Anti-gcdfp-15 monoclonal antibodies, methods of making and uses thereof

Abstract

The application relates to an anti-GCDFP-15 monoclonal antibody and a preparation method and application thereof, and belongs to the technical field of immunology. The anti-GCDFP-15 monoclonal antibody provided by the application has the amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 1 and the amino acid sequence of a light chain variable region as shown in SEQ ID NO. 2. The nucleotide sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are also provided, and the monoclonal antibody can be prepared by using genetic engineering technology. The monoclonal antibody prepared by the application has the advantages of stable property, high experimental repeatability and high titer, is suitable for immunological detection, and can specifically recognize the GCDFP-15 protein expressed in human skin and breast tissue.

Description

Technical Field

[0001] This invention relates to an anti-GCDFP-15 monoclonal antibody, its preparation method, and its application, belonging to the field of immunology technology. Background Technology

[0002] GCDFP-15 (Gross Cystic Disease Fluid Protein 15) is a secretory single-chain glycoprotein with a molecular weight of approximately 15 kDa, encoded by the PIP gene. It was initially isolated and identified from the cystic fluid of benign cystic lesions of the breast. In humans, it is mainly expressed in exocrine glands and related tissues such as sweat glands, mammary glands, salivary glands, lacrimal glands, and the prostate gland. Studies have found that GCDFP-15 expression is significantly upregulated in various pathological states, such as breast cancer, salivary gland tumors, and Sjögren's syndrome.

[0003] GCDFP-15 serves as a highly specific and sensitive biomarker for breast or other exosomes and their tumors. It exhibits high specificity in primary and recurrent / metastatic breast cancer; the positivity rate is even higher in mucinous breast carcinoma in older women, helping to differentiate age-related subtypes; for tumors of unknown origin, GCDFP-15 can help confirm breast origin, especially in cases of local recurrence or distant metastasis, where its specificity surpasses other biomarkers; GCDFP-15 is also positively expressed in salivary gland tumors (such as apocrine carcinoma), sweat gland tumors (such as vulvar Paget's disease), and prostate cancer, aiding in the identification of tumors in these tissues; the expression level of GCDFP-15 is also closely related to hormone receptor status, treatment response, and prognostic assessment in breast cancer patients, making it an indispensable target for clinicopathological diagnosis and basic research.

[0004] Clone 23A3 is one of the most representative classic clones of GCDFP-15 antibodies. Its core advantage lies in its extensive literature support and mature experimental application background, and it has long been regarded as one of the "gold standards" for GCDFP-15 detection. However, it is expensive, mostly prepared using tissue culture supernatant, and suffers from problems such as low antibody titers and batch-to-batch variability. At the same time, hybridoma cells are at risk of antibody gene loss, poor cell condition, and even cell death during preservation, which limits its further industrial production and application. Summary of the Invention

[0005] The first objective of this invention is to provide a monoclonal antibody against GCDFP-15 that is stable, has high experimental reproducibility, and exhibits good specificity and affinity.

[0006] A second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned anti-GCDFP-15 monoclonal antibody.

[0007] A third objective of this invention is to provide an expression cassette, expression vector, recombinant cell, or recombinant bacterium containing the aforementioned nucleic acid molecules.

[0008] A fourth objective of this invention is to provide the use of the above-mentioned expression cassette, expression vector, recombinant cells or recombinant bacteria in the preparation of anti-GCDFP-15 monoclonal antibodies.

[0009] The fifth objective of this invention is to provide a method for preparing an anti-GCDFP-15 monoclonal antibody.

[0010] The sixth object of the present invention is to provide the use of the above-mentioned anti-GCDFP-15 monoclonal antibody in the preparation of GCDFP-15 in vitro detection reagents or kits.

[0011] A seventh object of the present invention is to provide a detection reagent or kit comprising the above-described anti-GCDFP-15 monoclonal antibody.

[0012] The eighth object of the present invention is to provide an immunogenic fragment of the GCDFP-15 protein.

[0013] To achieve the above objectives, the technical solution adopted by the anti-GCDFP-15 monoclonal antibody in this invention is as follows: An anti-GCDFP-15 monoclonal antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region amino acid sequence as shown in SEQ ID NO.2.

[0014] The beneficial effects of the above technical solution are as follows: This invention provides an anti-GCDFP-15 monoclonal antibody and discloses the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the antibody. The antibody can be prepared by genetic engineering technology, avoiding the risk factors that occur in the traditional monoclonal antibody production and preservation process.

[0015] To achieve the above objectives, the technical solution adopted in this invention for a nucleic acid molecule encoding the aforementioned anti-GCDFP-15 monoclonal antibody is as follows: A nucleic acid molecule encoding the aforementioned anti-GCDFP-15 monoclonal antibody.

[0016] The beneficial effects of the above technical solution are as follows: This invention provides a nucleic acid molecule encoding an anti-GCDFP-15 monoclonal antibody, which preserves the anti-GCDFP-15 monoclonal antibody in the form of DNA, resulting in higher stability and facilitating subsequent exogenous expression and large-scale industrial production.

[0017] Specifically, the nucleotide sequence of the heavy chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.4.

[0018] To achieve the above objectives, the technical solution adopted in this invention for an expression cassette, expression vector, recombinant cell, or recombinant bacterium containing the aforementioned nucleic acid molecules is as follows: An expression cassette, expression vector, recombinant cell, or recombinant bacterium containing the aforementioned nucleic acid molecules.

[0019] The beneficial effects of the above technical solution are as follows: The present invention provides an expression cassette, expression vector, recombinant cell or recombinant bacteria containing a nucleic acid molecule encoding an anti-GCDFP-15 monoclonal antibody, which can rapidly, stably and on a large scale produce anti-GCDFP-15 monoclonal antibodies through genetic engineering.

[0020] Specifically, the expression vector is selected from prokaryotic or eukaryotic expression vectors.

[0021] Preferably, the expression vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0022] Preferably, the recombinant cells are selected from mammalian cells, insect cells, and plant cells.

[0023] Preferably, the recombinant bacteria are selected from bacteria, yeast, and filamentous fungi.

[0024] To achieve the above objectives, the technical solution adopted in this invention for the application of the expression cassette, expression vector, recombinant cells, or recombinant bacteria in the preparation of anti-GCDFP-15 monoclonal antibodies is as follows: The above-mentioned expression cassettes, expression vectors, recombinant cells or recombinant bacteria are used in the preparation of anti-GCDFP-15 monoclonal antibodies.

[0025] The beneficial effects of the above technical solution are as follows: This invention provides an expression cassette, expression vector, recombinant cells or recombinant bacteria containing a nucleic acid molecule encoding an anti-GCDFP-15 monoclonal antibody. Based on this, the anti-GCDFP-15 monoclonal antibody of this invention can be obtained by conventional genetic engineering methods, avoiding the risks that may exist in the hybridoma technology production process, such as gene mutation leading to decreased antibody stability, small fluctuations in culture conditions leading to cell population heterogeneity, and lack of antigen selection pressure leading to decreased antibody specificity.

[0026] To achieve the above objectives, the technical solution adopted in the preparation method of the anti-GCDFP-15 monoclonal antibody of the present invention is as follows: A method for preparing an anti-GCDFP-15 monoclonal antibody involves introducing the above-mentioned nucleic acid molecules into host cells, collecting the cell supernatant, and purifying and ultrafiltration the supernatant.

[0027] The beneficial effects of the above technical solution are as follows: The method for preparing anti-GCDFP-15 monoclonal antibodies provided by this invention, compared with traditional monoclonal antibody preparation methods, has advantages such as known antibody sequences, long-term preservation of antibody genes, stable antibody properties, and good experimental reproducibility. It is a standardized antibody production process that avoids the risk factors that occur in the traditional monoclonal antibody production and preservation process. Utilizing recombinant technology to prepare GCDFP-15 antibodies ensures the controllability and traceability of the antibody sequence, solves the batch-to-batch differences and preservation problems that may occur in traditional antibody production, and provides a more stable and reliable antibody tool suitable for long-term and standardized clinical diagnostic reagent development and basic research applications.

[0028] Preferably, the host cell includes mammalian cells; the mammalian cells include HEK293 cells and CHO cells.

[0029] HEK293 and CHO cells have accurate post-translational modification capabilities, are easy to transfect, have high tolerance to shear stress and osmotic pressure, express recombinant antibodies in near-native state, and can produce large amounts of recombinant proteins.

[0030] To achieve the above objectives, the technical solution adopted in this invention for the application of anti-GCDFP-15 monoclonal antibody in the preparation of GCDFP-15 in vitro detection reagents or kits is as follows: Application of anti-GCDFP-15 monoclonal antibody in the preparation of GCDFP-15 in vitro detection reagents or kits.

[0031] The beneficial effects of the above technical solution are as follows: Experimental verification shows that the anti-GCDFP-15 monoclonal antibody has good specificity and affinity, and can be used to prepare GCDFP-15 detection reagents or kits to detect the expression of GCDFP-15 in tissues and cells, and can further study the function of GCDFP-15.

[0032] To achieve the above objectives, the technical solution adopted in the present invention for a detection reagent or kit containing the above-mentioned anti-GCDFP-15 monoclonal antibody is as follows: A detection reagent or kit containing the above-mentioned anti-GCDFP-15 monoclonal antibody.

[0033] The beneficial effects of the above technical solution are as follows: the anti-GCDFP-15 monoclonal antibody of the present invention, which has good specificity and affinity, can be specifically prepared into detection reagents or kits for detecting GCDFP-15 in tissues or cells, such as immunohistochemical staining, immunocellular staining, Western blot or ELISA, according to the needs of users, which helps to meet diverse market demands.

[0034] Preferably, the detection reagent or kit includes an immunohistochemical detection reagent or kit.

[0035] To achieve the above objectives, the technical solution adopted in this invention for an immunogenic fragment of GCDFP-15 protein is as follows: An immunogenic fragment of the GCDFP-15 protein, the fragment having a nucleotide sequence as shown in SEQ ID NO.5.

[0036] The beneficial effects of the above technical solution are as follows: The immunogenic fragment of GCDFP-15 protein provided by the present invention is selected from amino acid 27 to 146 of the GCDFP-15 amino acid sequence as the design scheme, and GCDFP-15 recombinant protein is expressed. After animal immunization, it has good antigenicity. The immunogen prepared by it can produce highly specific and sensitive GCDFP-15 antibodies when immunized in animals. Attached Figure Description

[0037] Figure 1 The results of Western blot detection of the anti-GCDFP-15 monoclonal antibody in Example 4 of this invention are shown (M is the marker, and 1 is the electrophoresis result of the T47D cell lysis buffer as a sample). Figure 2 The results of immunohistochemical detection of skin tissue using the anti-GCDFP-15 monoclonal antibody in Example 4 of this invention (100×). Figure 3 The results of immunohistochemical detection of skin tissue using the anti-GCDFP-15 monoclonal antibody in Example 4 of this invention (100×). Figure 4 The images show the immunohistochemical detection results of breast tissue in Example 5 of this invention. The left image shows the detection results (100×) of a commercially available monoclonal antibody with clone number 23A3, and the right image shows the detection results (100×) of the anti-GCDFP-15 monoclonal antibody prepared in this invention. Detailed Implementation

[0038] GCDFP-15 is a highly specific and sensitive marker for breast tissue and its tumors, holding significant clinical value in breast pathology diagnosis, particularly for breast cancers with apocrine characteristics (such as invasive or mucinous carcinoma). Currently, one of the "gold standards" for GCDFP-15 detection in clinical / pathological settings is immunohistochemical detection using an antibody with clone number 23A3. However, this method suffers from high cost and significant batch-to-batch variability. Therefore, developing highly sensitive, highly specific GCDFP-15 antibodies suitable for industrial production is of great importance in breast pathology diagnosis.

[0039] Based on this, the present invention provides an anti-GCDFP-15 monoclonal antibody. The invention first selects amino acid fragments 27-146 from the GCDFP-15 amino acid sequence published by Uniprot as the design scheme. The designed GCDFP-15 antigen nucleotide sequence is artificially synthesized, inserted into a vector, transformed into *E. coli*, and expressed and purified to obtain the antigen. Then, mice are immunized with the antigen, and spleen cells are harvested from the mice after the initial shock immunization to prepare a single-cell suspension of spleen cells. This suspension is fused with myeloma cells to obtain hybridoma cells, and positive cell lines are obtained after cloning. Subsequently, total RNA is extracted from the positive cell lines, and the GCDFP-15 antibody gene is obtained using 5'RACE. The antibody heavy and light chain gene sequences are extracted, and the antibody is obtained through recombination and expression.

[0040] The amino acid sequence of the heavy chain variable region of the anti-GCDFP-15 monoclonal antibody provided by this invention is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. Based on the antibody amino acid sequence provided by this invention, modifications such as the addition, deletion, or substitution of one or more amino acids can be used to obtain its active fragment or conserved variant, laying the foundation for further improving the specificity and affinity of the antibody.

[0041] This invention also provides a nucleic acid molecule encoding the aforementioned anti-GCDFP-15 monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene is shown in SEQ ID NO.4. This nucleic acid molecule can be obtained through genetic engineering or chemical synthesis. The variant sequences of the heavy chain variable region nucleotide sequence and / or light chain variable region nucleotide sequence obtained by mutation of the above-mentioned nucleic acid molecule provided by this invention through one or more nucleotide additions, deletions, substitutions, modifications, etc., whose encoded amino acid sequences form single-chain antibodies, chimeric monoclonal antibodies, modified monoclonal antibodies, or other forms of monoclonal antibodies or antibody fragments, still retain the ability to specifically bind to GCDFP-15.

[0042] This invention provides the amino acid and nucleotide sequences of the variable regions of the heavy and light chains of an anti-GCDFP-15 monoclonal antibody. Based on these sequences, the monoclonal antibody of this invention can be obtained using conventional genetic engineering methods. Accordingly, this invention provides an expression cassette, expression vector, recombinant cells, and recombinant bacteria comprising the above-mentioned nucleic acid molecule encoding the anti-GCDFP-15 monoclonal antibody. The expression cassette, expression vector, recombinant cells, and recombinant bacteria are all commonly used tools in genetic engineering. The expression vector can be selected from prokaryotic or eukaryotic expression vectors, specifically including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors; the recombinant cells are selected from mammalian cells, insect cells, plant cells, etc.; and the recombinant bacteria are selected from bacteria, yeast, filamentous fungi, etc.

[0043] Using the above-mentioned nucleic acid molecules, expression cassettes, expression vectors, recombinant cells, and recombinant bacteria, the anti-GCDFP-15 monoclonal antibody of the present invention can be prepared by recombination technology.

[0044] This invention also provides the application of anti-GCDFP-15 monoclonal antibodies in the preparation of GCDFP-15 in vitro detection reagents or kits. The anti-GCDFP-15 monoclonal antibody provided by this invention has good specificity and affinity, and can be directionally prepared into detection reagents or kits for detecting GCDFP-15 in tissues or cells according to user needs. Specifically, the anti-GCDFP-15 monoclonal antibody of this invention can be made into a working solution, and combined with appropriate reagents to prepare detection reagents or kits for immunohistochemical staining, immune cell staining, Western blot, or ELISA.

[0045] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art, and the equipment and raw materials used are commercially available or commonly used in the art.

[0046] Example 1: An immunogenic fragment of GCDFP-15 protein This embodiment provides an immunogenic fragment of GCDFP-15 protein (GCDFP-15 antigen). The designed nucleotide sequence of GCDFP-15 antigen is artificially synthesized, inserted into a vector, transformed into E. coli, expressed, and then the bacterial cells are lysed and purified to obtain the antigen. The details are as follows: 1. Antigen design According to the GCDFP-15 amino acid sequence published by Uniprot (accession number P12273), GCDFP-15 consists of 146 amino acids. This invention selects the 27-146 amino acid fragment as the design scheme for recombinant protein expression.

[0047] 2. Construction of recombinant carriers The base sequence corresponding to amino acid fragment 27-146 of GCDFP-15 is shown in SEQ NO.5. An artificially synthesized antigen gene was added to both ends of the sequence with BamHI and XhoI restriction enzyme sites, and then recombinantly cloned into the pET-32a expression vector to construct the pET-32a-rGCDFP-15 recombinant vector.

[0048] 3. Pre-expression of antigens The pET-32a-rGCDFP-15 recombinant vector was transformed into BL21(DE3) Escherichia coli competent cells, and single colonies were picked and cultured in 4 mL of LB broth containing ampicillin (100 μg / mL) resistance until OD600. 600 Once the concentration reaches 0.4~0.8, add IPTG to a final concentration of 1mM for expression, analyze the expression product, and preserve the strain capable of expressing the target protein.

[0049] 4. Antigen expression The pre-expressed strain was inoculated into 350 mL of LB liquid medium containing ampicillin (100 μg / mL) resistance and cultured at 37°C until OD500. 600 Once the concentration reaches 0.4~0.8, add IPTG to a final concentration of 0.4mM and induce expression at 37℃ for 4h.

[0050] 5. Collection and disruption of bacterial cells The induced bacterial cells were collected by centrifugation, resuspended in 10mM PBS on ice, and sonicated (350W, 20min, 3s on, 3s off). The cells were then centrifuged at 12000g for 10min, and the supernatant was collected for further purification.

[0051] 6. Purification and identification of antigens The supernatant obtained in step 5 was filtered through a 0.45 μm filter membrane, and the filtrate was purified by nickel agarose gel electrophoresis. When loading the filtrate onto an equilibrated nickel column, the flow rate was controlled at 1 mL / min. Unadsorbed protein was removed by washing with 10 mM PBS, followed by gradient elution with PBS and PBS containing 0.5 M imidazole. Different elution peaks were collected, and the protein was identified by SDS-PAGE. Samples with a purity greater than 90% were selected for ultrafiltration, and the solution was replaced and stored in 10 mM PBS to obtain GCDFP-15 antigen at a concentration of 2 mg / mL.

[0052] Example 2: Variable region sequence information of anti-GCDFP-15 monoclonal antibody This embodiment provides a method for obtaining the amino acid and nucleotide sequences of the light and heavy chain variable regions of anti-GCDFP-15 monoclonal antibodies. Mice were immunized with the GCDFP-15 antigen prepared in Example 1. Mouse spleen cells were fused with sp2 / 0 to obtain hybridoma cells. After screening clones, monoclonal antibodies with high positive values ​​were obtained. Total RNA was extracted, and the GCDFP-15 antibody sequence information was obtained by 5'RACE. The specific operation is as follows: 1. Animal immunization The GCDFP-15 antigen prepared in Example 1 was mixed with an equal volume of Freund's complete adjuvant and administered subcutaneously to 6-8 week old BalB / c mice at a dose of 100 μg per mouse. Booster immunizations were given every 14 days using Freund's incomplete adjuvant, with the dose halved, administered intraperitoneally. Seven days after the third immunization, tail blood was collected and serum titers were determined using a serially diluted indirect ELISA method. Mice with the highest antibody titers were selected for tail vein pulse immunization at a dose of 50 μg per mouse.

[0053] 2. Cell fusion Myeloma cells were sp2 / 0 derived from BalB / c, and were in the logarithmic growth phase at fusion. Spleens were aseptically harvested from mice after a shock immunization, and single-cell suspensions of spleen cells were prepared. Mouse spleen cells and myeloma cells were mixed at a 1:5 ratio, and 1 mL of 50% PEG (pH 8.0) at 37°C was added. Incomplete culture medium was added, and after centrifugation and discarding the supernatant, HAT medium was added to resuspend the cells, and the mixture was brought to a final volume of 50 mL. The mixture was then added to 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator.

[0054] 3. Hybridoma cell screening and cloning Cell clones were selected after culturing in a constant temperature incubator for 7-10 days and then subjected to screening tests (ELISA, IHC, WB, and other methods). Positive wells were subjected to limiting dilutions, and ELISA values ​​were measured 5-6 days after each limiting dilution. OD values ​​were then collected. 450 Limiting dilutions were performed on wells with high positive values ​​for single clones until the entire 96-well plate was positive for ELISA. Single clones with high positive values ​​were then selected to confirm the cell line.

[0055] 4. Total RNA extraction The identified hybridoma cell lines were cultured and collected. 1 mL of Trizol was added, mixed by pipetting, and incubated at room temperature for 5-10 min until the cells were completely lysed. The cells were centrifuged at 12000 rpm for 5 min, and the precipitate was discarded. Then, 200 μL of chloroform was added, the tube was vigorously inverted, and the mixture was allowed to stand for 15 min. The tube was then centrifuged at 12000 rpm at 4℃ for 15 min, and the upper aqueous phase was collected. An equal volume of isopropanol was added, the mixture was gently mixed, and the mixture was allowed to stand for 10 min. The tube was then centrifuged at 12000 rpm at 4℃ for 10 min, and the supernatant was discarded. 1 mL of pre-chilled 75% ethanol was added, the bottom of the tube was gently tapped, and the tube was centrifuged at 12000 rpm at 4℃ for 5 min, and the supernatant was discarded. The tube was air-dried at room temperature for several minutes, and then 30-50 μL of pre-chilled DEPC water was added until dissolved to obtain the RNA product. The product was stored at -80℃ for a long time. Masks and gloves must be worn throughout the extraction process to prevent RNase contamination.

[0056] 5. Obtaining the GCDFP-15 antibody gene via 5'RACE The 5' sequence of the GCDFP-15 antibody was isolated using the SMARTer RACE 5' / 3' Kit (Clontech, Cat. No. 634859). Using 1 μg of total RNA as a template, first-strand cDNA synthesis was performed according to the manufacturer's instructions using the 5'-CDS primer A, SMART II A oligo, and 3'-CDS primer A provided in the kit, yielding 5'-RACE-Ready cDNA. PCR (Rapid Amplification of cDNA Ends) was then performed using these cDNAs as templates. 5'-RACE PCR used UPM (Universal Primer) primers, and GSP (gene-specific primer) primers designed based on the gene-specific sequence. The reaction volume for the first round of PCR was 50 μL, and the components are shown in Table 1.

[0057] Table 1 PCR reaction system The PCR reaction procedure is shown in Table 2.

[0058] Table 2 PCR reaction procedure Store PCR products at 4°C. Take 5 μL of the PCR product and perform electrophoresis on a 1.0% agarose gel. If the bands are uniform, excise and recover the product, construct it onto a pRACE array, and then select positive clones for sequencing. If the bands are not uniform, nested PCR is required for further amplification before constructing the product onto a pRACE array and selecting positive clones for sequencing.

[0059] 6. Identification of antibody heavy chain and light chain gene information and analysis of the IMGT database. Sequencing analysis revealed that the nucleotide sequence of the heavy chain variable region gene of the anti-GCDFP-15 monoclonal antibody of the present invention is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.1; the nucleotide sequence of the light chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.2.

[0060] Example 3: Preparation method of anti-GCDFP-15 monoclonal antibody This embodiment provides a method for preparing anti-GCDFP-15 monoclonal antibody. Based on the light and heavy chain sequence information of the anti-GCDFP-15 monoclonal antibody obtained in Example 2, the variable region genes of the heavy and light chains are amplified by PCR, constructed into an expression vector, transformed into host cells, and expressed and purified to obtain the anti-GCDFP-15 monoclonal antibody. The specific operations are as follows: 1. Construction of recombinant carriers Based on the light and heavy chain sequence information of the anti-GCDFP-15 monoclonal antibody obtained in Example 2, the variable regions of the heavy and light chains were amplified by PCR. The pTT5 vector was digested with EcoRI / HindIII for 3 hours, and then recovered by agarose gel electrophoresis. The antibody expression vector was then constructed by recombination. The positive clones with correct sequencing were extracted for plasmid extraction for transfection.

[0061] 2. Expression and purification of anti-GCDFP-15 monoclonal antibody 1) Cell culture before transfection and preparation of transfected cells HEK293 cells were placed in a 5% CO2 constant-temperature shaker and cultured at 37°C and 120 rpm. Before passage, cell counting was performed to confirm the density. After confirming the density, there was no need to centrifuge the cells; the cell suspension could be directly added to the culture medium in the required proportion. If an excessive number of dead cells were observed during culture, the cells should be discarded and new cells used.

[0062] Before transient transfection, cell density and viability need to be determined; cells do not need to be centrifuged, but can be directly added to HEK293 culture medium to dilute the cell density to 3 × 10⁻⁶. 6 Transfection was initiated after placing the shake flask in a 5% CO2 constant temperature shaker and incubating at 37℃ and 120rpm for 10 minutes.

[0063] KPM (Catalog No.: K03125), TA-293 (Catalog No.: K20001), KE-293 (Catalog No.: K30001), and KT-Feed (Catalog No.: K40001) used for transient transfection and expression were all purchased from Zhuhai Kerry Biotechnology Co., Ltd.

[0064] 2) Instantaneous transfection Taking 100mL cell suspension as an example: Prepare two 15mL sterile centrifuge tubes. Add 5mL KPM and 100μg sterile plasmid DNA (heavy chain: light chain plasmid ratio 1:1) to one tube and gently pipette to mix. Take the other centrifuge tube and add 5mL KPM and 500μL TA-293 transfection reagent, and gently pipette to mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently pipette to mix. Incubate at room temperature for 10min to prepare the plasmid-vector complex. Remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO2 constant temperature shaker for incubation. After 3 hours, add an appropriate amount of antibiotic as needed.

[0065] 3) Product expression 600 μL of KE-293 was added 24 hours after transfection, and KT-Feed was added at the same time to increase the expression level of the product. Cell supernatant was collected 6-8 days after transfection.

[0066] 4) Antibody purification Cell supernatant was collected, purified using an antibody affinity column, and the product was collected and ultrafiltered. The solution was changed and concentrated in PBS (pH 7.4) to obtain a 1 mg / mL anti-GCDFP-15 monoclonal antibody.

[0067] Example 4: Specificity Detection of Anti-GCDFP-15 Monoclonal Antibody This embodiment tests the anti-GCDFP-15 monoclonal antibody prepared in Example 3, using Western blotting and immunohistochemistry (IHC) to detect antibody specificity.

[0068] 1. Western blot detection of the specificity of anti-GCDFP-15 monoclonal antibody 1) Sample preparation Literature reports that GCDFP-15 is highly expressed in the T47D breast cancer cell line. Cultured T47D cells were collected, lysed with RAPI containing 1 mM PMSF, and then sampled with 5×SDS-PAGE Loading buffer.

[0069] 2) Electrophoresis and membrane transfer After protein loading, electrophoresis was performed at 80V for 20 minutes, followed by electrophoresis at 120V until the bromophenol blue reached the bottom of the gel. After electrophoresis, the membrane was transferred. The NC membrane and filter paper were soaked in transfer buffer beforehand and placed in a sandwich configuration in a wet transfer apparatus. The sequence was: electrode (-) - sponge - filter paper - gel - NC membrane - filter paper - sponge - electrode (+). Electrophoresis was performed at a constant voltage of 90V for 60 minutes.

[0070] 3) Closed Immerse the NC membrane in a blocking solution (TBST containing 5% skim milk) and incubate at 37°C for 2 hours.

[0071] 4) Primary antibody incubation The NC membrane was placed in the anti-GCDFP-15 monoclonal antibody obtained in Example 3 (the antibody was diluted 1:3000 with blocking buffer), incubated at 37°C for 1 hour, and then washed with TBST 4 times for 5 minutes each time.

[0072] 5) Secondary antibody incubation The NC membrane was placed in HRP-labeled goat anti-mouse IgG secondary antibody (antibody diluted 1:5000 with blocking buffer), incubated on a shaker at 37°C for 1 hour, and then washed 4 times with TBST for 5 minutes each time.

[0073] 6) Color development Components A and B in the ECL luminescent solution are mixed in a 1:1 ratio and then added to the NC film, which is then exposed in an exposure apparatus.

[0074] The results are as follows Figure 1 As shown, the target band at around 17 kDa was observed in the swimming lanes of the T47D cell lysate sample, indicating that the anti-GCDFP-15 monoclonal antibody of the present invention binds to the GCDFP-15 protein in T47D cells and has good antibody specificity.

[0075] 2. Specificity of IHC detection of GCDFP-15 monoclonal antibody 1) Sectioning: Take paraffin-embedded blocks of skin tissue, section them using a Leica tissue microtome, with a tissue thickness of 3μm, and dry-bake at 65℃ for 2 hours.

[0076] 2) Stain the sections using manual immunohistochemistry. The specific steps are as follows: A. High-temperature repair was performed using DAKO HIGH pH repair solution for 20 min. After cooling, the solution was incubated at room temperature for 5 min using 100 μL of endogenous peroxidase blocking solution. The solution was then used to soak the sample twice, for 5 min each time. B. The primary antibody used was the anti-GCDFP-15 monoclonal antibody obtained in Example 3. It was diluted with antibody dilution buffer at a ratio of 1:1000. 100 μL of the diluted antibody was added to the slide and incubated at 37°C for 30 min. The slide was then washed twice with washing buffer for 5 min each time. C. Use enzyme-labeled polymer for secondary antibody. Add 100 μL to the slice and incubate at room temperature for 20 min. Soak twice in washing buffer, 5 min each time. D. Add 100 μL of DAB colorimetric solution to the slide, incubate at room temperature for 5 min, and soak in purified water twice, 5 min each time; E. Add 100 μL of hematoxylin to the slice, incubate at room temperature for 5 min, and rinse with tap water to restore blue color.

[0077] 3) Sectioning, dehydration, clearing, and mounting Wash sections with deionized water for 3 min; soak in 85% ethanol for 1 min; soak in 95% ethanol for 1 min; soak in 100% ethanol for 1 min, twice in total; soak in xylene for 1 min, twice in total; mount with neutral resin.

[0078] 4) Observe the slides under a microscope The results are as follows Figure 2 and Figure 3 As shown. By Figure 2 As can be seen, skin tissue cells are stained dark, with only a small light spot in the central area. This indicates that the anti-GCDFP-15 monoclonal antibody of the present invention specifically binds to GCDFP-15 in the cytoplasm, consistent with the expected localization. Figure 3 It can be seen that GCDFP-15 exhibits specific expression in skin tissue, with clear staining results and no non-specific background staining, which is consistent with the expected expression localization characteristics.

[0079] Example 5: Application of anti-GCDFP-15 monoclonal antibody in the preparation of GCDFP-15 in vitro detection reagents or kits This embodiment provides the application of anti-GCDFP-15 monoclonal antibody in the preparation of GCDFP-15 in vitro detection reagents or kits, as detailed below: This embodiment provides an in vitro detection reagent for GCDFP-15, including a working solution of the anti-GCDFP-15 monoclonal antibody prepared in Example 3. The buffer used in the anti-GCDFP-15 monoclonal antibody working solution is TBS buffer. The concentration of anti-GCDFP-15 monoclonal antibody in the working solution is 0.1 μg / mL, the concentration of BSA is 10 mg / mL, and the preservatives Proclin 300 and Proclin 950 are added at a ratio of 1:1000 (v / v).

[0080] Based on the above-mentioned anti-GCDFP-15 monoclonal antibody working solution, a GCDFP-15 in vitro detection kit is constructed by combining DAKO HIGH pH restoration solution, endogenous peroxidase blocking solution, enzyme-labeled polymer, DAB chromogenic solution, hematoxylin, etc.

[0081] This embodiment also provides specific application examples of the above-mentioned detection reagents and kits. Currently, among GCDFP-15 monoclonal antibodies on the market, mouse monoclonal antibody GCDFP-15 (23A3) is widely used. The following is a detailed example of using mouse monoclonal antibody GCDFP-15 (clone number 23A3, purchased from Cell Marquee, catalog number 257M-16-RUO) and the above-mentioned detection reagents and kits to detect GCDFP-15 in breast tissue: Paraffin-embedded breast tissue was sectioned, and the sections were stained, dehydrated, cleared, and mounted using the above-mentioned GCDFP-15 detection reagent and 23A3 antibody (working concentration of 0.1 μg / mL). The specific process is as described in step 2 of Example 4, which detects the specificity of the GCDFP-15 monoclonal antibody by IHC.

[0082] The results are as follows Figure 4 As shown, by Figure 4 It can be seen that 23A3 ( Figure 4 (left) and the anti-GCDFP-15 monoclonal antibody of the present invention ( Figure 4 (Right) It exhibits specific expression in breast cancer tissue, with accurate staining localization, clear staining without non-specific staining, and a clean background. Compared with the 23A3 antibody, under the same working concentration conditions (both 0.1 μg / mL), the anti-GCDFP-15 monoclonal antibody provided by this invention showed a stronger positive signal and better results in immunohistochemical staining. The above results indicate that the anti-GCDFP-15 monoclonal antibody of this invention has comparable specificity and sensitivity to commercially available GCDFP-15 antibodies.

[0083] This invention provides the variable region amino acid sequences of the heavy and light chains of an anti-GCDFP-15 monoclonal antibody. Based on these sequences, the monoclonal antibody of this invention can be obtained using conventional antibody engineering methods, as detailed in Example 3. Western blotting and IHC specificity verification experiments demonstrate that the anti-GCDFP-15 monoclonal antibody provided by this invention can specifically recognize the GCDFP-15 protein and can be used for IHC immunohistochemical detection. Compared with traditional monoclonal antibody preparation methods, the anti-GCDFP-15 monoclonal antibody prepared by the genetic engineering method of this invention has advantages such as known sequence, stable antibody properties, and good reproducibility. The standardized antibody production process avoids the risk factors that occur during the production and storage of traditional monoclonal antibodies.

[0084] 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 therein. Such 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 monoclonal antibody against GCDFP-15, characterized in that, The anti-GCDFP-15 monoclonal antibody has the heavy chain variable region amino acid sequence as shown in SEQ ID NO.1 and the light chain variable region amino acid sequence as shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the anti-GCDFP-15 monoclonal antibody as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the heavy chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region gene of the anti-GCDFP-15 monoclonal antibody is shown in SEQ ID NO.

4.

4. An expression cassette, expression vector, recombinant cell, or recombinant bacterium comprising the nucleic acid molecule as described in claim 2 or 3.

5. The use of the expression cassette, expression vector, recombinant cell or recombinant bacteria as described in claim 4 in the preparation of anti-GCDFP-15 monoclonal antibody.

6. A method for preparing an anti-GCDFP-15 monoclonal antibody, characterized in that, The nucleic acid molecule described in claim 2 or 3 is introduced into a host cell, the cell supernatant is collected, and purified by ultrafiltration.

7. The use of the anti-GCDFP-15 monoclonal antibody as described in claim 1 in the preparation of GCDFP-15 in vitro detection reagents or kits.

8. A detection reagent or kit comprising the anti-GCDFP-15 monoclonal antibody as described in claim 1.

9. The detection reagent or kit according to claim 8, characterized in that, The detection reagents or kits include immunohistochemical detection reagents or kits.

10. An immunogenic fragment of GCDFP-15 protein, characterized in that, The fragment has a nucleotide sequence as shown in SEQ ID NO. 5.

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