A mouse anti-human prcc monoclonal paired antibody, hybridoma cell strain and application thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a mouse anti-human PRCC monoclonal paired antibody, a hybridoma cell strain and application thereof. The application provides hybridoma cell strains 5F9C2 (CCTCC NO: C2025312) and 15F7B5 (CCTCC NO: C2025341) which respectively secrete antibodies with unique heavy chain / light chain variable regions; the antibodies can be used for immunohistochemical and immunoblotting detection of PRCC alone, and can also be paired to establish a sandwich method system through an enzyme-linked immunosorbent assay. The antibodies provided by the application are high in specificity and sensitivity, and can be used for accurate qualitative and quantitative analysis of PRCC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mouse anti-human PRCC monoclonal paired antibody, its hybridoma cell line, and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, with persistently high incidence and mortality rates, and is the second leading cause of cancer-related deaths in specific sites. Clinically, the diagnosis of liver cancer mainly relies on imaging combined with serum biomarker testing. However, the diagnostic efficacy of alpha-fetoprotein (AFP) and de-γ-carboxythrombin (DCP) is currently insufficient for clinical predictive purposes. While advancements in science and technology have significantly improved the accuracy of ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), their use is limited due to issues such as cost, effectiveness, and reproducibility.

[0003] HCC is often asymptomatic in its early stages and is usually discovered at a late stage, which limits treatment options. Liver cancer patients can undergo various treatments such as surgical resection, liver transplantation, and local ablation. The 5-year survival rate after surgical treatment is 40%, but for patients diagnosed at an early stage, the 5-year survival rate can increase to 60%-70%, and for patients with micro-hepatocellular carcinoma, the 5-year survival rate can even reach 90%. Therefore, early diagnosis is of great significance for early intervention and treatment of HCC and for prolonging survival.

[0004] Current research has identified several genes whose abnormal expression is associated with the development and progression of liver cancer. However, the abnormal expression rate of these identified liver cancer-related genes in liver cancer is not high, and the sensitivity and specificity of these indicators are still insufficient to meet clinical prediction requirements. Therefore, there is an urgent need to find genes or proteins that can assist in the diagnosis of liver cancer and predict its condition and prognosis. Research in this area is of great significance for the clinical treatment of liver cancer and the prevention of tumor recurrence.

[0005] PRCC protein (proline-rich mitotic checkpoint control factor, Gene ID: 5546) is a protein element involved in the pre-mRNA cleavage process, which is one of its main biological functions. The applicant has previously completed research on the biological function and molecular mechanism of PRCC, and the corresponding research results have been published in the internationally renowned journal *Cell and Bioscience*. The results show that compared with AFP, PRCC has significant advantages in both sensitivity and specificity as a diagnostic marker for liver cancer. The level of PRCC protein in the serum of liver cancer patients is significantly higher than that in healthy individuals, and it is highly expressed in liver cancer cells and tissues. PRCC may be related to key factors in the DNA damage repair process, affecting DNA damage repair pathways and leading to the occurrence of liver cancer and poor prognosis. Based on this correlation between the relative expression level of PRCC protein and hepatocellular carcinoma, using this protein as a molecular marker to detect its expression level can guide the diagnosis and prognosis of liver cancer. Therefore, developing highly specific and sensitive monoclonal antibodies to identify PRCC protein for the differential diagnosis of related tumors is crucial. Summary of the Invention

[0006] The first aspect of the present invention provides a hybridoma cell line 5F9C2, which was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCCNO:C2025312.

[0007] A second aspect of the present invention provides a hybridoma cell line 15F7B5, which was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCCNO:C2025341.

[0008] A third aspect of the invention provides an anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 5F9C2.

[0009] Specifically, the amino acid sequence of the heavy chain variable region of the anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 5F9C2 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.

[0010] A fourth aspect of the invention provides an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 15F7B5.

[0011] Specifically, the amino acid sequence of the heavy chain variable region of the anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 15F7B5 is shown in SEQ ID No:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:4.

[0012] The fifth aspect of the present invention provides a gene encoding an anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 5F9C2, wherein the coding sequence of the heavy chain variable region is shown in SEQ ID No:5 and the coding sequence of the light chain variable region is shown in SEQ ID No:6.

[0013] A sixth aspect of the present invention provides a gene encoding an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 15F7B5, wherein the coding sequence of the heavy chain variable region is shown in SEQ ID No:7 and the coding sequence of the light chain variable region is shown in SEQ ID No:8.

[0014] The seventh aspect of the present invention provides the application of any of the above-described antibodies in the detection of human PRCC, wherein the detection includes, but is not limited to, immunohistochemical detection, Western blot detection, or enzyme-linked immunosorbent assay.

[0015] An eighth aspect of the present invention provides a human PRCC detection kit, the kit comprising at least one of the antibodies described above.

[0016] Specifically, the detection methods of the kit include immunohistochemical detection, Western blot detection, or enzyme-linked immunosorbent assay (ELISA).

[0017] More specifically, the detection method of the kit is a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), which includes the following steps: S1. Coating the first antibody onto a solid-phase support; S2. Add the sample to be tested and incubate. S3, Add the labeled second antibody and incubate; S4. Develop color and detect the signal; The first antibody and the second antibody are respectively an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 5F9C2 or an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 15F7B5.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. Stable and reliable cell lines: The hybridoma cell lines 5F9C2 and 15F7B5 have strong genetic stability and can stably secrete anti-human PRCC monoclonal antibodies for a long time, providing a continuous guarantee for the large-scale preparation of antibodies.

[0019] 2. Excellent antibody performance: The antibody has high specificity, low cross-reactivity, and outstanding sensitivity. It can be used alone or in pairs to achieve qualitative, localization and precise quantitative detection of PRCC protein, and is suitable for diverse detection scenarios.

[0020] Preservation Instructions 1 Culture name: Hybridoma cell line 5F9C2; Accession number: CCTCC NO: C2025312; Preservation period: October 22, 2025; Depository: China Center for Type Culture Collection; Location of the collection: Wuhan University, Wuhan, China.

[0021] Preservation Instructions 2 Culture name: Hybridoma cell line 15F7B5; Accession number: CCTCC NO: C2025341; Preservation period: October 22, 2025; Depository: China Center for Type Culture Collection; Location of the collection: Wuhan University, Wuhan, China. Attached Figure Description

[0022] Figure 1 This invention presents the SDS-PAGE analysis results of prokaryotic protein expression and identification. M represents the molecular weight standard of the protein, 1 represents pET30a induction (empty vector), 2 represents pET30a not induced, 3 represents pET30a induced, 4 represents the supernatant after pET30a induction and lysis, and 5 represents the precipitate after pET30a induction and lysis.

[0023] Figure 2 The image shows the SDS-PAGE analysis results of the purified PRCC protein expressed in this invention. M represents the molecular weight standard of the protein, 1 represents the sample after cleavage, 2 represents the eluent, and 3-5 represent the elution buffer.

[0024] Figure 3 The figure shows the results of Western Blot identification and analysis of the PRCC protein expressed in this invention. M is the molecular weight standard of the protein, 1 is the purified sample, and 2 is the His positive control.

[0025] Figure 4 The image shows the SDS-PAGE analysis results of the monoclonal antibody expression and purification of this invention. M represents the protein molecular weight standard, 1 represents the purification analysis results of the 5F9C2 antibody, and 2 represents the purification analysis results of the 15F7B5 antibody.

[0026] Figure 5 Linearity of the enzyme-linked immunosorbent assay (sandwich method) developed for paired antibodies 15F7B5 and 5F9C2; Figure 6The image shows the electrophoretic detection results of PCR amplification of the variable region of monoclonal antibody in this invention. M is DL2000 DNA Marker, 1 is 5F9C2-VH (heavy chain variable region), 2 is 5F9C2-VL (light chain variable region), 3 is 15F7B5-VH (heavy chain variable region), and 4 is 15F7B5-VL (light chain variable region). Figure 7 This image shows the results of monoclonal antibody IHC detection of PRCC protein expression in liver cancer and adjacent tissues for the present invention. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1 Expression of PRCC recombinant protein The target protein was expressed using IPTG-induced expression, and expression conditions were optimized by transforming pET30a-21231 (143-491) into BL21(DE3). After optimization, analysis showed that the target protein expressed by pET30a-21231 (143-491) was expressed in a soluble form. The target protein was purified by supernatant purification, and the PRCC target protein was obtained by Ni column affinity purification. Due to the protein structure, the molecular weight of the protein was larger than the theoretical molecular weight.

[0029] (1) Plasmid construction: The human PRCC gene sequence was retrieved from the GenBank database, and the amino acid fragment from position 143 to 491 was selected as the immunogen. Specific primers were designed, and NdeI and XhoI restriction enzyme sites were introduced on both sides of the DNA sequence encoding this fragment. The target fragment was obtained by PCR amplification and cloned into the pET-30a(+) expression vector digested with the same enzymes, thus successfully constructing the recombinant plasmid.

[0030] Protein 21231 (143-491) has a molecular weight of 39.34 kDa (including tag). MHHHHHHEPVKIAAPELHKGDSDSEEDEPTKKKTILQGSSEGTGLSALLPQPKNLTVKETNRLLLPHAFSRKPSDGSPDTKPSRLASKTKTSSLAPVVGTTTTTPSPSAIKAAAKSAALQVTKQITQEEDDSDEEVAPENFFSLPEKAEPPGVEPYPYPIPTVPEELPPGTEPEPAFQDD AANAPLEFKMAAGSSGAPWMPKPGDDYSYNQFSTYGDANAAGAYYQDYYSGGYYPAQDPALVPPQEIAPDASFIDDEAFKRLQGKRNRGREEINFVEIKGDDQLSGAQQWMTKSLTEEKTMKSFSKKKGEQPTGQQRRKHQITYLIHQAKERELELKNTWSENKLSRRQTQAKYGF (SEQ ID NO.9).

[0031] (2) Expression and identification of prokaryotic proteins: ① Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min. ② Heat shock at 42℃ for 90 sec, then quickly place on ice for 5 min, and add 600 μL of LB medium. ③ Shake at 37℃ and 220 r / min for 1 h, centrifuge, and spread the entire mixture onto LB plates containing 50 μg / mL Kan, and incubate upside down at 37℃ overnight. Pick single colonies from the transformation plates and inoculate them into test tubes containing 3 mL of LB medium containing 50 μg / mL Kan, and shake at 37℃ and 220 r / min overnight. ④ The next day, inoculate at a 1:100 ratio into 30 mL of TB medium containing 50 μg / mL Kan, and shake at 37℃ and 220 r / min until the bacterial cell OD reaches zero. 600⑤ Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. ⑥ Add IPTG to the remaining culture to a final concentration of 0.2 Mm, and incubate overnight at 15℃ and 220 r / min with shaking, or overnight at 37℃ and 220 r / min with shaking, to induce fusion protein expression. ⑦ Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in PBS; after sonication of the resuspended solution, take the supernatant and pellet respectively and resuspend them in loading buffer. ⑧ Perform 12% SDS-PAGE analysis and Coomassie Brilliant Blue staining for banding. Clones were selected, and protein expression was induced using IPTG. 12% SDS-PAGE analysis showed that the target protein was partially present in the supernatant. SDS-PAGE results indicated that pET30a-21231 (143-491) was partially expressed in the supernatant form (see [link to SDS-PAGE analysis]). Figure 1 ).

[0032] (3) Ni-column affinity purification of the fusion protein and result analysis: Ni-column purification: ① Using a low-pressure chromatography system, the supernatant was loaded into a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min. ② The column was washed with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the effluent OD 280 The value reached the baseline. ③ Wash with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min until the OD of the outflow reaches the baseline. 280 The value reached baseline. ④ Elute the target protein with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent. ⑤ Add the collected protein solution to a dialysis bag and dialyze overnight with PBS. ⑥ Perform 12% SDS-PAGE analysis. Purification result analysis: The target protein was obtained by Ni column affinity purification and analyzed by 12% SDS-PAGE (see...). Figure 2 ).

[0033] (4) Western Blot Method and Result Analysis: ① Sample Loading: Load 5 μL of sample. ② Electrophoresis: After sample loading, run the polyacrylamide gel at 70 V for the first time, then increase the voltage to 150 V until electrophoresis is complete. ③ Electrotransfer: After electrophoresis, remove the gel and transfer the membrane at a constant current of 250 mA for approximately 2 hours. ④ Blocking: After electrotransfer, remove the membrane and wash it 4 times with PBST, then place the membrane in 5% skim milk blocking buffer for 1 hour. ⑤ Primary Antibody Incubation: Dilute the primary antibody with blocking buffer and incubate the membrane overnight at 4°C. ⑥ Secondary Antibody Incubation: The next day, remove the membrane and wash it 4 times with PBST for 5 minutes each time. Dilute the secondary antibody with blocking buffer containing 5% milk and incubate the membrane in the secondary antibody for 1 hour. ⑦ Development: After the reaction is complete, remove the membrane and wash it 4 times in a clean box for 5 minutes each time. Develop with ECL and expose. The results of the Western Blot analysis are shown in ( Figure 3 ) Example 2: Preparation and purification of anti-PRCC monoclonal paired antibody This project used recombinant PRCC protein as an immunogen to immunize Balb / c mice. After obtaining high-titer immune serum, cell fusion and screening were performed to obtain multiple hybridoma cell lines that secrete anti-PRCC monoclonal antibodies. Further screening identified two hybridoma cell lines (5F9C2 and 15F7B5) that can recognize different PRCC antigenic epitopes. The monoclonal antibodies secreted by these cells were verified to be suitable as paired antibodies for establishing a highly sensitive sandwich assay system.

[0034] (1) Animal Immunization: Ten BALB / c mice were selected for immunization. For the first immunization, 100 μg of antigen was fully emulsified with an equal volume of Sigma complete Freund's adjuvant (CFA) to prepare a stable water-in-oil emulsion. The first immunization was performed by subcutaneous injection at multiple sites, with each mouse receiving 100 μg of antigen. Two weeks after the first immunization, booster immunizations were initiated. Each booster immunization used 50 μg of antigen, which was emulsified with an equal volume of Sigma incomplete Freund's adjuvant (IFA) and then injected subcutaneously at multiple sites. This booster immunization program was performed a total of four times.

[0035] (2) Antiserum titer assessment: Antiserum titer was assessed using an indirect ELISA method. ① PRCC antigen was diluted to 1 μg / mL with phosphate-buffered saline (PBS, pH 7.4), and 100 μL / well was used to coat a 96-well plate. The plate was incubated overnight at 4°C. ② The coating solution was discarded, the plate was washed three times with PBST, and 200 μL of blocking buffer was added to each well. The plate was then blocked at 37°C for 1 hour. ③ The blocking buffer was discarded, the plate was washed once, and 100 μL of the antiserum was serially diluted 3-fold at a starting concentration of 1:500. The plate was then incubated at 37°C for 1 hour. ④ The plate was discarded, the plate was washed three times with PBST, and 100 μL / well of a 1:20,000 dilution of goat anti-mouse-HRP secondary antibody was added. The plate was then incubated at 37°C for 1 hour. ⑤ The plate was discarded, the plate was washed four times, and 100 μL of TMB chromogenic solution was added to each well. The plate was then incubated at 37°C in the dark for 15 minutes. ⑥ Finally, 100 μL of 1 M HCl was added to terminate the reaction. ⑦ Immediately measure the OD value using an ELISA reader at a wavelength of 450 nm. The antiserum titer is determined by the maximum serum dilution factor where the OD value is greater than 2.1 times that of the negative control.

[0036] (3) Cell fusion screening stage: SP2 / 0 cells were amplified using DMEM medium containing 10% FBS. After approximately 6 T75 culture flasks were filled, cells were collected, centrifuged, resuspended, and counted. Mice with adequate titers were selected and intraperitoneally injected with 100 μg of antigen 3 days before fusion. Mice were sacrificed on the day of fusion, and spleens were aseptically harvested. Spleen cells were collected after grinding, washed with DMEM, and treated with erythrocyte lysis buffer before counting. Spleen cells were mixed with SP2 / 0 cells at an appropriate ratio, centrifuged, and resuspended in electrofusion buffer to a concentration of 2 × 10⁶ cells / mL. 7 Cells were electrofused at 1 μg / mL, incubated for 10 minutes, then added to HAT medium and seeded into 96-well plates. The plates were then incubated in a CO2 incubator. Screening began on day 10 post-fusion. Indirect ELISA was used to detect cell supernatant: the plates were coated with 1 μg / mL antigen and incubated overnight at 4°C. 100 μL of cell supernatant was added per well, and the plates were incubated at 37°C for 1 h. After washing, HRP-labeled secondary antibody diluted 1:20,000 was added. TMB staining was performed, and the reaction was stopped with HCl. OD was then measured. 450 A well is considered positive if the ratio of the sample OD value to the negative OD value is ≥2.1. After confirmation by retesting, preliminary positive wells proceed to the subcloning stage.

[0037] (4) Cell subcloning and lineage determination: Cell subcloning and lineage determination were accomplished using limiting dilution. Cells from positive wells were collected, counted, and their density adjusted through serial dilution: the cell suspension was first diluted sequentially to appropriate concentrations and then seeded into the AC, DF, and GH rows of a 96-well plate, with each well targeted for single-cell addition. The seeded cell plates were incubated at 37°C in a 5% CO2 incubator for 7–10 days. After cloning, the wells showing single-clone growth were observed under a microscope and labeled. The cell supernatant from each single-clone well was extracted, and antibody secretion was detected using an indirect ELISA method. The ELISA detection conditions were as follows: 0.1 μg / mL antigen coating was used, and the mixture was incubated overnight at 4°C; cell supernatant was added, and the mixture was incubated at 37°C for 1 hour; then, 1:20,000 diluted HRP-labeled secondary antibody was added, and the reaction was terminated with HCl after TMB development. The OD value was then read. 450 nm. A positive result is determined by a ratio of sample OD value to negative OD value ≥ 2.1.

[0038] (5) Antibody Expression and Purification Stage: Two monoclonal cell lines, 5F9C2 and 15F7B5, were selected for serum-free amplification to express antibodies. Cell supernatant was collected and purified using a Protein A / G affinity chromatography column. After loading, the bound antibodies were eluted with glycine buffer and immediately dialyzed overnight in PBS at 4°C. The purified antibody concentration was determined by the BCA method, and the titer was assessed by indirect ELISA. Purity analysis was performed using SDS-PAGE and Coomassie Brilliant Blue staining, showing that the antibody purity was above 90%. This procedure successfully obtained high-purity, high-titer antibody samples, meeting the requirements for subsequent applications. Figure 4 ).

[0039] (6) Antibody pairing detection: ① Biotin rapid labeling: 5F9C2 monoclonal antibody was labeled using the P0665 Biotin rapid labeling kit (Biotin-LC-NHS-Sulfo); ② Coating antibody: 15F7B5 monoclonal antibody was diluted to the required concentration of 1 μg / mL with PBS coating buffer, mixed well and added to the strip, 100 μL per well, and incubated overnight at 4℃; ③ Blocking: After coating, the coating buffer was discarded, the plate was washed 3 times with PBST, 200 μL of blocking buffer was added to each well, and the plate was incubated at 37℃ for 1 h. Remove the ELISA plate, discard the internal solution, and wash the plate once; ④ Protein reaction: Dilute PRCC protein at concentrations of (62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0) ng / mL, 100 μL per well, and incubate at 37℃ for 1 h; ⑤ Primary antibody incubation: Remove the ELISA plate, discard the internal solution, wash the plate 3 times with PBST, and add 100 μL of diluted biotin-labeled monoclonal antibody 5F7C2-Biotin (1:10000) to each well. ⑥ HRP labeling: Remove the microplate, discard the inner solution, wash the plate 3 times with PBST, and add 100 μL of diluted enzyme conjugate (SA-HRP) 1:10,000 to each well. Incubate at 37℃ for 1 hour; ⑦ Color development: Remove the microplate, discard the inner solution, wash the plate 4 times with PBST, add 100 μL of TMB color development solution to each well, and determine the color development time according to the color intensity; ⑧ Termination of reaction: Add 100 μL of 1M HCl solution to each well to terminate the reaction. Immediately read the absorbance at 450 nm on the microplate reader. Results: The enzyme-linked immunosorbent assay (sandwich method) developed using paired antibodies 15F7B5 and 5F9C2 showed a good linear relationship between PRCC protein concentration and absorbance at 450 nm in the range of 0–62.5 ng / mL. See ( ) for specific results. Figure 5 ⑦ Sensitivity testing: Select a negative standard of 0 ng / mL for multiple parallel tests, calculate the mean and standard deviation, and use the content corresponding to Mean+2×SD (0.041872 ng / mL) as the detection limit of the kit.

[0040] (7) Analysis of the variable region of monoclonal antibodies: Total RNA was extracted from cultured hybridoma cell lines 5F9C2 and 15F7B5: ① Cell lysis and phase separation: Hybridoma cells were treated with a lysis reagent containing phenolic compounds and denaturants to obtain cell homogenates. Chloroform was then added to the homogenates, and after thorough mixing and standing, the organic and aqueous phases were separated by centrifugation. ② RNA precipitation and purification: The upper aqueous phase containing RNA was collected, and pre-cooled isopropanol was added to precipitate the RNA. The RNA precipitate was collected by centrifugation and washed with an ethanol-water solution to obtain a high-purity RNA product. ③ RNA dissolution and quality assessment: The purified RNA was dissolved in RNase-free water. Subsequently, the integrity of the RNA was assessed by agarose gel electrophoresis, and its concentration and purity were determined by spectroscopic analysis. ④ Using the purified total RNA as a template, reverse transcription was performed using reverse transcriptase and appropriate primers to synthesize complementary DNA (cDNA). ⑤ Using cDNA as a template, the target fragment, including the light chain variable region (VL) gene and / or heavy chain variable region (VH) gene of the antibody, was amplified by polymerase chain reaction (PCR) using specific primer pairs designed for the variable and constant regions of the antibody gene. ⑥ The obtained PCR product was cloned into a TA cloning vector and transformed into host cells. After screening for positive clones, the recombinant plasmid was sequenced. By comparing and analyzing the obtained sequences, the coding sequence of the variable region of the monoclonal antibody was finally determined. See ( ) for specific results. Figure 6 ).

[0041] Through steps 1-6 above, the hybridoma cell line and antibody subtype were successfully identified. The sequences involved in this invention are as follows: SEQ ID No. 1 (5F9C2 heavy chain variable region amino acid sequence): EVKLEQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQAPGKGLEWMGWINTETGEPTYADVFKGRFVFSLETSASTAYLQINNLKNEDTATYFCDRGRPWGQGTTLTVSS.

[0042] SEQ ID No. 2 (5F9C2 light chain variable region amino acid sequence): DIVLTQSPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLLQRSGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHYPQTFGGGTNLEIK.

[0043] SEQ ID No. 3 (15F7B5 heavy chain variable region amino acid sequence): EVKLQESGAELVRPGALVKLSCKVSGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGNTIYDPKFQGKASLTADTSSNTAYLQLSSLTSEDTAVYYCAGGHYWGQGTTLTVSS。

[0044] SEQ ID No.4 (Amino acid sequence of the variable region of the light chain of 15F7B5): DIVMTQSPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWFLQKPGQSPKLLIYKVSNRFPGVPGRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK。

[0045] SEQ ID No.5 (DNA sequence of the variable region of the heavy chain of 5F9C2): GAGGTGAAGCTGGAGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATACACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGTCTTCAAGGGACGATTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAGAATGAGGACACGGCTACATATTTCTGTGATAGAGGGCGACCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0046] SEQ ID No.6 (DNA sequence of the variable region of the light chain of 5F9C2): GATATTGTGCTGACCCAGTCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTAATGGAAAGACATATTTGAATTGGTTGTTACAGAGGTCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTATCCTCAGACGTTCGGTGGAGGCACCAACCTGGAAATCAAA。

[0047] SEQ ID No.7 (DNA sequence of the heavy chain variable region of 15F7B5): GAGGTTAAGCTGCAGGAGTCAGGGGCTGAACTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGTTTCTGGCTTCAACATTAAAGACTACTATTTGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTAATACTATATATGACCCGAAGTTCCAGGGCAAGGCCAGTTTAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTGGTGGACACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0048] SEQ ID No.8 (DNA sequence of the light chain variable region of 15F7B5): GACATTTGTGATGACACAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTTCCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT TCCAACCGATTTCCTGGGGTCCCAGGCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0049] Example 3: Immunohistochemical detection of PRCC protein in human tissues The expression of PRCC protein in tumor tissue was detected by immunohistochemical experiments using the antibody from Example 2. The specific steps are as follows: (1) Dewaxing and hydration of sections: Hepatocellular carcinoma and adjacent tissue sections were baked in a 60°C oven for 60 minutes to melt the paraffin. They were then immersed sequentially in three xylene solutions for 10 minutes each to completely remove the paraffin. Gradient hydration was then performed: sections were immersed sequentially in anhydrous ethanol, 95% ethanol, and 75% ethanol for 10 minutes each, and finally in double-distilled water for 10 minutes to prepare for subsequent staining steps.

[0050] (2) Antigen retrieval: The hydrated sections were completely immersed in 0.01 M citrate buffer (pH 6.0) and antigen epitope retrieval was performed using microwave heating. The microwave was set to medium power and heated until the buffer was nearly boiling, then paused. This cycle was repeated four times, each time for six minutes. After retrieval, the sections were allowed to cool naturally to room temperature in the retrieval solution. The retrieval solution was discarded, and the sections were rinsed twice with double-distilled water and three times with phosphate-buffered saline (PBS), soaking for five minutes each time.

[0051] (3) Endogenous enzyme inactivation and blocking: Add sufficient endogenous peroxidase blocking agent to the tissue sections, ensuring complete coverage of the tissue, and incubate at room temperature for 15 minutes to eliminate endogenous peroxidase activity. Rinse three times with PBS, 5 minutes each time. After washing, add non-immune animal serum homologous to the secondary antibody and block at room temperature for 20 minutes to reduce non-specific background staining.

[0052] (4) Primary antibody incubation: Discard the blocking serum and add the primary antibody working solution (15F7B5 or 5F9C2) diluted at a ratio of 1:150 directly to the tissue area, ensuring that the liquid covers the tissue evenly. Place the slide flat in a humidified chamber and incubate overnight (about 16-18 hours) at 4°C.

[0053] (5) Secondary antibody incubation: Remove the slides from the refrigerator, recover the primary antibody, and wash thoroughly four times with PBS buffer, soaking for 5 minutes each time, to remove unbound primary antibody. Then add the corresponding horseradish peroxidase-labeled secondary antibody and incubate at 37°C for 30 minutes. After incubation, wash thoroughly four more times with PBS, 5 minutes each time.

[0054] (6) DAB staining: Prepare a diaminobenzidine (DAB) staining working solution at a ratio of 1:50 before use. Add the staining solution to the tissue and observe the staining in real time under an optical microscope, controlling the staining time between 3 and 10 minutes. When the specific staining is a clear brownish-yellow to brick-red color and the background staining is relatively light, immediately rinse with double-distilled water to terminate the reaction.

[0055] (7) Counterstaining, Differentiation, and Blue Reversal: Counterstain the cell nuclei with hematoxylin solution for 5-10 minutes. After washing away excess stain with double-distilled water, differentiate the cells for a few seconds with 1% hydrochloric acid-ethanol solution to remove non-specifically bound hematoxylin from the cytoplasm. Then, slowly rinse the sections with running tap water for 10 minutes, or perform a blue reversal treatment with preheated PBS to ensure the cell nuclei appear clearly blue. Finally, rinse in double-distilled water for 5 minutes.

[0056] (8) Dehydration, clearing and mounting: The sections were sequentially dehydrated by passing them through 75% ethanol, 95% ethanol and anhydrous ethanol, for about 2 minutes in each bath. Then they were cleared in xylene for 5 minutes. The sections were removed from the xylene and, when the solvent on the surface had slightly evaporated but was still moist, 1-2 drops of neutral resin were added and the sections were carefully covered with coverslips to seal them.

[0057] (9) Image acquisition and analysis: The sealed slide is placed under a digital slide scanner to perform a full-slide image scan and obtain a high-resolution digital image. Figure 7The results showed that the anti-human PRCC protein monoclonal antibody secreted by hybridoma cells, when detected by IHC in liver cancer tissue, appeared brown and was located in the cell nucleus, consistent with the theoretical subcellular localization of PRCC, indicating that the tumor cells in this tissue sample expressed PRCC protein. The concentrations of the monoclonal antibodies used in this detection were 5F9C2 (11 ng / ml) and 15F7B5 (6.6 ng / ml). Under these conditions, the positive signal intensity on the tumor cells met the clinical requirements for judgment, and no significant background staining was produced that would affect the diagnosis, indicating that the antibodies secreted by hybridoma cells 5F9C2 and 15F7B5 have good specificity and high sensitivity.

[0058] The above results indicate that the anti-human PRCC protein monoclonal antibodies secreted by the hybridoma cell lines 5F9C2 and 15F7B5 described in this application possess high specificity and sensitivity. Therefore, the anti-PRCC monoclonal antibodies secreted by the hybridoma cell lines 5F9C2 and 15F7B5 can be used for immunohistochemical detection to detect the expression of PRCC in related tumors, especially liver cancer. More importantly, the two can be used as paired antibodies to establish a highly sensitive sandwich detection system through enzyme-linked immunosorbent assay (ELISA), achieving accurate quantitative analysis of PRCC and providing a basis for studying the biological function of PRCC protein in tumors and for clinical diagnosis. Simultaneously, this invention provides the antibody sequence information, enabling large-scale and stable production of the antibody using recombinant DNA technology, ensuring the uniformity of product quality.

[0059] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hybridoma cell line 5F9C2, characterized in that, The hybridoma cell line 5F9C2 was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCCNO:C2025312.

2. A hybridoma cell line 15F7B5, characterized in that, The hybridoma cell line 15F7B5 was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCCNO:C2025341.

3. The anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 5F9C2 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 5F9C2 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.

4. The anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 15F7B5 according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human PRCC monoclonal antibody secreted by the hybridoma cell line 15F7B5 is shown in SEQ ID No:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

4.

5. The encoding gene of the anti-human PRCC monoclonal antibody according to claim 3, characterized in that, The coding sequence of the heavy chain variable region is shown in SEQ ID No:5, and the coding sequence of the light chain variable region is shown in SEQ ID No:

6.

6. The encoding gene of the anti-human PRCC monoclonal antibody according to claim 4, characterized in that, The coding sequence of the heavy chain variable region is shown in SEQ ID No:7, and the coding sequence of the light chain variable region is shown in SEQ ID No:

8.

7. The use of the antibody according to any one of claims 3-4 in the detection of human PRCC.

8. A human PRCC detection kit, characterized in that, The kit includes at least one of the antibodies described in any one of claims 3-4.

9. The human PRCC detection kit according to claim 8, characterized in that, The detection methods of the kit include immunohistochemical detection, Western blot detection, or enzyme-linked immunosorbent assay (ELISA).

10. The human PRCC detection kit according to claim 9, characterized in that, The detection method of the kit is a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), which includes the following steps: S1. Coating the first antibody onto a solid-phase support; S2. Add the sample to be tested and incubate. S3, Add the labeled second antibody and incubate; S4. Develop color and detect the signal; The first antibody and the second antibody are respectively an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 5F9C2 or an anti-human PRCC monoclonal antibody secreted by hybridoma cell line 15F7B5.

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