A PRRX2 antibody and its application in the detection of gemcitabine resistance in adenocarcinoma.

Abstract

This invention relates to the field of biomedical technology, specifically disclosing an anti-PRRX2 monoclonal antibody and its application in detecting gemcitabine resistance in adenocarcinoma. The antibody was prepared using hybridoma technology. BALB / c mice were immunized with recombinant human PRRX2 protein, and a high-affinity antibody strain was obtained through cell fusion, screening, and monoclonalization. The variable region sequences of the antibody's light and heavy chains are shown in SEQ ID No:7 and SEQ ID No:8, and the CDR region sequences are shown in SEQ ID Nos:1-6. After conjugation of the antibody with HRP, it was used in Western blotting to detect PRRX2 expression in samples from patients with pancreatic ductal adenocarcinoma (PDAC). The results showed a detection sensitivity of 96.6% and a specificity of 100% for gemcitabine-resistant samples. The antibody of this invention exhibits high specificity and stability, providing a reliable tool for clinical drug resistance detection and contributing to the development of personalized treatment strategies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a PRRX2 antibody and its application in the detection of gemcitabine resistance in adenocarcinoma. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a common malignant tumor of the pancreas, characterized by high invasiveness and low survival rates. Gemcitabine, as a first-line chemotherapy drug, is widely used to treat PDAC, but the emergence of drug resistance severely limits its efficacy. Statistics show that approximately half of patients develop resistance early in treatment, leading to disease progression. The mechanisms of drug resistance are complex, involving abnormal expression of multiple genes and proteins. Among them, the transcription factor PRRX2 (Paired Related Homeobox 2) has been reported to potentially participate in the regulation of epithelial-mesenchymal transition (EMT) and tumor stem cell characteristics, and is associated with chemotherapy resistance. However, current detection tools for PRRX2 lack high-affinity and specific antibodies. Existing commercial antibodies often exhibit cross-reactivity or insufficient sensitivity in application, making it difficult to accurately assess the association between PRRX2 expression levels and drug resistance. Therefore, developing a reliable PRRX2 detection antibody is of great significance for early diagnosis of drug resistance and personalized treatment. Summary of the Invention

[0003] The present invention first provides a monoclonal antibody that specifically binds to the human PRRX2 protein, comprising a light chain variable region sequence as shown in SEQ ID No:7 and a heavy chain variable region sequence as shown in SEQ ID No:8.

[0004] In some embodiments, the heavy chain variable region of the antibody comprises three complementarity-determining regions (CDRs) with amino acid sequences of SEQ ID No:1 (CDR1), SEQ ID No:2 (CDR2), and SEQ ID No:3 (CDR3), respectively.

[0005] In some embodiments, the light chain variable region of the antibody comprises three complementarity-determining regions (CDRs) with amino acid sequences of SEQ ID No:4 (CDR1), SEQ ID No:5 (CDR2), and SEQ ID No:6 (CDR3), respectively.

[0006] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody, comprising a nucleotide sequence encoding the light chain variable region shown in SEQ ID No:7 and a nucleotide sequence encoding the heavy chain variable region shown in SEQ ID No:8.

[0007] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, comprising the following steps: immunizing BALB / c mice with recombinant human PRRX2 protein, fusing spleen cells with SP2 / 0 myeloma cells, screening monoclonal hybridoma cells by limiting dilution method, and purifying the antibody after large-scale culture.

[0008] The present invention also provides an immunological detection reagent for detecting PRRX2 protein, comprising a conjugate formed by the above-mentioned monoclonal antibody and a detectable marker, wherein the detectable marker is selected from enzymes, fluorescent dyes, biotin or radioisotopes.

[0009] In some embodiments, the detectable marker is horseradish peroxidase (HRP), and the conjugate is prepared by glutaraldehyde crosslinking.

[0010] Finally, this invention provides a kit for detecting gemcitabine resistance, comprising: (a) The monoclonal antibody mentioned above; (b) Antibody dilution buffer; (c) Positive control: recombinant human PRRX2 protein; (d) Negative control: PRRX2 knockout cell lysate; (e) The instruction manual, which describes the standard operating procedure for detecting PRRX2 protein by Western blotting.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects: (1) High affinity and specificity: The monoclonal antibody C1 obtained through hybridoma technology has extremely high affinity for the PRRX2 protein, such as Figure 2 and Figure 3 The titer results shown indicate that the antibody maintains excellent binding activity even at low concentrations. Western blot analysis revealed that the antibody specifically recognizes the 25-37 kDa PRRX2 band, with extremely low non-specific binding. Figure 5 As shown.

[0012] (2) Excellent detection performance: In the validation of 60 pancreatic ductal adenocarcinoma patients, the antibody showed a sensitivity of 96.6% and a specificity of 100% for gemcitabine resistance detection, which is significantly better than existing commercial antibodies and provides a reliable tool for clinical drug resistance assessment.

[0013] (3) Good stability: After purification, the antibody showed a single clear band after SDS-PAGE verification, indicating that the antibody has high purity and good stability, and is suitable for long-term storage and clinical application.

[0014] (4) Clinical application value: This antibody provides a new biomarker detection method for the early diagnosis of gemcitabine resistance, which helps to realize individualized treatment, improve the effect of chemotherapy, and prolong the survival of patients.

[0015] (5) Feasibility of industrial production: Large-scale stable production of antibodies can be achieved through hybridoma technology, with controllable costs and good industrialization prospects. Attached Figure Description

[0016] Figure 1 This is a graph showing the antibody titer in mouse serum.

[0017] Figure 2 Image showing antibody titer detection in fusion cell supernatant.

[0018] Figure 3 This is a graph showing the antibody titer detection in monoclonal cell supernatant.

[0019] Figure 4 This is an SDS-PAGE detection image of AbP.

[0020] Figure 5 This is a Western blot image of AbP-HRP-bound PRRX2+ epithelial cells. Detailed Implementation

[0021] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0022] Example 1: Preparation of anti-PRRX2 monoclonal antibody By using cell fusion technology, B cells that secrete specific antibodies can be fused with myeloma cells that can proliferate indefinitely to form hybridoma cells, which can produce specific antibodies against a single antigenic epitope, i.e., monoclonal antibodies.

[0023] 1.1 Immunized animals Recombinant human PRRX2 protein (Abnova, H00051450-Q01) was selected as the antigen to immunize female BALB / c mice aged 6-8 weeks and weighing approximately 20g. The recombinant human PRRX2 protein was dissolved in PBS to prepare a 1mg / mL antigen solution. Freund's complete adjuvant was preheated at 37°C, and after gentle shaking to mix thoroughly, 500µL was drawn into a syringe, connected to a two-hole emulsification connector, and air was expelled. Another 500µL of the antigen solution was drawn into a separate syringe, air was expelled, and the syringe was secured to the two-hole emulsification connector. The plunger was smoothly pushed to continuously emulsify until a stable emulsion was formed. Emulsification was considered complete when small drops of emulsion formed stable oil droplets on the water surface. Mice were intraperitoneally injected with 100µL per syringe; additionally, multiple subcutaneous injections of 25µL per point on the back could be administered to enhance the immunization effect. A booster immunization was given 2 weeks after the initial immunization. The aforementioned antigen solution was emulsified with an equal volume of Freund's incomplete adjuvant using the same emulsification method as above. 100 µL of the emulsion was administered intraperitoneally to each mouse. Booster immunizations were then given weekly for a total of three weeks. Seven days after the final immunization, blood was collected via the marginal eye vein, serum was separated, and antibody titers were determined using ELISA. The antigen used for detection was recombinant human PRRX2 protein, and the secondary antibody was goat anti-mouse IgG-HRP. Serum was collected before the initial immunization as a negative control (Neg), and post-immunization serum was designated P1-P3. Antibody titer results are shown below. Figure 1 As shown, the horizontal axis represents the serum dilution factor (Log10), and the vertical axis represents the absorbance at 650 nm. The recombinant human PRRX2 protein is abbreviated as hPRRX2. All three mice produced specific antibodies against hPRRX2 after immunization, and the highest response was consistent, with mouse number 2 showing the highest antibody titer.

[0024] 1.2 Cell Fusion Mice (size 2) were sacrificed, and spleen cells were isolated. SP2 / 0 cells in logarithmic growth phase were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with serum-free medium. The cells were resuspended and counted, with viable cells counted. SP2 / 0 cells and the isolated spleen cells were mixed at a 1:10 ratio and centrifuged. The supernatant was discarded, and the tube was gently tapped to loosen the precipitate. 1 mL of preheated 45% PEG4000 was slowly added while agitating the tube. After standing for 90 seconds, the tube was centrifuged again, the supernatant was discarded, and the cells were resuspended in HAT selective medium containing 20% ​​fetal bovine serum. The cell suspension was diluted and added to 100 μL / well of a 96-well plate. Each spleen was diluted to make four 96-well plates. The cells were incubated for 5 days, then replaced with HT medium. After 2 weeks, the medium was replaced with RPMI 1640 basal medium. When hybridoma cells covered 10% of the bottom area of ​​the well, the supernatant was collected for ELISA to detect the affinity of the specific antibody. The experimental method was consistent with the serum titer determination, and the results were as follows: Figure 2 As shown. Figure 2 The vertical axis represents the absorbance at 650 nm, and the horizontal axis represents the dilution factor of the supernatant, taken as Log10. Hybridoma cells A and C showed good binding activity, with antibody titers greater than 2e5.

[0025] 1.3 Monoclonalization of hybridoma cells Hybridoma cells A and C, with high antibody affinity, were selected, and monoclonal antibodies were obtained using the limiting dilution method. The specific experimental steps are as follows: The hybridoma cells to be cloned were gently blown out of the culture wells, counted, and diluted with basal medium to 5 cells / mL. 100 μL of the diluted cell suspension was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator. Monoclonal antibodies were observed after 8 days. The supernatant was collected, and antibody activity was detected by ELISA. The experimental method was consistent with serum titer determination. The results are as follows: Figure 3 As shown. Figure 3 The x-axis represents the dilution factor of the cell culture supernatant, expressed as Log10. The single clone C1 showed the highest Emax, with the supernatant corresponding to 1 / 2 Emax exhibiting the largest dilution factor (820868). Therefore, it was selected for expansion culture and cryopreservation, and subsequent experiments were conducted. The cryopreservation solution was 10% DMSO + 90% fetal bovine serum.

[0026] Example 2: Purification of Monoclonal Antibodies Select monoclonal C1 antibodies with high affinity and good stability for amplification culture, expanding to two T75 flasks. The resulting monoclonal antibody is designated AbP. After 7 days, collect approximately 30 ml of supernatant, centrifuge at 8000 rpm at 4°C for 10 min, and transfer the supernatant to a new 50 ml centrifuge tube. Filter using a 0.22 µm filter membrane, measure the pH after filtration, and adjust to 7.0-7.5 using neutralization buffer. Add 500 µL of Protein G magnetic beads to the supernatant, mix thoroughly, and incubate overnight at 4°C. The next day, centrifuge at 1500 rpm at 4°C for 5 min and discard the supernatant. Resuspend the magnetic beads in 1 mL of cold PBS and transfer to a chromatography column. Wash the column with 10 mL of LPBS, repeating three times. Add 3 mL of elution buffer to the column, discard the residual PBS, and plug the bottom of the column. Incubate for 5-10 min. Repeat elution once more after the elution buffer has flowed out. Add 600 µL of neutralization buffer to the collected eluent and mix thoroughly. Transfer the eluent to a 30 kDa ultrafiltration tube and centrifuge at 4000 rpm and 4°C to concentrate the protein solution. Once the protein solution volume is reduced to 600 µL, add 5 mL of DPBS and centrifuge again under the same conditions. Repeat this process three times until the buffer is completely replaced with PBS. Determine the protein concentration using a spectrophotometer (mouse IgG mode). Take 3 µg of AbP protein for SDS-PAGE identification using an 8-12% gradient PAGE gel at 120V for 40 min. The results are shown below. Figure 4 As shown in the figure, the leftmost lane is the protein marker, lane 1 is the unreduced AbP protein without DTT treatment, and lane 2 is the reduced protein with DTT treatment. The figure shows that the molecular weight of the pre-purified protein is slightly larger than the theoretical molecular weight of mouse IgG, approximately 155 kDa, which may be due to glycosylation. The single band indicates no significant mismatch. In the reduced state, DTT breaks the disulfide bonds of the antibody, yielding a heavy chain with a molecular weight of approximately 55 kDa and a light chain with a molecular weight of approximately 25 kDa.

[0027] Example 3: Obtaining the variable region sequence of a monoclonal antibody Candidate hybridoma clones were lysed with Trizol and total RNA was extracted, which was then used as a template to synthesize first-strand cDNA. The specific steps are as follows: Single-clonal C1 cells were transferred into 6-well plates and cultured. When the cell confluence reached 70%, the cell culture medium was discarded, and the cells were washed twice with 1 mL PBS. 1 mL of Trizol was added to each well, ensuring complete coverage of the cells. The cells were incubated at room temperature for 5 min to allow for complete lysis, resulting in a clear solution without significant cell clumps. 200 μL of chloroform was added to the lysis buffer, the centrifuge tube was tightly capped, and the tube was vigorously shaken for 30 s. The solution was then incubated at room temperature for 2 min. The tube was centrifuged at 12000 g for 15 min at 4 °C. After centrifugation, the liquid separated into three layers: an upper colorless aqueous phase (containing RNA); a middle white protein phase; and a lower red organic phase (containing DNA and lipids). The upper aqueous phase was carefully aspirated using a pipette and transferred to a new RNase-free 1.5 mL centrifuge tube. Add an equal volume of pre-chilled isopropanol and gently invert to mix. Incubate at 4°C for 10 min. Centrifuge at 12000g for 10 min at 4°C, discard the supernatant, and a white or transparent RNA precipitate will be visible at the bottom of the tube. Add 1 mL of pre-chilled 75% ethanol to the precipitate, gently invert the centrifuge tube, and wash the RNA precipitate. Repeat the washing once. Open the centrifuge tube cap and air dry in a clean bench for 1-2 min to allow residual ethanol to evaporate. Add 50 μL of RNase-free ultrapure water and gently pipette until the precipitate is completely dissolved.

[0028] Using first-strand cDNA as a template, subsequent PCR amplification was performed with antibody variable region-specific primers to obtain the nucleic acids of the antibody light and heavy chain variable regions corresponding to hybridoma cells. After agarose gel electrophoresis, gel extraction and recovery, and Sanger sequencing by Suzhou Genewiz Technology Co., Ltd., the variable region sequences of the antibody were obtained. The sequences of the antibody heavy chain variable regions and light chain variable regions are shown in Table 1.

[0029] Table 1. Amino acid sequence of the variable region of AbP ; Example 4: Preparation of AbP-HRP conjugates The monoclonal antibody AbP was adjusted to a concentration of 1 mg / mL using PBS, and the HRP concentration was adjusted to 5 mg / mL. Both were dialyzed twice with PBS (pH 6.8) for 4 hours each time to remove impurities and ensure coupling efficiency. 1 mL of HRP was added to 20 μL of 0.25% glutaraldehyde solution and incubated at room temperature for 1 hour to allow the amino group of HRP to fully bind to the aldehyde group of glutaraldehyde. The HRP-aldehyde intermediate was then placed in a dialysis bag and dialyzed overnight with PBS at 4°C to completely remove unreacted free glutaraldehyde. 1 mL of AbP solution was added to the dialyzed HRP-aldehyde intermediate to make the antibody-HRP mass ratio 1:5, and PBS was added to a total volume of 3 mL. The mixture was then incubated at 4°C with slow stirring for 24 hours to allow the amino group of the antibody to bind to the aldehyde group of the HRP-aldehyde intermediate, forming an antibody-HRP covalent conjugate. Add 50 μL of 0.2 M glycine solution to the conjugation solution and incubate at room temperature for 1 h to block unreacted aldehyde groups, terminating the conjugation and protecting the antigen-binding activity of the antibody. Load the conjugation solution onto a Sephadex G-200 gel filter column and elute with PBS (pH=7.4, containing 0.15 M NaCl), collecting the first eluent peak. Measure the absorbance of the conjugate at 280 nm and 403 nm using a UV spectrophotometer to calculate the conjugation efficiency. The measured A403 / A280 = 0.48, indicating successful conjugation. Dilute the conjugate to 1 mg / mL with 50% glycerol, aliquot, and store at -20 °C.

[0030] Example 5: Application of AbP-HRP in gemcitabine resistance detection Sixty epithelial cell samples were obtained from tumor sites of patients with pancreatic ductal adenocarcinoma (PDAC), including 30 gemcitabine-resistant patients and 30 non-resistant patients. Pretreated tissues were placed in pre-chilled homogenizers on ice, and freshly prepared lysis buffer containing protease / phosphatase inhibitors was added. Homogenization was performed using an electric homogenizer at low speed for 30 seconds, with 1-minute intervals, for a total of three times, until a tissue suspension was formed. The homogenate suspension was transferred to pre-chilled 1.5 mL EP tubes and incubated on ice for 30 minutes, inverting the tubes every 10 minutes to ensure complete cell lysis. The cells were centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was carefully aspirated into a new pre-chilled EP tube, and the bottom precipitate was discarded. Protein concentration was determined and recorded using the BCA method. Based on the quantitative results, the protein concentration of all samples was adjusted to 2 mg / mL using lysis buffer, and then 5× loading buffer was added and mixed thoroughly by vortexing to prepare SDS-PAGE samples. AbP-HRP was then validated according to standard Western blotting procedures, and the AbP-HRP was used after dilution. Figure 5This image shows the detection results for one gemcitabine-sensitive sample and one gemcitabine-resistant sample. M represents the protein molecular weight marker. Lanes 1 and 2 represent gemcitabine-sensitive samples, while lanes 3 and 4 represent gemcitabine-resistant samples. Lanes 1 and 3 used AbP-HRP diluted 3000-fold to a final concentration of 0.33 μg / mL, while lanes 2 and 4 used AbP-HRP diluted 500-fold to a final concentration of 2 μg / mL. The image shows bands in lanes 3 and 4 within the 25-37 kDa range, with the band in lane 4 being more prominent. This indicates good antibody binding to the sample, with only a very low percentage of non-specific binding bands present. Furthermore, the antibody can be used for Western blotting detection within the concentration range of 0.33-2 μg / mL.

[0031] Following the steps described above, Western blotting was performed on all 60 patient samples. The results were scored and statistically analyzed. Samples from gemcitabine-resistant patients showing a clear band at 25-37 kDa were considered true positives, while samples from non-resistant patients showing no band were considered true negatives. Sensitivity and specificity were statistically analyzed, and the results are shown in Table 2.

[0032] Table 2. Statistics on the sensitivity and accuracy of AbP-HRP detection ; 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 monoclonal antibody that specifically binds to human PRRX2 protein, characterized in that, It includes a light chain variable region sequence as shown in SEQ ID No:7 and a heavy chain variable region sequence as shown in SEQ ID No:

8.

2. The monoclonal antibody according to claim 1, wherein the heavy chain variable region of the antibody comprises three complementarity-determining regions CDR1-CDR3, the amino acid sequences of which are SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3, respectively.

3. The monoclonal antibody according to claim 1, wherein the light chain variable region of the antibody comprises three complementarity-determining regions CDR1-CDR3, the amino acid sequences of which are SEQ ID No:4, SEQ ID No:5 and SEQ ID No:6, respectively.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1-3, characterized in that, It contains the nucleotide sequence encoding the light chain variable region shown in SEQ ID No:7 and the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID No:

8.

5. An immunological assay for detecting PRRX2 protein, comprising a conjugate of a monoclonal antibody as described in any one of claims 1-3 and a detectable marker, wherein the detectable marker is selected from enzymes, fluorescent dyes, biotin, or radioisotopes.

6. The immunological detection reagent according to claim 5, wherein the detectable marker is horseradish peroxidase (HRP), and the conjugate is prepared by glutaraldehyde cross-linking.

7. A kit for detecting gemcitabine resistance, characterized in that, Include: (a) The monoclonal antibody according to any one of claims 1-3; (b) Antibody dilution buffer; (c) Positive control: recombinant human PRRX2 protein; (d) Negative control: PRRX2 knockout cell lysate; (e) The instruction manual, which describes the standard operating procedure for detecting PRRX2 protein by Western blotting.

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