Anti-prdx3 monoclonal antibody and application thereof

Abstract

The application discloses an anti-PRDX3 monoclonal antibody and application thereof, and belongs to the technical field of biological detection. The amino acid sequence of a heavy chain variable region of the antibody is SEQ ID NO. 3, the coding gene is SEQ ID NO. 1, the amino acid sequence of a light chain variable region is SEQ ID NO. 4, and the coding gene is SEQ ID NO. 2. The antibody can specifically recognize a PRDX3 purified protein and a natural PRDX3 protein, and is not cross-reacted with a pET-28a empty vector irrelevant protein and a PRDX3 gene knockout sample. The antibody is suitable for indirect ELISA, Western-blot, immunohistochemistry and the like, can qualitatively / quantitatively detect PRDX3 in various biological samples, can be used for preparing a product for diagnosing a fibrosis related disease, and can assist in diagnosis by detecting PRDX3 expression in patient samples, thereby providing a reliable tool for PRDX3 related research, disease diagnosis and clinical transformation.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an anti-PRDX3 monoclonal antibody and its application. Background Technology

[0002] Thioredoxin-dependent peroxidase 3 (PRDX3) is a core member of the PRDX peroxidase family and a key effector molecule in the mitochondrial antioxidant defense system. Its functional specificity is characterized by its strict dependence on mitochondrial thioredoxin 2 (Trx2) as an electron donor. Through the redox cycle of cysteine ​​residues at its active sites (Cys47 and Cys168), it efficiently catalyzes the reduction of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and lipid peroxides produced by mitochondrial metabolism. This process directly maintains mitochondrial membrane potential stability, inhibits oxidative stress damage to mitochondrial lipids, proteins, and DNA, and thus regulates fundamental physiological activities such as apoptosis, proliferation, and energy metabolism, playing an irreplaceable role in maintaining cellular and tissue homeostasis.

[0003] Numerous studies have confirmed that abnormal PRDX3 function (abnormal expression, loss of activity, or peroxidation modification) is closely related to the development and progression of various major diseases, making it an important target for clinical diagnosis and treatment. In fibrotic diseases, PRDX3 effectively reduces the transformation of hepatic stellate cells into myofibroblasts by inhibiting the activation of the mitochondrial ROS / TGF-β1 / Smad2 / 3 signaling pathway and downregulates the expression of α-smooth muscle actin (α-SMA) and collagen I, thereby blocking the progression of liver fibrosis. In pulmonary fibrosis models, PRDX3 expression levels are significantly reduced, and the degree of downregulation is positively correlated with alveolar structural destruction and the degree of collagen deposition in the pulmonary interstitium, directly affecting the rate of disease progression. In ferroptosis-related diseases, when mitochondrial lipid peroxides accumulate, PRDX3 undergoes superoxidation modification, translocating from mitochondria to the cell membrane and inhibiting the function of the cystine / glutamate transporter (xCT), leading to insufficient glutathione (GSH) synthesis and exacerbating ferroptosis. This mechanism is particularly significant in chronic liver diseases such as iron deficiency anemia and non-alcoholic fatty liver disease. Furthermore, in neurodegenerative diseases, biallelic mutations in PRDX3 can completely eliminate its enzyme activity, leading to a decrease in the ability of nerve cells to clear ROS from mitochondria, resulting in oxidative damage and apoptosis of neurons. This phenomenon has been verified in samples from patients with spinocerebellar ataxia and Alzheimer's disease, further highlighting the core role of PRDX3 in maintaining nerve cell homeostasis.

[0004] Based on the key mechanisms of action of PRDX3 in fibrosis, ferroptosis-related diseases, and neurodegenerative diseases, it has become a diagnostic biomarker and therapeutic target with high clinical translational value. Accurate detection of PRDX3 expression levels (e.g., protein abundance in tissues / body fluids) and modification status (e.g., peroxidation modification) is crucial for early diagnosis, disease staging, and dynamic monitoring of treatment efficacy in related diseases. However, current PRDX3 detection mainly relies on traditional immunological methods such as Western blotting and immunohistochemistry (IHC). These techniques have significant limitations, including cumbersome procedures (e.g., WB requires multiple steps such as sample lysis, electrophoresis, and membrane transfer), reliance on specialized equipment and technicians, and long detection cycles (IHC requires 48-72 hours). More importantly, existing commercially available anti-PRDX3 antibodies are mostly polyclonal antibodies or recombinant antibodies that do not target the active site, which are prone to cross-reaction with other members of the PRDX family (e.g., PRDX1, PRDX2), leading to false positives or quantitative biases, making it difficult to meet the clinical needs for early, accurate diagnosis and dynamic monitoring of diseases.

[0005] Monoclonal antibodies, due to their high specificity (precisely identifying a single epitope), high batch stability, and scalable industrial production capabilities, have become a core tool for the development of biological detection and diagnostic reagents. Since Kohler and Milstein established B-lymphocyte hybridoma technology in 1975, the challenge of large-scale preparation of murine monoclonal antibodies has been overcome, laying a mature technological foundation for the development of highly specific detection reagents. However, to date, there are no mature patents or high-quality literature reports on highly specific murine monoclonal antibodies targeting PRDX3 active sites (such as key cysteine ​​residue regions) or unique conformational epitopes, and their application in the precise detection of PRDX3 (such as in the development of diagnostic kits). This technological gap directly results in the inability to achieve early screening and dynamic monitoring of PRDX3-related diseases in clinical practice through convenient and accurate immunological methods. Therefore, there is an urgent need to develop highly specific monoclonal antibodies targeting PRDX3 to fill this technological gap. Summary of the Invention

[0006] This invention aims to address the shortcomings of existing anti-PRDX3 detection tools, such as insufficient specificity, limited adaptation methods, difficulty in stably binding to natural PRDX3, and limitations in disease diagnostic applications. It proposes an anti-PRDX3 monoclonal antibody secreted by the hybridoma cell line 2A5F7. The heavy chain variable region of this antibody contains the amino acid sequence shown in SEQ ID NO.3 (encoding gene SEQ ID NO.1), and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.4 (encoding gene SEQ ID NO.2). This antibody is compatible with various detection technologies, including indirect ELISA, Western blot, IHC, and double-antibody sandwich ELISA, enabling qualitative / quantitative detection and subcellular localization analysis of PRDX3 in various sample types, such as animal tissues, cell lysates, and body fluids. It can also be used to prepare diagnostic products for diseases such as liver fibrosis, pulmonary fibrosis, and renal fibrosis, providing a reliable detection tool for basic research and clinical translation related to PRDX3.

[0007] This invention is achieved as follows: an anti-PRDX3 monoclonal antibody, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.3, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.4. Further, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.2.

[0008] This invention also protects the application of the above-mentioned anti-PRDX3 monoclonal antibody, specifically including:

[0009] 1. Application in the preparation of products for detecting PRDX3 antigen: The detection is a qualitative or quantitative detection of PRDX3 protein in biological samples, including animal tissue samples, cell lysate samples, or body fluid samples; the product is a detection reagent, detection kit, or test strip.

[0010] The method for detecting PRDX3 antigen includes:

[0011] (1) Indirect enzyme-linked immunosorbent assay (indirect ELISA): After the biological sample to be tested is coated with a solid-phase carrier, the anti-PRDX3 monoclonal antibody is used as the primary antibody to bind the PRDX3 antigen, and then an enzyme-labeled anti-mouse immunoglobulin secondary antibody is added. The qualitative or semi-quantitative detection of PRDX3 is achieved by enzyme-catalyzed colorimetric signal.

[0012] (2) Immunoblotting: After the protein of the sample to be tested is separated by electrophoresis and transferred to a solid membrane, the anti-PRDX3 monoclonal antibody is used as the primary antibody to bind PRDX3, and then an enzyme-labeled secondary antibody is added. The PRDX3-specific band at 28 kDa is identified by chemiluminescence or color development to achieve qualitative analysis of PRDX3 and expression level.

[0013] (3) Immunohistochemical detection method (IHC): After dewaxing, hydration and antigen retrieval, the tissue sections to be tested are bound to natural PRDX3 with the anti-PRDX3 monoclonal antibody as the primary antibody. Visible staining is formed by enzyme-labeled secondary antibody and chromogenic agent to detect the cellular localization and expression level of PRDX3.

[0014] (4) Double antibody sandwich ELISA method: The solid phase carrier is coated with the anti-PRDX3 monoclonal antibody as the capture antibody. After binding to PRDX3 in the sample to be tested, another enzyme-labeled anti-PRDX3 antibody that recognizes different antigenic epitopes of PRDX3 is added. The quantitative detection of PRDX3 is achieved by enzyme-catalyzed colorimetric signal.

[0015] 2. Application in the preparation of products for diagnosing fibrosis-related diseases: The fibrosis-related diseases refer to fibrosis diseases such as liver fibrosis, pulmonary fibrosis, or renal fibrosis; the diagnosis is based on detecting the expression level of PRDX3 protein in organ tissue samples or body fluid samples from patients.

[0016] Furthermore, this application also claims protection for a product for detecting PRDX3, the product comprising the aforementioned anti-PRDX3 monoclonal antibody. The product is a detection reagent, detection kit, or test strip.

[0017] Beneficial effects:

[0018] 1. The anti-PRDX3 monoclonal antibody 2A5F7 disclosed in this application (secreted by hybridoma cell line 2A5F7) has clearly defined amino acid sequences for its heavy chain variable region and light chain variable region (SEQ ID NO.3 and SEQ ID NO.4, respectively), and stable encoding gene sequences (SEQ ID NO.1 and SEQ ID NO.2, respectively). When it binds to PRDX3-expressing samples, it shows no cross-reactivity with PRDX3 knockout samples. Compared with existing antibodies that may have cross-reactivity, the high specificity and binding ability of this antibody to natural proteins can effectively avoid false positive or false negative results, laying the foundation for accurate detection of PRDX3 protein in biological samples.

[0019] 2. The anti-PRDX3 monoclonal antibody disclosed in this application can be applied to various detection technologies, including indirect enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemistry (IHC), and double-antibody sandwich ELISA. Furthermore, this antibody is compatible with a wide range of biological sample types, including animal tissue samples, cell lysate samples, and body fluid samples, meeting the needs of different scenarios such as basic research, preclinical experiments, and potential clinical testing. Its adaptability is far superior to existing PRDX3 detection tools that only target a single detection method or a single sample type.

[0020] 3. This application innovatively links anti-PRDX3 monoclonal antibodies with the diagnosis of fibrosis-related diseases. It proposes using PRDX3 as a biomarker to detect the expression level of PRDX3 in lung tissue or body fluid samples from patients, thereby achieving early diagnosis and monitoring of disease progression in fibrosis. Compared with traditional methods of pulmonary fibrosis diagnosis, this technical approach has advantages such as being minimally invasive, providing early warning, and offering objective results. It provides a brand-new solution for the diagnosis of fibrosis-related diseases and has significant clinical translational value.

[0021] 4. The hybridoma cell line 2A5F7 disclosed in this invention can stably secrete high-titer antibodies (ascites titer 1:51200) under standardized culture conditions, which facilitates the standardized production of antibodies, effectively reduces the risk of batch differences in the production process, and provides a stable and reliable source guarantee for the large-scale application of antibodies and subsequent product development. Attached Figure Description

[0022] Figure 1 These are the SDS-PAGE results for purified monoclonal antibodies; lane M represents the molecular weight standard of the pre-stained protein; lane 1 is the unpurified sample of 2A5F7 monoclonal antibody; lane 2 is the unpurified sample of 5G8 monoclonal antibody; lane 3 is the unpurified sample of 7G2D2 monoclonal antibody; lane 4 is the sample of 2A5F7 monoclonal antibody purified by affinity chromatography; lane 5 is the sample of 5G8 monoclonal antibody purified by affinity chromatography; and lane 6 is the sample of 7G2D2 monoclonal antibody purified by affinity chromatography.

[0023] Figure 2 The results are Western blot identification results for monoclonal antibodies; lane M is the molecular weight standard of pre-stained protein; lane 1 is the purified protein of PRDX3; lane 2 is the lysate after induction with pET-28a empty vector.

[0024] Figure 3 Immunohistochemical results of lung tissue from 6-week-old mice infected with Schistosoma japonicum and normal control mice. The left side shows the lung tissue of the infected group (SJ-1) mice; the right side shows the lung tissue of the normal control group (NC) mice.

[0025] Figure 4 The figure shows the results of Western blot detection of PRDX3 protein expression levels in lung tissues of different mouse models. Figure A shows the effect of bleomycin (BLM) treatment on PRDX3 protein expression in mouse lung tissues, and Figure B shows the expression of PRDX3 protein in lung tissues of mice under the Schistosoma japonicum infection model. β-Actin was used as an internal control in all experiments to verify the consistency of sample loading in each group. Detailed Implementation

[0026] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0027] Example 1

[0028] I. Immunogen Preparation and Immunization

[0029] 1) Immunogen preparation

[0030] Human PRDX3 protein was prepared and purified using a prokaryotic expression system. The specific procedure is as follows: The constructed pET-28a-PRDX3 recombinant expression plasmid was transformed into BL21(DE3) Escherichia coli competent cells, plated on LB solid medium containing kanamycin (final concentration 50 μg / mL), and cultured at 37℃ for 12-16 h. Positive single colonies were then picked and inoculated into LB liquid medium containing the same antibiotic, and cultured at 37℃ with shaking at 220 rpm until the bacterial culture reached OD. 600 =0.6-0.8, transfer to the scale-up culture system at a ratio of 1:100. When the OD of the scale-up culture medium... 600 When the concentration reached 0.8-1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L, and PRDX3 protein expression was induced at 30℃ and 220 rpm for 4 h. After induction, the bacterial cells were collected by centrifugation at 4℃ and 8000 rpm for 10 min, and resuspended in 0.01 M phosphate buffer (PBS, NaCl 8.0 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO2 0.24 g / L, ultrapure water to a final volume of 1 L, pH 7.4, the same below). The cells were then lysed by ultrasonic disruption (300W, 20 min), and the supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 min. The supernatant was then analyzed by chromatography using a HisTrap HP column (AKTA system) with binding buffer: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4. 7.9; Elution buffer: The same system contains 500 mM imidazole. Collect the elution fraction corresponding to the 280 nm absorption peak. Analyze the protein purity, and finally obtain human PRDX3 protein with a purity of over 90%, aliquot and store at -80℃ for later use.

[0031] 2) Animal immunization

[0032] The concentration of purified PRDX3 protein was determined using the BCA method and adjusted to 1.0 mg / mL with PBS buffer at pH 7.4. It was then mixed with Freund's complete adjuvant at a 1:1 volume ratio and emulsified using a vortex mixer (2000 rpm) for 30 minutes to prepare the PRDX3 immunogen emulsion.

[0033] Six 6-8 week old SPF-grade female BALB / c mice were selected. Each mouse was first immunized by subcutaneous injection of 0.1 mL of immunogen emulsion (containing 100 μg PRDX3 protein) at four points in the abdomen. Two weeks later, a second immunization was performed, using Freund's incomplete adjuvant and the same emulsification method, dosage, and route as the first immunization. Two weeks after the second immunization, a booster immunization was performed by intraperitoneal injection of 200 μg of adjuvant-free purified PRDX3 protein.

[0034] Three days after booster immunization, mice were euthanized by cervical dislocation, and the spleen was removed under sterile conditions. The spleen cells were ground and filtered through a 200-mesh cell sieve to prepare a single-cell suspension for subsequent cell fusion experiments.

[0035] II. Fusion of Hybridoma Cells

[0036] Hybridoma cell fusion was performed following a standardized experimental procedure, with the specific steps as follows:

[0037] 1) Serum sample preparation: First, before sacrificing mice and harvesting spleens for fusion experiments, whole blood was collected from the immunized BALB / c mice. After standing at 4°C for 2 h, the blood was centrifuged at 3000 rpm for 10 min to separate the serum, aliquoted, and stored at -20°C as a positive control for subsequent antibody screening. Serum from the same batch of mice (processed using the same method) was collected simultaneously for subsequent screening.

[0038] 2) Cell fusion procedure: Mouse spleens were aseptically obtained and single-cell suspensions of spleen cells were prepared. Simultaneously, cells in the logarithmic growth phase (OD) were collected. 600 SP2 / 0 myeloma cells (value 0.6-0.8) were washed twice with serum-free DMEM medium. Spleen cells were then mixed with SP2 / 0 myeloma cells at a cell ratio of 5:1. The mixture was centrifuged at 4°C and 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was dispersed. 50% PEG-2000 solution (Sigma-Aldrich) was added at 37°C, and the mixture was gently stirred and incubated at 37°C for 1 min. The fusion reaction was terminated by slowly adding 10 mL of serum-free DMEM medium over 5 min. Finally, the cell pellet was collected by centrifugation at 4°C and 100 rpm for 5 min.

[0039] 3) Preparation of control samples: 6-8 week old SPF grade ICR mice were pretreated with 3 mL of sterile liquid paraffin intraperitoneally for 72 h, then euthanized by cervical dislocation and peritoneal macrophages were aseptically collected. Feeder cell suspension was prepared in DMEM medium containing 10% fetal bovine serum, and their serum was used as a negative control.

[0040] 4) Hybridoma cell culture: Resuspend the fused cell pellet in HAT selective medium (2×10⁻⁶). 5The cells were dispensed into 96-well plates containing feeder cells (100 μL / well). After incubation at 37°C and 5% CO2 for 5-7 days, the medium was changed. Thereafter, the clones were examined under a microscope every 2 days (when the diameter was 1-2 mm and the coverage was 30%-50%), and the medium was changed. Once the cells were stable, the supernatant was collected for antibody screening.

[0041] III. Detection of Hybridoma Cells

[0042] 1) Optimization and operation of ELISA plate coating

[0043] After the hybridoma cells in step two have been cultured to the point where the supernatant can be collected, the coating parameters of the ELISA plate are first optimized using a square matrix assay (to lay the foundation for subsequent indirect ELISA screening of specific antibodies). Specifically, the human PRDX3 purified protein (prepared in step one, purity ≥90%) or PRDX3-specific synthetic peptide (KGKYLVLFFYPLDFTFVCPTEIVAFSDKANEFHDVN, SEQ ID NO.5) is used as the coating antigen and serially diluted with PBS buffer along the horizontal direction of the 96-well ELISA plate (dilution gradient: 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL). At the same time, the positive serum from the immunized mice frozen in step two is taken and serially diluted with PBS buffer at 100-fold, 200-fold, 400-fold, and 800-fold, respectively. The optimal coating concentration is determined to be 20 μg / mL based on the strength of the reaction signal by indirect ELISA detection.

[0044] The specific procedure for coating is as follows: First, prepare the coating buffer (i.e., carbonate buffer). The formula is as follows: Take 8 mL of 0.2 mol / L Na2CO3 solution and 17 mL of 0.2 mol / L NaHCO3 solution, add deionized water to make up to 100 mL, and adjust the pH to 9.6. Dilute the PRDX3 purified protein or specifically synthesized small peptide to the optimal coating concentration (20 μg / mL) with the coating buffer, add 100 μL / well to a 96-well microplate, and incubate at 37℃ for 3 h. After incubation, discard the coating solution in the wells, wash the microplate three times with PBST buffer (0.01 mol / L, pH 7.4 PBS containing 0.5% Tween-20), soaking for 5 min each time, and pat dry. Add 300 μL of PBS blocking buffer (0.01 mol / L, pH 7.4 PBS containing 5% skim milk) to each well. 7.3, PBS + 5% skim milk), incubate overnight at 4°C; after blocking, wash three times again with PBST buffer, pat dry, seal with sealing film, and store at 4°C for later use.

[0045] 2) Hybridoma cell positive screening

[0046] Indirect ELISA was used for specific screening of hybridoma cell culture supernatants. 50 μL of the test supernatant was added to the wells of a PRDX3 protein-coated ELISA plate. Positive controls (1:5000 dilution of immunized mouse serum) and negative controls (SP2 / 0 cell supernatant and 1:5000 dilution of ICR mouse serum) were also included. After incubation at 37°C for 1 h, the cells were washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added. The cells were incubated at 37°C for 45 min. After TMB staining for 15 min, the reaction was terminated with 2 mol / L sulfuric acid, and the absorbance at 450 nm was measured. The absorbance value of the reaction between the supernatant of the hybridoma cells to be tested and the purified protein / synthetic peptide of PRDX3 was recorded as P, and the absorbance value of the reaction between the supernatant of SP2 / 0 cell culture and the negative serum of ICR mice and the purified protein of PRDX3 was recorded as N. The P / N≥2.1 was used as the criterion for judging positive clones. All hybridoma cells corresponding to positive wells were marked for subsequent subclonal culture.

[0047] IV. Establishment of cell lines, serum-free culture, and antibody purification

[0048] 1) Cell line subcloning screening

[0049] Eighteen positive hybridoma cell lines obtained from the initial screening were subcloned using a three-stage limiting dilution method (final concentration 0.5 cells / well). Three stable monoclonal cell lines secreting anti-PRDX3 antibodies were ultimately selected and named 2A5F7, 5G8, and 7G2D2, respectively. Antibodies purified from these three cell lines were subsequently labeled as monoclonal antibodies 2A5F7, 5G8, and 7G2D2. The ELISA titer of 2A5F7 reached 1:51200. Strongly positive clones (2A5F7) with oval cell morphology and a doubling time ≤24h were selected and expanded to T25 culture flasks (passaged when cell density reached 80%). Simultaneously, they were cryopreserved in liquid nitrogen (the cryopreservation solution contained 90% fetal bovine serum + 10% DMSO, cooled to -80℃ and then transferred to liquid nitrogen).

[0050] 2) Serum-free culture process

[0051] Add the serum-free hybridoma medium preheated to 37℃ to the target cell culture dish, gently pipette and transfer to a 500 mL shake flask, add medium to 100-150 mL (adjust according to the initial cell volume), and incubate in a 37℃, 150 rpm, 5% CO2 shaker. Observe the cell status every 5-8 days by the color of the medium and the hanging drop on a glass slide to determine whether to add fresh medium.

[0052] 3) Antibody purification and quality control

[0053] After filtration through a 0.22 μm filter, the serum-free culture supernatant was equilibrated with a HiTrap Protein G HP chromatography column using 20 mM phosphate buffer (binding buffer, formulation: 1 L system contains 2.48 g disodium hydrogen phosphate dodecahydrate and 0.46 g potassium dihydrogen phosphate dihydrate, dissolved in ultrapure water, and adjusted to pH 7.0 with 1 M HCl or 1 M NaOH, then filtered through a 0.22 μm filter for sterilization). The flow rate was controlled at 1 mL / min. The collected serum-free culture supernatant was diluted 1:1 with binding buffer, filtered, and then loaded at a flow rate of 1 mL / min. After the protein peak subsided, the antibody was eluted with 0.1 M glycine buffer (formulation: 7.507 g glycine per L, adjusted to pH 2.7 with 1 M HCl, then brought to volume and sterilized by filtration through a 0.22 μm filter) and immediately neutralized to pH 7.0 with 1 M Tris-HCl (121.14 g Tris per L, adjusted to pH 9.0 with 1 M HCl, then brought to volume and sterilized by filtration through a 0.22 μm filter). The neutralized antibody solution was then concentrated by centrifugation at 4000 rpm in a Millipore Amicon Ultra-15 centrifuge tube (molecular weight cutoff 30 kDa) at 4°C, with the solution replaced three times with pH 7.4 PBS buffer. The concentration was increased to 10 mg / mL. After determining the antibody titer using an indirect ELISA method, the solution was aliquoted into sterile EP containers and stored at -20°C.

[0054] The purified antibody was analyzed by SDS-PAGE electrophoresis, and the results showed that ( Figure 1 Lane M is a pre-stained protein molecular weight standard (marker, molecular weight range 10-180 kDa), where clear step-like bands are visible. Comparing lanes 1-3 (unpurified 2A5F7, 5G8, 7G2D2 samples) with lanes 4-6 (purified 2A5F7, 5G8, 7G2D2 samples), it can be seen that a large number of contaminating protein bands were present in the serum-free culture supernatant before purification. However, after purification by Protein G affinity chromatography, all three monoclonal antibodies showed typical IgG dimer characteristic bands, namely two specific bands of heavy chain (about 55 kDa) and light chain (about 25 kDa), indicating that the purification method can effectively remove contaminating proteins and specifically enrich monoclonal antibodies.

[0055] V. Identification of Monoclonal Antibodies

[0056] 1. Antibody titer determination (indirect ELISA)

[0057] Antigen coating: The purified PRDX3 protein was diluted to 20 μg / mL with coating buffer (0.05 M carbonate buffer: 1 L system contains 1.59 g anhydrous sodium carbonate and 2.93 g sodium bicarbonate, dissolved in ultrapure water and adjusted to pH 9.6, then filtered through a 0.22 μm filter for sterilization). 100 μL was added to each well of the microplate and incubated at 37°C for 3 h. The liquid in the wells was discarded. The plate was washed three times for 5 min each time with 0.01 M phosphate buffer (PBST, pH 7.4, formulation: 1 L system contains 0.8 g sodium chloride, 0.02 g potassium chloride, 0.144 g disodium hydrogen phosphate, and 0.024 g potassium dihydrogen phosphate, dissolved in ultrapure water and adjusted to pH 7.4, then 5 mL of Tween-20 was added, mixed thoroughly, and filtered through a 0.22 μm filter for sterilization). The plate was then patted dry.

[0058] Blocking: Add 150 μL of blocking buffer (0.01M PBS containing 5% (w / v) skim milk to each well of the ELISA plate; formulation: first prepare 0.01M PBS with ultrapure water in the ratio of 0.8 g sodium chloride, 0.02 g potassium chloride, 0.144 g disodium hydrogen phosphate, and 0.024 g potassium dihydrogen phosphate in 1 L system and adjust the pH to 7.4, then add 50 g skim milk powder and stir until completely dissolved, then filter through a 0.22 μm filter membrane for sterilization), and incubate at 37℃ for 3 h; wash 3 times with PBST and pat dry.

[0059] Primary antibody incubation: Ascites fluid containing monoclonal antibodies 2A5F7, 5G8, and 7G2D2 was serially diluted with antibody diluent (0.01M PBS containing 5% (w / v) skim milk) at dilution ratios of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600, respectively. Serum from normal ICR mice was diluted to 1:200 with the same diluent as a negative control. 100 μL of serum was added to each well of the ELISA plate, with 3 parallel wells for each dilution. The plates were incubated at 37°C for 90 min. The plates were washed three times with PBST and patted dry.

[0060] Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse IgG antibody to 1:8000 with antibody dilution buffer, add 100 μL / well to the microplate, and incubate at 37℃ for 90 min; wash 5 times with PBST and pat dry.

[0061] Color development and termination: Add 100 μL of TMB chromogenic solution to each well of the microplate and incubate at room temperature in the dark for 5-10 min; once the positive wells turn blue, add 50 μL / well 2 M H2SO4 to terminate the reaction. Measure the absorbance (OD) of each well at 450 nm using a microplate reader. 450(nm), record the OD value of each parallel well for each dilution.

[0062] The results showed that monoclonal antibodies 2A5F7, 5G8, and 7G2D2 could specifically recognize the PRDX3 protein and showed no cross-reactivity with the negative control (serum from normal ICR mice). The antibody titer results are shown in Table 1. 2A5F7 had the highest titer (1:51200), followed by 5G8 (1:25600), and 7G2D2 had a titer of 1:12800, indicating that all three monoclonal antibodies could specifically recognize the PRDX3 protein and had high reactivity.

[0063] Table 1. Results of titer determination of purified monoclonal antibodies

[0064] Antibody name antibody titer 2A5F7 1:51200 5G8 1:25600 7G2D2 1:12800

[0065] 2. Specificity Validation (Western-blot)

[0066] 1) Protein sample preparation

[0067] PRDX3 protein purification: After purification by nickel column affinity chromatography, the protein was denatured by boiling in protein loading buffer containing β-mercaptoethanol for 5 min, and the final concentration was adjusted to 1 μg / μL.

[0068] pET-28a empty vector lysate: E. coli BL21(DE3) transformed with pET-28a empty vector was induced to express by IPTG, then lysed by sonication and centrifuged to collect the supernatant, which was then mixed with protein loading buffer and boiled for denaturation.

[0069] 2) SDS-PAGE and transfer

[0070] Take 20 μg of each of the above protein samples, separate them by 12% SDS-PAGE, and then equilibrate the gel and nitrocellulose (NC) membrane in transfer buffer (25 mmol / L Tris, 192 mmol / L glycine, 20% methanol, pH 8.0) for 5 min; then transfer the protein from the gel to the NC membrane by constant current transfer at 90 mA for 20 min using a BIO-RAD semi-dry transfer device.

[0071] 3) Immunoblotting reaction

[0072] Blocking: The NC membrane after transfer was placed in PBST (0.01 M PBS containing 0.05% Tween-20) containing 5% (w / v) skim milk and blocked overnight at 4°C;

[0073] Primary antibody incubation: Discard the blocking solution, wash the NC membrane 3 times (5 min each time) with PBST, add the anti-PRDX3 monoclonal antibody (2A5F7 monoclonal antibody prepared in this application, dilution ratio 1:1000) diluted with blocking solution, and incubate on a shaker at room temperature for 2 h;

[0074] Secondary antibody incubation: After washing the NC membrane 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody (diluted 1:5000 in blocking buffer) and incubate at room temperature for 1 h;

[0075] Colorimetric imaging: After washing the NC membrane thoroughly with PBST 5 times (5 min each time), the membrane was developed with an ECL chemiluminescence kit, and images were acquired using a gel imaging system.

[0076] 4) Results Analysis

[0077] The results are as follows Figure 2 (Western blot results) show that a specific band of 28 kDa appeared in the purified PRDX3 protein (lane 1), while no cross-reaction was observed in the empty vector lysate (lane 2). This indicates that the anti-PRDX3 monoclonal antibody 2A5F7 prepared in this example can not only specifically bind to the purified PRDX3 protein, but also does not cross-react with irrelevant proteins expressed by the pET-28a empty vector, demonstrating good specificity.

[0078] 3. Verification of natural protein binding (immunohistochemistry)

[0079] Liver tissue from mice infected with Schistosoma japonicum (6 weeks old) and lung tissue from normal mice were used as controls for immunohistochemical staining. The steps are as follows:

[0080] ① Tissue processing: Paraffin-embedded tissue sections were dewaxed with xylene and hydrated with graded ethanol;

[0081] ② Antigen retrieval: Microwave antigen retrieval was performed using citrate buffer (pH 6.0) (high heat for 5 min, medium heat for 10 min).

[0082] ③ Blocking: Incubate with 3% H2O2 at room temperature for 10 min to block endogenous peroxidase, then block with 5% BSA at room temperature for 1 h;

[0083] ④ Primary antibody incubation: Add anti-PRDX3 monoclonal antibody (2A5F7 monoclonal antibody prepared in this application, dilution ratio 1:1000) diluted with 5% BSA, and incubate overnight in a humidified chamber at 4°C;

[0084] ⑤ Secondary antibody and staining: After washing with PBST, add biotin-labeled goat anti-mouse IgG secondary antibody and incubate at 37℃ for 1 h; then add streptavidin-biotin protein-peroxidase complex and incubate at 37℃ for 30 min; develop color with DAB staining solution for 5-10 min, counterstain cell nuclei with hematoxylin, differentiate with hydrochloric acid and ethanol, dehydrate with graded ethanol, clear with xylene, and mount with neutral resin.

[0085] The results are as follows Figure 3 As shown: The lung tissue of mice in the left schistosomiasis-infected group (SJ-1) showed weak positivity, with only a small number of cells exhibiting brownish-yellow positive staining; the lung tissue of mice in the right normal control group (NC group) showed strong positivity, with a greater number of positively stained cells and deeper staining. This result indicates that schistosomiasis infection can lead to local downregulation of PRDX3 expression in target cells, reflecting differences in PRDX3 expression levels and tissue distribution under different pathological models (such as schistosomiasis-related lung injury). Combined with parallel test results of pulmonary fibrosis tissue, this further confirms that the anti-PRDX3 monoclonal antibody prepared in this embodiment can specifically bind to the natural PRDX3 protein in tissues and can effectively distinguish the differences in PRDX3 expression under different pathological conditions.

[0086] 4. Verification of natural protein binding (Western-blot)

[0087] Sample source:

[0088] (1) Lung tissue of mice treated with bleomycin (BLM) (BLM treatment group) and lung tissue of mice in the PBS control group (WT group) at the same time; (2) Lung tissue of wild-type mice infected with Schistosoma japonicum (SJ-I group) and lung tissue of PRDX3 gene knockout mice infected with Schistosoma japonicum (KO group).

[0089] The expression of PRDX3 protein in the lung tissue of mice in the different treatment or infection groups was detected by routine Western blot.

[0090] The results show:

[0091] (1) Comparison of PRDX3 protein expression in lung tissue of mice in the BLM-treated group and the WT group: Figure 4 As shown in Figure A, the lung tissue samples from the WT group mice exhibited a clear and bright PRDX3 protein-specific band at approximately 28 kDa, with a high gray value, indicating stable expression of PRDX3 protein in mouse lung tissue under normal physiological conditions. In contrast, the PRDX3 protein band at the same molecular weight position in the BLM-treated group mice was significantly weaker, and the gray value was significantly lower than that in the WT group, suggesting that bleomycin treatment can downregulate the expression level of PRDX3 protein in mouse lung tissue, consistent with the expected trend of PRDX3 protein expression changes in the lung injury model.

[0092] (2) Comparison of PRDX3 protein expression in lung tissue of SJ-I and KO mice infected with Schistosoma japonicum: In the Schistosoma japonicum infection model, a strong positive PRDX3 protein band was observed in the lung tissue samples of SJ-I mice at 28 kDa, indicating that acute inflammatory stress induced by Schistosoma japonicum infection can induce high expression of total PRDX3 protein in mouse lung tissue; while no PRDX3 protein-specific band was detected in the lung tissue samples of KO mice at 28 kDa, only the internal reference β-Actin protein band was detected, proving that PRDX3 protein was completely absent in this sample, further excluding the interference of non-specific bands. Figure 4 (B in the middle).

[0093] Specificity verification of the anti-PRDX3 monoclonal antibody (2A5F7): Based on the comparative results of the two groups above, all samples showing detected PRDX3 protein expression (WT group, SJ-I group) exhibited a specific band at 28 kDa. In contrast, the bands at the corresponding positions in samples with downregulated PRDX3 protein expression (BLM treatment group) or absent PRDX3 protein expression (KO group) showed a weakening or disappearance trend, respectively. Furthermore, the internal control β-Actin band was clear and uniformly bright in all samples, indicating consistent sample loading and no significant operational errors during the experiment. Simultaneously, no non-specific bands were observed, demonstrating that the prepared anti-PRDX3 monoclonal antibody (2A5F7) can specifically recognize and bind to PRDX3 protein expressed in its native state, with no cross-reactivity, indicating good antibody specificity.

[0094] In summary, this experiment, using Western blot analysis of lung tissues from different groups (WT, BLM, SJ-I, KO) of mice under bleomycin-treated and Schistosoma japonicum infection models, fully demonstrates that the anti-PRDX3 monoclonal antibody (2A5F7) prepared in this application possesses excellent natural protein binding ability: it can accurately identify PRDX3 protein expressed in mouse lung tissue under natural conditions with high specificity and no obvious non-specific binding; it can effectively distinguish between samples with normal, downregulated, and absent PRDX3 protein expression, meeting the needs of subsequent detection, quantitative analysis, and related biological function research of natural PRDX3 protein, and providing reliable experimental evidence for the further application of this antibody.

[0095] 5. Preparation method of heavy chain variable region (VH) and light chain variable region (LH) genes of anti-PRDX3 monoclonal antibody (2A5F7 strain)

[0096] 1) Cell culture and RNA extraction

[0097] Hybridoma cell line 2A5F7 was seeded in DMEM medium containing 10% fetal bovine serum and cultured at 37°C in a 5% CO2 incubator. Cell status was observed daily using an inverted microscope until the cell density reached 80%-90% (approximately 5 × 10⁻⁶ cells / year). 6 Cells were collected when they were uniform in shape (round / oval, with good refractive properties) and free of obvious debris (cells / mL). Total RNA was extracted using the one-step TRIzol reagent method, with strict adherence to aseptic techniques to avoid RNase contamination.

[0098] 2) cDNA first-strand synthesis

[0099] Using SuperScript II reverse transcriptase, with Oligo(dT) 18 Using primers, reverse transcription was completed in a 42°C water bath for 50 min. The reaction system contained 1 μg total RNA (template), 10 mM dNTPs, 40 U RNase inhibitor, and 5× reverse transcription buffer, ultimately obtaining the first strand of cDNA from the PRDX3 gene.

[0100] 3) Variable region gene amplification

[0101] VH gene amplification primers:

[0102] F: GAGGTGAAGCTGATGGAGTCTGGACCT (SEQ ID NO.6);

[0103] R: TGAGGAGACGGTGACTGAGGTCCCTGC (SEQ ID NO.7);

[0104] VL gene amplification primers:

[0105] F: CAAATTGTTCTCACCCAGTCTCCAGCAAT (SEQ ID NO.8);

[0106] R: TCTGATTTCCAGCTTGGTGCCTCCAC (SEQ ID NO. 9).

[0107] VH gene amplification: Using this cDNA as a template, after pre-denaturation at 94℃ for 2 min, 27 cycles were performed (denaturation at 94℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 2 min), followed by a final extension at 72℃ for 10 min. Primer design was based on the conserved sequence of the variable region of the mouse IgG heavy chain.

[0108] VL gene amplification: Using the same cDNA as a template, the annealing temperature was optimized to 52℃, followed by 27 cycles of pre-denaturation at 94℃ for 2 min. The cycle parameters were adjusted as follows: denaturation at 94℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 1 min, and a final extension at 72℃ for 10 min. The amplified products were verified by 1% agarose gel electrophoresis. Both VH and VL bands were located around 400 bp. After gel purification, the amplified products were dissolved in TE buffer (pH 8.0).

[0109] 4) VH and VL gene sequencing

[0110] The purified VH and VL gene products were bidirectionally sequenced using the Sanger sequencing method and universal primers. The results are as follows:

[0111] VH gene: The coding region sequence (SEQ ID NO.1) is 357 bp in length and encodes 119 amino acids (SEQ ID NO.3).

[0112] VL gene: The sequence (SEQ ID NO.2) is 312 bp in length and encodes 104 amino acids (SEQ ID NO.4).

[0113] Comparative analysis with the IMGT / V-QUEST database confirmed that both VH and VL genes contain complete complementarity-determining regions (CDR1-3) and framework regions (FR1-4), thus identifying them as functional immunoglobulin variable region sequences.

[0114] SEQ ID NO.1:

[0115] GAGGTGAAGCTGATGGAGTCTGGACCTGACCTGGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATA TGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACGTCTGAGGACACTGCCGTCTATTACTGTACTATGATTACGACAGGGTACTGGTACTTCGATGTCTGGGGCGCAGGGACCTCAGTCACCGTCTCCTCA.

[0116] SEQ ID NO.2:

[0117] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGCAGTGCCAGCTCGAGTGTATCTTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATACCACATCCAAC CTGGCTTCTGGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTTTCTCACAATCAGCAGTGTGGAGCTGAACGCTGCCGATTACTGCCATCAGTGGGGTAGTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGA.

[0118] SEQ ID NO.3:

[0119] EVKLMESGPDLVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTMITTGYWYFDVWGAGTSVTVSS.

[0120] SEQ ID NO.4:

[0121] QIVLTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSPKLLIYTTSNLASGVPSRFSGSGSGTFYFLTISSVEAEDAADYYCHQWGSWTFGGGTKLEIR.

[0122] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the protection scope of the present invention.

Claims

1. An anti-PRDX3 monoclonal antibody, characterized in that, The heavy chain variable region is the amino acid sequence shown in SEQ ID NO. 3, and the light chain variable region is the amino acid sequence shown in SEQ ID NO.

4.

2. The anti-PRDX3 monoclonal antibody of claim 1, wherein, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

2.

3. The use of the anti-PRDX3 monoclonal antibody according to any one of claims 1-2 for the preparation of a product for the detection of PRDX3 antigen, characterized in that, The detection is qualitative detection or semi-quantitative detection of PRDX3 protein in a biological sample, which is an animal tissue sample, a cell lysate sample, or a body fluid sample.

4. The use according to claim 3, wherein the compound is ###0002### The product is a detection reagent, a detection kit, or a detection test strip.

5. The use according to claim 3, wherein the compound is ###0002### The method for detecting the PRDX3 antigen comprises: (1) indirect enzyme-linked immunosorbent assay: after the biological sample to be detected is coated on a solid carrier, the anti-PRDX3 monoclonal antibody is used as a primary antibody to bind to the PRDX3 antigen, and an enzyme-labeled anti-mouse immunoglobulin secondary antibody is added, so that qualitative or semi-quantitative detection of PRDX3 is realized through an enzyme-promoted color development signal; (2) immunoblotting: after the proteins in the sample to be detected are separated by electrophoresis and transferred to a solid-phase membrane, the anti-PRDX3 monoclonal antibody is used as a primary antibody to bind to PRDX3, and an enzyme-labeled secondary antibody is added, so that qualitative and expression level analysis of PRDX3 are realized by chemiluminescence or color recognition of a PRDX3-specific band at 28 kDa; (3) immunohistochemical detection: after a tissue section to be detected is dewaxed, hydrated, and antigen-repaired, the anti-PRDX3 monoclonal antibody is used as a primary antibody to bind to the native PRDX3, and a visible stain is formed through an enzyme-labeled secondary antibody and a color developing agent, so that the cell localization and expression level of PRDX3 are detected.

6. A product for detecting PRDX3, characterized by, The product is a detection reagent, a detection kit, or a detection test strip.

7. The product of claim 6, wherein the product is for detecting PRDX3. The product is a detection reagent, a detection kit, or a detection test strip.

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