Anti-c-met protein monoclonal antibodies and related products and uses thereof

Abstract

The present application relates to a kind of anti-C-MET protein monoclonal antibody and its related products and purposes, belong to biological medicine technical field, the anti-C-MET protein monoclonal antibody includes heavy chain and light chain, the heavy chain includes heavy chain variable region, the heavy chain variable region includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1, CDR2, CDR3 in heavy chain variable region is as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 respectively;The light chain includes light chain variable region, the light chain variable region includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1, CDR2, CDR3 in light chain variable region is as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 respectively;The present antibody has higher specificity and sensitivity, can specifically recognize the cell expressing C-MET protein, not only suitable for immunology detection, but also can effectively prevent the phenomenon of false positive and false negative in immunology detection, both can solve the deficiency of existing C-MET antibody, and be conducive to further improve the accuracy of detection and diagnosis result.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-C-MET protein monoclonal antibody and its uses. Background Technology

[0002] C-MET (Mesenchymal-Epithelial Transition factor, MET) is a receptor tyrosine kinase (RTK) encoded by the MET proto-oncogene, belonging to the receptor class of hepatocyte growth factor (HGF, also known as scatter factor). C-MET plays a crucial role in physiological processes such as embryonic development, tissue repair, and regeneration; however, its abnormal activation (e.g., mutation, amplification, or overexpression) is closely related to the occurrence, invasion, and metastasis of various cancers. C-MET antibodies are antibodies that specifically recognize and bind to the C-MET protein.

[0003] Aberrant activation of C-MET is a key driver of the occurrence, progression, and metastasis of various malignant tumors. Its oncogenic mechanism is complex, involving gene mutation, amplification, overexpression, and ligand-dependent activation, and is closely related to tumor invasiveness, drug resistance, and poor prognosis. C-MET is a key biomarker in the treatment of non-small cell lung cancer, and therapeutic drugs targeting this marker include carmatinib, tepoltinib, crizotinib, cabozantinib, cerivtinib, and gumetinib. Early drug development primarily targeted patients with C-MET gene amplification. In recent years, clinical studies of drugs targeting C-MET protein overexpression (such as cerivtinib and gumetinib) have emerged, creating an objective need for methods to detect C-MET protein overexpression.

[0004] The primary clinical method for detecting C-MET protein overexpression is immunohistochemistry (IHCC). This method is a traditional detection technique that utilizes the principle of specific antigen-antibody binding to detect the expression level of the target protein in tissues. Immunohistochemical detection mainly relies on antibodies that specifically bind to the target antigen. Currently, there are relatively few C-MET antibodies available, and their affinity is not high enough. Specifically, C-MET (MET receptor tyrosine kinase) belongs to the receptor tyrosine kinase (RTK) family, which contains 58 known members and regulates key biological processes such as cell proliferation, differentiation, survival, and migration. C-MET shares a high degree of structural and functional overlap with other RTKs (such as the common EGFR and HER2). Existing C-MET antibodies may exhibit abnormal expression or cross-reactivity, leading to false positive results. Simultaneously, some tumor cells expressing low levels of C-MET may show weak staining or be undetectable, resulting in false negatives. For example, in current C-MET therapy for non-small cell lung cancer, a strong staining cell percentage of ≥90% is required to meet the treatment criteria. If antibody affinity is insufficient, staining intensity may be downgraded, leading to missed detections, affecting diagnostic accuracy, and causing patients to miss treatment opportunities. Therefore, how to avoid false positives and false negatives in C-MET antibody immunological testing is an urgent problem to be solved. Summary of the Invention

[0005] The first aspect of this invention addresses the aforementioned technical problems by providing a monoclonal antibody against C-MET protein, which has higher specificity and sensitivity. It can specifically recognize cells expressing C-MET protein, making it suitable not only for immunological detection but also helping to avoid false positives and false negatives, thus significantly improving the accuracy of detection and diagnosis.

[0006] A monoclonal antibody against C-MET protein comprises a heavy chain and a light chain. The heavy chain includes a variable region comprising CDR1, CDR2, and CDR3, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region being shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The light chain includes a variable region comprising CDR1, CDR2, and CDR3, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region being shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. Compared to existing C-MET antibodies, the anti-C-MET protein monoclonal antibody provided in this protocol has higher specificity and sensitivity. It can specifically recognize cells expressing C-MET protein, which helps to further prevent false positives and false negatives in immunological testing. This not only solves the shortcomings of existing C-MET antibodies, but also helps to further improve the accuracy of detection and diagnostic results, so that it can be applied to scientific research and non-disease diagnostic purposes.

[0007] Furthermore, the heavy chain variable region also includes FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0008] Furthermore, the light chain variable region also includes FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 in the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO:8.

[0010] Furthermore, the amino acid sequence of the light chain is shown in SEQ ID NO:16.

[0011] A second aspect of the present invention also provides a reagent for detecting C-MET protein, comprising the aforementioned anti-C-MET protein monoclonal antibody. Because this anti-C-MET protein monoclonal antibody has higher specificity and sensitivity, it can specifically recognize cells expressing C-MET protein and can be used in reagents for detecting C-MET protein, thus facilitating more accurate detection results.

[0012] A third aspect of the present invention also provides a detection tool for detecting C-MET protein, comprising the anti-C-MET protein monoclonal antibody or comprising the reagent. Because the anti-C-MET protein monoclonal antibody has higher specificity and sensitivity, it can specifically recognize cells expressing C-MET protein, and can be used as a detection tool for detecting C-MET protein, which is beneficial for obtaining more accurate detection results.

[0013] Preferably, the detection tool is a reagent kit, a test strip, an antibody chip, or a detection plate. In this embodiment, when the detection tool is a reagent kit, the kit contains the anti-C-MET protein monoclonal antibody or the reagent for detecting C-MET protein; when the detection tool is a test strip, the test strip contains the anti-C-MET protein monoclonal antibody or the reagent for detecting C-MET protein; when the detection tool is an antibody chip, the antibody chip contains the anti-C-MET protein monoclonal antibody or the reagent for detecting C-MET protein; and when the detection tool is a detection plate, the detection plate contains the anti-C-MET protein monoclonal antibody or the reagent for use in scientific research and non-disease diagnostic C-MET protein detection.

[0014] Furthermore, the kit also includes a container in which the anti-C-MET protein monoclonal antibody or reagent is disposed.

[0015] A fourth aspect of this invention provides the use of the aforementioned anti-C-MET protein monoclonal antibody in the preparation of tools for detecting C-MET protein. Because this anti-C-MET protein monoclonal antibody has higher specificity and sensitivity, and can specifically recognize cells expressing C-MET protein, it can be applied to tools for detecting C-MET protein, which is beneficial for obtaining more accurate detection results.

[0016] Furthermore, the tool for detecting C-MET protein includes reagents, kits, test strips, antibody chips, or detection plates. In this embodiment, when the tool includes reagents, the reagents include the anti-C-MET protein monoclonal antibody; when the tool includes a kit, the anti-C-MET protein monoclonal antibody can be contained within the kit; when the tool includes a test strip, the anti-C-MET protein monoclonal antibody can be contained within the test strip; when the tool includes an antibody chip, the anti-C-MET protein monoclonal antibody can be contained within the antibody chip; when the tool includes a detection plate, the anti-C-MET protein monoclonal antibody can be contained within the detection plate; all of these methods can achieve the function of detecting C-MET protein.

[0017] Compared with existing technologies, the anti-C-MET protein monoclonal antibody and its related products and uses provided by this invention have higher specificity and sensitivity. It can specifically recognize cells expressing C-MET protein. It is not only suitable for immunological detection, but also can effectively prevent false positives and false negatives in immunological detection, which is conducive to further improving the accuracy of detection and diagnosis results. It can be widely used in scientific research and non-disease diagnosis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows a comparison of immunohistochemical staining results for a case of non-small cell lung cancer. The left image uses the recombinant rabbit monoclonal antibody against C-MET (LBP4-C-MET) prepared in this invention; the right image uses the commercially available antibody C-MET (Roche, SP44).

[0020] Figure 2 The images show a comparison of immunohistochemical staining results of esophageal squamous epithelium. The left image uses the recombinant rabbit monoclonal antibody against C-MET (LBP4-C-MET) prepared in this invention, while the right image uses the commercially available antibody C-MET (Roche, SP44).

[0021] Figure 3 The image shows the kinetic curve of the interaction between the anti-C-MET recombinant rabbit monoclonal antibody (LBP4-C-MET) and the antigen.

[0022] Figure 4 The image shows the kinetic curves of the interaction between the commercially available antibody C-MET (Roche, SP44) and the antigen.

[0023] Figure 5 The images show a comparison of the results of the immunoblotting assay. The left image uses the commercially available antibody C-MET (Roche, SP44), while the right image uses the recombinant rabbit monoclonal antibody against C-MET (LBP4-C-MET) prepared in this invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] This embodiment provides a method for preparing a monoclonal antibody against C-MET protein, comprising the following steps:

[0027] I. Antigen preparation.

[0028] The antigen is the intracellular region (AA 956-1390) of recombinant human C-MET (Uniport code: P08581, detailed information such as its amino acid sequence, domains, and functional annotations can be found in the UniProt database based on this number). The antibody binds to the intracellular region (AA 956-1390) of the C-MET antigen. The amino acid sequence of the antigen is as follows:

[0029] KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELV QAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNE THNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGA PPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS

[0030] For the aforementioned antigens, by comparing similar sequences within the same family, highly specific regions were selected as antigens and conjugated with highly immunogenic haptens to enhance the immune response in experimental rabbits, which is beneficial in increasing the likelihood of obtaining specific antibodies. Since antigens are broken down and delivered to B cells in the form of short peptides by antigen-presenting cells, and randomly encoded within the B cells to form antibodies that recognize these peptides and are secreted into body fluids, maximizing antigen specificity increases the probability of producing highly specific antibodies. Therefore, screening for highly specific antigens can improve the specificity of the obtained antibodies. Simultaneously, highly specific antigens often have shorter amino acid sequences (shorter sequences have a lower probability of overlap with other antigens) and lower molecular weights, while low molecular weight antigens are less likely to elicit an immune response. Therefore, in this embodiment, conjugating highly immunogenic haptens can improve the efficiency of immunization.

[0031] II. Immunity.

[0032] The C-MET antigen obtained in step one above was mixed with and emulsified with complete Freund's adjuvant, and then administered subcutaneously to multiple rabbits, with each immunization performed three times. Blood samples were collected after each immunization, and serum titers were determined using ELISA and immunohistochemistry. Rabbits with the highest antibody titers against the C-MET antigen were selected for further single B cell screening.

[0033] III. Single B cell screening and sequencing.

[0034] Spleens were harvested from target animals, and B cells secreting specific antibodies were isolated by antigen coating adsorption. After culture, the supernatant of the B cells was collected, and B cells capable of secreting C-MET-binding antibodies were identified by ELISA. These B cells were lysed to obtain total RNA (ribonucleic acid). cDNA (complementary DNA) was obtained using a reverse transcription kit, and the reverse transcription product was used as a template for PCR (polymerase chain reaction). The antibody-encoded heavy chain variable region (VH) and light chain variable region (VL) sequences were amplified using the corresponding primers. The reaction program was as follows: VH: 95℃, 5 min; 95℃, 30 s; 70℃, 30 s; 72℃, 1 min; 72℃, 10 min for 35 cycles; VL: 95℃, 5 min; 95℃, 30 s; 55℃, 30 s; 72℃, 1 min; 72℃, 10 min for 35 cycles. The amplification products were detected by 1% agarose gel electrophoresis, and then a single target band was selected for gel recovery. The target band recovered from the gel and a mammalian cell expression vector containing the constant region gene were transformed into TOP10 competent cells via homologous recombination. The cells were cultured at 37°C for 12 hours, and single clones were selected for sequencing. The sequencing results were analyzed using existing software (such as Snapgene software) to screen for expression plasmids with VH and VL sequences that match the characteristics of rabbit-derived antibody sequences.

[0035] In this embodiment, the use of single B-cell sequencing technology is beneficial to improving the efficiency of screening high-affinity antibodies and the cloning success rate.

[0036] IV. Preparation and purification of monoclonal antibodies on cells.

[0037] 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 high-purity antibody. Finally, the purified monoclonal antibody was determined to be of high concentration, aliquoted, and stored at 4°C. The monoclonal antibody is an anti-C-MET recombinant rabbit monoclonal antibody, belonging to the anti-C-MET protein monoclonal antibody category.

[0038] The obtained anti-C-MET recombinant rabbit monoclonal antibody was sequenced, and it includes a heavy chain and a light chain. The heavy chain includes a heavy chain variable region and a heavy chain constant region. The light chain includes a light chain variable region and a light chain constant region. The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 of the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively. See Table 1 for details. The amino acid sequence of the complete heavy chain is shown in SEQ ID NO:8. See Table 2 for details.

[0039] Table 1. Sequence of the heavy chain variable region (VH) in the anti-C-MET recombinant rabbit monoclonal antibody.

[0040]

[0041] Table 2. Heavy chain sequence in anti-C-MET recombinant rabbit monoclonal antibody

[0042]

[0043] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; the amino acid sequences of FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, as detailed in Table 3; the amino acid sequence of the complete light chain is shown in SEQ ID NO:16, as detailed in Table 4.

[0044] Table 3. Sequence of the light chain variable region (VL) in the anti-C-MET recombinant rabbit monoclonal antibody

[0045]

[0046] Table 4. Light chain sequences in anti-C-MET protein monoclonal antibodies

[0047]

[0048] Example 2

[0049] This example demonstrates immunohistochemical detection using anti-C-MET recombinant rabbit monoclonal antibody as the primary antibody, as follows:

[0050] (1) Sample preparation: HELA cells, HT-29 cells, SK-BR-3 cells, non-small cell lung cancer and various normal tissue sections fixed in formalin and embedded in paraffin were baked in a constant temperature oven at 60℃ for 1-2 hours and stored for later use.

[0051] (2) Use an automated repair instrument to dewax, hydrate, and repair the sections. During this process, the temperature is preferably controlled at 95℃ and the time is preferably controlled at 30min.

[0052] (3) Immerse in hydrogen peroxide for 10 minutes to block the reaction, then rinse with pure water 3 times. Then circle the tissue to be tested with an immunohistochemical pen, rinse with pure water 2 times, and immerse in phosphate buffer (PBST) for 2-3 minutes.

[0053] (4) Primary antibody incubation: Add 100 μL of anti-C-MET recombinant rabbit monoclonal antibody to completely cover the tissue, incubate at room temperature for 1 h, and then wash with PBST 3 times for 3 min each time.

[0054] (5) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. The incubation time can be 30 min. Rinse with PBST 3 times, 3 min each time.

[0055] (6) DAB color development: Prepare DAB color development solution according to the kit instructions. Drop an appropriate amount of the prepared DAB color development solution to completely cover the tissue. The incubation time can be 10 min. Rinse with pure water 3 times, 3 min each time.

[0056] (7) Hematoxylin counterstaining: Follow the instructions and suggestions of the hematoxylin manufacturer to counterstain the sections for 2 minutes, and then perform PBST blueing for 5 minutes.

[0057] (8) Dehydrate and become transparent: Soak in alcohol for 2-3 minutes and air dry.

[0058] (9) Mounting: Mount the sample with neutral resin.

[0059] (10) Perform slice scanning.

[0060] (11) Results analysis and statistics.

[0061] Immunohistochemical staining results are categorized as positive or negative. Positive expression must occur at a specific antigenic site on the cell or tissue to be considered positive. When tissue staining distribution is clear and cell localization is accurate, the staining results are further subdivided based on differences in staining intensity. For example, in this embodiment, the staining results are further subdivided based on the H-score, as follows:

[0062] 1. Samples with an H-score in the range of 200-300 are defined as having strong C-MET expression;

[0063] 2. Samples with an H-score in the range of 100-199 are defined as having moderate C-MET expression.

[0064] 3. Samples with an H-score in the range of 1-99 are defined as having weak C-MET expression;

[0065] 4. A sample with an H-Score of 0 is defined as C-MET negative;

[0066] The recombinant rabbit monoclonal antibody against C-MET prepared in this invention (labeled LBP4-C-MET) and the commercially available antibody C-MET (Roche, SP44) were used to simultaneously detect and compare the results in 108 cases of non-small cell lung cancer. The entire experiment employed a double-blind design. The immunohistochemical results of C-MET were statistically analyzed, and the results are shown in the table below:

[0067] Table 5. Statistical analysis of C-MET immunohistochemical results

[0068]

[0069] First, the results showed that LBP4-C-MET staining was accurate in localization, clear in staining, and free of nonspecific staining, with a clean background. In immunohistochemical detection, the positive rate was 10% higher than that of commercially available antibodies. Specifically, LBP4-C-MET monoclonal antibody showed higher staining rates in the moderately positive (8 cases) and strongly positive (6 cases) ranges compared to commercially available antibodies, indicating that LBP4-C-MET monoclonal antibody has higher sensitivity than commercially available antibodies.

[0070] second, Figure 1 This is a comparison of immunohistochemical staining results for a case of non-small cell lung cancer. The left image uses the LBP4-C-MET of this invention; the right image uses commercially available C-MET (SP44). Figure 1As can be seen, for the same non-small cell lung cancer tissue, the staining intensity of the anti-C-MET recombinant rabbit monoclonal antibody (LBP4-C-MET) of this invention is significantly stronger than that of commercially available C-MET (SP44), and it is less likely to cause false negatives due to the degradation of staining intensity, which is beneficial to improving the accuracy of diagnosis.

[0071] Third, results of normal tissue microarray analysis: The normal tissue microarray included 24 types of normal tissue samples, primarily selected from fresh, promptly fixed surgical specimens. These 24 normal tissues included: skin, tonsils, appendix, lung, kidney, thyroid gland, liver, pancreas, stomach, testis, cervix, endometrium, fallopian tube, mammary gland, placenta, prostate, small intestine, large intestine, thymus, spleen, salivary glands, esophagus, brain, and skeletal muscle. Results showed that the commercially available antibody C-MET (SP44) exhibited non-specific staining in esophageal squamous epithelial tissue, resulting in weak to moderate C-MET expression and false positives. C-MET antibody should not be expressed in esophageal squamous epithelial tissue. Furthermore, the LBP4-C-MET monoclonal antibody prepared in this invention showed almost no non-specific staining, indicating that the LBP4-C-MET monoclonal antibody of this invention has higher specificity than commercially available antibodies, effectively reducing the probability and risk of false positives and improving accuracy. Specifically, as follows... Figure 2 The image shows a comparison of immunohistochemical staining results of esophageal squamous epithelium. The left image uses the LBP4-C-MET monoclonal antibody prepared in this invention, while the right image uses commercially available C-MET (SP44). Figure 2 It can be seen that the LBP4-C-MET monoclonal antibody prepared in this invention does not stain esophageal squamous epithelial tissue and no false positive results occur; while commercially available C-MET (SP44) stains esophageal squamous epithelial tissue, resulting in false positive results. This proves that the LBP4-C-MET monoclonal antibody prepared in this invention has better specificity than currently commercially available similar antibodies.

[0072] Example 3

[0073] This example demonstrates the affinity determination of the anti-C-MET recombinant rabbit monoclonal antibody, using the following method:

[0074] The binding ability of C-MET monoclonal antibodies to human C-MET antigen was characterized using Biacore SPR (surface plasmon resonance). Using a protein A microarray (channel 1 as the reference channel and channel 2 as the ligand channel), the antibody was immobilized as a ligand at a concentration of 10 μg / ml into channel 2 of the protein A microarray. The binding time was 30 s, and the flow rate was 10 μL / min. During the assay, the antigen was used as the analyte, and the LBP4-C-MET monoclonal antibody was used as the antibody. The antibody was diluted with running buffer to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, 7.81 nM, 3.91 nM, and 0 nM, respectively.

[0075] During implementation, the analytical stream was routed through channels 1 and 2, with binding time controlled at 150 s, dissociation time controlled at 600 s, and flow rate controlled at 30 μL / min. After analyte loading, the chip surface was regenerated using glycine buffer at pH 1.5. Ligand immobilization was required before each analyte loading, and the chip surface was regenerated with regeneration solution after analyte loading.

[0076] The affinity of LBP4-C-MET monoclonal antibody for human C-MET antigen at different concentrations was determined, and the affinity curves of LBP4-C-MET monoclonal antibody for human C-MET antigen were fitted using a 1:1 kinetic model with appropriate analytical software. The results are as follows: Figure 3 As shown.

[0077] Simultaneously, a control group was set up, using commercially available C-MET (SP44) antibody. All other test parameters and conditions were the same as described above. The results are as follows: Figure 4 As shown, the comparison of affinity test results is shown in Table 6.

[0078] Table 6 Comparison of Affinity Measurement Results

[0079]

[0080] In Table 6, KD represents the dissociation constant, a comprehensive indicator of affinity. A lower KD value indicates higher intermolecular affinity, meaning a lower concentration is needed to achieve a certain degree of binding, indicating that the two molecules are more likely to combine and form a more stable complex. kon represents the binding rate constant, reflecting the speed at which the antibody binds to the antigen; a higher value indicates a more efficient binding process. Kdis represents the dissociation rate constant, reflecting the speed of complex dissociation and is a core parameter for measuring the stability of the antibody-antigen complex; a lower value indicates a more stable complex and higher affinity. Therefore, the data comparison in Table 6 shows that the affinity of the LBP4-C-MET monoclonal antibody in this invention to the human C-MET antigen is much higher than that of commercially available C-MET (SP44) to the human C-MET antigen, revealing that the LBP4-C-MET monoclonal antibody in this invention has higher sensitivity.

[0081] Example 4

[0082] This example demonstrates the detection of immunoblotting using the recombinant rabbit monoclonal antibody LBP4-C-MET as the primary antibody. The method is as follows:

[0083] (1) Activate the polyvinylidene fluoride (PVDF) membrane with HELA / HT-29 / SK-BR-3 cell lysate by activating it with methanol for 1 min and washing it three times with TBST; block it with 5% skim milk for 1 h; TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (sodium chloride) and surfactant Tween-20.

[0084] (2) Primary antibody incubation: Place the blocked PVDF membrane in a solution containing diluted LBP4-C-MET antibody and incubate at 4°C overnight. After incubation, remove the membrane and wash it with TBST 5 times for 5 minutes each time.

[0085] (3) Secondary antibody incubation: Place the cleaned PVDF membrane in a diluted HRP-anti-rabbit IgG (1:10000) solution and shake at room temperature for 1 hour. After incubation, remove the membrane and wash it with TBST 5 times for 5 minutes each time.

[0086] (4) Prepare the developing solution according to the Immobilon Western Developing Kit instructions, and evenly drop it onto the membrane. Develop the membrane using an image workstation (such as a GelView 6000ProⅡ multi-functional image workstation) according to the instructions. The results are as follows: Figure 5 As shown, the left figure uses commercially available C-MET (Roche, SP44), and the right figure uses the LBP4-C-MET monoclonal antibody prepared in this invention.

[0087] The theoretical molecular weight of C-MET protein is 155 kDa, and it also has low molecular weight isoforms in different tumor cells. Figure 5 HELA is a high-expression cell line, HT-29 is a low-expression cell line, and SK-BR-3 is a negative cell line. In the Western blotting results, the LBP4-C-MET monoclonal antibody prepared in this invention and the commercially available C-MET (Roche, SP44) antibody showed no band of the theoretical molecular weight of C-MET protein in SK-BR-3, while the band size was consistent in HELA and HT-29. This indicates that the anti-C-MET recombinant rabbit monoclonal antibody (i.e., LBP4-C-MET) of this invention can recognize C-MET protein with high specificity.

[0088] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against C-MET protein, characterized in that, It comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, the heavy chain variable region includes CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The light chain includes a light chain variable region, which includes CDR1, CDR2 and CDR3. The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively.

2. The anti-C-MET protein monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region also includes FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

3. The anti-C-MET protein monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:

8.

4. The anti-C-MET protein monoclonal antibody according to claim 1, characterized in that, The light chain variable region also includes FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 in the light chain variable region are shown as SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively.

5. The anti-C-MET protein monoclonal antibody according to claim 4, characterized in that, The amino acid sequence of the light chain is shown in SEQ ID NO:

16.

6. A reagent for detecting C-MET protein, characterized in that, It comprises the anti-C-MET protein monoclonal antibody as described in any one of claims 1-5.

7. A detection tool for detecting C-MET protein, characterized in that, It comprises the anti-C-MET protein monoclonal antibody as described in any one of claims 1-5, or the reagent as described in claim 6.

8. The detection tool according to claim 7, characterized in that, The detection tool is a reagent kit, test strip, antibody chip, or detection plate.

9. Use of an anti-C-MET protein monoclonal antibody as described in any one of claims 1-5 in the preparation of a tool for detecting C-MET protein.

10. The use according to claim 9, characterized in that, The tools used for detecting C-MET protein include reagents or kits or test strips or antibody chips or detection plates.

Images