Monoclonal antibodies targeting krt8 and their detection applications

Abstract

The application provides a monoclonal antibody targeting KRT8 and a detection application thereof, and belongs to the field of antibodies. The antibody provided by the application has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9; the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, and the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 are respectively shown as SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17. The application is proved by experiments that the antibody provided by the application has high affinity and strong specificity. The application also proves, through immunohistochemistry, flow cytometry and immunocytochemistry experiments, that the antibody provided by the application has high application value in the detection of KRT8.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to monoclonal antibodies targeting KRT8 and their detection applications. Background Technology

[0002] The KRT8 gene, also known as keratin 8, is a key gene located on human chromosome 12q13.13. It belongs to type II of the keratin family, whose proteins primarily play a structural support role within the cell. The protein encoded by the KRT8 gene is an intermediate filament protein that forms part of the cytoskeleton and is essential for maintaining cell shape and stability.

[0003] Abnormal KRT8 expression and function have been observed in various cancers. For example, KRT8 expression levels are significantly elevated in certain types of liver and breast cancer. This abnormal expression may be associated with tumor growth and spread. KRT8 abnormalities may also affect the invasiveness and metastatic ability of tumor cells. Besides cancer, mutations or abnormal expression of the KRT8 gene are also associated with a variety of hereditary and acquired diseases. For example, in some hereditary skin diseases, KRT8 deficiency leads to loss of skin barrier function, increasing the risk of infection and inflammation. Therefore, the development of highly specific and high-affinity KRT8 antibodies is of great significance for the detection of diseases with abnormal KRT8 expression.

[0004] However, during the preparation of natural monoclonal antibodies, issues such as immunogen purification and impurities, interference from tag / fusion proteins, extensive cross-linking of antigenic epitopes, differences in the quaternary structure between immunoantigens and natural antigens, and the fact that commonly used fusion chaperones such as Sp2 / O secrete endogenous κ light chains that combine with the heavy chains of spleen cells to form "mixed antibodies," potentially altering the microenvironment of the antibody complementarity-determining region, lead to insufficient antibody specificity. Existing literature reports that "there are over 6 million commercially available antibodies, but less than half possess reliable specificity." The gold standard for antibody specificity verification is KD / KO verification; currently published literature on the same target mainly focuses on antibody affinity verification, showing a significant deficiency in antibody specificity verification. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0006] The first aspect of the present invention provides a KRT8 antibody or an antigen-binding fragment thereof, the KRT8 antibody or the antigen-binding fragment thereof having a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 being shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 being shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively.

[0007] Furthermore, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 6; the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 14.

[0008] In this invention, the antibody includes monoclonal antibody, polyclonal antibody, and recombinant antibody.

[0009] In this invention, the antibody includes monospecific antibodies and multispecific antibodies. The multispecific antibody is bispecific, trispecific, tetraspecific, or even more specific.

[0010] In this invention, antibodies include non-human antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0011] In this invention, the antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, single-chain antibody scFv, and nanobody VHH.

[0012] Furthermore, the antibody also includes a constant region, and according to the structure of the constant region, the antibody includes IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0013] Furthermore, the constant region can be the human IgG constant region or the mouse IgG constant region.

[0014] Furthermore, the constant region is a mouse constant region, including but not limited to IgG1 and IgG2a.

[0015] A second aspect of the present invention provides a polynucleotide molecule that encodes the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0016] "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA having a backbone modified for stability or other reasons.

[0017] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiesters present between the nucleotides of the unmodified oligonucleotide.

[0018] Furthermore, the nucleotide does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be appropriate for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions; therefore, nucleotides formed by such insertions, deletions, inversions, and / or substitutions are also within the scope of protection of this invention.

[0019] Furthermore, the polynucleotide molecule contains a nucleotide sequence as shown in SEQ ID NO.2.

[0020] Furthermore, the polynucleotide molecule contains a nucleotide sequence as shown in SEQ ID NO.10.

[0021] A third aspect of the present invention provides a carrier comprising the polynucleotide molecule described in the second aspect of the present invention.

[0022] Examples of vectors that can be used in this invention include, but are not limited to: plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0023] Various vectors known in the art can be used, such as commercially available vectors, and then the polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. Further, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papillomavirus vectors.

[0024] Furthermore, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0025] Furthermore, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of this invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0026] Furthermore, the expression vector is selected from pcDNA3.1.

[0027] A fourth aspect of the present invention provides a modified host cell comprising the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the polynucleotide molecule as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention.

[0028] Furthermore, the modified host cell is obtained by introducing the vector described in the third aspect of the present invention into the host cell.

[0029] As used herein, the term "host cell" includes a cell in which a foreign (exogenous or transgenic) nucleic acid has been introduced. The foreign nucleic acid may include an expression vector operatively linked to a transgene, and the host cell may be used to express the nucleic acid and / or a polypeptide encoded by the foreign nucleic acid (transgene). The host cell may be a cultured cell or may be extracted from an individual. Host cells include primary cells of an individual and their progeny, regardless of passage number. Host cells include immortalized cell lines. Progeny cells may or may not contain the same genetic material as the parent cells. The term "host cell" encompasses progeny cells. In one embodiment, "host cell" describes any cell (including its progeny) that has been modified, transfected, transduced, hosted, and / or manipulated in any way to express an antibody, as disclosed herein. In one example, an expression vector operatively linked to a nucleic acid, as described herein, encoding a desired antibody or an antigen-binding fragment thereof, may be introduced into a host cell.

[0030] Furthermore, the host cell can be a prokaryote, such as *Escherichia coli*, or a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), a mammalian cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma. In one embodiment, the host cell comprises non-human cells, including CHO, BHK, NSO, SP2 / 0, and YB2 / 0. In one embodiment, the host cell comprises human cells, including HEK293, HT-1080, Huh-7, and PER.C6. Examples of host cells include the COS-7 strain of monkey kidney cells (ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, or DHFR-deficient CHO strain DX-B 11, HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70); human embryonic kidney cells, such as 293, 293EBNA, or MSR 293; human epidermal A431 cells, human Colo 205 cells, transformed other primate cell lines, normal diploid cells, cell lines derived from major tissues cultured in vitro, primary explants, HL-60, U937, HaK, or Jurkat cells. In one embodiment, host cells include lymphocytes, such as Y0, NSO, or Sp20.

[0031] The vector can be introduced into the host cell using methods suitable for the host cell. Various methods for introducing nucleic acids into the host cell are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; viral transfection; non-viral transfection; microbolite bombardment; liposome transfection; and infection (e.g., where the vector is an infectious agent).

[0032] A fifth aspect of the present invention provides a hybridoma cell comprising the ability to produce the KRT8 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0033] The sixth aspect of the present invention provides derivatives comprising complexes formed by directly or indirectly conjugating the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention to a detectable marker.

[0034] As used in this invention, the detectable label may be radioactive, colorimetric, antigenic, enzymatic, detectable beads (e.g., magnetic or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels may be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens).

[0035] The seventh aspect of the present invention provides a product for detecting KRT8, the product comprising one or more of the following: the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention; the polynucleotide molecule as described in the second aspect of the present invention; the vector as described in the third aspect of the present invention; the modified host cell as described in the fourth aspect of the present invention; the hybridoma cell as described in the fifth aspect of the present invention; and the derivative as described in the sixth aspect of the present invention.

[0036] Furthermore, the product includes one or more of the following: reagent kits, test strips, reagents, and chips.

[0037] Furthermore, the kit also includes, but is not limited to, one or more of the following: a container for holding KRT8 antibody or its antigen-binding fragment when not in use; instructions for use; KRT8 antibody or its antigen-binding fragment attached to a solid support; known standards; and reference samples.

[0038] The term "known standard" can refer to a solution containing a known amount or concentration of KRT8, wherein such solution can be a naturally occurring solution; or such solution can be a synthetic solution, such as a buffered aqueous solution in which a known amount of KRT8 is diluted. Known standards described herein may include KRT8 isolated from subjects, recombinant or purified KRT8 protein, or KRT8 associated with disease symptoms.

[0039] The term "reference sample" is a sample that can be compared with another sample, such as a test sample, to characterize the sample being compared. A reference sample will have certain characteristic properties that serve as the basis for comparison with the test sample. For example, a reference sample might be used as a benchmark for KRT8 levels indicating whether a subject has cancer. The reference sample does not necessarily have to be analyzed in parallel with the test sample; therefore, in some cases, the reference sample can be a previously determined value or range used to characterize a given condition, such as KRT8 levels indicating whether a subject has a disease.

[0040] Furthermore, the kits include, but are not limited to, ELISA detection kits, immunofluorescence detection kits, FACS kits, Western Blot kits, IHC detection kits, and ICC kits.

[0041] The eighth aspect of the present invention provides the use of the KRT8 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the polynucleotide molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the modified host cell described in the fourth aspect of the present invention, the hybridoma cell described in the fifth aspect of the present invention, and / or the derivative described in the sixth aspect of the present invention in the preparation of products for detecting KRT8.

[0042] Furthermore, the product used to detect KRT8 is the product described in the seventh aspect of this invention.

[0043] Furthermore, the product is used to diagnose KRT8-related diseases. These KRT8-related diseases include, but are not limited to, diseases caused by abnormal KRT8 expression, including, but not limited to, colon cancer.

[0044] The ninth aspect of the present invention provides a method for preparing the KRT8 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising culturing the modified host cells as described in the fourth aspect of the present invention or the hybridoma cells as described in the fifth aspect of the present invention.

[0045] The antibodies or antigen-binding fragments described herein can be purified using protein isolation / purification methods commonly known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reverse-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination of these methods. Following purification, the peptides are exchanged in different buffers and / or concentrated using any of a variety of methods known in the art, including but not limited to filtration and dialysis.

[0046] The tenth aspect of the present invention provides a method for detecting KRT8 or a fragment thereof in a test sample for non-diagnostic or diagnostic purposes, the method comprising contacting the test sample with the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with the derivative as described in the sixth aspect of the present invention, or contacting the test sample with the product as described in the seventh aspect of the present invention, and detecting the formation of a complex of the KRT8 antibody or its antigen-binding fragment or an antibody derivative of the KRT8 antibody or its antigen-binding fragment with KRT8 or a fragment thereof.

[0047] The eleventh aspect of the present invention provides a method for diagnosing KRT8-related diseases, the method comprising contacting a test sample with the KRT8 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with the derivative as described in the sixth aspect of the present invention, or contacting the test sample with the product as described in the seventh aspect of the present invention, and detecting the formation of a complex of the KRT8 antibody or its antigen-binding fragment or an antibody derivative of the KRT8 antibody or its antigen-binding fragment with KRT8 or its fragment.

[0048] The advantages and beneficial effects of this invention are as follows: This invention provides a monoclonal antibody targeting KRT8 and its detection application. Experiments demonstrate that the antibody provided by this invention exhibits high affinity and specificity, with a KD value of 4.93 × 10⁻⁶. -9 M does not bind to other proteins on the cell. This invention also demonstrated the application of the antibody provided in detecting KRT8 through immunohistochemistry, flow cytometry, and immunocytochemistry experiments, showing it to have high application value. Attached Figure Description

[0049] Figure 1 The curve is the kinetic fit curve for the KRT8 monoclonal antibody.

[0050] Figure 2 Figure showing the experimental results of immunoblotting detection of KRT8 protein expression in wild-type / KD stable cell lines.

[0051] Figure 3 This is an image showing the immunohistochemical (IHC) results of co-staining hematoxylin and KRT8 protein.

[0052] Figure 4 The graph shows the flow cytometry (FCM) results of the isotype control group and the KRT8 staining experimental group.

[0053] Figure 5 This is an image showing the immunocytochemical (ICC) fluorescence results of DAPI and KRT8 protein co-stained. Detailed Implementation

[0054] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0055] Example 1: Preparation of Antibody 1. Experimental Methods 1. Expression and purification of KRT8 recombinant protein (1) Construction of eukaryotic plasmid expression vector The Uniport number P13647 protein was truncated for expression by adding a 6×Histag and a thrombin restriction site to the N-terminus of the target protein sequence. After adding the KOZAK sequence and restriction site to the above sequence, the humanized codons were optimized by Anhui General Company, and the protein was cloned into a eukaryotic expression vector (Thermo Fisher, pcDNA3.1) for subsequent transfection and expression experiments. The full length of the target protein sequence (SEQ ID NO.1) is as follows: MGSSHHHHHHSSGLVPRGSHMQEKEQIKTLNNKFASFIDKVRFLEQQNKMLETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEMENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQ.

[0056] (2) Transfection of KRT8 plasmid After thawing HEK 293F cells from the liquid nitrogen tank, they were continuously passaged until the cell mass reached the specified density and the viability exceeded 90%. Then, the prepared HEK 293F cells were used for subsequent transfection experiments. The cells were transferred to a new 125ml shake flask, and 30ml of HEK 293F cells were added, resulting in a density of 0.5 × 10⁻⁶ cells / mL. 6 Cells / ml. Following the instructions for the Lipo2000 transfection reagent, the eukaryotic expression vector was mixed with Lipo 2000 at a ratio of 1:3 and added to the prepared HEK 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. Cell viability was monitored during culture by adding 10% volume of feed medium (e.g., PolyTool™ 293 D) + 0.5% Enhancer + 1% 293 B. When cell viability approached 70%, cells were removed by centrifugation, and the supernatant was used for target protein purification.

[0057] (3) Purification of KRT8 recombinant protein Collect the supernatant, filter it through a 0.45 μm filter membrane to remove residual cell debris, and adjust the pH to 7.5-8.0 (if the culture medium contains phenol red, the color should be light pink). Add imidazole to a final concentration of 10 mM and adjust the NaCl to 150 mM.

[0058] Pour the nickel packing material into a gravity column, allow it to settle naturally, wash with 5 column volumes of deionized water, and equilibrate with 5 column volumes of binding buffer. Maintain a flow rate of 1-2 mL / min (natural flow rate for the gravity column is acceptable). Add the processed sample to the chromatography column at a flow rate of 1.0 mL / min and collect the flow-through. Wash with 10 column volumes of binding buffer to remove unbound proteins, then wash with 10 column volumes of wash buffer to remove non-specific bound proteins, monitoring the OD280 of the eluent until it reaches baseline. Elute in one step with 10 column volumes of elution buffer (250-500 mM imidazole), collecting 1 mL of eluent per tube and labeling the tubes. Finally, elute with 5 column volumes of deionized water + 5 column volumes of 0.1 M EDTA (to strip Ni²⁺). + Wash with 5 column volumes of deionized water to remove impurities remaining in the nickel column. Then regenerate the nickel column with 5 column volumes of 0.1 M NiSO4 + 5 column volumes of deionized water + 5 column volumes of binding buffer. Finally, equilibrate the column with 20% ethanol and store at 4°C.

[0059] The purified protein was concentrated by ultrafiltration, filtered through a 0.45 μm filter membrane for sterilization, and then the protein concentration was measured using nanodrop before being stored at -20°C.

[0060] (4) Detection of KRT8 recombinant protein Take an appropriate amount of sample (2 μg) and mix it with 2× reducing loading buffer in the specified ratio. Heat at 100°C for 5 minutes, then briefly centrifuge before loading the sample. After electrophoresis, place the gel in Coomassie Brilliant Blue staining solution and stain on a shaker at room temperature for 2 hours. Then transfer it to destaining solution (40% methanol, 10% acetic acid) and destain overnight or change the destaining solution until the background is transparent. Take a picture in white light mode, save the image, and label the position of the molecular weight standard and sample information. Estimate the molecular weight of the target protein based on the molecular weight standard.

[0061] 2. Preparation of mouse hybridoma antibodies (1) Immunity The recombinant protein of SEQ ID NO.1 was mixed with complete Freund's adjuvant (1:1) and emulsified. Five BALB / c mice were immunized subcutaneously. Two weeks later, the antigen containing the above recombinant protein (SEQ ID NO.1) was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After the three immunizations, blood was collected and serum titers were determined by serial dilution using ELISA. Mice with the highest antibody titer against the SEQ ID NO.1 antigen were selected for the next step of cell fusion.

[0062] (3) Cell fusion Prepare mouse-derived sp2 / 0 myeloma cells in advance, ensuring they are in the logarithmic growth phase at the time of fusion. Obtain spleens from immunized mice and prepare a single-cell suspension of lymphocytes. Mix the mouse spleen lymphocytes with the myeloma cells and perform electrofusion according to the program set on a BTX 2001 LITE cell fusion instrument. After fusion, allow the cells to stand for 4 minutes, then dilute them in 50 ml centrifuge tubes to 40 ml of HAT-containing medium and incubate at 37°C for 1 hour. Then, aliquot the diluted solution into 10 wells of 96-well plates and incubate at 37°C with 5% CO2. Observe the fused cell status in the 96-well plates 6-9 days after fusion, change the medium with HT, and continue incubation at 37°C with 5% CO2.

[0063] (4) Screening and cloning Seven to ten days after fusion, clonal cells were screened using an ELISA test with the antigen (SEQ ID NO.1). The corresponding cell line numbers were labeled, and the cells in the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive ELISA result. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies.

[0064] (5) Perform antibody sequencing on the selected hybridoma cell lines. Total RNA was isolated from hybridoma cells according to the TriZol reagent instructions. The total RNA was reverse transcribed into cDNA according to the Vazyme first-strand cDNA synthesis kit instructions. The nucleotide sequences of the heavy chain variable region and light chain variable region of the KRT8 monoclonal antibody were amplified using specific primers. The nucleotide sequences of the heavy chain variable region and light chain variable region were then cloned into a plasmid expression vector (Thermo Fisher, pcDNA3.1) containing the light and heavy chain constant regions of mouse IgG1 for cell transfection.

[0065] (6) Cell transfection and screening Prepare the 293F cells to be transfected in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 6-well plates at a density of 2 ml per well (0.5 × 10⁻⁶ cells / well). 6 Cells / ml. The eukaryotic expression vector was mixed with Lipo2000 at a ratio of 1:3 and added to the prepared 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. After 3-5 days of culture, the transfected cell supernatant was subjected to ELISA to screen positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. If the immunohistochemical detection was positive, the detected antibody sequence was confirmed to be correct.

[0066] (7) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect large numbers of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4°C.

[0067] Ultimately, the heavy chain variable region nucleotide sequence of the KRT8 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID NO.2, and the heavy chain complementarity-determining region (CDR) of the KRT8 monoclonal antibody is encoded by the DNA sequences shown in SEQ ID NO.3 (CDR1), SEQ ID NO.4 (CDR2), and SEQ ID NO.5 (CDR3).

[0068] The nucleotide sequence of the light chain variable region is encoded by the DNA sequence shown in SEQ ID NO.10, and the light chain complementarity-determining region of the KRT8 monoclonal antibody is encoded by the DNA sequences shown in SEQ ID NO.11 (CDR1), SEQ ID NO.12 (CDR2), and SEQ ID NO.13 (CDR3).

[0069] The obtained base sequence was translated into amino acid sequence analysis, and the amino acid sequence of the heavy chain variable region of the KRT8 monoclonal antibody was obtained as shown in SEQ ID NO.6. The heavy chain complementarity-determining region of the KRT8 monoclonal antibody was encoded by the DNA sequences shown in SEQ ID NO.7 (CDR1), SEQ ID NO.8 (CDR2), and SEQ ID NO.9 (CDR3).

[0070] The amino acid sequence of the light chain variable region of the KRT8 monoclonal antibody is shown in SEQ ID NO.14, and the complementarity-determining region of the light chain of the KRT8 monoclonal antibody is encoded by the DNA sequences shown in SEQ ID NO.15 (CDR1), SEQ ID NO.16 (CDR2), and SEQ ID NO.17 (CDR3).

[0071] 2. Experimental Results (1) ELISA screening of isolated strains and determination of subtypes are shown in Table 1 below.

[0072] Table 1. Results of ELISA screening and subtype determination

[0073] Ten hybridoma cell lines that stably secrete anti-KRT8 antibodies were successfully obtained through cell fusion and screening. Their cell numbers are: 1-B11-H5, 2-G2-B3, 3-F8-G6, 4-C8-E1, 5-E11-E7, 6-B7-D1, 7-E7-B7, 8-H3-H9, 9-G8-E6, and 10-F9-A11. Indirect ELISA results showed that the supernatant of all 10 cell lines exhibited significant binding signals to the KRT8 antigen. Several cell lines showed significantly higher binding signals than the blank control (0.04), demonstrating the high affinity of the secreted antibodies. Antibody subtype detection results show that the monoclonal antibody obtained in this invention contains two IgG subtypes: IgG1 ((1-B11-H5, 2-G2-B3, 4-C8-E1, 6-B7-D1, 7-E7-B7) and IgG2a (3-F8-G6, 5-E11-E7, 8-H3-H9, 9-G8-E6, 10-F9-A11)). This provides a diverse selection basis for the subsequent application of the antibody in functional experiments (such as complement activation and binding to different Fc receptors).

[0074] (2) Sequence analysis results: One of the antibodies was sequenced, and the sequence information is shown in Table 2 below.

[0075] Table 2. Amino acid and nucleotide sequences of antigens and antibodies

[0076] Example 3: Monoclonal Antibody Affinity Test 1. Experimental Methods (1) Sample testing process: Ligand (KRT8 monoclonal antibody) concentration: Dilute the antibody to 0.5 μg / mL or 1 μg / mL with 1×PBST buffer, and take 400 μL of each for the experiment.

[0077] Analyte (KRT8 antigen) concentration: Dilute the antigen to 0 nM, 1.56 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM with 1×PBST buffer.

[0078] (2) Parallel method for detecting affinity and kinetics In the opened Biacore 8K Control Software, click New method in the upper right corner of the main interface (or click the Methods window and then click +NEW), select the Multi-cycle kinetics / affinity using capture template in the Kinetics / affinity > Antibody / general > Kinetics / affinity menu, and double-click to open it.

[0079] In the Method definition interface, select "set to fixed 25℃" for the sample chamber temperature, select the corresponding unit for the concentration, and leave other settings unchanged. In the Analysis window below, under the Analyte tab, enter the default 25℃ for the analysis temperature, 120s for the Contact time, and 180s for the Dissociation time (300s or longer for slow dissociation samples), and 30 μL / min for the Flow rate; under the Capture tab, set the Contact time to 60s and the Flow rate to 10 μL / min. Enter the same numbers as in the Analysis window for each item in Startup (or leave the system default values ​​unchanged).

[0080] In the 2. Variables and positioning interface, under Use channels, only check option 1 (Channels 2-7 are not used in this experiment). Keep the Startup default settings, click Analysis, and in the pop-up table, enter the sample name in the solution field. Click Add cycle to add up to 8 cycles. Set the Concentration for the first cycle to 0 nM. Enter all the above concentrations from low to high (note that you need to set the replicate concentration and zero concentration). On the right, select the 96-well plate type in Type according to the sample volume, and set and change the sample position. You can click the settings icon on the far right of the screen to check the corresponding pooling.

[0081] 2. Experimental Results Based on the above test analysis, the KD value of the KRT8 antibody is 4.93 × 10⁻⁶. -9 M. Other test parameters are shown in Table 3 below, and the dynamic fitting curve is as follows. Figure 1 As shown.

[0082] Table 3. Results of KRT8 antibody affinity test

[0083] Example 3: KD-specific detection of monoclonal antibodies 1. Experimental Methods In this embodiment, the anti-KRT8 monoclonal antibody of the present invention was used as the primary antibody. Western blotting (WB) was used to detect the expression level of KRT8 protein in wild-type / KD stable cell lines, verifying its specificity against KRT8 protein. The method is as follows: (1) Prepare wild-type HeLa (mouse myoblast cell line) cell line and KRT8 gene knockdown cell lysate (20 μg), prepare gel, spot, electrophoresis, transfer membrane. PVDF membrane needs to be activated. Activate with methanol for 1 min, wash the membrane twice with pure water and then wash it three times with TBST. Block: Place the membrane in blocking solution prepared with PBST containing 5% skim milk and shake at room temperature for 1 h. (2) Primary antibody incubation: Dilute 1 mg / mL of KRT8 monoclonal antibody at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: HRP anti-mouse IgG (0.4 mg / mL, 1 μL) was diluted with antibody diluent at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 h; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0084] 2. Experimental Results The results are as follows Figure 2 As shown, lane 1: Western blotting (WB) was performed using HeLa wild-type cell lysate incubated with GAPDH antibody as the primary antibody; lane 2: Western blotting was performed using HeLa KRT8 knockdown cell lysate incubated with GAPDH antibody as the primary antibody; lane 3: Western blotting was performed using HeLa wild-type cell lysate incubated with KRT8 antibody as the primary antibody; lane 4: Western blotting was performed using HeLa KRT8 knockdown cell lysate incubated with KRT8 antibody as the primary antibody. In lane 3, the anti-KRT8 monoclonal antibody of this invention showed a signal with a molecular weight of approximately 53 kDa, while no signal was observed in lane 4. This indicates that the KRT8 knockdown cell line does not express protein, and the anti-KRT8 monoclonal antibody of this invention also showed no recognition band. Furthermore, the WB band was relatively pure. Therefore, the KRT8 monoclonal antibody can specifically recognize the KRT8 protein.

[0085] Example 3 Immunohistochemical Specific Detection of Monoclonal Antibodies 1. Experimental Methods (1) Sample preparation: The human colon cancer tissue sections fixed in formalin and embedded in paraffin were baked in a constant temperature oven at 60℃ for 12 hours and stored for later use; (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time; (3) Hydration of sections: The sections were hydrated by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes in sequence, and then rinsed twice with purified water for 3 minutes each time. (4) Antigen retrieval: It is recommended to use the high temperature heat retrieval method for 3 min (if using an automatic retrieval instrument, the high temperature retrieval at 98℃ for 20 min can be set). After the slides are naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen and rinsed twice with purified water for 3 min each time. (5) Inactivation of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent to completely cover the tissue, incubate at room temperature for 10 min, rinse twice with purified water for 3 min each time, and rinse once with PBST. (6) Primary antibody incubation: Add 100 μL of 0.5 μg / mL KRT8 monoclonal antibody to completely cover the tissue, incubate in a 37℃ incubator for 1 h, and wash with PBST 3 times for 5 min each time; (7) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. After incubation, rinse the slides with PBST 3 times for 5 minutes each time, and rinse with purified water once. (8) DAB staining: Prepare DAB staining solution according to the instructions of the DAB staining solution kit. Drop an appropriate amount of the prepared DAB staining solution to completely cover the tissue. Stop staining when the color does not deepen. Rinse 3 times with purified water. (9) Hematoxylin counterstaining: Counterstain the sections according to the operating steps and suggestions in the instructions of the hematoxylin manufacturer, and rinse with PBST or tap water to return to blue; (10) Dehydration and clearing: Soak in 70%, 85%, 95%, 100%, and 100% graded alcohols sequentially for 3 minutes each time; clear with xylene twice for 5 minutes each time; (11) Mounting: Mount the sample with neutral resin.

[0086] 2. Experimental Results KRT8 forms part of the cytoplasmic skeletal network of epithelial cells, maintaining cellular mechanical strength, and is located within the cytoplasm. Furthermore, while hematoxylin stains the cell nucleus blue, KRT8 antibody staining produces a brown color. From... Figure 3As can be seen, the brown color is distributed around the cell nucleus, while the cell nucleus itself is not stained, indicating that the KRT8 antibody staining site is correctly located, proving its staining specificity.

[0087] Example 4: Monoclonal antibody cell flow cytometry specific detection 1. Experimental Methods (1) Cell preparation Collect HepG2 cells in the logarithmic growth phase, discard the old culture medium, and gently wash the cells twice with PBS. Add an appropriate amount of trypsin to digest the cells. Once the cells have completely detached, add complete culture medium to stop the digestion. Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 4°C and 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in pre-cooled PBS, and repeat the centrifugation and washing once. Resuspend the cells in flow cytometry staining buffer and adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 6 Cells / 100μL, dispensed into flow cytometry tubes, 100μL per tube, divided into experimental group and isotype control group, with 3 biological replicates in each group.

[0088] (2) Cell fixation and permeabilization (KRT8 is a nuclear protein and requires permeabilization treatment) Add 1 mL of pre-chilled 4% paraformaldehyde to each tube of cells, vortex to mix, and fix at 4°C in the dark for 15 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, add 1 mL of pre-chilled PBS to resuspend the cells, and centrifuge and wash once to remove residual fixative. Add 1 mL of pre-chilled 0.1% Triton X-100 permeabilization buffer to the cell pellet, vortex to mix, and permeabilize at 4°C in the dark for 10 min to allow the antibody to enter the cell nucleus and bind to the KRT8 antigen. Centrifuge, discard the supernatant, and wash twice with pre-chilled PBS to completely remove the permeabilization buffer.

[0089] (3) Closed and primary antibody incubation Add 100 μL of 1% BSA-PBS blocking buffer to each tube of cells, vortex to mix, and block at 4°C for 30 min to block non-specific binding sites. Centrifuge and discard the supernatant: Experimental group: Add 100 μL of anti-KRT8 antibody diluted 1:2000 (with 1% BSA-PBS), vortex to mix. Isotype control group: Add 100 μL of homologous isotype control IgG diluted at the same concentration, vortex to mix. Incubate at 4°C in the dark for 1 h (or overnight at 4°C to improve binding specificity). After incubation, add 1 mL of pre-cooled flow cytometry staining buffer to each tube, centrifuge at 4°C and 1000 rpm for 5 min, discard the supernatant, and repeat washing 3 times to completely remove unbound primary antibody and reduce background fluorescence.

[0090] (4) Incubation with fluorescent secondary antibody Add 100 μL of Alexa Fluor® 647-labeled secondary antibody (diluted with 1% BSA-PBS) diluted according to the instructions to each cell pellet and vortex to mix. Incubate at 4°C in the dark for 30 min, strictly avoiding light throughout the process to prevent fluorescence quenching. After incubation, wash three times with pre-cooled flow cytometry buffer, centrifuging for 5 min each time to completely remove unbound secondary antibody.

[0091] (5) On-machine testing and data analysis Add 300-500 μL of pre-chilled flow cytometry staining buffer to each tube of cells, resuspend the cells, vortex to mix, and analyze within 2 hours. Flow cytometer settings: Forward scatter (FSC) / Side scatter (SSC) gate, select intact viable cell populations, and exclude cell debris and clusters. Select the corresponding detection channel for the Alexa Fluor® 647 (usually the APC channel), adjust the voltage with an isotype control group, and set the negative gate. Collect ≥10,000 viable cells per sample and record fluorescence intensity data.

[0092] 2. Experimental Results Data analysis: FlowJo software was used to analyze the data, and fluorescence intensity histograms were plotted. The fluorescence signal shift between the experimental group (red) and the isotype control group (green) was compared. The proportion of KRT8 positive cells and the mean fluorescence intensity (MFI) were calculated. Results are as follows: Figure 4 As shown, the green curve represents the isotype control, and the red curve represents the KRT8-stained group. This indirectly proves that the KRT8 antibody can bind to KRT8-positive cells, producing a signal shift.

[0093] Example 5: Specific detection of monoclonal antibody immunofluorescence 1. Experimental Methods (1) Cell sample preparation Remove the spread / confocal culture dishes inoculated with HepG2 cells from the CO2 incubator, disinfect the surface of the culture dishes by wiping with alcohol swabs, and place them in a laminar flow hood. Aspirate the old culture medium from the culture dishes, and gently rinse the cell surface twice with PBS buffer. After each rinse, gently absorb excess liquid with absorbent paper (avoid vigorous rinsing to prevent cell detachment).

[0094] (2) Cell fixation Add an appropriate amount of 4% paraformaldehyde fixative to the culture dish, completely covering the cell surface, and incubate at room temperature for 15-20 minutes (do not fix for too long to avoid damaging the antigen epitopes). After fixation, aspirate the fixative, rinse the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to ensure complete removal of any residual fixative.

[0095] (3) Cell permeability Add an appropriate amount of 0.1% Triton X-100 permeabilization buffer to the culture dish, completely covering the cells, and incubate at room temperature for 10 minutes for permeabilization. After permeabilization, aspirate the permeabilization buffer, wash the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to remove any residual permeabilization buffer.

[0096] (4) Blocking treatment (reducing non-specific binding) Discard the PBS buffer, add an appropriate amount of 5% BSA blocking solution to the culture dish, completely covering the cell surface, and incubate at room temperature for 30 minutes (during the incubation period, gently shake on a shaker to ensure even incubation). After incubation, do not rinse; directly discard the blocking solution (retain a small amount of blocking solution to prevent cell drying).

[0097] (5) Primary antibody incubation (specific binding of antigen) Slowly add the pre-diluted anti-KRT8 antibody (1:1000) dropwise to the cell surface, ensuring complete coverage. Incubate overnight at 4°C in the dark (or at room temperature for 2 hours, preferably overnight at 4°C to enhance binding specificity). During incubation, ensure even antibody coverage to avoid air bubbles or antibody loss. Set up a blank control (blocking buffer only, no primary antibody) to exclude non-specific binding by the secondary antibody. After incubation, discard the primary antibody and wash the cells three times with PBS buffer for 5 minutes each time, shaking gently on a shaker to thoroughly remove unbound primary antibody and avoid background fluorescence interference.

[0098] (6) Secondary antibody incubation (fluorescent labeling) Slowly add pre-diluted Alexa Fluor 647-labeled secondary antibody to the cell surface, completely covering the cells. Incubate at room temperature in the dark for 1 hour (avoid light throughout to prevent fluorescence quenching). During incubation, gently shake the cell on a shaker to ensure uniform binding of the secondary antibody; avoid excessively high antibody concentrations, which can lead to nonspecific fluorescence enhancement. After incubation, discard the secondary antibody and wash the cells three times with PBS buffer for 5 minutes each time, shaking gently on a shaker to remove unbound secondary antibody and reduce background fluorescence.

[0099] (7) Nuclear staining (DAPI staining) Discard the PBS buffer, add an appropriate amount of diluted DAPI staining solution to the culture dish, completely covering the cells, and stain at room temperature in the dark for 5-10 minutes (the staining time should not be too long to avoid excessive nuclear fluorescence masking the target protein signal). After staining, discard the DAPI staining solution, rinse the cells twice with PBS buffer, 5 minutes each time, and shake gently on a shaker to remove residual DAPI staining solution and avoid fluorescence interference.

[0100] (8) Mounting and microscopic observation Gently absorb excess liquid from the surface of the slide with absorbent paper. Add 1-2 drops of anti-fluorescence quencher to the center of the slide, and gently cover the slide with the cell side down on the anti-fluorescence quencher, avoiding air bubbles. If using a confocal culture dish, the anti-fluorescence quencher can be added directly without mounting. Place the mounted slide / confocal culture dish in a light-proof box and immediately observe using a Leica Stellaris 5 laser confocal microscope. Adjust the laser intensity and smart gain parameters to acquire fluorescence images. Acquire single-channel and merged images of DAPI (blue fluorescence, excitation wavelength 405 nm) and Alexa Fluor 647 (magenta fluorescence, excitation wavelength 647 nm), respectively.

[0101] 2. Experimental Results Experimental results are as follows Figure 5 As shown, cells were stained with anti-KRT8 antibody (1:1000) using immunocytochemical staining. Cell nuclei stained blue with DAPI (first from left); KRT8 stained magenta with Alexa Fluor 647 (second from left); and cells co-stained with DAPI and Alexa Fluor 647 showed a reddish-blue color (third from left), indicating that the KRT8 antibody can specifically stain the cytoplasm.

[0102] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A KRT8 antibody or its antigen-binding fragment, characterized in that, The KRT8 antibody or its antigen-binding fragment has a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. The light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. The amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. The amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively.

2. The KRT8 antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 6; the light chain variable region has an amino acid sequence as shown in SEQ ID NO.

14.

3. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the KRT8 antibody or its antigen-binding fragment as described in any one of claims 1-2.

4. A carrier, characterized in that, The carrier comprises the polynucleotide molecule of claim 3.

5. A modified host cell, characterized in that, The modified host cell comprises the KRT8 antibody or its antigen-binding fragment as described in any one of claims 1-2, the polynucleotide molecule as described in claim 3, or the vector as described in claim 4.

6. A hybridoma cell, characterized in that, The hybridoma cells include those that produce the KRT8 antibody or its antigen-binding fragment as described in any one of claims 1-2.

7. A derivative, characterized in that, The derivatives include complexes formed by directly or indirectly conjugating the KRT8 antibody or its antigen-binding fragment according to any one of claims 1-2 to a detectable marker.

8. A product for detecting KRT8, characterized in that, The product for detecting KRT8 includes one or more of the following: the KRT8 antibody or its antigen-binding fragment as described in any one of claims 1-2, the polynucleotide molecule as described in claim 3, the vector as described in claim 4, the modified host cell as described in claim 5, the hybridoma cell as described in claim 6, and the derivative as described in claim 7.

9. The product according to claim 8, characterized in that, The products include one or more of the following: reagent kits, test strips, reagents, and chips.

10. The use of the KRT8 antibody or antigen-binding fragment thereof according to any one of claims 1-2, the polynucleotide molecule according to claim 3, the vector according to claim 4, the modified host cell according to claim 5, the hybridoma cell according to claim 6, and / or the derivative according to claim 7 in the preparation of a product for detecting KRT8.

11. A method for preparing the KRT8 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The method includes culturing the modified host cells of claim 5 or the hybridoma cells of claim 6.

Images