Monoclonal antibody against PSA protein and cell strain, preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human PSA antigen, secreting cell strain, its preparation method and its use in immunodetection.The above technical scheme selects the amino acid of 31-124 of PSA protein as antigen peptide, is codon optimized, becomes the gene fragment suitable for expression in escherichia coli BL21, and finally the recombinant protein includes TRX protein label, PSA protein fragment and histidine protein label.The recombinant protein is immunized to mouse, is fused by cell, screening and subcloning, obtains the mouse hybridoma cell strain 16F1 of high-efficiency secreted anti-PSA protein monoclonal antibody and the anti-PSA protein monoclonal antibody secreted by the cell strain.The antibody obtained in the present application has high specificity, sensitivity, and can specifically recognize the cell expressing PSA protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to a monoclonal antibody against PSA protein, its cell line, preparation method, and application. Background Technology

[0002] Prostate-specific antigen (PSA), also known as kallikrein 3 (KLK3 / hK3), is a biomarker for prostate cancer diagnosis and is widely used in prostate cancer screening, recurrence detection after treatment of carcinoma in situ, and efficacy monitoring of systemic therapy for metastatic cancer. Epidemiological studies have shown that serum PSA levels and changes in serum PSA levels (PSA rate) are associated with an increased risk of prostate cancer development. PSA is an independent risk factor for prostate cancer progression to extraprostatic regions, pelvic lymph node extension, distant metastasis, and death. These studies suggest that PSA is not only a biomarker for prostate cancer but may also play a role in the development of prostate cancer and / or the progression of prostate carcinoma in situ to metastatic cancer.

[0003] Currently, early diagnosis of prostate cancer mainly includes serum PSA level detection, early tumor biopsy, and pathological diagnosis, which has greatly improved the detection rate of prostate cancer. However, due to the characteristics of prostate cancer, such as insidious onset, inconspicuous early symptoms, and slow early disease progression, further improvements in detection rate are needed in clinical practice to achieve early diagnosis and treatment, thereby improving the survival rate of prostate cancer patients. PSA immunohistochemistry has broad application value in the diagnosis of prostate cancer, especially metastatic prostate cancer.

[0004] In conclusion, besides being an effective biomarker, PSA may also be a potential therapeutic target for prostate cancer. Summary of the Invention

[0005] The inventors provide a monoclonal antibody against PSA protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.4; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.5.

[0006] Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.3.

[0007] Furthermore, the monoclonal antibody specifically recognizes the PSA protein.

[0008] Furthermore, the monoclonal antibody is a mouse IgG1κ subtype monoclonal antibody.

[0009] Furthermore, the monoclonal antibody was produced by a hybridoma cell line with accession number CGMCC NO 21968.

[0010] The inventors have also provided a method for preparing a monoclonal antibody against PSA protein, wherein the antigen used to immunize mice is a recombinant protein, which is recombinantly expressed by Escherichia coli and contains a TRX protein tag, a PSA protein fragment and a histidine protein tag.

[0011] Furthermore, the PSA protein fragment is the amino acid fragment from position 25 to position 261 of the PSA protein, and its amino acid sequence is the amino acid sequence shown in SEQ ID NO.1.

[0012] The inventors also provided a hybridoma cell line that secretes anti-PSA protein molecules. The cell line is mouse hybridoma cell line 16F1. The cell line was deposited on March 24, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO 21968, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0013] The inventors also provide a PSA protein immunoassay reagent, wherein the immunoassay reagent contains an anti-PSA protein monoclonal antibody whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4 and whose amino acid sequence of the light chain variable region is shown in SEQ ID NO.5 as its active ingredient.

[0014] Furthermore, the immunoassay includes immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0015] Unlike existing technologies, the beneficial technical effects of this invention are as follows: The above technical solution selects amino acids 25-261 of the PSA protein as the antigenic peptide, performs codon optimization to create a gene fragment suitable for expression in *E. coli* BL21, and the resulting recombinant protein contains a TRX protein tag, a PSA protein fragment, and a histidine protein tag. The recombinant protein is used to immunize mice, and through cell fusion, selection, and subcloning, a mouse hybridoma cell line 16F1 that efficiently secretes anti-PSA protein monoclonal antibodies is obtained, along with the anti-PSA protein monoclonal antibody secreted by this cell line. The antibody obtained by this method has high specificity and sensitivity, can specifically recognize cells expressing PSA protein, and is suitable for immunological detection, especially immunohistochemical detection. Attached Figure Description

[0016] Figure 1The image shows the gel image of the recombinant PSA protein expression results fused with histidine tag in Example 1, where M represents Marker; 1 is the whole bacterial sample; 2 is the supernatant sample after sonication; and 3 is the precipitate sample after removing the supernatant.

[0017] Figure 2 Comparison of immunohistochemical staining results for prostate cancer; the left image shows PSA secreted by 16F1, and the right image shows commercially available PSA. Detailed Implementation

[0018] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0019] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0020] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0021] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.

[0022] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.

[0023] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0024] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.

[0025] Example 1: Preparation of recombinant PSA protein fragments

[0026] I. Gene Optimization and Synthesis

[0027] Based on the PSA protein sequence accessed in the Uniprot database (accession number P07288), a protein fragment consisting of amino acids 25-261 was selected and directly optimized into a gene fragment suitable for expression in *E. coli* BL21. During PCR, EcoRI and XhoI restriction enzyme sites were added to the 5' and 3' ends of the gene, respectively.

[0028] PCR products were separated by agarose gel electrophoresis and recovered. The recovered fusion protein gene and the plasmid vector pET32a used for expression were digested with EcoRI and XhoI, respectively, and recovered by electrophoresis again. Ligation was performed using T4 DNA ligase. The ligation products were transformed into *E. coli* competent cells BL21. Clones were picked from plates and inoculated for colony PCR identification. Clones with positive PCR results were selected for sequencing analysis; clones with completely correct sequences were used.

[0029] Choosing different antigens for immunization may produce antibodies with different binding properties. The molecule also exhibits multiple variants due to alternative splicing, ultimately leading to different antibodies recognizing cells expressing the antigen in different ways. The PSA molecule was analyzed according to its published sequence. Based on its cell membrane structure, antigenicity, hydrophilicity / hydrophobicity of its constituent amino acids, and secondary structure, a suitable region with good immunogenicity and soluble expression was selected for recombinant expression. Codon optimization was performed on amino acid residues 25-261 of PSA, resulting in a molecular weight of approximately 45 kDa. The PSA protein was obtained using a prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of an antigenic PSA protein fragment and protein tags for recombinant protein purification, namely TRX and HIS.

[0030] II. Protein Expression and Purification

[0031] Overnight single-colony cultures were transferred to 100 mL LB medium at a ratio of 1:100. Kanamycin was added to a final concentration of 10 μg / mL, and the culture was incubated at 37°C with shaking until the OD600 reached 0.6-0.8. 0.5 mmol / L IPTG was added, and the culture was incubated at 37°C with shaking for 2 h. After harvesting, the cells were sonicated. The cells were centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to another tube for temporary storage. The precipitate was resuspended in 10 mM Tris-HCl (pH 8.0) solution, and the supernatant was discarded. This process was repeated, and the supernatant was discarded. 5-10 mL of 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea was added to dissolve the protein. The cells were centrifuged at 12000 rpm for 10 min, and the supernatant was collected.

[0032] Figure 1 This is a gel image showing the purification results of the recombinant PSA protein with a fused histidine tag. The protein concentration is 3 mg / mL, suitable for animal immunization and antibody screening and identification.

[0033] Example 2: Establishment of hybridoma cell lines

[0034] I. Immunity

[0035] The recombinant protein from Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881) and immunized with 4-6 week old female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Each mouse received a subcutaneous injection at 6 points in the abdomen, with a dose of 60 μg / mouse. A booster immunization was administered every 14 days, with the antigen emulsified using Freund's incomplete adjuvant (Sigma, F5506), at a dose of 30 μg / mouse. Seven days after the third booster immunization, the polyclonal antibody titer against the immunogen in mouse serum was detected by indirect ELISA (wavelength 450 nm). Mice with the highest titer were given a tail vein injection for pulse immunization, with the antigen mixed with physiological saline at a dose of 50 μg / mouse.

[0036] II. Cell Fusion

[0037] Aseptically prepare a suspension of immunocompromised mouse spleen cells and mix it with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. Centrifuge at 1500 rpm for 5 min. Discard the supernatant and place the centrifuge tube in a 37°C water bath. Slowly add 1 ml of PEG1500 (Roche) over 1 minute while stirring the cells. After incubating in warm water for 1 min, add 10 ml of serum-free IMDM (Sigma), mix well, and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 10 ml of serum (PAA) to carefully agitate the cells. Add 5 ml of thymocytes mixed with 10x HAT (Sigma) and mix well. Add 25 ml of semi-solid culture medium containing 2.1% nitrocellulose (Sigma) and mix thoroughly. Then pour the mixture evenly into 20 cell culture dishes. Place the cell culture dishes in a humidified chamber and incubate at 37°C in a 5% CO2 incubator.

[0038] III. Cloning and ELISA screening of positive hybridoma cells

[0039] Seven days after fusion, the clonal cell clusters reached a suitable size and density. Under a dissecting microscope, round, solid, and large clonal clusters were aspirated and transferred into 96-well plates containing pre-prepared culture medium, and incubated at 37°C in a 5% CO2 incubator. After three days, the cell mass occupied approximately 2 / 3 of the bottom area. 100 μL of the supernatant was collected and screened using ELISA with immunogen and synthetic peptides, respectively. Positive clones were completely replaced with 200 μL of complete culture medium containing feeder cells and 1% HT (Sigma). Two days later, a second ELISA screening was performed, and positive clones were transferred to 24-well plates containing pre-prepared culture medium (containing feeder cells and HT). Five days later, 100 μL of the supernatant was collected for a third ELISA screening. Positive clones were successively transferred to 6-well plates and cell culture flasks for expansion culture and cryopreservation. Example 3: Preparation of monoclonal antibodies using ascites induction method.

[0040] I. Preparation of Ascites

[0041] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, and counted at approximately 5 × 10⁶ cells. 5 1 ml of suspended cells was injected intraperitoneally into mice sensitized with paraffin oil. Ascites fluid was collected starting 7 days later. The collected ascites fluid was centrifuged at 4000 rpm for 10 min at 4°C. The middle ascites fluid was carefully aspirated and collected in centrifuge tubes and stored at 4°C or -20°C.

[0042] II. Purification of Monoclonal Antibodies

[0043] The antibody was purified from ascites fluid using HiTrap rProtein A FF (GE) affinity chromatography according to the manufacturer's instructions. Purity was determined by SDS-PAGE gel chromatography, and concentration was determined by the Bradford method. The purified antibody was stored at -20°C.

[0044] Example 4: Characterization of Monoclonal Antibodies

[0045] I. Subclass Identification

[0046] Dilute the coated goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) with 100 mM PBS (pH 7.4) to a final concentration of 0.5 μg / ml. Add 100 μl to each well and incubate overnight at 4°C. Empty the liquid and wash three times with PBS containing 0.05% Tween (PBS-T). Add 200 μl of blocking buffer (PBS containing 2% BSA and 3% sucrose) to each well and incubate at 37°C for 1 h. Empty the liquid and wash three times with PBS-T. Add 0.1 ml of hybridoma supernatant to each well and incubate at 37°C for 1 h. Empty the liquid and wash three times with PBS-T. Add 0.1 ml of HRP-labeled goat anti-mouse (κ,λ) antibody (1:1000) or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) (1:2000) to each well and incubate at 37°C for 1 h. Pour off the liquid and wash three times with PBS-T. Add 50 μl of citrate buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 to each well for colorimetric reaction. Measure the OD value at 405 nm within 10–20 min.

[0047] The results showed that the monoclonal antibody of the present invention is an IgG1κ type murine monoclonal antibody.

[0048] II. Determination of Affinity Constant

[0049] The PSA recombinant protein prepared in Example 1 was coated at a concentration of 2 μg / ml, 100 μl / well, and incubated overnight at 4°C, followed by three washes with PBS-T. 200 μl of blocking buffer was added to each well, and the mixture was blocked at 37°C for 2 h, followed by three washes with PBS-T. The monoclonal antibody purified in Example 3 was serially diluted 2-fold starting at 1:200, with the last well containing a blank control. The mixture was incubated at 37°C for 1 h, followed by three washes with PBS-T. HRP-labeled goat anti-mouse secondary antibody was diluted 1:20000, 100 μl per well, and incubated at 37°C for 1 h, followed by three washes with PBS-T. 100 μl of citrate-phosphate buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 was added to each well for color development for 10 min, and the reaction was terminated by adding 50 μl of 0.5M sulfuric acid solution. The absorbance was measured at 450 nm using a microplate reader. Plot the curve of OD value corresponding to antibody dilution factor, find the dilution factor A corresponding to half of the maximum binding OD value, and calculate the affinity constant of the antibody as 3.84 × 10⁻⁶ using the following formula. 9 .

[0050]

[0051] III. Monoclonal antibody reaction specificity and application efficacy

[0052] The PSA recombinant protein prepared in Example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, followed by 12% polyacrylamide gel electrophoresis. The gel protein band was transferred to a PVDF membrane (Millipore) using a Bio-Rad electrotransfer system according to standard procedures. The membrane was incubated overnight at 4°C in TBS-T blocking buffer containing 5% skim milk powder. Monoclonal antibody (1:1000 dilution) secreted by 41B1 hybridoma was added and incubated overnight at 4°C. After washing the membrane with TBS-T, goat anti-mouse secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) diluted 1:5000 was added, and the membrane was incubated at room temperature for 1 hour. After washing again with TBST, ECL ultrasensitive chromogenic solution (Beijing Pulilai Gene Technology Co., Ltd.) was added, and chemiluminescence image data were acquired using a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

[0053] Example 5: Determination of the variable region sequence of the antibody

[0054] Freshly cultured hybridoma cells were collected, and the supernatant was used to verify antigen-binding properties. This confirmed that the cell line used for cloning could indeed secrete the required antibodies. After confirmation, 10 cells were collected by centrifugation. 6The above hybridoma cells were used. Total RNA was extracted from hybridoma cells using the Trizol method. 9 μL of total RNA was added to 2.5 μL Loligo(dT)12–18 primer (10 mM) and 5 μL dNTPs. The mixture was thoroughly incubated at 70°C for 5 minutes, then placed on ice for 5 minutes, or denatured using the reverse transcriptase used. Subsequently, 5 μL RT buffer (5X), 2.5 μL DTT (0.1 M), and 1 μL reverse transcriptase were added, and the reaction was carried out at 42°C for 1 hour. The reaction was terminated by incubation at 70°C for 15 minutes, and the obtained cDNA was stored at -20°C. The first-strand cDNA was then subjected to PCR amplification. 25 pmol of each primer was added to a 50 μL reaction system. The primer sequences for amplifying the heavy and light chain variable regions were designed and synthesized according to the mouse monoclonal antibody primer sequences in the book *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen.

[0055] The remaining dNTPs and buffer were added as usual. Finally, 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set to 94℃ for 40 seconds, 52℃ for 40 seconds, and 72℃ for 40 seconds, for 20 to 25 cycles, with a final extension at 72℃ for 3 minutes. The product could be stored at 4℃ for later use or directly electrophoresed. 20 μL of the PCR product was taken for electrophoretic analysis. Separation was performed on a 1.5% agarose gel. The length of the light chain (κ light chain) was between 320-340 bp, and the length of the heavy chain was between 340-370 bp. If products specific to this region were found, the gel was excised and recovered, cloned into a T vector or expression vector, and sequenced.

[0056] Example 6. Immunohistochemical tissue microarray staining and identification

[0057] I. Chip fabrication process

[0058] Each sample was first stained with hematoxylin and eosin (HE) to determine the tumor location. Tissue microarrays were fabricated using a fully automated tissue microarray system from 3DHISTECH. The fabricated tissue microarray blocks were placed in a wax block mold and placed in a 68°C oven for 10 minutes to fuse the tissue core with the recipient wax block. The mold was then gently removed from the oven, and the semi-molten paraffin was allowed to cool at room temperature for about 30 minutes, followed by freezing at -20°C for 6 minutes. The tissue microarray blocks were then removed from the mold and sectioned or stored at 4°C for later use. After trimming, serial sections were prepared with a thickness of 3 μm. The serial sections were then immersed in 40% alcohol and allowed to spread naturally. The separated sections were then transferred to 50°C warm water for 30 seconds for further spreading. The sections were mounted on poly-L-lysine-treated slides, and the fabricated tissue microarrays were baked in a 68°C oven for 2 hours. After removal, they were cooled to room temperature and stored at -4°C.

[0059] II. IHC staining and analysis

[0060] The slides were dewaxed three times with xylene for 6 minutes each time, then hydrated in a gradient of 100%, 100%, 95%, and 85% ethanol for 3 minutes each time, and finally rinsed with tap water. Antigen retrieval was performed, and the slides were then placed in a humidified chamber and rinsed with PBS for 3 x 3 minutes. Incubation with 3% H2O2 for 10 minutes was followed by PBS rinsing for 3 x 3 minutes. The slides were then dehydrated, and a suitable diluted primary antibody (the initial dilution should be designed based on the antibody concentration) was added. Incubation was performed at room temperature (25°C) for 1 hour, followed by PBS rinsing for 3 x 3 minutes. Secondary antibody was added and incubated at room temperature for 15-30 minutes, followed by PBS rinsing for 3 x 3 minutes. The PBS was discarded, and the slides were developed with freshly prepared DAB chromogenic solution for 3-10 minutes. Counterstaining with hematoxylin for 25 seconds and then blue staining with PBS for 30 seconds was performed. Dehydration was then performed sequentially with an ethanol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes). Finally, the slides were cleared with xylene for 3 minutes and mounted with neutral resin.

[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, as follows:

[0062] 1. The sample is weakly positive; it is marked as "+";

[0063] 2. The sample is moderately positive; marked as "++";

[0064] 3. The sample is highly positive; it is marked as "+++".

[0065] 4. If the sample is negative, mark it as "-".

[0066] III. Data Statistics

[0067] 1. Tumor tissue microarray detection results:

[0068] The antibody PSA (16F1) and the commercially available antibody PSA (ER-PR8) were simultaneously detected in 31 patients with benign prostatic hyperplasia and 24 patients with prostate cancer, and the results were compared.

[0069] The immunohistochemical results of PSA were statistically analyzed. The entire trial was conducted using a double-blind design, and the statistical results are shown in the table below:

[0070]

[0071] The results showed that the anti-PSA protein monoclonal antibody secreted by the 16F1 cell line stained accurately, clearly, and without non-specific staining, with a clean background. In immunohistochemical detection, the positive rate and positive intensity were both higher than those of commercially available antibodies. This indicates that the PSA secreted by the 16F1 cell line has higher sensitivity and effectively avoids false negative results.

[0072] Figure 2 Comparison of immunohistochemical staining results for prostate cancer (left: PSA secreted by 16F1, right: commercially available PSA).

[0073] 2. Results of microarray analysis of normal tissue:

[0074] The normal tissue microarray includes 30 types of normal tissue samples, primarily selected from fresh, promptly fixed surgical specimens; each tissue type includes three different case samples. The 30 normal tissue types include: brain, heart, cerebellum, esophagus, adrenal glands, stomach, ovary, small intestine, pancreas, colorectal region, parathyroid glands, liver, pituitary gland, salivary glands, testes, kidneys, thyroid gland, prostate, breast, uterus, spleen, bladder, tonsils, skeletal muscle, thymus (in young children), skin, bone marrow, peripheral nerves, lungs, and mesothelial cells.

[0075] Simultaneous detection of this antibody (16F1) and commercially available antibodies on normal tissue microarrays showed consistent positive and negative results, indicating that the specificity of this antibody in normal tissues is comparable to that of commercially available antibodies.

[0076] Finally, it should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.

Claims

1. A monoclonal antibody against PSA protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody according to claim 1, characterized in that, The DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.

3.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes the PSA protein.

4. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a mouse IgG1κ subtype monoclonal antibody.

5. A monoclonal antibody against PSA protein, produced by a hybridoma cell line with accession number CGMCC NO 21968.

6. A hybridoma cell line that secretes anti-PSA protein molecules, wherein the cell line is mouse hybridoma cell line 16F1, and the cell line is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO21968.

7. A PSA protein immunoassay reagent, characterized in that, The immunoassay reagent contains the monoclonal antibody against PSA protein as described in claim 1 as its active ingredient.

8. The immunoassay reagent according to claim 7, characterized in that, The immunoassays include immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

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