A ca15-3 antigen binding protein and uses thereof

Abstract

This invention provides a CA15-3 antigen-binding protein and its application. The CA15-3 antigen-binding protein includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDR3 having the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 13; the light chain variable region includes CDR3 having the sequence shown in SEQ ID NO. 10 or SEQ ID NO. 16. The CA15-3 antigen-binding protein of this invention is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity. Furthermore, the combined antibody pair exhibits high specificity and accuracy. The obtained CA15-3 antigen-binding protein can be paired for the diagnosis of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a CA15-3 antigen-binding protein and its applications. Background Technology

[0002] CA15-3 (cancer antigen 15-3, also known as Mucin1, MUC1, EMA, MCD, PEM, PUM, KL-6, and MAM6) is a member of the mucin family, a type I transmembrane protein with a highly glycosylated extracellular domain extending 200-500 nm from the cell surface. CA15-3 consists of two subunits: an N-terminal subunit of the extracellular domain and a C-terminal subunit of the cytoplasmic domain. The core protein has a molecular weight of 120-220 kDa, which increases to over 400 kDa when the glycosylated portion is included.

[0003] CA15-3 was the first breast cancer-associated antigen to be discovered. It is located on the surface of tumor cells. When cells become cancerous, the activity of proteases and salivary enzymes on the cell membrane increases, the cytoskeleton is destroyed, and cell antigens are released into the blood, resulting in an increase in serum CA15-3 levels.

[0004] Elevated CA15-3 levels are observed in breast cancer patients, with a sensitivity of 60% in early-stage breast cancer and 80% in late-stage breast cancer. CA15-3 is of significant value in monitoring treatment efficacy, predicting prognosis, and diagnosing recurrence and metastasis in breast cancer. CA15-3 also shows a certain positive rate in other malignant tumors, such as lung cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, and primary liver cancer.

[0005] The development process of CA15-3 detection antibodies mainly includes the following steps: (1) Preparation of main immunogens: generally using natural antigens, peptides or cells expressing CA15-3, etc.; (2) Immunization of immunized animals: select suitable immunized animals, usually mice, rabbits, etc.; (3) Inject the immunogen into the immunized animals to stimulate them to produce CA15-3 specific antibodies; (4) Screening and identification: screen CA15-3 specific antibodies with specificity and affinity by screening methods such as phage display and single cell sorting; (5) Further identification and evaluation of candidate antibodies, including affinity determination, flow cytometry analysis, cloning, antibody optimization and improvement.

[0006] However, there are currently few anti-CA15-3 protein antibodies available, and imported brands are expensive. Furthermore, CA15-3 requires a pair of antibodies for high sensitivity. Therefore, developing a monoclonal antibody that specifically binds to the CA15-3 protein would have significant value in the diagnosis of breast cancer. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a CA15-3 antigen-binding protein and its applications. The CA15-3 antigen-binding protein described in this invention is a rabbit monoclonal antibody. Compared to mouse antibodies and polyclonal antibodies, rabbit antibodies naturally possess higher affinity and stronger specificity, and the resulting antibody pair exhibits high specificity and accuracy. The obtained antibody can be paired for the diagnosis of breast cancer.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a CA15-3 antigen-binding protein, characterized in that the CA15-3 antigen-binding protein includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13.

[0010] The light chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.10 or SEQ ID NO.16.

[0011] Preferably, the heavy chain variable region includes CDR1 having a sequence as shown in SEQ ID NO.5 or SEQ ID NO.11.

[0012] Preferably, the heavy chain variable region includes CDR2 having a sequence as shown in SEQ ID NO.6 or SEQ ID NO.12.

[0013] Preferably, the light chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.8 or SEQ ID NO.14.

[0014] Preferably, the light chain variable region includes CDR2 having a sequence as shown in SEQ ID NO.9 or SEQ ID NO.15.

[0015] Preferably, the heavy chain variable region includes CDR1 having the sequence shown in SEQ ID NO. 5, CDR2 having the sequence shown in SEQ ID NO. 6, and CDR3 having the sequence shown in SEQ ID NO. 7; and the light chain variable region includes CDR1 having the sequence shown in SEQ ID NO. 8, CDR2 having the sequence shown in SEQ ID NO. 9, and CDR3 having the sequence shown in SEQ ID NO. 10; or,

[0016] The heavy chain variable region includes CDR1 having the sequence shown in SEQ ID NO. 11, CDR2 having the sequence shown in SEQ ID NO. 12, and CDR3 having the sequence shown in SEQ ID NO. 13; and the light chain variable region includes CDR1 having the sequence shown in SEQ ID NO. 14, CDR2 having the sequence shown in SEQ ID NO. 15, and CDR3 having the sequence shown in SEQ ID NO. 16.

[0017] Preferably, the heavy chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3; and / or,

[0018] The light chain variable region includes a sequence that has at least 90% identity with the sequence shown in SEQ ID NO.2 or SEQ ID NO.4.

[0019] Preferably, the sequence of the heavy chain variable region includes SEQ ID NO.1 or SEQ ID NO.3.

[0020] Preferably, the sequence of the light chain variable region includes SEQ ID NO.2 or SEQ ID NO.4.

[0021] Preferably, the sequence of the heavy chain variable region includes that shown in SEQ ID NO.1, and the sequence of the light chain variable region includes that shown in SEQ ID NO.2; or,

[0022] The sequence of the heavy chain variable region includes that shown in SEQ ID NO.3, and the sequence of the light chain variable region includes that shown in SEQ ID NO.4.

[0023] Preferably, the antigen-binding protein is an antibody.

[0024] Preferably, the antibody is a monoclonal antibody.

[0025] Preferably, the constant region of the monoclonal antibody is a rabbit-derived constant region.

[0026] In this invention, New Zealand white rabbits were immunized with the CA15-3 natural antigen. After four immunizations, the antiserum titer was detected by enzyme-linked immunosorbent assay (ELISA). Once the serum titer reached a certain dilution, a shock immunization was performed. Serum was collected for polyclonal antibody purification, and spleens were collected and ground. Total RNA was extracted from the spleen and reverse transcribed to synthesize first-strand cDNA. Using a set of primers matching the variable regions FR1 and FR4 of the rabbit antibody light and heavy chains, the variable region fragments (VH / Vκ) of the immunized rabbit light and heavy chains were amplified. A second amplification was performed using another set of primers, adding a linker sequence with complementary overlapping regions to the antibody light and heavy chain variable fragments. The VH and Vκ fragments were assembled into a single-stranded antibody fragment (scFv) by overlap extension PCR (SOE PCR).

[0027] After digesting the scFv fragment and phage display vector using the same restriction endonuclease, the ligation was performed via gel recovery and recombinant vector ligation. The ligation products were cleaned, and the ligation was electroporated into *E. coli* TG1 to obtain an scFv phage display bacterial library. The bacterial library was cultured, and overinfection with helper phage M13K07 was performed to aid in packaging and rescuing the phage library, displaying the scFv fragment on the phage surface. The CA15-3 natural antigen was coated onto an immunoassay plate, blocked with blocking buffer, and then the phage library was added. Phages displaying the specific scFv fragment were captured by binding to the CA15-3 protein. Unbound phages were removed by rinsing. The phages were eluted from the immunoassay plate and used to infect logarithmically growing TG1 hosts. After culturing, helper phages could be used again for overinfection and packaging rescue. After several rounds of screening, the TG1 bacterial suspension infected with the selected phage was serially diluted, spread on plates, and then single clones were picked and cultured in 96-well plates. Helper phages were added to package the single clones, and the binding activity of phages to CA15-3 in the supernatant of the single clones was detected by ELISA to obtain positive single clones that specifically bind to CA15-3.

[0028] Candidate positive clones were sequenced and constructed into the eukaryotic expression vector pcDNA3.1(+) containing the constant regions of the rabbit antibody heavy and light chains, resulting in a complete light and heavy chain expression vector. The light and heavy chain vector plasmids were co-transfected into HEK293E cells for expression, and the positive clone antibody proteins were purified from the culture supernatant using Protein A. The specificity and affinity of the positive clone antibody proteins for CA15-3 were verified by ELISA, ultimately yielding two antibodies that specifically bind to CA15-3.

[0029] In this invention, two antibodies that specifically bind to the CA15-3 protein were screened and designated CF24 and CF128. The amino acid sequences are shown below, with the underlined sequences representing the antibody's CDR region.

[0030] SEQ ID NO.1 (Amino acid sequence of the heavy chain variable region of CF24)

[0031] QSVEESGGRLVTPGTPLTLTCTVFGFSLS DFGVS WVRQAPGKGLEWIG YIWIDGNTDYASWAKG RFTISKASTTVDLKITSPTTEDTATYFCAR MRDIRTYAYAGYNI WGPGTLVTVSS.

[0032] SEQ ID NO.5 (heavy chain CDR1 of CF24): DFGVS.

[0033] SEQ ID NO.6 (heavy chain CDR2 of CF24): YIWIDGNTDYASWAKG.

[0034] SEQ ID NO.7 (heavy chain CDR3 of CF24): MRDIRTYAYAGYNI.

[0035] SEQ ID NO.2 (Amino acid sequence of the light chain variable region of CF24)

[0036] YVMMTQTPASVSEPVGGTVTIKC QASQSIYSYLA WYQQKPGQPPKLLIY KASTLAS GVPSRFKGSGSGTQFTLTISDLECDDAATYYC QSTYDSEDNT FGGGTKVVVE.

[0037] SEQ ID NO.8 (CDR1 of the light chain of CF24): QASQSIYSYLA.

[0038] SEQ ID NO.9 (CDR2 of the light chain of CF24): KASTLAS.

[0039] SEQ ID NO.10 (CDR3 of the light chain of CF24): QSTYDSEDNT.

[0040] SEQ ID NO.3 (Amino acid sequence of the heavy chain variable region of CF128)

[0041] QSVKESEGGLFKPTDTLTLTCTVSGFSLS SNAIY WVRQAPGNGLEWIG AIYSYGGTYYANWAKS RSTITRNTNLNTATLKMTSLTAADTATYFCAR GGVGDSDSAINI WGPGTLVTVSS.

[0042] SEQ ID NO.11 (heavy chain CDR1 of CF128): SNAIY.

[0043] SEQ ID NO.12 (heavy chain CDR2 of CF128): AIYSYGGTYYANWAKS.

[0044] SEQ ID NO.13 (heavy chain CDR3 of CF128): GGVGDSDSAINI.

[0045] SEQ ID NO.4 (Amino acid sequence of the light chain variable region of CF128)

[0046] ALVMTQTPASVSEPVGGTVTIKC QASQSIGSNLA WYQQKPGQRPKLLIY YASTLES GVSSRFKGSRSGTEFTLTISDLECDDAATYYC QQGYSSGGVDNV FGGGTKLEIK.

[0047] SEQ ID NO.14 (light chain CDR1 of CF128): QASQSIGSNLA.

[0048] SEQ ID NO.15 (light chain CDR2 of CF128): YASTLES.

[0049] SEQ ID NO.16 (light chain CDR3 of CF128): QQGYSSGGVDNV.

[0050] In a second aspect, the present invention provides a nucleic acid molecule that encodes the CA15-3 antigen-binding protein described in the first aspect.

[0051] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0052] Fourthly, the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect; or the host cell has the nucleic acid molecule described in the second aspect integrated into its genome.

[0053] Fifthly, the present invention provides a reagent for detecting CA15-3 protein in a sample, the reagent comprising the CA15-3 antigen-binding protein described in the first aspect.

[0054] In a sixth aspect, the present invention provides a kit comprising the CA15-3 antigen-binding protein described in the first aspect; or the kit comprising the reagent for detecting CA15-3 protein in a sample as described in the fifth aspect.

[0055] In a seventh aspect, the present invention provides the application of the CA15-3 antigen-binding protein described in the first aspect in the preparation of tumor detection products.

[0056] The CA15-3 antigen-binding protein described in this invention can be applied to the diagnosis of CA15-3 lymphocyte-related diseases, such as the early diagnosis of breast cancer.

[0057] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The CA15-3 antigen-binding protein obtained in this invention can specifically bind to the CA15-3 target. This protein can be applied to the diagnosis of diseases related to changes in CA15-3 concentration, such as the early diagnosis of breast cancer; it also has great potential in immunological research and the development of novel treatment strategies. The CA15-3 antigen-binding protein obtained in this invention can be used directly or packaged into kits with various labels. Attached Figure Description

[0060] Figure 1 This is the result of the potency test.

[0061] Figure 2 This is the result of SDS-PAGE electrophoresis.

[0062] Figure 3 These are the results of affinity testing.

[0063] Figure 4 This is the result of antibody pairing assay. Detailed Implementation

[0064] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0065] 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.

[0066] Example 1: Animal Immunity and Serum Titer Test

[0067] In this embodiment, each polypeptide (CA15-3 polypeptide) was used to immunize two rabbits (purchased from Guangdong Provincial Medical Laboratory Animal Center, name: ordinary grade New Zealand rabbit, size: about 1.8kg). The polypeptide was synthesized by Genscript.

[0068] 1. Collect 1 mL of blood from the ears of two rabbits and use the collected serum as a control. Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 mL of PBS, mix 1.5 mL of antigen with 1.5 mL of complete adjuvant, and shake on a shaker for at least 15 minutes until it reaches a water-in-oil state; immunize each rabbit with 1.5 mL of the antigen on its back.

[0069] 2. Two weeks later, administer a second immunization. Each rabbit is immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant. Dilute 1 mg of antigen in 1 mL of PBS and mix 1 mL of antigen with 1 mL of incomplete adjuvant. Shake on a shaker for at least 15 minutes until it reaches a water-in-oil state. Immunize 1 mL of the antigen on the back of each rabbit.

[0070] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, 1 mg of antigen was diluted in 1 mL of PBS, and mixed with 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on a shaker for more than 15 minutes until it reached a water-in-oil state; 1 mL of the mixture was immunized on the back of each rabbit.

[0071] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, 1 mg of antigen was diluted in 1 mL of PBS, and mixed with 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on a shaker for more than 15 minutes until it reached a water-in-oil state; 1 mL of the mixture was immunized on the back of each rabbit.

[0072] 5. One week later, take 1 mL of blood from the ear and test the titer with ELISA. If the titer reaches 1:100,000 or higher, then perform a shock immunization.

[0073] 6. Shock immunization: Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 mL of PBS, mix 1.5 mL of antigen + 1.5 mL of complete adjuvant, shake on a shaker for more than 15 minutes until it reaches a water-in-oil state; immunize each rabbit with 1.5 mL on its back.

[0074] 7. Three days later, blood was collected from the ears. 5 mL of blood was collected from each rabbit to test the titer. The titer met the expectations. Whole blood was collected using anticoagulant tubes and serum was separated.

[0075] In this embodiment, the materials involved in the above operations are all commonly used reagents and consumables.

[0076] Example 2: ELISA detection of antiserum titer

[0077] 1. Dilute the CA15-3 natural antigen (purchased from XEMA, R229C) with PBS to 1 μg / mL, add 100 μL to each well of the immunoassay plate, and incubate overnight at 4°C.

[0078] 2. Wash the microplate with PBST 3 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0079] 3. Add 300 μL of 3% BSA blocking solution to each well and incubate at 37°C for 1 hour.

[0080] 4. Wash the microplate with PBST 3 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0081] 5. Dilute the antiserum with PBS and add 100 μL to each well of the immunoassay plate. Incubate at 37°C for 1 hour.

[0082] 6. Wash the microplate with PBST 6 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0083] 5. Add 100 μL of Anti-rabbit Antibody (HRP) diluted in PBS (anti-rabbit secondary antibody, Beijing Yiqiao Shenzhou, catalog number SSA003) to each well and incubate at room temperature for 1 hour.

[0084] 6. Wash the microplate with PBST 6 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0085] 7. Remove solutions A and B of the TMB substrate chromogenic solution from 4°C, allow them to return to room temperature, mix them in equal proportions to prepare an appropriate amount of chromogenic solution, add 100 μL to each well of the microplate, and react in the dark for 10 minutes.

[0086] 8. Add 50 μL of 1 M HCl to each well to stop the colorimetric reaction, and read the OD450 value using a microplate reader.

[0087] Potency test results as follows Figure 1 As shown, the titer is greater than 1:102400, indicating successful immunization.

[0088] Example 3: Screening of rabbit antibody scFv display library

[0089] Rabbit spleen cells were ground and RNA was extracted to construct a cDNA library. The variable region sequences of antibodies in the library were amplified using literature-based methods, and a phage display library was constructed for antibody screening. After three rounds of panning, 192 single clones were selected, yielding 34 positive clones, of which CF24 and CF128 were preferred.

[0090] The amino acid sequence of the heavy chain variable region of CF24 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of CF24 is shown in SEQ ID NO.2. The amino acid sequence of the heavy chain variable region of CF128 is shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of CF128 is shown in SEQ ID NO.4.

[0091] Example 4: Expression and purification of positive clone antibodies

[0092] Recombinant antibody expression: The CF24 and CF128 light and heavy chain sequences were synthesized by Guangzhou Liuhe and the codon sequences were optimized before being constructed into the pCDNA3.1(+) vector. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed at a molar ratio of 1:1. 300 μg of plasmid was taken and added to 12.5 mL of OPM 293 CD05 medium and 800 μL of 1 mg / mL PEI (polyethyleneimine) solution. The mixture was incubated at room temperature for half an hour and then added to 250 mL of HEK293F cells that were being cultured. The cells were placed in a shaker and cultured at 37℃, 8% CO2, and 110 rpm. OPM 293 cells were added every other day, and the cells were cultured for 7 days. The cell supernatant was collected by centrifugation.

[0093] Recombinant antibody purification: 3 mL of Protein A packing material (Sino Biotech) was added to the received supernatant and incubated at room temperature for half an hour. The packing material was then removed and loaded into an empty purification column. 20 mL of PBS solution was added for washing, followed by elution with 10 mL of 100 mM glycine solution (pH 3.0). The eluent was neutralized to pH 7.0 with 1 M Tris solution. The neutralized eluent was concentrated to 1 mL, and the protein concentration was determined. Representative antibodies were then subjected to SDS-PAGE gel electrophoresis for purity assessment. Electrophoresis results are shown below. Figure 2 As shown, Figure 2 This is a diagram showing the purification results of positive antibodies. Figure 2 In the diagram, lane M represents the molecular weight standard, lane 1 contains recombinant antibody CF24, and lane 2 contains recombinant antibody CF128. Figure 2 As can be seen, the antibody meets the electrophoretic purity standard, and no other obvious bands are observed besides the main band.

[0094] Example 5: Positive clone affinity ELISA assay

[0095] 1. Coat the enzyme strip with CA15-3 natural antigen (purchased from XEMA, R229C) overnight. Wash the plate three times with PBST, 300 μL per well, patting off any remaining liquid on a paper towel after the last wash. 2. Add 200 μL of 3% BSA-PBS and block at 37°C for 2 hours. Wash the plate three times with PBST, 300 μL per well, patting off any remaining liquid on a paper towel after the last wash. 3. Dilute the antibody to different concentrations and add 100 μL to each well, incubating at 37°C for 1 hour. Wash the plate three times with PBST, 300 μL per well, patting off any remaining liquid on a paper towel after the last wash. 4. Add 100 μL of diluted commercial horseradish peroxidase-conjugated goat anti-rabbit antibody (HRP) and incubate at 37°C for 1 hour. Wash the plate six times with PBST, 300 μL per well, patting off any remaining liquid on a paper towel after the last wash. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as the substrate for color development for 10 minutes. 6. After terminating the reaction by adding 50 μL of 0.1 mol / L sulfuric acid, measure the absorbance at 450 nm.

[0096] Affinity test results are shown in Figure 3 The affinity of the two antibodies was calculated using Origin software based on the ELISA results. -11 about.

[0097] Example 6 Antibody Pairing Range Test

[0098] 1. Coat the CA15-3 antibody onto the immunoassay plate overnight. Wash the plate three times with PBST, 300 μL per well, and blot off any remaining liquid on a paper towel on the last wash.

[0099] 2. Add 200 μL of 3% BSA-PBS and block at 37°C for 2 hours. Wash the microplate three times with PBST, 300 μL per well, and blot off any remaining liquid on a paper towel after the last wash.

[0100] 3. Dilute the CA15-3 natural antigen to different concentrations, add 100 μL, and incubate at 37°C for 2 hours. Wash the ELISA plate 6 times with PBST, 300 μL per well, and blot off any remaining liquid on a paper towel after the last wash. 4. Add 100 μL of diluted anti-CA15-3-HRP antibody and incubate at 37°C for 1 hour. Wash the ELISA plate 6 times with PBST, 300 μL per well, and blot off any remaining liquid on a paper towel after the last wash. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as the substrate for color development for 10 minutes. 6. After stopping the reaction by adding 50 μL of 0.1 mol / L sulfuric acid, measure the absorbance at 450 nm.

[0101] The results of the antibody pairing assay are shown below. Figure 4 Its linear range is 6.25-100 U / mL (R0).2 >0.99).

[0102] In summary, the anti-CA15-3 protein monoclonal antibody (CA15-3 antigen-binding protein) provided by this invention is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity. The affinity between the two antibodies is 10. -11 The antibody pairing assay results showed that the linear range was 6.25-100 U / mL, and the combined antibody pairs had high specificity and accuracy.

[0103] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-CA15-3 monoclonal antibody or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO.7; and the light chain variable region includes CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.

10. Alternatively, the heavy chain variable region may include CDR1 of the sequence shown in SEQ ID NO.11, CDR2 of the sequence shown in SEQ ID NO.12, and CDR3 of the sequence shown in SEQ ID NO.13; and the light chain variable region may include CDR1 of the sequence shown in SEQ ID NO.14, CDR2 of the sequence shown in SEQ ID NO.15, and CDR3 of the sequence shown in SEQ ID NO.

16.

2. The anti-CA15-3 monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.2; or, The sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.3, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.

4.

3. The anti-CA15-3 monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The constant region of the monoclonal antibody is a rabbit-derived constant region.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CA15-3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 5; or the host cell genome is integrated with the nucleic acid molecule of claim 4.

7. A reagent for detecting CA15-3 protein in a sample, characterized in that, The reagent includes any one of the anti-CA15-3 monoclonal antibodies or antigen-binding fragments thereof as described in any one of claims 1-3.

8. A reagent kit, characterized in that, The kit includes the anti-CA15-3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3; or the kit includes the reagent for detecting CA15-3 protein in a sample as described in claim 7.

9. The use of the anti-CA15-3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3 in the preparation of breast cancer detection products.

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