heavy chain variable region (VH) contains 112 amino acids, the sequence of which is shown in SEQ ID NO.3.

[0007] The rabbit monoclonal antibody specifically recognizes the MUC6 protein.

[0008] The antibody specifically recognizes the amino acid sequence shown in SEQ ID NO.1.

[0009] The inventors provide a method for preparing the antibody, which involves synthesizing a polypeptide and conjugating it to the carrier protein KLH as an immunogen for immunization of New Zealand white rabbits. Peripheral blood mononuclear cells (PBMCs) are obtained from the immunized animal, and specific B cells are sorted, then molecularly cloned and transfected into mammalian cells using a recombinant vector. Cells are cultured to obtain a supernatant containing secreted antibodies. The supernatant is then purified using affinity chromatography to obtain a rabbit monoclonal antibody against MUC6.

[0010] This invention also provides the application of anti-MUC6 rabbit monoclonal antibody in the preparation of an immunoassay tool for detecting MUC6 protein. The immunoassay tool may include a reagent kit, chip, or test strip, etc.

[0011] The present invention also provides an immunohistochemical detection kit, comprising an anti-MUC6 rabbit monoclonal antibody, which can detect the expression status of MUC6 protein in tissue cells. The tissues include normal gastric tissue and gastric cancer tissue. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0013] Figure 1 IHC results of anti-MUC6 rabbit monoclonal antibody OTIR4C11 in gastric tissue.

[0014] Figure 2 IHC results of anti-MUC6 rabbit monoclonal antibody OTIR4C11 in gastric cancer. Detailed Implementation

[0015] This invention discloses a method and application of anti-MUC6 rabbit monoclonal antibody for immunoassay. The technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0016] Example 1: Preparation of anti-MUC6 rabbit monoclonal antibody

[0017] 1. Antigen preparation and animal immunization

[0018] The MUC6 protein sequence NP_005952.2, containing 2439 amino acids, was obtained from the NCBI website and used as the standard sequence. The sequence TSSTHHAEATSTS, amino acid positions 1698 to 1710, was selected and coupled to the carrier protein KLH as an immunogen. The peptide was synthesized by Zhongtai Biochemical Co., Ltd.

[0019] 2. Animal immunization

[0020] The synthesized MUC6 peptide was emulsified with complete Freund's adjuvant and administered subcutaneously to approximately 2 kg New Zealand white rabbits at a dose of 500 μg per rabbit. A second immunization was performed two weeks later, using incomplete Freund's adjuvant emulsification at a dose of 250 μg per rabbit. After three immunizations, tail blood was collected and serum titers were determined using serial dilutions with ELISA and IHC. The criteria for determining whether to collect PBMCs or continue immunization were based on an OD450 greater than 1.0 at an ELISA titer of 128,000 and the presence of staining in the stomach and gastric cancer cells detected by IHC.

[0021] 3. PBMC isolation, specific B cell sorting, and clonal recombination

[0022] New Zealand white rabbits that met the serum testing criteria after immunization were fixed supine on the operating table. The fur around the heart was shaved, and the skin was disinfected with alcohol swabs. A 50ml syringe was used to puncture the heart at the site of strongest heartbeat. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn, and the whole blood was transferred to a sterile 50ml tube. This was mixed with an equal volume of PBS and slowly added dropwise onto the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, yielding rabbit PBMCs. Antigen-specific B cells were then sorted and cultured, and positive clones were screened using antigen-coated ELISA plates. Positive clones were lysed and the lysate was collected. RNA was extracted from the lysate and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were determined by sequencing.

[0023] 4. Preparation and purification of monoclonal antibodies

[0024] To obtain a rabbit monoclonal antibody that specifically recognizes the human MUC6 protein, this invention loads the heavy and light chain genes of the rabbit monoclonal antibody into an expression vector, and transfects the recombinant plasmid into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contains the recombinant rabbit monoclonal antibody recognizing the human MUC6 protein. The cell suspension is collected, the supernatant is obtained by centrifugation, and the antibody is purified by affinity chromatography. The concentration of the purified monoclonal antibody is determined by the BCA method.

[0025] Example 2: Analysis of the variable region gene and amino acid sequence of rabbit monoclonal antibody OTIR4C11

[0026] Using the recombinant plasmid of the OTIR4C11 antibody as a DNA template, sequencing primers for the light and heavy chain variable regions were designed based on the 5' vector sequences of the light and heavy chains on the templates. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody OTIR4C11 was obtained by sequencing. The sequencing results of the light and heavy chains were analyzed using IMGT / V-QUEST analysis software at http: / / www.imgt.org. The amino acid sequence of the light chain of the rabbit monoclonal antibody OTIR4C11 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.3. The light chain variable region (VL) is 112 amino acids in length. Its four domains in the FR have 26, 17, 36, and 10 amino acids respectively, and its three domains in the CDR have 8, 3, and 12 amino acids respectively. The regions of CDR1, CDR2, and CDR3 are 27-34 aa, 52-54 aa, and 91-102 aa respectively, with amino acid sequences of: QSVYDNKN; KAS; AGQGYYNDYSFA. The heavy chain variable region (VH) contains 112 amino acids. Its four domains in the FR have 24, 17, 37, and 11 amino acids respectively, and its three domains in the CDR have 8, 7, and 8 amino acids respectively, corresponding to amino acid regions of 25-32 aa, 50-56 aa, and 94-101 aa, with sequences of: GIDLSRYA; MGSSGDT; GYGGGFDP.

[0027] Example 3: IHC detection of rabbit monoclonal antibody OTIR4C11

[0028] 1. Experimental Methods:

[0029] (1) Formalin-fixed gastric and gastric cancer tissue blocks were embedded in paraffin and sectioned using a Leica tissue slicer with a tissue thickness of 4 μm.

[0030] (2) Dewaxing and hydration: analytical grade xylene for 10 min × 3 times, anhydrous ethanol for 10 min × 3 times, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and deionized water for 3 min × 3 times.

[0031] (3) Add antigen retrieval solution [1mM EDTA, 10mM Trisbuffer (pH 8.0)] and autoclave for 2.5 min. When the autoclave temperature drops to about 90℃, open the autoclave, remove the specimen, and then allow it to cool naturally to room temperature. Immerse in deionized water for 3 min × 3 times.

[0032] (4) Inactivate endogenous peroxidase in tissues with 3% hydrogen peroxide and let stand at room temperature for 10 min. Soak in deionized water for 5 min × 3 times.

[0033] (5) Add the monoclonal antibody secreted by OTIR4C11 (1.15 μg / ml) and place in a humidified chamber. Incubate at 37°C for 60 min. Wash twice with PBST (0.1% Tween-20) for 5 min each time.

[0034] (6) Add ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer (Catalog No. PV-8000) and incubate at 37°C for 30 min. Wash with PBS for 5 min × 3 times.

[0035] (7) Develop color with DAB solution for 5 minutes. Wash with distilled water.

[0036] (8) Counterstain cell nuclei with hematoxylin for 2 min, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinse 3 times with distilled water and let stand at room temperature for 1 min.

[0037] (9) Dehydration and clearing: 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 3×5 min; xylene for 3×5 min, neutral resin for mounting.

[0038] (10) Microscopic examination.

[0039] 2. Experimental Results:

[0040] The expression of MUC6 protein in normal stomach and gastric cancer was detected using the rabbit monoclonal antibody OTIR4C11 against MUC6. Results for normal stomach tissue are as follows: Figure 1 As shown, the left image is the result under low magnification, and the right image is the result under high magnification. The results show that the surface mucus cells on the mucosal lumen and the surface of the gastric pits are unstained, indicating a negative staining, meaning there is no expression of MUC6 protein. However, the cells in the neck and base of the gastric fundic glands in the lamina propria are positive, indicating MUC6 protein expression. These results are consistent with literature. The results for gastric cancer are as follows... Figure 2 The gastric cancer cells shown exhibit diffuse granular staining. sequence list <110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd. <120> Anti-human mucoprotein 6 (MUC6) rabbit monoclonal antibody and its uses <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 13 <212> PRT <213> Homo sapiens <400> 1 Thr Ser Ser Thr His His Ala Glu Ala Thr Ser Thr Ser 1 5 10 <210> 2 <211> 112 <212> PRT <213> Oryctolagus cuniculus <400> 2 Gln Ala Val Val Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asp Asn 20 25 30 Lys Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Glu Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gln Gly Tyr Tyr 85 90 95 Asn Asp Tyr Ser Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 3 <211> 112 <212> PRT <213> Oryctolagus cuniculus <400> 3 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile Gly 35 40 45 Ile Met Gly Ser Ser Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Ile Glu Asp Thr Ala Thr Tyr Phe Cys Gly Tyr Gly 85 90 95 Gly Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu Ala Thr Val Ser Ser 100 105 110

Claims

1. A monoclonal antibody against MUC6 rabbits, characterized in that: The rabbit monoclonal antibody is OTIR4C11, and it includes a light chain variable region and a heavy chain variable region. The light chain variable region contains three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid sequences of the antigenic determinants CDR1, CDR2, and CDR3 are QSVYDNKN, KAS, and AGQGYYNDYSFA, respectively. The heavy chain variable region contains three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid sequences of the antigenic determinants CDR1, CDR2, and CDR3 are GIDLSRYA, MGSSGDT, and GYGGGFDP, respectively.

2. The anti-MUC6 rabbit monoclonal antibody according to claim 1, characterized in that: The sequence of the light chain variable region is shown in SEQ ID NO.2, and the sequence of the heavy chain variable region is shown in SEQ ID NO.

3.

3. The use of the anti-MUC6 rabbit monoclonal antibody according to claim 1 or 2 in the preparation of an immunoassay tool for detecting MUC6 protein, characterized in that, The immunoassay tools include, but are not limited to, reagent kits, chips, and test strips.

Images