A rabbit anti-mouse secondary antibody, and a preparation method and application thereof

Abstract

The application belongs to the technical field of immunochemistry, and particularly relates to a rabbit anti-mouse secondary antibody, a preparation method and application thereof, and particularly application in immunohistochemical detection. The rabbit anti-mouse secondary antibody comprises a plurality of monoclonal antibodies which specifically bind to a constant region of a mouse primary antibody. The antigen epitopes recognized by the plurality of monoclonal antibodies do not overlap with each other, and jointly cover mouse IgG1, IgG2a and IgG2b subtypes. The plurality of monoclonal antibodies comprise a heavy chain variable region sequence as shown in SEQ ID NO: 17-22, and a light chain variable region sequence as shown in SEQ ID NO: 23-28. The application also relates to a nucleotide sequence encoding the rabbit anti-mouse secondary antibody, an expression vector, a preparation method, and application of the new rabbit anti-mouse secondary antibody in an IHC detection method or device. The rabbit anti-mouse secondary antibody can be applied to the fields of immunohistochemistry, indirect enzyme-linked immunosorbent assay, immunoblotting, antibody chip preparation, flow cytometry and other detection and screening fields.

Description

Technical Field

[0001] This invention belongs to the field of immunochemical technology, and particularly relates to a rabbit anti-mouse secondary antibody, its preparation method and application, especially its application in immunohistochemical detection. Background Technology

[0002] Secondary antibodies play a crucial auxiliary role in pathological diagnosis and are indispensable reagents for key diagnostic techniques such as immunohistochemistry and immunofluorescence. Their core function is to specifically bind to the constant region of the primary antibody, amplifying the signal and accurately locating, characterizing, and semi-quantitatively detecting target antigens within tissue cells. They are widely used in tumor subtyping, determining the benign or malignant nature of lesions, screening for targeted therapy biomarkers, and assessing disease prognosis, providing pathologists with precise diagnostic evidence and directly influencing the development of clinical treatment plans.

[0003] Currently, polyclonal secondary antibodies remain commonly used reagents in some clinical institutions, but they have significant limitations and shortcomings: these secondary antibodies originate from multiple B lymphocytes, have diverse antigen-binding sites, low specificity, and are prone to cross-reaction with irrelevant antigens in the sample, leading to cluttered detection background and increasing the risk of false positives; furthermore, they are affected by individual differences in animal immunization and batch-by-batch immunization, resulting in poor batch-to-batch consistency and unreproducible test results, making it difficult to adapt to standardized pathological testing procedures; simultaneously, they cannot accurately identify primary antibody subtypes, have insufficient adaptability, and can easily interfere with diagnostic judgment, failing to meet the needs of accurate pathological diagnosis. In existing technologies, some improved solutions have emerged to balance the broad-spectrum recognition capability and batch-to-batch stability of secondary antibodies. For example, Chinese patent (CN120058954A) uses gene fusion technology to construct bispecific nanobodies against mouse IgG and rabbit IgG; Abcam has developed recombinant polyclonal antibody technology. However, the aforementioned existing technologies still have shortcomings in achieving comprehensive coverage of different murine subtype primary antibodies (IgG1, IgG2a, IgG2b, etc.) and synergistic binding of multiple epitopes of the same murine primary antibody molecule, making it difficult to simultaneously meet the detection requirements of high specificity, high sensitivity, and broad-spectrum adaptability.

[0004] Therefore, there is an urgent need to develop a novel rabbit anti-mouse secondary antibody reagent that retains the broad-spectrum recognition capability of polyclonal secondary antibodies while possessing the high specificity and batch-to-batch stability of monoclonal antibodies. This would enable accurate and convenient detection of different subtypes of mouse primary antibodies and allow the reagent to directly replace existing traditional polyclonal secondary antibodies without altering the existing detection process. Summary of the Invention

[0005] In view of the aforementioned shortcomings and deficiencies of the prior art, this invention provides a widely applicable rabbit anti-mouse secondary antibody that can accurately identify the constant region of mouse primary antibodies, along with its preparation method and application. This effectively replaces traditional rabbit anti-mouse polyclonal secondary antibodies, improving the accuracy, stability, and compatibility of detection methods such as IHC, WB, and ELISA, and meeting the needs of precise clinical testing and standardized scientific research testing. This invention also relates to the nucleotide sequence encoding the rabbit anti-mouse secondary antibody, the encoding gene, the nucleic acid molecule, the expression vector, the preparation method, and the application of the rabbit anti-mouse secondary antibody in IHC detection methods or devices.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include: In a first aspect, the present invention provides a rabbit anti-mouse secondary antibody comprising multiple monoclonal antibodies that specifically bind to the constant region of a mouse primary antibody. The antigenic epitopes recognized by the multiple monoclonal antibodies do not overlap and collectively cover mouse IgG1, IgG2a, and IgG2b subtypes. The multiple monoclonal antibodies include a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17-22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:23-28.

[0007] Furthermore, the multiple monoclonal antibodies that specifically bind to the constant region of the murine primary antibody are a mixture of antibodies A, B, C, D, E, and F, with a mass ratio of 1:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5. Preferably, the mass ratio of antibodies A, B, C, D, E, and F is 1:3:1:1:3:2.

[0008] This rabbit anti-mouse secondary antibody is prepared by mixing six different strains of rabbit anti-mouse monoclonal antibodies (antibody A, antibody B, antibody C, antibody D, antibody E, and antibody F) in a mass ratio of 1:3:1:1:3:2. The rabbit anti-mouse monoclonal secondary antibodies specifically bind to the constant region of the mouse primary antibody, and the antigenic epitopes recognized by each group of monoclonal secondary antibodies do not overlap. At the same time, they cover commonly used primary antibody subtypes such as mouse IgG1, IgG2a, and IgG2b.

[0009] The rabbit anti-mouse monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the rabbit anti-mouse monoclonal antibody provided by this invention was produced through rabbit hybridoma fusion screening and eukaryotic expression in 293 cells. In preparing the rabbit anti-mouse monoclonal antibody, the antigen used to immunize the rabbits (New Zealand white rabbits) was a mixture of different mouse monoclonal antibody subtypes. After immunizing the rabbits, cell fusion and clonal screening were performed to obtain a positive hybridoma cell line that could efficiently secrete monoclonal antibodies. Molecular cloning technology was used to obtain the nucleotide sequences encoding the heavy and light chain amino acid sequences of the antibody. The nucleotide sequences were constructed on a eukaryotic expression vector, transfected into the 293 cell line using a transfection reagent, and the cell supernatant was collected. The cell supernatant was purified by protein A column affinity chromatography to obtain the rabbit anti-mouse monoclonal antibody. Monoclonal antibodies binding to different mouse antibody subtypes were screened and isolated from the obtained rabbit anti-mouse monoclonal antibody. Six different rabbit anti-mouse monoclonal antibodies (antibody A, antibody B, antibody C, antibody D, antibody E, and antibody F) were selected and mixed in an optimal mass ratio of 1:3:1:1:3:2. The mixture can simultaneously cover commonly used primary antibody subtypes such as mouse IgG1, IgG2a, and IgG2b.

[0010] The rabbit anti-mouse monoclonal antibody can specifically recognize mouse-derived primary antibodies and has no cross-reaction with human IgG.

[0011] Secondly, the present invention provides a coding gene for encoding the above-mentioned rabbit anti-mouse secondary antibody.

[0012] Preferably, the encoding gene includes a DNA sequence as shown in SEQ ID NO:5-10 or a complementary sequence thereof, respectively used to encode the heavy chain variable region of the rabbit anti-mouse secondary antibody; and a DNA sequence as shown in SEQ ID NO:11-16 or a complementary sequence thereof, respectively used to encode the light chain variable region of the rabbit anti-mouse secondary antibody.

[0013] Thirdly, the present invention provides a nucleic acid molecule comprising encoding genes for encoding the rabbit anti-mouse secondary antibody.

[0014] Fourthly, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0015] Fifthly, the present invention provides a host cell that has been transformed or transfected with the above-mentioned expression vector.

[0016] In a sixth aspect, the present invention provides a method for preparing a rabbit anti-mouse monoclonal antibody, wherein the above-mentioned expression vector is used to transform or transfect the above-mentioned host cells, the transformed or transfected cells are cultured, the cell supernatant is collected and purified, and the rabbit anti-mouse monoclonal antibody is obtained.

[0017] The seventh aspect concerns the application of the rabbit anti-mouse secondary antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in a mouse primary antibody detection device, wherein the detection device includes, but is not limited to, reagent kits, antibody chips, etc.

[0018] Eighthly, the present invention provides an immunoassay kit for detecting primary antibodies against mouse IgG1, IgG2a, and IgG2b subtypes, comprising horseradish peroxidase-labeled rabbit anti-mouse secondary antibody as described in claim 1 or 2.

[0019] The rabbit anti-mouse secondary antibody provided by this invention employs a mixture of multiple specific rabbit anti-mouse monoclonal secondary antibodies. All monoclonal secondary antibodies undergo rigorous screening to effectively avoid non-specific binding and reduce cross-reactivity. The binding to mouse primary antibodies exhibits high specificity and sensitivity, enabling specific identification and detection of mouse primary antibody reagents. Through the rational combination of multiple monoclonal secondary antibodies, this invention can cover commonly used primary antibody subtypes such as mouse IgG1, IgG2a, and IgG2b, with a binding spectrum comparable to traditional polyclonal secondary antibodies. Simultaneously, it can accurately identify primary antibodies for each subtype, adapting to the detection needs of different subtypes without requiring secondary antibody replacement for different subtypes, making it more convenient to use. Furthermore, it can directly replace traditional rabbit anti-mouse polyclonal secondary antibodies without adjusting existing detection procedures. This antibody can be applied in immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chip preparation, flow cytometry, and other detection and screening fields, maintaining excellent detection performance in different detection methods. Its strong versatility facilitates accurate assessment and detection results. Attached Figure Description

[0020] Figure 1The images show the immunohistochemical detection results of the rabbit anti-mouse secondary antibody prepared in this invention and the commercially available rabbit anti-mouse polyclonal antibody (Abcam, ab6728) against CD10 antibody (IgG1 subtype) in tonsil tissue, CD20 antibody (IgG2a subtype) in tonsil tissue, and TROP2 antibody (IgG2b subtype) in breast cancer tissue; where a is the immunohistochemical detection result of the rabbit anti-mouse secondary antibody against CD10 antibody (IgG1 subtype) in tonsils, and b is the immunohistochemical detection result of the commercially available rabbit anti-mouse polyclonal antibody against CD10 antibody (IgG1 subtype) in tonsils (a). (c and d are at the same magnification). c is the immunohistochemical detection result of rabbit anti-mouse secondary antibody against CD20 antibody (IgG2a subtype) in tonsils; d is the immunohistochemical detection result of commercially available rabbit anti-mouse polyclonal antibody against CD20 antibody (IgG2a subtype) in tonsils (c and d are at the same magnification); e is the immunohistochemical detection result of rabbit anti-mouse secondary antibody against TROP2 antibody (IgG2b subtype) in breast cancer; f is the immunohistochemical detection result of commercially available rabbit anti-mouse polyclonal antibody against TROP2 antibody (IgG2b subtype) in breast cancer (e and f are at the same magnification). Figure 2 The figure shows the statistical results of the rabbit anti-mouse secondary antibody and the commercially available antibody (Abcam, ab6728) of the present invention against the titers of the three mouse primary antibodies of IgG1, IgG2a and IgG2b at eight different concentration gradients. Figure 3 The results of the immunoblotting test were used to verify the ability of the rabbit anti-mouse secondary antibody of the present invention to recognize the IgG1 subtype mouse primary antibody after HRP labeling. Figure 4 The results of the immunoblotting test were used to verify the ability of the rabbit anti-mouse secondary antibody of the present invention to recognize the IgG2a subtype mouse primary antibody after HRP labeling. Figure 5 The results of the immunoblotting test were used to verify the ability of the rabbit anti-mouse secondary antibody of the present invention to recognize the IgG2b subtype mouse primary antibody after HRP labeling. Detailed Implementation

[0021] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0022] 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 conventional products that can be purchased through legitimate channels. Human tissue samples are formalin-fixed and paraffin-embedded human tissue samples, all of which have been pathologically verified, and informed consent has been obtained from the patients.

[0023] Example 1 This embodiment describes the preparation and screening of rabbit anti-mouse secondary antibodies, including the following steps: (1) Antigen preparation The mouse primary antibody antigen is a mixed antigen. The specific mixing method is as follows: Mouse IgG1, IgG2a, and IgG2b subtype primary antibodies were selected and mixed in a mass ratio of 1:1:1. The concentration of the mixed mouse primary antibodies was then adjusted to 1 mg / ml with PBS buffer and used as an immunogen to immunize rabbits.

[0024] (2) Immunity The mixed antigen obtained in step (1) was mixed with complete Freund's adjuvant (volume ratio 1:1) and emulsified. Multiple New Zealand white rabbits were immunized by subcutaneous injection. Two weeks later, the antigen was emulsified with incomplete Freund's adjuvant (volume ratio 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. The rabbit with the highest antibody titer against the antigen was selected for the next step of cell fusion.

[0025] (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. Immunized rabbit spleens were used to prepare a single-cell suspension of lymphocytes. The rabbit spleen lymphocytes were mixed with the myeloma cells, and 50% PEG1500 was added. IMDM medium was added, and after centrifugation and discarding the supernatant, the cells were gently resuspended and mixed in HAT medium. The volume was adjusted to 800 mL, and the mixture was aliquoted into 96-well plates and incubated at 37°C with 5% CO2. The fused cell status in the 96-well plates was observed 6-9 days after fusion. The medium was changed with HT, and the plates were continued to be incubated at 37°C with 5% CO2.

[0026] (4) Screening and cloning Seven to ten days after fusion, clonal cells were screened using an ELISA test with the immunogen (obtained in step 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.

[0027] (5) Perform antibody sequencing on the selected hybridoma cell lines. Total RNA was isolated from selected positive hybridoma cells according to the TriZol RNA extraction reagent instructions. Following the instructions of the TIANScript first-strand cDNA synthesis kit, the total RNA was reverse transcribed into cDNA. The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were amplified using specific primers. These sequences were then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk1) for cell transfection. The specific primers used included: heavy chain variable region primers VH-F and VH-R, and light chain variable region primers VK-F and VK-R.

[0028] The sequence of the heavy chain variable region primer VH-F is shown in SEQ ID NO:1, and the sequence of VH-R is shown in SEQ ID NO:2. The specific sequences of SEQ ID NO:1 and SEQ ID NO:2 are as follows: The sequence of SEQ ID NO:1 is: AGACTGGGCTGCGCTGGCTTC.

[0029] The sequence of SEQ ID NO:2 is: GTGAGGGTGCCCGAG.

[0030] The sequence of the light chain variable region primer VH-F is shown in SEQ ID NO:3, and the sequence of VH-R is shown in SEQ ID NO:4. The specific sequences of SEQ ID NO:3 and SEQ ID NO:4 are as follows: The sequence of SEQ ID NO:3 is: ATGGACAYGAGGGCCCCCACTC.

[0031] The sequence of SEQ ID NO:4 is: GGTGGGAAGATGAGGACAGTAGG.

[0032] (6) Cell transfection and screening Prepare 293 cells for transfection in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 24-well plates at a density of 1.5 ml per well (3 × 10⁻⁶ cells / well). 6 per ml.

[0033] The eukaryotic expression vector was mixed with polyethyleneimine (PEI) at a mass ratio of 1:6 and added to the prepared 293 cells. The cells 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 for positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. A positive immunohistochemical test confirmed the correct antibody sequence.

[0034] (7) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography with protein A to obtain antibodies with a purity >95%. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4℃-8℃.

[0035] Screening and Combination of Positive Monoclonal Antibodies Alternatively labeled microplates were coated with antibodies against mouse IgG1, IgG2a, and IgG2b subtypes. The selected positive antibodies were then detected separately using ELISA for each mouse subtype to determine the specific subtype bound to each antibody. After differentiating the selected rabbit anti-mouse antibodies according to their bound mouse antibody subtypes, paired ELISA assays were performed on antibodies that bound to the same subtype. Antibodies with non-overlapping binding sites were then selected. Ultimately, six different antibodies—Antibody A, Antibody B, Antibody C, Antibody D, Antibody E, and Antibody F—were identified from the positive rabbit anti-mouse antibodies.

[0036] Immunohistochemical staining was performed on six different antibodies to determine the optimal concentration for each. Based on the optimal concentration for each antibody, the six antibodies were mixed in a mass ratio ranging from: Antibody A: Antibody B: Antibody C: Antibody D: Antibody E: Antibody F = 1:1~5:1~5:1~5:1~5:1~5. Within this range, different mixing combinations were designed to ensure that the mixed antibodies could simultaneously recognize and cover commonly used primary antibody subtypes, including but not limited to murine IgG1, IgG2a, and IgG2b. Through experimental screening, the optimal mass ratio of the six antibodies was determined to be 1:3:1:1:3:2, based on the criteria of "clear color development of each subtype with no nonspecific background" while meeting subtype coverage requirements.

[0037] Finally, the heavy chain variable region nucleotide sequences of the six rabbit anti-mouse monoclonal antibodies are shown in SEQ ID NO:5-10.

[0038] The nucleotide sequence of the heavy chain variable region of antibody A is SEQ ID NO:5, and the sequence of SEQ ID NO:5 is as follows: cagagcctggaggagagcggcggaaggttggtgacacctggcacacctcttaccctgacctgcaccgccagcatcctggtgatcagcgactacgccgttacctgggtgaggcaagcccctggcaagggcttagagtggatcggcttcatcgagagggacggcaccacctacta cgccagctggcctaagggcaggttcaccatcagcaggaccagcaccacagtggacctggagatgaccagcctgaccaccgaggacaccgctacctacttctgcggcaggatctactacgccggcgctagcgctgacatttggggacctggaaccctggtgacagtgagcagc.

[0039] The nucleotide sequence of the heavy chain variable region of antibody B is SEQ ID NO:6, and the sequence of SEQ ID NO:6 is as follows: cagagcgtggaggagagcggcggaaggctggttacacctggaacacccccttaccctgacctgcaccgtgagcggcttcagcctgagcaactactacatgagctgggtgaggcaggcccctggcaagggcctggagtatattggctggatcagcgacaccgccaccacctactac gctagctgggctaagggcagattcaccatcagcaagaccagcagcaccaccgtggacctgaagatgaccagcctgaccaccgaggacaccgctacctacttctgcgccagggtgtactacaccggctggggagctgacatttggggacctggaaccctggtgaccgtgtctagc.

[0040] The nucleotide sequence of the heavy chain variable region of antibody C is SEQ ID NO:7, and the sequence of SEQ ID NO:7 is as follows: cagagcctggaggagagcggcggcaggctggttacacccggaacacctttaaccctgacctgcaccgccagcggcttcagcctgagcagctacaagatgggatgggtgaggcaggcccctggaaagggcctggagtacatcggatggatcagcgacaccgccaccacctactacgctagctgggccaagggaaggttcaccatcagcaagaccagcagcaccaccgtggacctgaagatgaccagcctgaccaccgaggacaccgctacctacttctgcgccagggtgtactacaccggctggggagctgacatttggggacctggaaccctggtgaccgtgtctagc。

[0041] The nucleotide sequence of the heavy chain variable region of antibody D is SEQ ID NO:8, and the sequence of SEQ ID NO:8 is: cagagcgtggaggagagcggcggccacctggttacacctggaacccctttaaccctgacctgcaccgtgagcggcatcgacctgagcagctacgccatgatctgggtgaggcaggctcctggcaagggcttggagtggatcggcatcatctacgctggcgaggacacctactacgccaactgggccaagggcaggttcaccatcagcaagaccagcagcaccaccgtggacctgaggatgaccagccccaccacagaagacaccgctacctacttctgcgccagggtgttctacggcggcgagatcggagacatttggggacctggaaccctggtgaccgtgagcagc。

[0042] The nucleotide sequence of the heavy chain variable region of antibody E is SEQ ID NO:9, and the sequence of SEQ ID NO:9 is: cagagcgtggaggagagcggcggcaggctggttacacctggaacacctctgacactgacctgcaccgtgagcggcatcgacctgagcagctatgctatgggctgggtgaggcaagctcccggcaagggcttggagtacatcggcatcatcagcaggagcggcagcacctactac gccagctgggctaagggcaggttcacaatcagcaagaccagcagcaccacgtggacctgaggatcaccagccccacacctgaagacaccgctacctacttctgcgccagggacgctagcagcggcgattctcctgacatttggggacctggaaccctggtgaccgtgagcagc.

[0043] The nucleotide sequence of the heavy chain variable region of antibody F is SEQ ID NO:10, and the sequence of SEQ ID NO:10 is as follows: cagagcctggaggagagcggcggcaggcttgtttctcctggaacccctctgacactgacctgcaccgtgagcggcatcgacctgagcagctactggatgagctgggtgaggcaggctcctggcaagggcttggagtacatcggcatcatcaccgtgatcggcagcac ctactacgccagctgggtgaagggaaggttcaccatcagcaagaccagcaccaccgtggacctgaagatcaccagccccaccaccgaggacacagctacctacttctgcgcttctggcgactactacaccatctggggccccggcacccttgtgacagtgagcagc.

[0044] The light chain variable region nucleotide sequences of the six rabbit anti-mouse monoclonal antibodies are shown in SEQ ID NO:11-16.

[0045] The nucleotide sequence of the light chain variable region of antibody A is SEQ ID NO:11, and the sequence of SEQ ID NO:11 is as follows: gcccaggtgctgacccagacagcttctcctgtttctgccgctgtgggaggcaccgtgaccatcaagtgccagagcagccagaacgtgtggaacaacaacgagctgagctggtaccagcagaagcccggccagcctcccaagttgctgatctacctggccagcaccttggctagcggcgttccttctaggttcaagggcagcggcagcggaacccacttcaccctgaccatctctgacctggagtgcgacgacgccgccacatactactgccagggcggatatggagtgggcatcaaccccttcggcggaggaacagaggtggtggtgaag。

[0046] The nucleotide sequence of the light chain variable region of antibody B is SEQ ID NO:12, and the sequence of SEQ ID NO:12 is: gccatcgtgatgacccagacacccagcagcaagagcgtgcccgttggcgataccgtgaccatcaactgccaggcctctcagagcatcaacaacaactggctgagctggttccagcagaagcccggccagcctcccaagttgctgatctacctggcctctaccctggctagcgaggtgcccagcaggttcaaaggctctggctctggaacccagttcaccctgaccatcaccgacgtggtgtgcgacgacgctgccacatactactgcgccggatacaagcccgacagcatcgacgcctacggctttggaggaggaaccgaggtggttgtgaag。

[0047] The nucleotide sequence of the light chain variable region of antibody C is SEQ ID NO:13, and the sequence of SEQ ID NO:13 is: gcccaggtgctgacccagaccccttctcctgtttctgctgccgttggaggcaccgtgaccatcagctgccagagcagccagacagtgtactacggcaacaggctgagctggtaccagcagaagcccggccaacctcccaagttgctgatctacgccaccagcagcctggctagcggcgttcccagcaggttcaaaggatctgagagcggcaccgagttcaccctgaccatcagcgacgtggagtgcgacgatgctgccacctactactgcgctggctactacagcggcgacatcttcgccttcggcggaggaacagaggtggtggtgaag。

[0048] The nucleotide sequence of the light chain variable region of antibody D is SEQ ID NO:14, and the sequence of SEQ ID NO:14 is: gcccaggtgctgacccagaccccttctcctgtttctgctgctgtgggaggcaccgtgaccatcagctgccagagcagccagacagtgtactacggcaacaggctgagctggtaccagcagaagcccggccagcctcctaagttgctgatctacgccaccagcagcctggctagcggcgtgccttctaggtttaagggaagcgagagcggcaccgagttcaccctgaccatcagcgacgtggagtgtgatgacgccgccacctactactgcgccggatactacagcggcgacatcttcgccttcggcggaggaacagaggtggtggtgaag。

[0049] The nucleotide sequence of the light chain variable region of antibody E is SEQ ID NO:15, and the sequence of SEQ ID NO:15 is: gcccaggtgctgacccagacccctagcagcgtttctgctgctgttggaggcaccgtgaccatcaactgccagagcagcgagagcgtgtacagcgacaagaggctggcctggtaccagcagaagcccggacagagccctaagctgctgatctacctggccagcacc ctggctagcggagttcccagcaggttctctggatctggctctggcacccagttcaccctgaccatcagcgacctggagtgcgacgatgctgccacctactactgcgccggagcttacagcatcagcatgcacatcttcggcggcggcacagaggtggtggtgaag.

[0050] The nucleotide sequence of the light chain variable region of antibody F is SEQ ID NO:16, and the sequence of SEQ ID NO:16 is as follows: gcccaggtgctgacccagacccctctcctgtttctgccgccgttggaagcaccgtgaccatcagctgccagtctagccagagcgtgtacgacaacaactggctgagctggttccagcagatccccggccacagccccaagttgctgatgtacaccgccagcacc ttggctagcggcgtgcctagcaggttcaagggatctggaagcggcacccagttcaccctgaccatcagcgacctgcagtgcgatgatgccgccacctactattgcgccggcggatacagcggcaacatcttcaccttcggcggaggaacagaggtggtggtgaag.

[0051] The obtained base sequences were translated into amino acid sequences, and the amino acid sequences of the heavy chain variable region of six rabbit anti-mouse monoclonal antibodies were analyzed and obtained as shown in SEQ ID NO:17-22.

[0052] The amino acid sequence of the heavy chain variable region of antibody A is SEQ ID NO:17, and the sequence of SEQ ID NO:17 is as follows: QSLEESGGRLVTPGTPLTLTCTASILVISDYAVTWVRQAPGKGLEWIGFIERDGTTYYASWPKGRFTISRTSTTVDLEMTSLTTEDTATYFCGRIYYAGASADIWGPGTLVTVSS.

[0053] The amino acid sequence of the heavy chain variable region of antibody B is SEQ ID NO:18, and the sequence of SEQ ID NO:18 is as follows: QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYYMSWVRQAPGKGLEYIGWISDTATTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARVYYTGWGADIWGPGTLVTVSS.

[0054] The amino acid sequence of the heavy chain variable region of antibody C is SEQ ID NO:19, and the sequence of SEQ ID NO:19 is as follows: QSLEESGGRLVTPGTPLTLTCTASGFSLSSYKMGWVRQAPGKGLEYIGWISDTATTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARVYYTGWGADIWGPGTLVTVSS.

[0055] The amino acid sequence of the heavy chain variable region of antibody D is SEQ ID NO:20, and the sequence of SEQ ID NO:20 is as follows: QSVEESGGHLVTPGTPLTLTCTVSGIDLSSYAMIWVRQAPGKGLEWIGIIYAGEDTYYANWAKGRFTISKTSSTTVDLRMTSPTTEDTATYFCARVFYGGEIGDIWGPGTLVTVSS.

[0056] The amino acid sequence of the heavy chain variable region of antibody E is SEQ ID NO:21, and the sequence of SEQ ID NO:21 is as follows: QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIISRSGSTYYASWAKGRFTISKTSSTTVDLRITSPTPEDTATYFCARDASSGDSPDIWGPGTLVTVSS.

[0057] The amino acid sequence of the heavy chain variable region of antibody F is SEQ ID NO:22, and the sequence of SEQ ID NO:22 is as follows: QSLEESGGRLVSPGTPLTLTCTVSGIDLSSYWMSWVRQAPGKGLEYIGIITVIGSTYYASWVKGRFTISKTSTTVDLKITSPTTEDTATYFCASGDYYTIWGPGTLVTVSS.

[0058] The obtained base sequences were translated into amino acid sequences, and the amino acid sequences of the light chain variable region of six rabbit anti-mouse monoclonal antibodies were analyzed and obtained as shown in SEQ ID NO:23-28.

[0059] The amino acid sequence of the light chain variable region of antibody A is SEQ ID NO:23, and the sequence of SEQ ID NO:23 is as follows: AQVLTQTASPVSAAVGGTVTIKCQSSQNVWNNNELSWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTHFTLTISDLECDDAATYYCQGGYGVGINPFGGGTEVVVK.

[0060] The amino acid sequence of the light chain variable region of antibody B is SEQ ID NO:24, and the sequence of SEQ ID NO:24 is as follows: AIVMTQTPSSKSVPVGDTVTINCQASQSINNNWLSWFQQKPGQPPKLLIYLASTLASEVPSRFKGSGSGTQFTLTITDVVCDDAATYYCAGYKPDSIDAYGFGGGTEVVVK.

[0061] The amino acid sequence of the light chain variable region of antibody C is SEQ ID NO:25, and the sequence of SEQ ID NO:25 is as follows: AQVLTQTPSPVSAAVGGTVTISCQSSQTVYYGNRLSWYQQKPGQPPKLLIYATSSLASGVPSRFKGSESGTEFTLTISDVECDDAATYYCAGYYSGDIFAFGGGTEVVVK.

[0062] The amino acid sequence of the light chain variable region of antibody D is SEQ ID NO:26, and the sequence of SEQ ID NO:26 is as follows: AQVLTQTPSPVSAAVGGTVTISCQSSQTVYYGNRLSWYQQKPGQPPKLLIYATSSLASGVPSRFKGSESGTEFTLTISDVECDDAATYYCAGYYSGDIFAFGGGTEVVVK.

[0063] The amino acid sequence of the light chain variable region of antibody E is SEQ ID NO:27, and the sequence of SEQ ID NO:27 is as follows: AQVLTQTPSSVSAAVGGTVTINCQSSESVYSDKRLAWYQQKPGQSPKLLIYLASTLASGVPSRFSGSGSGTQFTLTISDLECDDAATYYCAGAYSISMHIFGGGTEVVVK.

[0064] The amino acid sequence of the light chain variable region of antibody F is SEQ ID NO:28, and the sequence of SEQ ID NO:28 is as follows: AQVLTQTPSPVSAAVGSTVTISCQSSQSVYDNNWLSWFQQIPGHSPKLLMYTASTLASGVPSRFKGSGSGTQFTLTISDLQCDDAATYYCAGGYSGNIFTFGGGTEVVVK.

[0065] Example 2 This embodiment describes the immunohistochemical detection of three mouse-derived primary antibodies against different subtypes of rabbit anti-mouse secondary antibodies: CD10 antibody (IgG1 subtype) in tonsil tissue, CD20 antibody (IgG2a subtype) in tonsil tissue, and TROP2 antibody (IgG2b subtype) in breast cancer tissue. The method is as follows: (1) Sample preparation: The tonsils and breast cancer tissue sections that have been fixed in formalin and embedded in paraffin are baked in a constant temperature oven at 60℃ for 1-2 hours and stored for later use; (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 minutes 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 minutes (if using an automatic retrieval instrument, you can set it to 98℃ for 20 minutes). After the slides have cooled to room temperature, circle the tissue to be tested with an immunohistochemical pen and rinse twice with purified water for 3 minutes 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 phosphate buffer (PBST); (6) Primary antibody incubation: Add 100 μL of 1 μg / mL mouse primary 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.

[0066] Depend on Figure 1 The results showed that CD10 antibody and CD20 antibody both showed specific positive staining in tonsil tissue, and TROP2 antibody showed specific positive staining in breast cancer tissue. Furthermore, the rabbit anti-mouse secondary antibody exhibited better staining performance and a deeper color than commercially available rabbit anti-mouse polyclonal antibodies. This indicates that the rabbit anti-mouse secondary antibody of this invention, due to its high specificity and strong positive signal, is easier to evaluate and has higher identification accuracy in IHC staining, making it more accurate for detecting and differentiating cancers.

[0067] Example 3 This embodiment describes the determination of the affinity of rabbit anti-mouse secondary antibody. The determination method is as follows: (1) Remove the mouse primary antibodies for IgG1, IgG2a and IgG2b subtypes from 4℃ and bring them to room temperature. Dilute them to a concentration of 1 μg / ml and add 100 μL / well to each well of a 96-well microplate. Incubate overnight at 4℃, then block with 2% bovine serum albumin (BSA) overnight at 4℃. (2) The rabbit anti-mouse secondary antibody was diluted to an initial concentration of 0.5 μg / mL, and then serially diluted 2-fold, with a total of 8 concentration gradients for comparison; (3) Add the diluted rabbit anti-mouse secondary antibody at a rate of 100 μL / well to a 96-well microplate containing peptides, cover with a sealing film, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium. (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry. (5) Dilute horseradish peroxidase (HRP) labeled goat anti-rabbit IgG according to the instructions for use of the secondary antibody, add 100 μL / well to the microplate, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium. (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry. (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes; (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development; (9) Read the OD value at 450 nm on the microplate reader, organize the data, and analyze the results as follows. Figure 2 As shown.

[0068] The results showed that, in eight concentration gradient tests, compared with commercially available rabbit anti-mouse polyclonal antibodies, the rabbit anti-mouse secondary antibody of the present invention had strong affinity and high sensitivity for the three subtypes of IgG1, IgG2a and IgG2b mouse primary antibodies, and could still achieve a high OD value under low antibody concentration conditions, which can save experimental and detection costs.

[0069] Example 4 This embodiment describes the detection of HRP-labeled rabbit anti-mouse secondary antibody against mouse-derived primary antibodies for three subtypes of IgG1, IgG2a, and IgG2b using Western blotting. The method is as follows: (1) Human cervical cancer cell lysate was selected to verify TGM2 antibody (IgG1 subtype); 293 cell and human cervical cancer cell lysate were selected to verify PRKAA2 antibody (IgG2a) subtype; human brain tissue lysate and human cerebellum tissue lysate were selected to verify GFAP antibody (IgG2b) subtype. A polyvinylidene fluoride (PVDF) membrane containing the above cell lysates was activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in a blocking solution prepared with 5% bovine serum albumin (BSA) and shaken at room temperature for 2 h. TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and the surfactant Tween-20.

[0070] (2) Primary antibody incubation: Dilute the above primary antibody to a concentration of 0.5 μg / mL, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min). (4) Secondary antibody incubation: Dilute HRP-rabbit anti-mouse secondary antibody with FG solution at 1:5000, mix well and add to membrane strip, and shake at room temperature for 1 hour; (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min). (6) Substrate: Mix equal volumes of luminol / enhancer solution and hydrogen peroxide solution diluted 5 times with pure water in the same container, add the membrane strip, and incubate for 2 min; (7) Exposure: Place the film in the dark box and expose the X-ray film for different time periods according to the fluorescence intensity; then perform the operation in the order of 1 min development, 1 min cleaning, and 1 min fixing, and finally clean and dry; the results are as follows. Figures 3 to 5 As shown.

[0071] The theoretical molecular weight of the TGM2 protein is around 77 kDa. Figure 3 The results showed that a TGM2 positive band appeared in the lysate of human cervical cancer cells, with the band located at approximately 77 Da.

[0072] The theoretical molecular weight of PRKAA2 protein is around 62 kDa. Figure 4 Cell 293 represents human renal epithelial cells, composed of... Figure 4 The results showed that PRKAA2 positive bands appeared in the lysates of 293 cells and human cervical cancer cells. Due to possible glycosylation modification on the protein surface, the band position was higher, approximately 70 Da.

[0073] The theoretical molecular weight of GFAP protein is around 50 kDa, composed of... Figure 5 The results showed that GFAP-positive bands appeared in the lysate of human brain tissue and human cerebellum tissue, with the bands located at approximately 50 Da.

[0074] In summary, in the swimming lane, the HRP-labeled rabbit anti-mouse secondary antibody can specifically recognize three mouse primary antibodies: TGM2, PRKAA2, and GFAP. This indicates that the rabbit anti-mouse secondary antibody of the present invention can highly specifically recognize three subtypes of mouse primary antibodies: IgG1, IgG2a, and IgG2b.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rabbit anti-mouse secondary antibody, characterized in that, The invention comprises multiple monoclonal antibodies that specifically bind to the constant regions of murine primary antibodies. The antigenic epitopes recognized by these multiple monoclonal antibodies do not overlap and collectively cover murine IgG1, IgG2a, and IgG2b subtypes. The multiple monoclonal antibodies include a heavy chain variable region and a light chain variable region. The amino acid sequences of the heavy chain variable region are shown in SEQ ID NO:17-22, and the amino acid sequences of the light chain variable region are shown in SEQ ID NO:23-28.

2. The rabbit anti-mouse secondary antibody according to claim 1, characterized in that, The monoclonal antibodies that specifically bind to the constant region of the murine primary antibody are a mixture of antibodies A, B, C, D, E, and F, with a mass ratio of 1:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5.

3. A gene encoding a gene, characterized in that, Used to encode the rabbit anti-mouse secondary antibody as described in claim 1 or 2.

4. The encoding gene according to claim 3, characterized in that, It includes: DNA sequences as shown in SEQ ID NO:5-10 are used to encode the heavy chain variable region of the rabbit anti-mouse secondary antibody, and DNA sequences as shown in SEQ ID NO:11-16 are used to encode the light chain variable region of the rabbit anti-mouse secondary antibody.

5. A nucleic acid molecule, characterized in that, It contains the encoding gene as described in claim 4.

6. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. Transform or transfect the host cells of the expression vector according to claim 6.

8. A method for preparing a rabbit anti-mouse monoclonal antibody, characterized in that, The rabbit anti-mouse monoclonal antibody is obtained by transforming or transfecting the host cell using the expression vector described in claim 6, culturing the transformed or transfected cells, collecting the cell supernatant and purifying it.

9. The application of the rabbit anti-mouse secondary antibody of claim 1 or 2, the encoding gene of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, and the host cell of claim 7 in a mouse primary antibody detection device.

10. An immunoassay kit for detecting primary antibodies against murine IgG1, IgG2a, and IgG2b subtypes, characterized in that, The rabbit anti-mouse secondary antibody of claim 1 or 2, including horseradish peroxidase-labeled antibodies.

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