Anti-human rb protein monoclonal antibody and its application, hybridoma cell strain and kit

Abstract

The application relates to the technical field of immunoglobulin, in particular to an anti-human RB protein monoclonal antibody and application thereof, a hybridoma cell strain and a kit. The monoclonal antibody is secreted by a hybridoma cell strain OTI12B5, and the hybridoma cell strain OTI12B5 is preserved in the China General Microbiological Culture Collection Center on April 26, 2025, and the preservation number is CGMCC No.46357. The hybridoma cell strain OTI12B5 provided in the application can stably secrete the anti-human RB protein monoclonal antibody, and the anti-human RB protein monoclonal antibody can be specifically combined with the RB protein, significantly improves the specificity, accuracy and reliability of RB protein immunodetection, can be used for labeling of the RB protein, and therefore can detect the RB protein content in tissues and dynamically evaluate the change of the RB protein in targeted treatment.

Description

Technical Field

[0001] This application relates to the technical field of immunoglobulins, and more particularly to an anti-human RB protein monoclonal monomer and its applications, hybridoma cell lines, and reagent kits. Background Technology

[0002] The RB gene was discovered in 1971 by German oncologist Alfred Knudson in retinoblastoma (RB) of the eye in children. It was the first tumor suppressor gene to be cloned and sequenced. Located on chromosome 13q14.2, it contains 27 exons and encodes a protein of 928 amino acids with a molecular weight of 110 kDa. It includes a pocket domain (responsible for binding to E2F) and a C-terminal domain. Expressed in the cell nucleus, it is a DNA-binding protein. The RB protein is involved in multiple related pathways, such as cell cycle progression, regulation of differentiation, maintenance of genome stability, and immune evasion.

[0003] RB proteins are also involved in complex post-transcriptional modifications and related downstream signaling, including both E2F-dependent and E2F-independent modes. In normal cells in a non-proliferative state, RB proteins are in an unphosphorylated and hypophosphorylated state. Unphosphorylated RB proteins bind to the transcription factor E2F, preventing cells from transitioning from G1 phase to S phase. When cells are in the proliferative cycle, RB proteins are phosphorylated and dissociate from E2F, restoring E2F activity and allowing cells to transition from G1 phase to S phase. pRB inhibits cell cycle proliferation by suppressing the action of the transcription factor E2F, thus playing a role in inhibiting tumor cell proliferation. Mutations or deletions of RB disrupt cell cycle regulation, potentially inducing tumors.

[0004] Statistics show that approximately 40% of retinoblastoma patients carry germline mutations in the RB gene. Furthermore, RB deletions are observed in various types of tumors; about 5.1% of tumors show RB gene alterations, with higher prevalence in tumors such as lung adenocarcinoma, small cell lung cancer, invasive ductal carcinoma of the breast, urothelial carcinoma of the bladder, glioblastoma, and osteosarcoma. RB gene mutations and deletions can be detected using methods such as PCR and sequencing (e.g., NGS) to identify molecular causes, assess familial genetic risk, and provide guidance on eugenics.

[0005] Whole-genome sequencing remains costly and cannot fully and accurately reflect the functional state of proteins; even with normal genes, RB proteins may be abnormally phosphorylated, degraded, or inactivated. Immunohistochemical detection methods can determine the abundance or absence of RB protein expression; this technology is mature and less expensive. However, this method requires high-quality anti-RB protein antibodies to ensure the accuracy of the results.

[0006] Currently, there is a lack of highly specific anti-RB protein antibodies for clinical diagnosis. Therefore, developing highly specific and sensitive monoclonal antibodies against RB protein is of great significance for the detection of RB protein in tissues and for the dynamic assessment of changes in RB protein during targeted therapy. Summary of the Invention

[0007] This application provides an anti-human RB protein monoclonal monomer and its application, a hybridoma cell line, and a reagent kit. The hybridoma cell line OTI12B5 provided in this application can stably secrete an anti-human RB protein monoclonal antibody, and this anti-human RB protein monoclonal antibody can specifically bind to RB protein, significantly improving the specificity, accuracy, and reliability of RB protein immunoassay. It can be used for RB protein labeling, thus enabling the detection of RB protein content in tissues and dynamic assessment of changes in RB protein during targeted therapy.

[0008] In a first aspect, this application provides an anti-human RB protein monoclonal monomer, employing the following technical solution:

[0009] A monoclonal monomer against human RB protein, the monoclonal antibody comprising a light chain variable region and a heavy chain variable region; the light chain variable region comprising LCDR1, LCDR2, and LCDR3;

[0010] The LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, the LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:6;

[0011] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3;

[0012] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:8, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0013] Optionally, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence with more than 85% homology obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:3.

[0014] Optionally, the light chain variable region comprises an amino acid sequence having 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 97% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:3.

[0015] Optionally, the light chain variable region has a total length of 106 amino acids, with the number of amino acids in the four domains of its FR being 26, 17, 36 and 11, respectively, and the number of amino acids in the three domains of its LCDR being 8, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 52aa-54aa and 91aa-95aa, respectively, and their amino acid sequences are: QSLLNSGN (SEQ ID NO:4), RMS (SEQ ID NO:5) and AGNDH (SEQ ID NO:6), respectively.

[0016] Optionally, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence with more than 85% homology obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:7.

[0017] Optionally, the heavy chain variable region comprises an amino acid sequence having 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 97% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:7.

[0018] Optionally, the total length of the heavy chain variable region is 107 amino acids, with the number of amino acids in the four domains of its FR being 24, 16, 36 and 11, the number of amino acids in the three domains of HCDR being 8, 7 and 5, and the number of amino acids in HCDR1, HCDR2 and HCDR3 being 25aa-32aa, 49aa-55aa and 92aa-96aa, respectively. Their amino acid sequences are: GYTFTSTY (SEQ ID NO:8), IEPSSYT (SEQ ID NO:9) and TRDGT (SEQ ID NO:10).

[0019] Optionally, the monoclonal monomer is secreted by the hybridoma cell line OTI12B5, which was deposited on April 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46357.

[0020] Secondly, this application provides the application of an anti-human RB protein monoclonal antibody in the preparation of a kit for detecting the content of RB protein in tissues and dynamically assessing changes in RB protein during targeted therapy.

[0021] Thirdly, this application provides a polynucleotide. This polynucleotide encodes the aforementioned anti-human RB protein monoclonal antibody.

[0022] Fourthly, this application provides a hybridoma cell line, employing the following technical solution:

[0023] A hybridoma cell line that secretes the aforementioned anti-human RB protein monoclonal antibody; the hybridoma cell line is named hybridoma cell line OTI12B5, and was deposited on April 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46357.

[0024] Fifthly, this application provides an immunohistochemical detection kit for human RB protein, employing the following technical solution:

[0025] An immunohistochemical detection kit for human RB protein, the kit comprising a primary antibody reagent; the primary antibody reagent comprising the aforementioned anti-human RB protein monoclonal antibody.

[0026] Optionally, the kit further includes antigen retrieval solution, endogenous peroxidase inhibitor, hypersensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

[0027] Optionally, the secondary antibody reagent is a hypersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0028] Preservation information:

[0029] Hybridoma cell line: Mouse anti-human RB monoclonal hybridoma cell line OTI12B5.

[0030] Accession number: CGMCC No.46357.

[0031] Date of preservation: April 26, 2025.

[0032] Preservation institution: China General Microbiological Culture Collection Center (CGMCC).

[0033] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] In summary, this application includes at least one of the following beneficial technical effects:

[0035] The anti-human RB protein monoclonal antibody provided in this application can specifically bind to human RB protein, significantly improving the specificity, accuracy, and reliability of RB protein immunoassay. It can be used for RB protein labeling, thus enabling the detection of RB protein content in tissues and dynamic assessment of RB protein changes during targeted therapy. Simultaneously, the hybridoma cell line OTI12B5 provided in this application can stably secrete the anti-human RB protein monoclonal antibody.

[0036] Using the anti-human RB protein monoclonal antibody and kit of this application, the expression status of RB protein was detected by IHC method. No expression of RB protein was detected in RB-deficient tumors, but RB protein was clearly detected in the internal control cells of the same tissue. This indicates that the anti-human RB protein monoclonal antibody of this application has good specificity and can be well applied to the immunohistochemical detection of RB protein in tissues. Attached Figure Description

[0037] 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.

[0038] Figure 1 This is a Western blot result of recombinant RB protein in Example 2. The expression of recombinant RB protein in E. coli cells was detected using anti-HIS antibody. Lane L represents the detection results using E. coli cell lysates transfected with the empty vector as the antigen, and lane R represents the detection results using E. coli cell lysates transfected with pET23a-rRB plasmid as the antigen.

[0039] Figure 2 The image shows the SDS-PAGE detection results of the recombinant RB protein in Example 2. The recombinant RB protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0040] Figure 3 A schematic diagram showing the results of IHC detection of normal tissues using monoclonal antibodies secreted by the hybridoma cell line OTI12B5 (a. tonsil tissue; b. appendix tissue; c. placental tissue; and d. pancreatic tissue).

[0041] Figure 4 Schematic diagram of the results of IHC detection of RB expression in tumor tissues using monoclonal antibodies secreted by hybridoma cell line OTI12B5 (a. lung cancer tissue; b. lung cancer tissue; c. papillary thyroid carcinoma tissue; d. colorectal cancer tissue; and e. leiomyosarcoma tissue).

[0042] Figure 5 A schematic diagram showing the results of IHC detection of RB-deficient tumor tissue using monoclonal antibodies secreted by hybridoma cell line OTI12B5 (both sides are lung cancer tissue). Detailed Implementation

[0043] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0044] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0045] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0047] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0049] The present application will be further described in detail below with reference to the embodiments and test results.

[0050] Example 1

[0051] This embodiment provides the construction of a recombinant expression plasmid for the RB protein.

[0052] Specifically, the following steps are included:

[0053] The RB gene NM_000321.3 was selected from Genebank. Based on the amino acid sequence characteristics of RB, the amino acid fragment from RB to 590 was designed as an immunogen (its corresponding nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2). Primers were designed, and restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene, respectively. The gene was inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rRB.

[0054] Example 2

[0055] This embodiment provides the expression and purification of recombinant RB protein. Specifically, it includes the following steps:

[0056] (1) Transformation and screening

[0057] After thawing 100 μl of competent cells on ice, add the recombinant plasmid pET23a-rRB and mix gently. Incubate on ice for 30 min, then heat shock at 42°C for 90 sec, and then place on ice for 1-2 min. Add 500 μl of antibiotic-free LB medium to a sterile environment and incubate at 37°C for 45 min. Spread an appropriate amount of bacterial culture evenly onto an antibiotic-containing plate and incubate overnight at 37°C. Pick 24 single clones and culture at 37°C and 200 rpm. Monitor the OD value of the bacterial culture regularly, and separate the cultures. One portion is stored for later use, while the other portion is induced with 1 mM IPTG and cultured for another 7 h. Centrifuge to collect the cells, add lysis buffer to prepare lysis buffer, and perform Western blotting (WB) on the samples using anti-His antibody. Select strains with the target band, such as... Figure 1 As shown.

[0058] Depend on Figure 1The test results showed that the E. coli cell lysate transfected with pET23a-rRB plasmid had a distinct specific band at 35 kDa in lane R, while the control lysate transfected with empty vector did not have a band of the corresponding size in lane L, indicating that the cells specifically expressed recombinant RB protein.

[0059] (2) Protein expression

[0060] The selected bacterial strains were expanded into a 2L culture medium system. The growth status of the strains was monitored. When the OD number reached about 0.6, the strains were induced with 1mM IPTG for 7 hours. The bacterial cells were collected by centrifugation, and lysis buffer was added. The cells were sonicated for 20 minutes and then centrifuged at 12000rpm for 20 minutes at 4℃. The supernatant was collected.

[0061] (3) Affinity chromatography purification

[0062] The target protein was purified and enriched using a nickel ion column. Unbound proteins were removed by elution with buffer. Finally, the product was eluted with eluents containing different concentrations of imidazole. The eluents were collected, and the products were analyzed by SDS-PAGE electrophoresis. Results are shown below. Figure 2 The deproteinized protein that meets the requirements is added to 10% glycerol and stored for later use.

[0063] Depend on Figure 2 The test results showed that the purified recombinant RB protein had a distinct specific band at 35kD in the SDS-PAGE gel image, indicating that the obtained protein met the requirements for use as an antigen for animal immunization.

[0064] Example 3

[0065] This embodiment provides the process for preparing animal immune and hybridoma cell lines.

[0066] BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with the recombinant RB protein prepared in Example 2 according to standard methods.

[0067] (1) Animal immunization

[0068] The synthesized recombinant RB protein was emulsified with complete Freund's adjuvant and immunized 6-8 week old BALB / c mice via intraperitoneal injection at a dose of 50 μg / mouse. A second round of immunization was administered every two weeks, emulsified with incomplete Freund's adjuvant at a dose of 30 μg / mouse. After three immunizations, tail blood was collected and serum titers were determined using a serially diluted ELISA method. Based on the results, an immune response was determined in the immunized animals. Spleens from mice with antibody titers meeting the requirements were selected for cell fusion.

[0069] (2) Cell fusion

[0070] Hybridoma technology was used to prepare fusion cells, with myeloma cells being sp2 / 0 cells in the logarithmic growth phase. Spleens from mice that met the required immune response were collected and used to prepare a single-cell suspension of lymphocytes. Mouse spleen lymphocytes and myeloma cells were mixed at a 1:7 ratio, and 1 mL of preheated 50% PEG (pH 8.0) at 37°C was added. The mixture was allowed to stand for 1 min, and then 10 mL of incomplete culture medium was slowly added to terminate the reaction. The supernatant was discarded after centrifugation, and the mixture was resuspended in HAT medium and mixed thoroughly. The volume was adjusted to 50 mL with MC medium containing methylcellulose, and the mixture was aliquoted into 3.5 cm culture dishes and placed in a humidified chamber at 37°C with 5% CO2 for incubation. The growth of the clonal clusters was observed daily, and after approximately 8 days, clones were selected under a dissecting microscope and transferred to 96-well plates for cell culture.

[0071] Example 4

[0072] This embodiment provides the screening process for hybridoma cell lines that specifically recognize RB protein and the antibody preparation process.

[0073] ELISA and IHC assays were used to screen monoclonal cell lines that specifically recognize RB. The supernatants from the ELISA-screened hybridoma cell lines were then subjected to IHC assays.

[0074] (I) ELISA detection method for screening specific hybridoma cell lines

[0075] The process is as follows:

[0076] (1) Detection of the original coating: Based on the number of clones in the detection hybridoma supernatant, the recombinant RB protein prepared in Example 2 was diluted to 0.4 μg / mL with coating buffer and added to a 96-well plate at a rate of 100 μL / well, and incubated overnight at 4°C.

[0077] (2) Sealing: Add 5% skim milk powder at a volume of 100 μL / well, seal at 37℃ for 1 hour, discard the supernatant, wash with deionized water and spin dry for later use.

[0078] (3) Primary antibody incubation: The supernatant of hybridoma cell culture was used as the primary antibody. 100 μL / well was added to the recombinant RB protein coated plate and incubated at 37°C for 1 h. The supernatant was discarded and the plate was washed 3 times with deionized water.

[0079] (4) Secondary antibody incubation: Add HRP-labeled goat anti-mouse IgG at 100 μL / well, incubate at 37℃ for 1 h, discard the supernatant, and wash 3 times with deionized water.

[0080] (5) Substrate color development: Add 100 μL of substrate A solution and substrate B solution in a 1:1 ratio at 1 well, and incubate at 37°C for 15 min.

[0081] (6) Termination of reaction: Add 50 μL of 2M sulfuric acid per well to terminate the reaction.

[0082] (7) Data analysis: Set the wavelength to 450nm on the microplate reader and read the data.

[0083] The recombinant RB protein-coated plate showed a positive result, which was interpreted as specific recognition of RB. The supernatant was then subjected to IHC analysis.

[0084] (II) Screening of specific hybridoma cell lines using IHC detection method

[0085] The process is as follows:

[0086] (1) Take formalin-fixed tonsil and appendix tissue blocks and embed them in paraffin. Set the thickness of the Leica tissue slicer to 4μm and prepare the slides.

[0087] (2) Dewaxing and hydration: The tablets treated above are sequentially subjected to analytical grade xylene for 10 min × 3 times, anhydrous ethanol for 1 min × 3 times, 95% ethanol for 1 min, 85% ethanol for 1 min, 75% ethanol for 1 min, and deionized water for 2 min × 3 times to achieve dewaxing and hydration.

[0088] (3) Antigen repair: Antigen repair is performed using high temperature and high pressure. Antigen repair solution (EDTA antigen repair solution, pH 8.0) is added to the pressure cooker and heat repair is performed for 2.5 min. When the pressure cooker temperature drops to about 90℃, the pressure cooker is opened, the slices are taken out, and then naturally cooled to room temperature. They are then soaked in deionized water for 2 min × 3 times.

[0089] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in tissues, let stand at room temperature in the dark for 15 min, and soak in deionized water for 2 min × 3 times.

[0090] (5) Incubation with primary antibody: Draw a border around the tissue using an immunohistochemical pen to define the reagent loading area, and wash with 0.1% PBST for 2 min × 1 time. Use hybridoma cell culture supernatant as the primary antibody, add 200 μl of supernatant to the reaction area, place the slide in a humidified chamber, and incubate at 37°C for 60 min. Wash with 0.1% PBST for 2 min × 3 times.

[0091] (6) Incubation with secondary antibody: Add 100 μl of hypersensitive HRP-labeled goat anti-mouse / rabbit IgG polymer to the reaction area and incubate at 37°C for 30 min. Wash with 0.1% PBST for 2 min × 3 times.

[0092] (7) DAB color development: Add 120 μl of freshly prepared DAB color development solution, react at room temperature for 5 min and then stop the color development, and wash 3 times.

[0093] (8) Hematoxylin counterstaining, differentiation and blueing: stand in hematoxylin solution for 12s, wash 3 times to stop the color development, differentiate in 1% hydrochloric acid ethanol solution, rinse 3 times with deionized water to stop the reaction, put into boiling pH 8.0 Tris-EDTA disodium solution for blueing, then put into room temperature pH 8.0 Tris-EDTA disodium solution for a few seconds, and rinse 3 times with tap water.

[0094] (9) Dehydration and clearing: After blueing, the slides were treated with 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min × 3 times, and xylene for 1 min × 3 times, and then mounted with neutral resin.

[0095] (10) Microscopic examination: Observe the staining results under a microscope.

[0096] Nuclear staining was observed in cells of squamous epithelium and germinal centers in tonsil tissue and in cells of glandular epithelium and germinal centers in appendix tissue. The staining pattern conformed to the expression pattern of RB, indicating that the monoclonal antibody secreted by the hybridoma could bind to the RB protein, and the result was considered positive. After multiple rounds of limiting dilution, IHC analysis was performed on the supernatants of different clones. Comparison of the results showed that the antibody secreted by the hybridoma cell line with clone number OTI12B5 better met the quality control standards for the RB test. Therefore, it was determined that this cell line should be expanded for culture to prepare a high-purity antibody, namely, an anti-human RB protein monoclonal monomer.

[0097] The hybridoma cell line with clone number OTI12B5 was deposited on April 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46357.

[0098] (III) Expression and purification of monoclonal antibodies

[0099] The hybridoma cell line OTI12B5 was cultured in DMEM containing 15% serum in 10 cm culture dishes and expanded to approximately 4 × 10⁶ cells / year. 7 Centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L roller flask. Add serum-free culture medium to bring the cell density to approximately 3 × 10⁶ cells / min. 5 Cells / ml. After culturing for another 1.5 weeks, when the cell death rate dropped to 60%, the cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was collected and purified by affinity chromatography. The appropriate column material was selected according to the antibody subtype for purification (for subtype IgG2b, protein G column material was used for purification). The concentration of the purified monoclonal antibody was determined, and after adding antibody protectant, it was stored at 2-8℃.

[0100] Example 5

[0101] This embodiment provides the gene and amino acid sequence analysis of the variable region of the anti-human RB protein monoclonal monomer.

[0102] The SMARTer® RACE 5' / 3' kit was purchased from Takara Bio USA. The variable region light and heavy chain gene sequences of hybridoma cell functional antibodies 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.

[0103] Based on the fact that the monoclonal monomer of the anti-human RB protein is of the IgG2b subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of its Ig and Kappa constant regions were designed. The primer sequences are as follows:

[0104] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11);

[0105] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12).

[0106] mRNA was extracted from the hybridoma cell line OTI12B5 and reverse transcribed into cDNA. The heavy and light chain DNA fragments of the anti-human RB protein monoclonal antibody were amplified using RACE. The amplified light and heavy chains were ligated into the cloning vector PUC119 by enzyme digestion. Positive clones were selected using blue-white screening, and the positive plasmids were purified and sequenced using an ABI 3730 sequencer with universal primers M13F and M13R.

[0107] M13F: 5'-TGTAAAACGACGCGCCAGT-3' (SEQ ID NO: 13);

[0108] M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 14).

[0109] Using the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the sequencing results of the nucleotide sequences of the light and heavy chains of the anti-human RB protein monoclonal antibody were analyzed. The amino acid sequence of the variable region of the light chain of the anti-human RB protein monoclonal antibody is shown in SEQ ID NO:3, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:7.

[0110] The light chain variable region is 106 amino acids in length. The number of amino acids in the four domains of its FR are 26, 17, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 8, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 52aa-54aa and 91aa-95aa, respectively. Their amino acid sequences are: QSLLNSGN (SEQ ID NO:4), RMS (SEQ ID NO:5) and AGNDH (SEQ ID NO:6), respectively.

[0111] The heavy chain variable region is 107 amino acids in length. The number of amino acids in the four domains of its FR are 24, 16, 36 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 5, respectively. HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 49aa-55aa and 92aa-96aa, respectively. Their amino acid sequences are: GYTFTSTY (SEQ ID NO:8), IEPSSYT (SEQ ID NO:9) and TRDGT (SEQ ID NO:10).

[0112] Example 6

[0113] This embodiment provides an immunohistochemical detection kit containing a monoclonal antibody against human RB protein.

[0114] The above-mentioned immunohistochemical detection kit containing anti-human RB protein monoclonal antibody includes antigen retrieval solution [1mM EDTA, 10mM Tris buffer (pH 8.0)], anti-human RB protein monoclonal antibody prepared in Example 4, endogenous peroxidase inhibitor (hydrogen peroxide), ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

[0115] Example 7

[0116] This embodiment provides immunohistochemical detection using an anti-human RB protein monoclonal antibody as the primary antibody.

[0117] The immunohistochemical detection kit of Example 6 was used to detect RB protein in samples from different tissue sources. The specific steps were as follows in the "IHC detection method for screening specific hybridoma cell lines" in Example 4. In step (1), during the slide preparation process, the types and quantities of tissue samples were increased. The tissues used included tonsils, appendix, pancreas, placenta, lung cancer (4 cases), intestinal cancer, papillary thyroid carcinoma, and leiomyoma. Some reagents in steps (2) to (8) were derived from the kit prepared in Example 5. In step (6), the concentration of the primary antibody (anti-human RB protein monoclonal antibody) was 1.38 μg / mL.

[0118] The experimental results are as follows:

[0119] Figure 3 The staining results of the anti-human RB protein monoclonal antibody immunohistochemical detection kit in tonsil (a), appendix (b), placenta (c) and pancreas (d) tissues are shown.

[0120] Depend on Figure 3 The results showed that the staining results in the tonsil and appendix tissues were basically consistent with the detection results of the hybridoma cell culture supernatant during the screening stage of the monoclonal antibody hybridoma. Different signal intensities were observed in the nuclei of cells such as the germinal centers and squamous epithelium of the tonsils and the glandular epithelium of the appendix. Brown staining was observed in the nuclei of cells such as the trophoblast cells and vascular endothelial cells of the placenta, and the acinar epithelium, islet cells, and ductal epithelium of the pancreatic tissue, indicating positive expression of RB protein.

[0121] Figure 4 The results of the immunohistochemical assay kit for anti-human RB protein monoclonal antibody staining in two lung cancer (a and (b), papillary thyroid carcinoma (c), colorectal cancer (d), and leiomyoma (e) tissues are presented.

[0122] Depend on Figure 4 The results showed that the tumor cells from the two lung cancers, which were from different sources, exhibited different staining intensities, suggesting that there may be differences in RB protein expression between the two cases. The tumor cell nuclei of papillary thyroid carcinoma, colorectal cancer, and leiomyosarcoma cells also showed brown staining, indicating the presence of RB protein.

[0123] Figure 5 The staining results of the anti-human RB protein monoclonal antibody immunohistochemical detection kit on two lung cancer cases are shown.

[0124] Depend on Figure 5The results show that the tumor cells indicated by the star (for example only) in both the left and right images did not show brown staining, indicating the absence of RB protein expression in the tumor cells, and the result was negative. The vascular endothelial cells indicated by the triangle (for example only) showed positive nuclear staining, and the right image shows positive staining in the nuclei of lymphocytes and other stromal cells, which can be used as internal controls. These results are consistent with the clinical diagnosis of the case. The concentration of the monoclonal antibody used in this test was 1.38 μg / mL. Under these conditions, the positive signal intensity on the tumor cells met the clinical interpretation requirements, and there was no background staining that would affect the results, indicating that the antibody secreted by the hybridoma cell OTI12B5 has good specificity and high sensitivity.

[0125] In summary, the anti-human RB protein monoclonal antibody secreted by the hybridoma cell line OTI12B5 provided in this application exhibits high specificity and sensitivity. Therefore, the anti-human RB monoclonal antibody secreted by the hybridoma cell line OTI12B5 can be used for immunohistochemical detection to detect the expression of RB in related tumors.

[0126] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A monoclonal antibody against human RB protein, characterized in that, The monoclonal antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises LCDR1, LCDR2, and LCDR3; The LCDR1 is the amino acid sequence shown in SEQ ID NO:4, the LCDR2 is the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 is the amino acid sequence shown in SEQ ID NO:6; The heavy chain variable region includes HCDR1, HCDR2, and HCDR3; The HCDR1 is the amino acid sequence shown in SEQ ID NO:8, the HCDR2 is the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 is the amino acid sequence shown in SEQ ID NO:

10.

2. The anti-human RB protein monoclonal antibody according to claim 1, characterized in that, The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

3.

3. The anti-human RB protein monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:

7.

4. The anti-human RB protein monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line OTI12B5, which has the accession number CGMCC No. 46357.

5. The use of any monoclonal antibody according to any one of claims 1-4 in the preparation of a kit for detecting the content of RB protein in tissues and dynamically assessing changes in RB protein during targeted therapy.

6. A polynucleotide, characterized in that, The polynucleotide encodes the monoclonal antibody according to any one of claims 1-4.

7. A hybridoma cell line, characterized in that, The hybridoma cell line secretes the monoclonal antibody of claim 1; the name of the hybridoma cell line is hybridoma cell line OTI12B5, and the preservation number is CGMCCNo.46357.

8. An immunohistochemical detection kit for human RB protein, characterized in that, The kit includes a primary antibody reagent; the primary antibody reagent includes any one of claims 1-4.

9. The reagent kit according to claim 8, characterized in that, The kit also includes antigen retrieval solution, endogenous peroxidase inhibitor, hypersensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

Images