Anti-FAP antibody-magnetic bead conjugate kit and application thereof

Abstract

The invention discloses an anti-FAP antibody-magnetic bead conjugate kit and application thereof, and belongs to the field of biological reagents for diagnosis. The method comprises the following steps: preparing a high-affinity monoclonal antibody by taking FAP protein as a target, selecting carboxyl magnetic beads as a solid-phase carrier, activating carboxyl on the surfaces of the magnetic beads through an EDC / Sulfo-NHS cross-linking agent, so that the carboxyl and amino on FAP antibody molecules form a stable amido bond, and constructing a'carboxyl magnetic bead-amido bond-FAP antibody 'covalent coupling structure. In the structure, the carboxyl magnetic beads provide a stable solid-phase carrier and efficient magnetic responsiveness, the EDC / Sulfo-NHS cross-linking system ensures firm combination of the antibody and the magnetic beads, the anti-FAP antibody ensures targeted specificity to FAP + EVs, and efficient and specific capture and rapid magnetic separation of the FAP + EVs are realized through synergism of the carboxyl magnetic beads, the EDC / Sulfo-NHS cross-linking system and the anti-FAP antibody, so that the magnetic beads are used for early diagnosis of tumor patients.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic biological reagents, and more specifically, relates to a diagnostic reagent for FAP-related diseases, and more particularly to an anti-FAP antibody-magnetic bead conjugate kit and its application. Background Technology

[0002] In the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) are core stromal cells that regulate tumor proliferation, metastasis, and immune escape. CAFs are formed from normal fibroblasts (NFs) activated by tumor signals, and their functional specificity stems from differences in the expression of surface markers. Fibroblast activating protein (FAP) is a specific target of CAFs. As a transmembrane serine protease, it is highly expressed on the surface of CAFs in epithelial tumors such as lung cancer and gastric cancer, but almost not expressed in normal NFs. Exosomes secreted by CAFs carry active substances such as FAP, serving as both TME communication carriers and possessing diagnostic value for tumors.

[0003] Extracellular vesicles (EVs) are a type of membrane-bound vesicle released by cells, playing important roles in cell communication and disease development. The biomolecules carried by EVs (such as proteins, RNA, and DNA) can serve as biomarkers for the early detection of cancer. These specific molecular markers can be identified through blood tests, offering high sensitivity and specificity, and can also be used to monitor treatment response and disease progression.

[0004] Depending on their secretion pathway, endosomes (EVs) can be classified into exosomes, microvesicles, and apoptotic bodies. Microvesicles are released directly from the cell membrane due to exocytosis, while apoptotic bodies are vesicles that shrink or divide due to apoptosis. Unlike other EVs, exosomes originate from the invagination of endosomes in the parent cell to form multivesicular bodies (MVBs). Through early and late endosomes, MVBs can dynamically communicate with other organelles or cellular compartments (such as the transGolgi network, endoplasmic reticulum, mitochondria, phagosomes, RNA particles, and micronuclei). Different types of cargo, such as proteins, RNA, DNA, or lipids, are classified into MVBs. After MVBs mature, they can fuse with lysosomes and be degraded, or fuse with the cell membrane to release endovesicles (EVs), i.e., exosomes. It is evident that exosomes may carry some membrane proteins from the parent cell; therefore, we reasonably hypothesize that the lipid bilayer membrane of EVs such as exosomes released by CAFs also contains the expression of the membrane protein FAP.

[0005] The detection of serum EVs provides a potential non-invasive biomarker for liquid biopsy of tumors, as well as a target for molecular therapy; however, serum EVs are derived from various types of cells in the body, and how to capture tumor-derived EVs and then detect protein markers related to tumor progression is the key to accurate diagnosis, disease monitoring and prognostic assessment of cancer patients.

[0006] In recent years, the role of CAF-derived exosomes (EVs) in tumor progression has attracted attention. Studies have shown that CAFs can release EVs, which, through their carried proteins, miRNAs, and other components, play important roles in tumor growth, metastasis, and treatment resistance by regulating metabolism, suppressing immune responses, promoting angiogenesis, and maintaining tumor cell stemness. Based on these studies, we have reason to believe that enriching serum CAF-derived exosomes and other EVs and then detecting tumor progression-related EV protein biomarkers could help provide new serological biomarkers and potential molecular therapeutic targets for tumor prevention and treatment.

[0007] Existing technologies only use FAP as a detection biomarker. For example, patent CN 115166242 A only uses anti-FAP reagents to detect exosome FAP, without addressing targeted separation. Among other technologies, single antibody methods are prone to destroying exosomes or are costly, while nucleic acid aptamer methods have limited target recognition and are susceptible to interference, neither of which meets the requirements. The core issue lies in the fact that the physical properties of CAF-derived EVs overlap with those of other EVs, and there is a lack of efficient conjugation systems for CAF-specific biomarkers. Therefore, developing FAP-targeted, high-purity, and highly stable EV separation tools is key to overcoming the bottleneck in tumor liquid biopsy. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the first objective of the present invention is to provide an anti-FAP antibody-magnetic bead conjugate based on FAP specific targeting, so as to solve the problem that the prior art cannot efficiently and specifically capture CAF-derived EVs from complex serum samples, thereby achieving high-purity enrichment of FAP+ EVs, while ensuring the structural integrity of EVs to meet subsequent detection requirements.

[0009] To achieve the above objectives, the inventors conducted extensive experimental research and tireless exploration, and finally obtained the following technical solution: an anti-FAP antibody-magnetic bead conjugate, wherein the anti-FAP antibody-magnetic bead conjugate is formed by covalently coupling an anti-FAP antibody with a carboxyl magnetic bead via an amide bond.

[0010] More preferably, in the anti-FAP antibody-magnetic bead conjugate described above, the anti-FAP antibody and carboxyl magnetic beads are covalently coupled through an EDC / Sulfo-NHS dual crosslinking agent system as a medium.

[0011] It should be noted that this invention prepares high-affinity monoclonal antibodies targeting FAP protein. Carboxyl magnetic beads are selected as the solid-phase carrier. The carboxyl groups on the surface of the magnetic beads are activated by an EDC / Sulfo-NHS cross-linking agent, forming stable amide bonds with the amino groups on the FAP antibody molecules, thus constructing a covalently coupled structure of "carboxyl magnetic beads-amide bonds-FAP antibody". In this structure, the carboxyl magnetic beads provide a stable solid-phase carrier and efficient magnetic responsiveness; the EDC / Sulfo-NHS cross-linking system ensures a strong binding between the antibody and the magnetic beads; and the anti-FAP antibody ensures targeted specificity for FAP+ EVs. These three components synergistically achieve efficient and specific capture and rapid magnetic separation of FAP+ EVs.

[0012] Secondly, the present invention also provides a method for preparing the above-mentioned anti-FAP antibody-magnetic bead conjugate, the method comprising the following steps:

[0013] (1) Activation of carboxyl magnetic beads: Take carboxyl magnetic beads, add 0.1M pH 5.5 MES buffer, place them in an ultrasonic cleaner and ultrasonically clean for 2-6 minutes, magnetically separate and discard the liquid, repeat the cleaning 2-4 times; then add 0.1M pH 5.5 MES buffer, ultrasonically mix to fully suspend the magnetic beads, and obtain a magnetic bead suspension; weigh EDC and Sulfo-NHS at a mass ratio of 1:1, dissolve them in 0.1M pH 5.5 MES buffer to make 8-12 mg / ml stock solutions, immediately add them to the magnetic bead suspension, ultrasonically mix for 0.5-3 minutes, place them in a constant temperature shaker at 24-26℃ and react for 0.8-1.5h at a speed of 100-150r / min; after the reaction is completed, add 0.1M pH 5.5 MES buffer and ultrasonically clean for 2-6 minutes, magnetically separate and discard the liquid to obtain activated carboxyl magnetic beads;

[0014] (2) Antibody conjugation, blocking, purification, and preservation: Add 0.1M pH 9.5 carbonate buffer to the activated carboxyl magnetic beads and sonicate for 1-4 minutes; add FAP monoclonal antibody at a ratio of 1mg magnetic beads to 20μg antibody, gently invert the centrifuge tube to mix, and place it in a constant temperature shaker at 24-26℃ in the dark for 2-3 hours at a speed of 100-150r / min; after the reaction, place the centrifuge tube on a magnetic rack for magnetic separation and discard the supernatant; add pH 7.4 PBS buffer containing 0.5% BSA, mix, and place it in a constant temperature shaker at 36.5-37.5℃ for 1.5-3 hours at a speed of 100-150r / min; after blocking, magnetically separate and discard the liquid, and sonicate and wash 2-4 times with pH 7.4 PBS buffer containing 0.5% BSA, magnetically separating and discarding the liquid after each wash; finally, use pH 7.4 PBS buffer containing 0.5% BSA. Resuspend the conjugate in PBS buffer, aliquot, and store in a sealed container at 2-8°C.

[0015] In addition, the present invention also provides a tumor diagnostic kit, the kit comprising the above-mentioned anti-FAP antibody-magnetic bead conjugate.

[0016] Because the "carboxylated magnetic bead-amide bond-FAP antibody" covalently coupled structure constructed in this invention can achieve efficient capture and rapid separation of FAP+ EVs in serum samples of tumor patients, it can be used for the early diagnosis of tumor patients. Therefore, this invention also provides the application of the above-mentioned anti-FAP antibody-magnetic bead conjugate in capturing and separating FAP+ EVs in serum samples of tumor patients; and the application of the above-mentioned anti-FAP antibody-magnetic bead conjugate in the preparation of kits for early tumor diagnosis.

[0017] Compared with existing technologies, this invention provides an anti-FAP antibody-magnetic bead conjugate based on FAP specific targeting, its preparation method, and its application, which has the following advantages and advancements:

[0018] 1. Significantly improved specificity: A targeting system designed for the CAF-specific biomarker FAP is used to achieve precise capture of CAF-derived EVs by using high-affinity anti-FAP antibodies, effectively avoiding non-specific contamination caused by traditional physical methods and general biomarkers (such as CD63 / CD9), resulting in a significant improvement in separation purity.

[0019] 2. Excellent capture efficiency and stability: By optimizing reaction conditions and material ratios based on the covalent coupling principle of carboxyl magnetic beads and antibodies, high coupling efficiency, antibody activity, conjugate stability and EV binding efficiency are ensured, while low background interference is minimized.

[0020] 3. Preserving the complete structure and function of EVs: The gentle immunomagnetic bead method avoids strong physical shearing or chemical treatment, maximizing the preservation of EV membrane integrity and content activity, providing reliable samples for downstream molecular detection, functional studies and clinical diagnosis.

[0021] 4. Easy to operate and highly applicable: The magnetic beads are superparamagnetic, which can be separated quickly. The process is simplified, time is short, and it is easy to automate. It is suitable for complex body fluid samples such as serum, and provides an efficient tool for the clinical translation of FAP+EVs as liquid biopsy biomarkers.

[0022] 5. Breakthrough in existing technological bottlenecks: For the first time, FAP is targeted for EV separation, solving the problem of specific enrichment of CAF EVs, making up for the shortcomings of existing methods that only use FAP for detection, and promoting the development of tumor microenvironment research and precision diagnosis. Attached Figure Description

[0023] Figure 1SDS-PAGE spectrum; As shown in the figure, under reducing conditions, the sample shows main bands at 25kDa and 55kDa, while under non-reducing conditions, a single main band is shown at 150kDa; Conditions: Sample processing: Non-reducing: 20 μL 1mg / ml sample, add 5 μL non-reducing 5× loading buffer; Reducing: 20 μL 1mg / ml sample, add 5 μL reducing 5× loading buffer, heat at 100℃ for 5 min; Sample loading volume: 10μg; Electrophoresis conditions: ① 80V, 5min ② 145V, 42min.

[0024] Figure 2 : SEC-HPLC chromatogram; purity 100%; conditions: column: AdvanceBio SEC 300Å 2.7μm 7.8×300mm; mobile phase: 150mM NaH2PO4-NaOH+10%IPA, pH7.0; column temperature: 30.0℃; flow rate: 0.50 mL / min; detection wavelength: 220 nm; injection volume: 5μL.

[0025] Figure 3 : Expression and localization of FAP in CAF-derived exosomes; where: (A) Western blot detection of FAP expression; (B) Immunogold electron microscopy detection of FAP localization.

[0026] Figure 4 FAP was highly expressed in the serum exosomes of LUAD patients; (A) Western blot analysis of FAP expression in serum exosomes of LUAD patients and healthy controls; (B) Comparison of FAP expression levels in serum exosomes of LUAD patients and healthy controls. **: P<0.01.

[0027] Figure 5 : Anti-FAP antibody-magnetic bead conjugate capture of exosomes and Evs; where: (A) Western blot detection of serum exosome capture; (B) Transmission electron micrograph of serum exosomes captured by anti-FAP antibody-magnetic bead conjugate; (C) Transmission electron micrograph of serum EVs captured by anti-FAP antibody-magnetic bead conjugate. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, experimental methods described in the embodiments are performed under conventional conditions.

[0029] Example 1: Preparation of FAP rabbit monoclonal antibody

[0030] This embodiment employs a four-step preparation method: "immunization-sorting-recombinant expression-purification." The core optimizations include the immunization strategy and expression system, enhancing antibody affinity and yield. The specific implementation is as follows:

[0031] 1. Targeted Immunization Induction: Recombinant FAP protein (purchased from MCE, catalog number HY-P72659) was selected as the specific immunogen, and 3-month-old ordinary New Zealand white rabbits were used as immunization animals. For the first immunization, 500 μg / 1 ml of FAP antigen was fully emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites to elicit an immune response. A booster immunization was performed 4 weeks later, with an equal dose of antigen emulsified with Freund's incomplete adjuvant before injection to reduce the stimulation of the adjuvant on the animals while maintaining immune memory. Seven days after the second immunization, ear vein blood was collected, and antibody titers were detected by indirect ELISA. Serum diluted 2500-40000 times was used as the test sample. When the titer reached 10000 times or more, the next step was performed; otherwise, a booster immunization was given.

[0032] 2. High-affinity monoclonal B cell sorting: Peripheral blood was collected 3 days after the third immunization, and peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. A fluorescent probe was prepared by conjugating FAP protein with phycoerythrin (APC) and directly staining PBMCs. Monoclonal B cells binding to the fluorescent signal were sorted using a flow cytometer (BD FACSAria III) to ensure a purity of over 99%. The sorted monoclonal B cells were seeded into 96-well cell culture plates and cultured for 7 days in RPMI 1640 medium containing 10% fetal bovine serum. The culture supernatant was used for ELISA detection. Wells with positive clones (OD450 ≥ 2.0) were selected, the medium was changed, and the cells were cultured for another 2 days. Binding stability was verified again by ELISA. Finally, three high-affinity monoclonal cell lines were obtained, and the variable region sequence of the antibody was extracted.

[0033] 3. CHO Cell Recombinant Expression: Targeting the codon bias of CHO cells, the retrieved antibody light and heavy chain gene sequences were optimized, and an IL-2 signal peptide was introduced to enhance protein secretion efficiency. The optimized sequence was cloned into the pcDNA3.4 expression vector. The recombinant plasmid was transformed into DH5α competent cells, and single colonies were picked and expanded. Endotoxin-free plasmids were extracted using the OMEGA EndoFree PlasmidMaxi Kit (catalog number D6950-02) to ensure an endotoxin content <0.1 EU / μg. Using a Celerix electroporator (model CTX-1500A), the light and heavy chain plasmids were co-transfected at a 1:1 ratio into CHO-S cells in logarithmic growth phase. CHO feed was added starting 24 hours post-transfection, every 48 hours for a total of 4 additions. On day 10 of cell culture, when the viable cell rate dropped below 60%, the fermentation broth was collected, centrifuged at 8000 r / min for 10 minutes to remove the cell pellet, and then filtered through a 0.22 μm filter membrane to obtain a clear antibody expression supernatant.

[0034] 4. Affinity chromatography purification: The AKTA Pure 25 protein purification system (GE Healthcare) was used with a Mabselect Sure pre-packed column (1 ml, catalog number 17-5438-01) for antibody purification. Before purification, the column was equilibrated with 10 column volumes (CV) of equilibration buffer (20 mM PBS, pH 7.4) until the 280 nm UV absorption baseline stabilized. The expression supernatant was loaded at a flow rate of 1 ml / min. After loading, the column was washed with 20 CV of equilibration buffer to remove unbound contaminants until the UV absorbance returned to baseline. Then, elution was performed with elution buffer (20 mM Gly-HCl, pH 2.7) at a flow rate of 0.5 ml / min. The UV absorption peak was monitored in real time, and the eluent in the peak range was collected and immediately neutralized to pH 7.0-7.4 with 1 M Tris-HCl buffer (pH 9.0). The collected antibody samples were placed in dialysis bags and dialyzed in 20 mM PBS buffer (pH 7.4) at 4°C for 24 hours, with the buffer changed three times during this period. The antibody molecular weight (approximately 50 kDa for the heavy chain and approximately 25 kDa for the light chain) was verified by SDS-PAGE electrophoresis. The antibody concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific) to ensure that the final product concentration was ≥1 mg / ml and the purity was >95%.

[0035] In this embodiment, recombinant FAP protein was selected as the specific immunogen. By optimizing the immunization strategy (initial Freund's complete adjuvant stimulation followed by subsequent Freund's incomplete adjuvant enhancement), both sufficient activation of the immune response and reduced adjuvant stimulation on the experimental animals were ensured. Indirect ELISA results showed that effective antibodies were still detectable in serum 7 days after the second immunization, even after dilutions of 2500-40000 times, with titers exceeding the preset standard of 10000 times. This indicates successful targeted immunization induction, and the experimental animals have developed specific immune memory against the FAP antigen, providing a sufficient source of immune cells for subsequent screening of high-affinity B cells. After isolating PBMCs, specific B cells were captured by FAP-APC labeling and flow cytometry sorting (purity >99%). After culturing in 96-well plates, OD cells were screened by ELISA. 450 Positive clones with ≥2.0 were obtained, ultimately yielding three high-affinity stable cell lines to meet antibody preparation requirements. The antibody gene sequence was optimized, and an IL-2 signal peptide was introduced to improve secretion efficiency; endotoxin-free plasmid electroporation ensured safe and efficient expression. SDS-PAGE validation after purification showed that the 25kDa (light chain) and 55kDa (heavy chain) main bands were clear under reducing conditions, while the 150kDa dimer remained intact under non-reducing conditions. Figure 1 Nanodrop detection concentration ≥1 mg / ml, purity >95% Figure 2 High-purity FAP rabbit monoclonal antibody was successfully obtained.

[0036] Example 2: Preparation of anti-FAP antibody-magnetic bead conjugate

[0037] 1. Preparation of experimental materials: carboxyl magnetic beads, 0.1M pH 5.5 MES buffer, 0.1M pH 9.5 carbonate buffer, EDC, Sulfo-NHS, FAP rabbit monoclonal antibody (prepared in Example 1, concentration 1mg / ml), pH 7.4 PBS buffer containing 0.5% BSA, 25℃ constant temperature shaker, magnetic rack, ultrasonic cleaner.

[0038] 2. Activation of Carboxyl Magnetic Beads: Accurately weigh 10 mg of carboxyl magnetic beads into a sterile centrifuge tube. Place the centrifuge tube on a magnetic rack for magnetic separation for 5 minutes, and discard the supernatant. Add 1 ml of 0.1 M pH 5.5 MES buffer, and sonicate for 3 minutes. Magnetic separation and discard the liquid. Repeat the washing process 3 times. Finally, add 0.6 ml of 0.1 M pH 5.5 MES buffer and sonicate to fully suspend the magnetic beads. Weigh 2 mg of EDC and 2 mg of Sulfo-NHS, and dissolve them separately in 50 μL of 0.1 M pH 5.5 MES buffer to prepare a 10 mg / ml stock solution. Immediately add the dissolved stock solution to the magnetic bead suspension, sonicate for 1 minute, and react in a 25°C shaker at 120 rpm for 1 hour. After the reaction, add 1 ml of 0.1 M pH 5.5 MES buffer, sonicate for 3 minutes, magnetic separation and discard the liquid to obtain activated carboxyl magnetic beads.

[0039] 3. Antibody conjugation, blocking, purification, and preservation: Add 1 ml of 0.1 M pH 9.5 carbonate buffer to the activated carboxyl magnetic beads and sonicate for 2 minutes. Add 200 μg of FAP rabbit monoclonal antibody (200 μL, 1 mg / ml) at a ratio of 1 mg magnetic beads to 20 μg antibody. Gently invert the centrifuge tube to mix, and incubate at 25°C in the dark for 2 hours at 120 rpm. After the reaction, place the centrifuge tube on a magnetic rack for magnetic separation for 5 minutes, discard the supernatant (take a small amount of supernatant and use Nanodrop to detect protein concentration to verify antibody binding). Add 1 ml of pH 7.4 PBS buffer containing 0.5% BSA, mix well, and incubate at 37°C in the shaker for 2 hours at 100 rpm. After sealing, the solution was magnetically separated and discarded. The conjugate was ultrasonically washed three times with pH 7.4 PBS buffer containing 0.5% BSA, and magnetically separated and discarded after each wash. Finally, the conjugate was resuspended in pH 7.4 PBS buffer containing 0.5% BSA to a concentration of 2 mg / ml, aliquoted into 100 μL tubes, labeled, and stored in a sealed container at 2–8°C.

[0040] This embodiment, through key process optimization, confirmed that the FAP antibody and carboxyl magnetic beads formed a robust covalent coupling structure. The magnetic bead activation stage employed an EDC / Sulfo-NHS dual crosslinking agent system, achieving efficient activation of the carboxyl groups in 0.1M pH 5.5 MES buffer, providing the necessary prerequisite for the formation of amide bonds between the antibody amino group and the magnetic bead carboxyl group. The coupling reaction used 0.1M pH 9.5 carbonate buffer to regulate the reaction environment, while strictly adhering to the optimal ratio of 1mg magnetic beads to 20μg antibody to ensure uniform distribution of antibody molecules on the magnetic bead surface. The preparation process exhibits significant stability and reproducibility, providing support for the large-scale production and clinical application of the conjugate. All reaction conditions in the experiment (such as temperature control for the 25℃ coupling reaction and 37℃ blocking reaction, and reaction time of 1-2 hours) were optimized and validated. Furthermore, the dosage of key reagents (such as 2mg of EDC and 2mg of Sulfo-NHS per 10mg of magnetic beads), buffer formulation, and other parameters were clearly controllable, avoiding the impact of process fluctuations on product quality.

[0041] Example 3: Experimental study on the expression and localization of FAP in CAF-derived exosomes

[0042] 1. Extraction and culture of tumor-associated fibroblasts

[0043] Cancerous tissue and adjacent tissue were collected from patients with lung adenocarcinoma who underwent surgical resection at the Department of Thoracic Surgery, Fujian Provincial Hospital Affiliated to Fuzhou University. The tissue was washed and cut into 1-2 mm pieces. 3 Small pieces of tissue were digested with trypsin, filtered, and centrifuged. The resulting tissue pellet was then seeded into DMEM / F-12 medium containing 10% FBS. After culturing at 37°C for 48 hours, the medium was changed. Once the cells reached 70% confluence, they were re-coated onto plates. 2-3 hours after coating, non-adherent cells were removed to obtain primary fibroblasts. Adherent CAFs and paired NFs were then passaged.

[0044] 2. Isolation of exosomes

[0045] Primary fibroblasts were used at 8 × 10 5 Cells were seeded in 6 cm culture dishes and allowed to adhere and grow to approximately 70%–80% confluence. They were then washed twice with PBS. Serum-free medium was added, and the cells were cultured for another 24 h. The conditioned medium was collected and centrifuged at 3000g for 30 minutes at 4°C to remove cell debris, followed by filtration through a 0.22 μm filter. Exosomes were isolated from the filtrate using the EXODUS system. The concentrate was collected, and the isolated exosomes were resuspended in PBS to a final volume of 400 μL and stored at -80°C. All 1×PBS used in this study was pre-filtered through a 0.22 μm filter.

[0046] 3. Western blot (WB)

[0047] Cells were lysed on ice for 30 min with non-denaturing lysis buffer containing protease / phosphatase inhibitors, then centrifuged at 12000g at 4℃ for 15 min, and the supernatant was collected. Protein quantification was performed using the BCA method, followed by boiling for 10 min in 5× loading buffer to denature the proteins. Separating and stacking gels of appropriate molecular weights were prepared and subjected to constant voltage electrophoresis (80 V for stacking gel, 120 V for separating gel) until bromophenol blue migrated to the bottom of the gel. A transfer apparatus was assembled using a methanol-activated PVDF membrane in a sandwich configuration, and wet-transferred at 400 mA for 1 h. After transfer, the membrane was blocked with 5% skim milk at room temperature for 1 h, followed by incubation with primary antibody at 4℃ overnight, then with secondary antibody at room temperature for 1 h. After each incubation, the membrane was washed three times with TBST for 10 min each time. ECL chemiluminescent substrate was added, and the membrane was exposed for imaging and analysis of the relative expression level of the target protein.

[0048] 4. Immunogold electron microscopy

[0049] 20 μL of exosome sample was mixed with an equal volume of 4% paraformaldehyde and dropped onto a hydrophobic paraffin membrane. A Formvar carbon membrane coated with a copper mesh was floated and incubated for 20 min, then air-dried for 1 h. After blocking with 1% BSA-PBS for 30 min, the sample was incubated overnight with rabbit anti-human FAP primary antibody at 4℃, washed three times with 0.05% Tween-20 PBS for 5 min each time, and then incubated with colloidal gold-labeled goat anti-rabbit secondary antibody at room temperature in the dark for 1 h. The washing and rinsing with deionized water were repeated twice. After negative staining with 2% uranium acetate for 30 s, the sample was air-dried and observed under a Helios Nanolab DualBeam transmission electron microscope at 80 kV.

[0050] Western blot analysis of FAP in exosomes from CAF culture supernatant of lung adenocarcinoma (LUAD) patients showed high expression of FAP in CAF-derived exosomes, while low expression of FAP was observed in normal fibroblast (NF) exosomes, suggesting high FAP expression in CAF-derived exosomes (see [link to article]). Figure 3 A), and further immunogold electron microscopy confirmed that FAP is a membrane protein present in CAF exosomes (see A). Figure 3 B).

[0051] Example 4: Experimental study on the high expression of FAP in serum exosomes of LUAD patients

[0052] 1. Isolation of serum exosomes

[0053] After collecting serum samples, centrifuge at 1500g for 15 minutes at 4℃, collect the supernatant, and then centrifuge again under the same conditions to further remove platelets and cell debris. Take 400μL of supernatant, dilute to 30 mL with phosphate-buffered saline (PBS), and filter through a 0.22μm filter membrane. Use the EXODUS system to separate exosomes from the above filtrate, collect the concentrate, resuspend the separated exosomes in PBS to 400μL, and store at -80℃.

[0054] 2. Western blot (WB)

[0055] Cells were lysed on ice for 30 min with non-denaturing lysis buffer containing protease / phosphatase inhibitors, then centrifuged at 12000g at 4℃ for 15 min, and the supernatant was collected. Protein quantification was performed using the BCA method, followed by boiling for 10 min in 5× loading buffer to denature the proteins. Separating and stacking gels of appropriate molecular weights were prepared and subjected to constant voltage electrophoresis (80 V for stacking gel, 120 V for separating gel) until bromophenol blue migrated to the bottom of the gel. A transfer apparatus was assembled using a methanol-activated PVDF membrane in a sandwich configuration, and wet-transferred at 400 mA for 1 h. After transfer, the membrane was blocked with 5% skim milk at room temperature for 1 h, followed by incubation with primary antibody at 4℃ overnight, then with secondary antibody at room temperature for 1 h. After each incubation, the membrane was washed three times with TBST for 10 min each time. ECL chemiluminescent substrate was added, and the membrane was exposed for imaging and analysis of the relative expression level of the target protein.

[0056] We conducted serum sample analysis on patients with lupus ulcerative colitis (LUAD). The results showed that FAP was significantly highly expressed in the serum exosomes of LUAD patients, with a positive rate of 100% (8 / 8); while in healthy controls, serum exosomes showed low or no FAP expression, with a positive rate of 25.0% (2 / 8) (see...). Figure 4 A, Figure 4 (B) This suggests that there is a significant accumulation of exosomes released from the tumor microenvironment CAF in the serum of LUAD patients; capturing serum CAF-derived exosomes / EVs using FAP can help obtain tumor-specific exosome / EVs-related liquid biopsy biopsy markers.

[0057] Example 5: Application of anti-FAP antibody-magnetic bead conjugate in capturing FAP+ EVs

[0058] This embodiment provides a standardized FAP+EVs capture process, adapted for clinical serum sample testing. Specific steps are as follows:

[0059] 1. Sample pretreatment: Take 1 mL of serum sample, centrifuge at 4℃ and 12000 r / min for 15 minutes to remove cell debris, and collect the supernatant for later use;

[0060] 2. Activation of the conjugate: Take 250 μL of anti-FAP antibody-magnetic bead conjugate (prepared in Example 2) and add it to a 1.5 mL EP tube. Wash the tube once with 500 μL of PBST and twice with 500 μL of PBS.

[0061] 3. Targeted capture: Add the pretreated serum supernatant to an EP tube containing the conjugate, gently invert to mix, and incubate at 37°C for 30 minutes;

[0062] 4. Separation and washing: Place the EP tube on a magnetic rack for 3 minutes to adsorb, discard the supernatant, wash 3 times with washing buffer, adsorb for 2 minutes after each wash, and resuspend in 30 μL PBS.

[0063] The inventors constructed an anti-FAP antibody-magnetic bead conjugate, successfully captured FAP(+) exosomes from the serum of LUAD patients, and verified the results by Western blot and electron microscopy (see [link to article]). Figure 5 A, Figure 5 B). Based on this result, it was further successfully applied to the capture of serum FAP(+) EVs in LUAD patients (see B). Figure 5 C).

[0064] This embodiment removes impurities such as cell debris from serum to avoid interfering with targeted capture and is suitable for the complexity of clinical samples. A magnetic rack rapidly separates the complex in 3 minutes, followed by three washes to remove impurities, yielding purified FAP+ EVs. Based on antigen-antibody specific binding, FAP+ EVs can be accurately captured, with specificity superior to traditional ultracentrifugation. The standardized procedure is adapted for batch testing, successfully establishing a reliable capture method.

Claims

1. An anti-FAP antibody-magnetic bead conjugate, characterized in that, The anti-FAP antibody-magnetic bead conjugate is formed by covalently coupling an anti-FAP antibody with a carboxyl magnetic bead via an amide bond.

2. The anti-FAP antibody-magnetic bead conjugate according to claim 1, characterized in that, The anti-FAP antibody and carboxyl magnetic beads are covalently coupled through an EDC / Sulfo-NHS dual crosslinking agent system as a medium.

3. A method for preparing the anti-FAP antibody-magnetic bead conjugate according to claim 1, characterized in that, The method includes the following steps: (1) Activation of carboxyl magnetic beads: Take carboxyl magnetic beads, add 0.1M pH 5.5 MES buffer, place them in an ultrasonic cleaner and ultrasonically clean for 2-6 minutes, magnetically separate and discard the liquid, repeat the cleaning 2-4 times; then add 0.1M pH 5.5 MES buffer, ultrasonically mix to fully suspend the magnetic beads, and obtain a magnetic bead suspension; weigh EDC and Sulfo-NHS at a mass ratio of 1:1, dissolve them separately in 0.1M pH 5.5 MES buffer to make 8-12 mg / ml stock solutions, immediately add them to the magnetic bead suspension, ultrasonically mix for 0.5-3 minutes, place them in a constant temperature shaker at 24-26℃ and react for 0.8-1.5h at a speed of 100-150r / min; after the reaction is completed, add 0.1M pH 5.5 MES buffer and ultrasonically clean for 2-6 minutes, magnetically separate and discard the liquid to obtain activated carboxyl magnetic beads; (2) Antibody conjugation, blocking, purification, and preservation: Add 0.1M pH 9.5 carbonate buffer to the activated carboxyl magnetic beads and sonicate for 1-4 minutes; add FAP monoclonal antibody at a ratio of 1mg magnetic beads to 20μg antibody, gently invert the centrifuge tube to mix, and place it in a constant temperature shaker at 24-26℃ in the dark for 2-3 hours at a speed of 100-150r / min; after the reaction, place the centrifuge tube on a magnetic rack for magnetic separation and discard the supernatant; add pH 7.4 PBS buffer containing 0.5% BSA, mix, and place it in a constant temperature shaker at 36.5-37.5℃ for 1.5-3 hours at a speed of 100-150r / min; after blocking, magnetically separate and discard the liquid, and sonicate and wash 2-4 times with pH 7.4 PBS buffer containing 0.5% BSA, magnetically separating and discarding the liquid after each wash; finally, use pH 7.4 PBS buffer containing 0.5% BSA. Resuspend the conjugate in PBS buffer, aliquot, and store in a sealed container at 2–8°C.

4. A tumor diagnostic kit, characterized in that, The kit contains the anti-FAP antibody-magnetic bead conjugate as described in claim 1 or 2.

5. The use of the anti-FAP antibody-magnetic bead conjugate of claim 1 or 2 in capturing and separating FAP and EVs from serum samples of tumor patients.

6. The use of the anti-FAP antibody-magnetic bead conjugate according to claim 1 or 2 in the preparation of a kit for early tumor diagnosis.

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