Chemiluminescence method-based pancreatic ductal adenocarcinoma exosome marker detection kit and preparation method and application thereof

Abstract

The invention relates to a pancreatic ductal adenocarcinoma exosome marker detection kit based on a chemiluminescence method as well as a preparation method and application of the pancreatic ductal adenocarcinoma exosome marker detection kit. The kit comprises an exosome targeted capture processing component, an REG1A detection component, an REG1B detection component and a common component, the exosome targeted capture treatment component consists of EphA2 immunocapture magnetic beads, a high-efficiency capture buffer solution and an exosome lysis solution; the REG1A detection component is composed of REG1A capture magnetic particles, a horse radish peroxidase labeled anti-REG1A detection antibody, a REG1A calibration product and a REG1A quality control product; the REG1B detection component is composed of REG1B capture magnetic particles, a horse radish peroxidase labeled anti-REG1B detection antibody, a REG1B calibration product and a REG1B quality control product; the kit is composed of a 20 * concentrated washing solution, a sample diluent and a chemiluminescent substrate solution. The detection kit can effectively eliminate interference of free protein and other exosomes, the detection specificity reaches 98.3%, the sensitivity reaches 1.6-1.9 pg / mL, and a more reliable auxiliary detection basis is provided for clinical decision making.

Description

Technical Field

[0001] This invention relates to a chemiluminescence immunoassay-based detection kit for pancreatic ductal adenocarcinoma exosome markers, its preparation method, and its application, belonging to the field of clinical laboratory technology. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant gastrointestinal tumor with a long-standing five-year survival rate of less than 10% and a median survival of less than six months. The core challenge to this prognosis lies in the extreme difficulty of early diagnosis: over 80% of patients are diagnosed at a locally advanced stage (stage III) or with distant metastases (stage IV), missing the opportunity for radical surgery. Data shows that the five-year survival rate for early-stage (stage I) patients can reach 30-40%, while for advanced-stage patients it is less than 5%, a difference of 8-10 times. PDAC has a biological evolution window of 10-15 years from gene mutation initiation to the formation of metastatic lesions; this is the golden opportunity for "early detection, early intervention, and early treatment," but current screening methods cannot effectively utilize this window.

[0003] The most commonly used serum tumor marker for pancreatic ductal adenocarcinoma (PDAC) in clinical practice currently suffers from three major fatal flaws: First, its early sensitivity is severely insufficient. In resectable PDAC tumors <2cm in diameter, the positive rate of CA19-9 is only 27-39%, and the level increase is limited (median value approximately 50-80 U / mL), making it highly susceptible to missed diagnoses. Second, its specificity is low. The positive rate is as high as 15-20% in benign diseases such as biliary obstruction, chronic pancreatitis, and cirrhosis, and the cross-positive rate is approximately 10-15% in other gastrointestinal tumors such as colorectal cancer and gastric cancer, leading to false positives and overdiagnosis costs exceeding hundreds of millions of yuan annually. Third, it has a genetic blind spot. Approximately 5-10% of Lewis antigen-negative (Le(ab-)) individuals cannot synthesize the CA19-9 antigen epitope, so even in advanced PDAC, their serum CA19-9 levels may remain completely normal, resulting in 100% missed diagnoses. These three flaws mean that CA19-9 is only suitable for monitoring treatment efficacy and lacks value for early screening.

[0004] In recent years, liquid biopsy technology, focusing on exosome analysis, has brought revolutionary hope for the early diagnosis of tumors. Exosomes are 30-150 nm membrane vesicles actively secreted by cells. The combination of their intraluminal and membrane proteins exhibits high tumor specificity, accurately reflecting the molecular characteristics of in situ tumors. However, exosome detection faces a dual technical bottleneck: firstly, its abundance in blood is extremely low (approximately 10% in healthy individuals). 8 ~10 9The concentration of exosomes (particles / mL, 2-5 times higher in early-stage PDAC patients) is a significant concern. Traditional ultracentrifugation methods take over 8 hours, and immunoprecipitation methods have poor specificity and are easily affected by free proteins. Furthermore, the concentration of free tumor-associated proteins in serum is typically 10²-10³ times higher than that in exosomes. If tumor exosomes cannot be specifically captured and total protein is directly measured, tissue specificity cannot be guaranteed. Therefore, achieving efficient, specific, and automated exosome capture and quantification of intravesical biomarkers is a key obstacle to clinical translation.

[0005] REG1A and REG1B, as members of the regeneration gene family, play a unique role in the development and progression of pancreatic cancer cell dysplasia (PDAC). Multicenter prospective cohort studies (n>10,000) and Mendelian randomization analysis have confirmed a causal association between elevated serum REG1A / REG1B levels and the risk of PDAC (OR 3.2–4.5). Abnormal expression can be traced back 3–5 years before clinical diagnosis, and the expression level is positively correlated with tumor burden. Molecular mechanism studies have shown that REG1A / REG1B promotes pancreatic cancer cell proliferation and drug resistance by activating the PI3K / Akt pathway. Their exosome-encapsulated form avoids protease degradation and exhibits more than 10-fold increased stability compared to free proteins. However, the clinical translation of REG1A / REG1B faces a dilemma: in serum, free REG1A / REG1B proteins lack tissue specificity, with positive rates of 45-60% in chronic pancreatitis, approximately 25% in diabetes, and only 18% and 12% in colorectal and gastric cancer, respectively. This results in a single-indicator detection specificity of less than 80% and a positive predictive value of <15%, limiting their clinical application value. Direct detection of exosomal REG1A / REG1B, however, faces challenges such as low capture efficiency, weak detection signals, and a lack of standardized threshold systems.

[0006] Studies have shown that Eph receptor tyrosine kinase A2 (EphA2) exhibits "superexpression" characteristics on the surface of PDAC exosomes: the expression level of EphA2 in exosomes secreted by PDAC cells is more than 100 times higher than that in exosomes derived from normal pancreatic cells, and 10 to 50 times higher than that in exosomes from other tumors (such as liver cancer and lung cancer). However, linking EphA2 with PDAC detection and diagnosis, and using it for REG1A / REG1B detection, still faces and requires overcoming three major technical challenges: First, capture efficiency, which requires improving the capture efficiency of specific exosomes to ensure early detection of low-abundance samples; second, detection sensitivity barriers, which require achieving pg / mL level protein quantification; and third, clinical interpretation challenges, which require establishing a threshold system based on a large sample cohort and a clear, reliable, and clinically interpretable set of interpretation rules to transform the dual-indicator detection results into accurate diagnostic conclusions, which is the key to the development of multi-indicator joint detection kits.

[0007] In the existing technology, there are no reports of commercially available kits that optimize the EphA2 capture system to efficiently enrich pancreatic ductal adenocarcinoma exosomes, and jointly detect their REG1A / REG1B proteins, and are equipped with complete and transparent single indicators and combined diagnostic thresholds. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a chemiluminescence-based exosome biomarker detection kit for pancreatic ductal adenocarcinoma, its preparation method, and its application.

[0009] This invention proposes for the first time an innovative strategy of "targeted capture-intracystic biomarker detection": using EphA2 to specifically capture PDAC exosomes, and then using REG1A / REG1B proteins as biomarkers to detect exosome intracystic biomarkers, thereby achieving "tissue tracing" and "signal amplification" at the protein level, and then using it for early screening and auxiliary diagnosis of pancreatic ductal adenocarcinoma.

[0010] The technical solution of the present invention is as follows:

[0011] A chemiluminescence immunoassay-based exosome biomarker detection kit for pancreatic ductal adenocarcinoma includes an exosome-targeted capture processing component, a REG1A detection component, a REG1B detection component, and a common component;

[0012] The exosome targeted capture processing component consists of EphA2 immunocapture magnetic beads, high-efficiency capture buffer, and exosome lysis buffer.

[0013] The REG1A detection component consists of REG1A capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1A detection antibody, REG1A calibrator, and REG1A quality control.

[0014] The REG1B detection component consists of REG1B capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1B detection antibody, REG1B calibrator, and REG1B quality control.

[0015] The common components consist of 20× concentrated washing solution, sample diluent, and chemiluminescent substrate solution.

[0016] According to a preferred embodiment of the present invention, the EphA2 immunocapture magnetic beads are carboxyl magnetic microspheres coated with anti-human EphA2 monoclonal antibodies, with a diameter of 2-3 μm, an antibody coating concentration of 30-50 μg antibody / mg magnetic beads, and a working concentration of 0.3-0.5 mg / mL;

[0017] Prepared according to the following method:

[0018] (1) Activation of magnetic beads: Take carboxyl magnetic microspheres, wash them twice with coupling buffer, add a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and gently shake at room temperature for 28~32 min to activate.

[0019] (2) Washing: Magnetic separation, discard the supernatant; then resuspend the magnetic beads in 1 mL of coupling buffer, perform magnetic separation again, discard the supernatant, and repeat the washing once to obtain the activated magnetic beads;

[0020] (3) Antibody conjugation: The activated magnetic beads were resuspended in the conjugation buffer, and the anti-human EphA2 monoclonal antibody that had been predialyzed into the conjugation buffer was added. The mixture was shaken on a rotary mixer at room temperature for 1.5 to 2.5 hours.

[0021] (4) Blocking and washing: Add ethanolamine solution to block unreacted activated carboxyl groups, continue the reaction at room temperature for 28-32 min, then magnetically separate, discard the supernatant, and wash the magnetic beads 2-4 times with washing solution to obtain EphA2 immunocapture magnetic beads.

[0022] According to a preferred embodiment of the present invention, the formulation of the high-efficiency capture buffer is as follows: 1000 mL of sterile 10 mM phosphate buffer (PBS) containing 30-50 g of polyethylene glycol 6000 (PEG6000), 0.1-0.2 mL of Tween-20, and 0.22-0.55 g of calcium chloride (CaCl2), with the pH adjusted to 6.5-7.0 using hydrochloric acid or sodium hydroxide.

[0023] According to a preferred embodiment of the present invention, the exosome lysis buffer is formulated as follows: 20-50 mM Tris-HCl buffer (pH=7.4-8.0), 1-2% (v / v) of nonionic detergent, 100-200 mM NaCl, and a mixture of 1× concentration protease inhibitors.

[0024] More preferably, the nonionic detergent is Triton X-100 or ethyl phenyl polyethylene glycol (NP-40); the protease inhibitor mixture is an EDTA-free protease inhibitor mixture.

[0025] According to a preferred embodiment of the present invention, the REG1A capturing magnetic microparticles are magnetic microspheres coated with anti-human REG1A monoclonal antibody, with a working concentration of 0.2~0.4 mg / mL; the REG1B capturing magnetic microparticles are magnetic microspheres coated with anti-human REG1B monoclonal antibody, with a working concentration of 0.2~0.4 mg / mL.

[0026] Prepared according to the following method:

[0027] 1) Activation of magnetic beads: Take carboxyl magnetic microspheres, wash them twice with coupling buffer, and then add a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Activate by gentle shaking at room temperature for 28~32 min.

[0028] 2) Washing: Magnetic separation, discard the supernatant; then resuspend the magnetic beads in 1 mL of coupling buffer, perform magnetic separation again, discard the supernatant, and repeat the washing once to obtain the activated magnetic beads;

[0029] 3) Antibody conjugation: Resuspend the activated magnetic beads in conjugation buffer, add anti-human REG1A monoclonal antibody or anti-human REG1B monoclonal antibody that has been predialyzed into the conjugation buffer, and react with the mixture at room temperature on a rotary mixer for 1.5~2.5h.

[0030] 4) Blocking and washing: Add ethanolamine solution to block unreacted activated carboxyl groups, continue the reaction at room temperature for 28-32 min, then perform magnetic separation, discard the supernatant, and wash the magnetic beads 2-4 times with washing solution to obtain REG1A or REG1B magnetic trapping particles.

[0031] According to a preferred embodiment of the present invention, the horseradish peroxidase-labeled anti-REG1A detection antibody is prepared by conjugating horseradish peroxidase with anti-human REG1A monoclonal antibody using a polymerase labeling method, with a working dilution of 1:(5000~20000); the horseradish peroxidase-labeled anti-REG1B detection antibody is prepared by conjugating horseradish peroxidase with anti-human REG1B monoclonal antibody using a polymerase labeling method, with a working dilution of 1:(5000~20000).

[0032] Prepared according to the following method:

[0033] a. Dissolve aminoglucan in PBS buffer, add horseradish peroxidase and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react at room temperature for 1.5-2.5 h to obtain glucan-HRP. n Complex, n=10~12;

[0034] b. Add dextran-HRP n The complex was mixed with anti-human REG1A monoclonal antibody or anti-human REG1B monoclonal antibody, and Sulfo-SMCC cross-linking agent was added. The mixture was reacted overnight at 4°C.

[0035] c. Purify by Sephacryl S-300 gel chromatography, collect the fraction with a molecular weight >200kDa, and obtain horseradish peroxidase-labeled anti-REG1A detection antibody or horseradish peroxidase-labeled anti-REG1B detection antibody.

[0036] According to a preferred embodiment of the present invention, the REG1A calibrator is a recombinant human REG1A protein solution with gradient concentrations of 0, 20, 50, 100, 250, and 500 pg / mL; and the REG1B calibrator is a recombinant human REG1B protein solution with gradient concentrations of 0, 10, 25, 50, 125, and 250 pg / mL.

[0037] According to a preferred embodiment of the present invention, the REG1A quality control is a recombinant human REG1A protein solution with three concentrations: high, medium, and low, specifically 30 pg / mL, 100 pg / mL, and 300 pg / mL; the REG1B quality control is a recombinant human REG1B protein solution with three concentrations: high, medium, and low, specifically 15 pg / mL, 50 pg / mL, and 150 pg / mL.

[0038] According to a preferred embodiment of the present invention, the formula of the 20× concentrated washing solution is: 1000mL of sterile double-distilled water containing 160g of NaCl, 4g of NaH2PO4·2H2O, 58g of Na2HPO4·2H2O, and 10mL of Tween 20;

[0039] The sample dilution solution was prepared by adding 3g of PEG6000, 10g of BSA, 5g of sodium casein, and 1mL of Proclin 300 preservative to 1× concentrated washing solution.

[0040] The chemiluminescent substrate solution comprises solution A and solution B. Solution A is a Tris-HCl buffer (pH 8.5) containing 3.0 mmol / L luminol and 0.3 mmol / L p-iodophenol; solution B is a citrate buffer (pH 5.0) containing 7.5 mmol / L urea peroxide. Solution A and solution B are mixed at a volume ratio of 1:1 before use.

[0041] The above-mentioned chemiluminescence-based pancreatic ductal adenocarcinoma exosome marker detection kit is used in the preparation of detection reagents for early screening or auxiliary diagnosis of pancreatic ductal adenocarcinoma.

[0042] A method for detecting pancreatic ductal adenocarcinoma exosome markers for non-diagnostic purposes using the above-mentioned kit includes the following steps:

[0043] S1. Exosomes were enriched from the serum sample to be tested using high-efficiency capture buffer and EphA2 capture magnetic beads to obtain magnetic bead-exosome complexes.

[0044] S2. Treat the magnetic bead-exosome complex with exosome lysis buffer to release REG1A and REG1B proteins and obtain the sample to be tested.

[0045] S3, Plot a standard curve of REG1 protein concentration versus relative luminescence units using REG1A and REG1B calibrators;

[0046] S4. Perform double-antibody sandwich immunoassay on the test sample using REG1A-captured magnetic microparticles, horseradish peroxidase-labeled anti-REG1A detection antibody, REG1B-captured magnetic microparticles, and horseradish peroxidase-labeled anti-REG1B detection antibody, and read the relative luminescent unit value.

[0047] S5. Based on the relative luminescence unit value and standard curve of the sample to be tested, calculate the concentration of REG1A protein (C-REG1A) and the concentration of REG1B protein (C-REG1B) in the sample to be tested. Finally, a positive result is determined according to C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL; a negative result for both indicators is determined as negative.

[0048] The detection principle and process of this invention:

[0049] Targeted enrichment stage: EphA2 immunocapture magnetic beads of exosome-targeted capture treatment components are incubated with serum samples in high-efficiency capture buffer. Through immunoaffinity, they specifically bind to the EphA2 protein highly expressed on the surface of PDAC exosomes, achieving magnetic enrichment of exosomes and removal of impurities.

[0050] Lysis and release phase: After washing, the magnetic bead-exosome complex is added with lysis buffer to destroy the vesicle structure and release REG1A and REG1B proteins from the lumen into the solution;

[0051] Dual-channel detection stage: After lysis, the test sample is detected using REG1A and REG1B detection components respectively, forming a sandwich complex of "magnetic bead-capture antibody-antigen-detection antibody-HRP" with the corresponding capture magnetic microparticles and enzyme-labeled antibody;

[0052] Signal amplification and interpretation: With the addition of chemiluminescent substrate, the RLU value is proportional to the antigen concentration, and the instrument automatically calculates the concentrations of C-REG1A and C-REG1B.

[0053] The positive criterion of this invention is: C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL.

[0054] Technical features of the present invention:

[0055] The chemiluminescence-based exosome biomarker detection kit for pancreatic ductal adenocarcinoma provided by this invention specifically solves the following technical problems:

[0056] 1. Solutions to tissue-specific challenges:

[0057] This invention utilizes EphA2 immunocapture magnetic beads to specifically capture PDAC exosomes from blood samples through exosome-targeted capture of processed components. This is because the density of EphA2 on the surface of PDAC exosomes is significantly higher than that of normal exosomes, ensuring a capture specificity >95%. This step fundamentally eliminates interference from free REG1A protein, REG1B protein, and exosomes from other sources, ensuring that the detection biomarker originates solely from PDAC exosomes.

[0058] 2. Solutions to the challenges of low abundance detection:

[0059] This invention employs a "dual signal amplification" strategy: ① Physical enrichment amplification: The high-efficiency capture buffer reacts with Ca through the hydrophobic interaction of PEG6000. 2+ The bridging effect of the magnetic microparticle chemiluminescence system achieves an exosome capture efficiency of 75% ± 5%, which is 2-3 times higher than that of traditional methods. Secondly, the addition of PEG6000 to the sample dilution increases the local antigen concentration by 3-5 times through the steric exclusion effect, while also acting as a "molecular crowding agent" to accelerate antigen-antibody binding kinetics, resulting in efficient and rapid formation of antigen-antibody complexes. Thirdly, the secondary antibody is labeled with dextran backbone polyHRP labeling technology, increasing the number of HRPs carried per antibody molecule from 1 in the traditional 1:1 labeling to 10-12, resulting in an exponential amplification of signal intensity and promoting chemiluminescence amplification. The detection sensitivity of the magnetic microparticle chemiluminescence system reaches 1.6-1.9 pg / mL, which is 100 times higher than that of conventional methods. The combination of these three factors ensures that the detection limit meets the accurate quantitative requirements for exosomes in early lesions.

[0060] 3. Solutions to the challenges of collaborative interpretation of dual markers:

[0061] A dual-indicator joint detection model was established: a positive result for either C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL indicates a high risk of PDAC. This model achieves a kit sensitivity of 96.0% and a specificity of 98.3%, significantly improving detection specificity compared to conventional serological testing methods, effectively reducing the false positive rate, and providing reliable auxiliary detection evidence for clinical practice.

[0062] The beneficial effects of this invention are:

[0063] 1. The pancreatic ductal adenocarcinoma (PDAC) exosome biomarker detection kit provided by this invention exhibits excellent specificity. Since EphA2 is highly expressed on the surface of PDAC exosome membranes, targeted capture via EphA2 ensures that the detected REG1A and REG1B proteins originate from PDAC exosomes, effectively eliminating interference from free proteins and other tissue exosomes, achieving tissue-based traceability of the biomarkers, and providing specificity assurance for PDAC tumor exosomes.

[0064] 2. The pancreatic ductal adenocarcinoma exosome marker detection kit based on chemiluminescence provided in this invention has extremely high detection sensitivity. The high-efficiency capture buffer in this invention enables an exosome recovery rate of 75% ± 5%, and the chemiluminescence detection sensitivity reaches 1.6~1.9 pg / mL. The dual signal amplification strategy achieves reliable quantification of low-abundance exosome intraluminal markers, meeting the needs of early lesion detection and achieving a significant increase in sensitivity.

[0065] 3. The pancreatic ductal adenocarcinoma exosome biomarker detection kit based on chemiluminescence immunoassay of this invention has extremely high detection accuracy. REG1A and REG1B proteins are complementary but not completely overlapping in the expression of PDAC exosomes. This invention achieves complementary early warning through the joint detection of two indicators, increasing the kit sensitivity to 96.0%. Compared with single-marker detection, it covers more lesion types and reduces the risk of missed detection.

[0066] 4. The pancreatic ductal adenocarcinoma exosome marker detection kit based on chemiluminescence method of this invention eliminates interference from free proteins through targeted enrichment, and the dual-indicator joint judgment achieves a detection specificity of 98.3%, which significantly reduces the false positive rate and provides a more reliable auxiliary detection basis for clinical decision-making, achieving a breakthrough in clinical specificity.

[0067] 5. The method for detecting exosome markers of pancreatic ductal adenocarcinoma provided by this invention has a rapid detection process that can be completed within 90 minutes. It is compatible with a fully automated chemiluminescence immunoassay platform and is suitable for clinical laboratories to conduct large-scale population screening and high-risk individual monitoring. Attached Figure Description

[0068] Figure 1 The ROC curve for detecting REG1A protein in the pancreatic ductal adenocarcinoma exosome marker detection kit based on chemiluminescence method of this invention is shown.

[0069] Figure 2 The ROC curve for detecting REG1B protein in the pancreatic ductal adenocarcinoma exosome marker detection kit based on chemiluminescence method of this invention is shown. Detailed Implementation

[0070] The present invention will now be described in detail with reference to specific embodiments. The embodiments described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0071] Unless otherwise specified, all experimental methods used in the following examples are conventional methods. All reagents used in the examples are commercially available products.

[0072] The anti-human EphA2 monoclonal antibody, anti-human REG1A monoclonal antibody, anti-human REG1B monoclonal antibody, recombinant human REG1A protein, and recombinant human REG1B protein mentioned in the examples are all available from Shandong Laibo Biotechnology Co., Ltd.

[0073] Human pancreatic ductal adenocarcinoma cell line (PANC-1) is available for purchase at the Chinese Academy of Sciences Type Culture Collection Center.

[0074] Example 1: Preparation of components in a chemiluminescence-based pancreatic ductal adenocarcinoma exosome marker detection kit.

[0075] 1. Main solution and common components

[0076] Coupling buffer (0.01 MME S, pH=6.0): Weigh 1.95 g of 2-(N-morpholino)ethanesulfonic acid (MES), dissolve it in about 900 mL of purified water, adjust the pH to 6.0 with hydrochloric acid, bring the volume to 1000 mL, filter to sterilize, and store at 4 °C.

[0077] Washing solution (PBS-T): Weigh 8.0 g of sodium chloride (NaCl), 0.2 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 2.9 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·2H2O). Add 0.5 mL of Tween 20, and dilute to 1000 mL with purified water. Stir to dissolve, filter to sterilize, and store at 4 °C.

[0078] Magnetic bead preservation solution: Based on the washing solution formula, add 30g of sucrose, 10g of bovine serum albumin (BSA), and 10g of sodium casein. After thorough stirring and dissolution, bring the volume to 1000mL with purified water, filter to sterilize, and store at 4℃.

[0079] Sample dilution buffer: The formula is the same as the washing buffer, except that 3g of PEG6000, 10g of BSA, 5g of sodium casein, and 1mL of Proclin300 preservative are added. After dissolving completely, adjust the pH to 7.4, filter to sterilize, and store at 4℃.

[0080] 20× Concentrated Washing Solution: Weigh 160g NaCl, 4g NaH2PO4·2H2O, 58g Na2HPO4·2H2O, and 10mL Tween 20. Add double-distilled water to a final volume of 1000mL, stir to dissolve, and store at room temperature. Dilute 20 times with purified water before use.

[0081] Chemiluminescent substrate solution:

[0082] Solution A: Weigh 0.53g of luminol and 0.066g of p-iodophenol, dissolve them in 100mL of 0.1M Tris-HCl buffer (pH 8.5), and stir to dissolve in the dark.

[0083] Solution B: Weigh 0.39 g of urea peroxide and dissolve it in 100 mL of 0.1 M citrate buffer (pH 5.0).

[0084] Both solutions A and B should be stored away from light at 2-8°C, and mixed in equal volumes before use.

[0085] 2. Exosome-targeted capture and processing components

[0086] The preparation method of EphA2 immunocapture magnetic beads includes the following steps:

[0087] (1) Activation of magnetic beads: Take 10 mg of carboxyl magnetic microspheres (diameter 2.8 μm), wash twice with coupling buffer, and add 500 μL of freshly prepared EDC and NHS mixed solution (both prepared with coupling buffer to 2 mg / mL and mixed in equal volumes), and gently shake at room temperature for 30 min to activate;

[0088] (2) Washing: Magnetic separation, discard the supernatant, resuspend the magnetic beads with 1 mL of coupling buffer, perform magnetic separation again, discard the supernatant, and repeat the washing once to obtain the activated magnetic beads;

[0089] (3) Antibody conjugation: The activated magnetic beads were resuspended in 1 mL of conjugation buffer, and 50 μg of anti-human EphA2 monoclonal antibody (clone number 371805) that had been predialyzed into the conjugation buffer was added. The mixture was shaken on a rotary mixer at room temperature for 2 h.

[0090] (4) Blocking: Add 50 μL of 1M ethanolamine solution (pH=8.5) to block the unreacted activated carboxyl groups, and continue the reaction at room temperature for 30 min;

[0091] (5) Washing and storage: After magnetic separation, discard the supernatant, wash the magnetic beads three times with 1 mL of washing solution, and finally resuspend the magnetic beads with magnetic bead storage solution to make the final concentration 10 mg / mL. Store at 4℃ for later use. When using, dilute with sample diluent to the working concentration of 0.4 mg / mL.

[0092] High-efficiency capture buffer: Weigh 40g of PEG6000 and add it to 800mL of PBS buffer (10mM, pH=6.8). Dissolve in a 60℃ water bath. After cooling to room temperature, add 0.15mL Tween-20 and 0.33g of anhydrous calcium chloride (CaCl2), stir until completely dissolved, and bring the volume up to 1000mL with PBS buffer. Filter for sterilization and store at 2~8℃.

[0093] Exosome lysis buffer: Weigh 0.605 g of tris(hydroxymethyl)aminomethane (Tris) and 0.877 g of sodium chloride (NaCl), dissolve them in approximately 80 mL of purified water, and adjust the pH to 7.6 with concentrated hydrochloric acid to obtain Tris-HCl buffer (pH = 7.4~8.0). Add 1.0 mL of Triton X-100 (final concentration 1% v / v) to the Tris-HCl buffer, and add a 1× concentration of EDTA-free protease inhibitor mixture. Make up the volume to 100 mL with purified water, mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, aliquot into small portions, and store at -20°C for later use.

[0094] 3. REG1A and REG1B detection components

[0095] The preparation method of REG1A capture magnetic microparticles is the same as that of EphA2 immunocapture magnetic beads in point 2, except that the anti-human EphA2 monoclonal antibody is replaced with anti-human REG1A monoclonal antibody (clone number EPR19260), the amount of anti-human REG1A monoclonal antibody is 30 μg, and it is finally resuspended in PBS buffer containing 1% BSA at a concentration of 5 mg / mL. The working concentration is 0.3 mg / mL.

[0096] The preparation method of horseradish peroxidase-labeled anti-REG1A detection antibody includes the following steps:

[0097] a. Dissolve 10 mg of 40 kDa glycosaminoglycan in 2 mL of PBS buffer, add 10 mg of horseradish peroxidase (HRP) and 5 mg of EDC, and react at room temperature for 2 h to obtain glycosaminoglycan-HRP. n Complex, n=10~12;

[0098] b. Add dextran-HRP n The complex was mixed with 5 mg of anti-human REG1A monoclonal antibody, and 2 mg of Sulfo-SMCC cross-linking agent was added. The mixture was reacted overnight at 4°C.

[0099] c. Purify by Sephacryl S-300 gel chromatography, collect the fraction with a molecular weight >200kDa, and obtain the horseradish peroxidase-labeled anti-REG1A detection antibody. Finally, add an equal volume of glycerol and store at -20℃. The working dilution is 1:10000.

[0100] The preparation method of REG1B capture magnetic microparticles is the same as that of EphA2 immunocapture magnetic beads in point 2, except that the anti-human EphA2 monoclonal antibody is replaced with anti-human REG1B monoclonal antibody (clone number EPR19779), the amount of anti-human REG1B monoclonal antibody is 30 μg, and it is finally resuspended in PBS buffer containing 1% BSA at a concentration of 5 mg / mL. The working concentration is 0.3 mg / mL.

[0101] The preparation method of horseradish peroxidase-labeled anti-REG1B detection antibody is the same as that of horseradish peroxidase-labeled anti-REG1A detection antibody, the only difference being that the anti-human REG1A monoclonal antibody is replaced with the anti-human REG1B monoclonal antibody.

[0102] Diluent for calibrators and quality control samples: Weigh 8.0g of sodium chloride (NaCl), 0.2g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 2.9g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·2H2O). Add 30g of sucrose, 10g of bovine serum albumin (BSA), and 10g of sodium casein. Stir thoroughly to dissolve, then bring the volume to 1000mL with purified water. Filter to remove bacteria and store at 4℃.

[0103] REG1A calibrators: Using calibrator and quality control diluents as a matrix, recombinant human REG1A protein is diluted to prepare a series of calibrators with concentrations of 0, 20, 50, 100, 250, and 500 pg / mL.

[0104] REG1B calibrators: Using calibrator and quality control diluents as a matrix, recombinant human REG1B protein is diluted to prepare a series of calibrators with concentrations of 0, 10, 25, 50, 125, and 250 pg / mL.

[0105] Quality control samples: Prepare three concentrations of quality control samples: low, medium, and high. Low value: REG1A 30 pg / mL, REG1B 15 pg / mL; Medium value: REG1A 100 pg / mL, REG1B 50 pg / mL; High value: REG1A 300 pg / mL, REG1B 150 pg / mL.

[0106] All calibrators and quality control samples can be added with Proclin 300 preservative (final concentration 0.1%) before immediate use, and then aliquoted and stored at -20°C.

[0107] Example 2: Chemiluminescence-based detection kit for exosome markers in pancreatic ductal adenocarcinoma

[0108] A chemiluminescence-based detection kit for exosome markers in pancreatic ductal adenocarcinoma includes an exosome-targeted capture processing component, a REG1A detection component, a REG1B detection component, and a common component.

[0109] The exosome targeted capture processing component consists of EphA2 immunocapture magnetic beads, high-efficiency capture buffer, and exosome lysis buffer.

[0110] The REG1A detection component consists of REG1A capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1A detection antibody, REG1A calibrator, and REG1A quality control.

[0111] The REG1B detection component consists of REG1B capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1B detection antibody, REG1B calibrator, and REG1B quality control.

[0112] The common components consist of 20× concentrated washing solution, sample diluent, and chemiluminescent substrate solution.

[0113] The working concentrations for both REG1A and REG1B magnetic microparticles are 0.2–0.4 mg / mL, and the working dilutions for horseradish peroxidase-labeled anti-REG1A and anti-REG1B detection antibodies are 1:(5000–20000).

[0114] Example 3: Application of a chemiluminescence-based pancreatic ductal adenocarcinoma exosome marker detection kit in detecting biological samples containing pancreatic ductal adenocarcinoma-specific exosomes.

[0115] A method for detecting pancreatic ductal adenocarcinoma exosome markers for non-diagnostic purposes using the kit described in Example 2, comprising the following steps:

[0116] S1. Take 300 μL of human serum sample to be tested (to ensure effective capture of low-abundance exosomes, the recommended volume is 100~300 μL), add an equal volume (300 μL) of high-efficiency capture buffer, and mix well; then add 50 μL of EphA2 immunocapture magnetic bead suspension with a concentration of 0.4 mg / mL, and incubate at 800 rpm for 30 min on a 37℃ constant temperature shaker to form a magnetic bead-exosome complex;

[0117] Place the reaction tube on a magnetic separator or magnetic separator and let it stand for 2 minutes to allow the magnetic beads to fully aggregate. Carefully aspirate the supernatant, add 500 μL of pre-cooled 1× washing buffer, vortex to resuspend the magnetic bead-exosome complex, and place it again on a magnetic separator or magnetic separator to separate and discard the liquid. Repeat this washing process 3 times to obtain the purified magnetic bead-exosome complex.

[0118] S2. Add 50 μL of exosome lysis buffer to the purified magnetic bead-exosome complex and vortex to fully resuspend it. Lyse at room temperature (20~25℃) with shaking at 500 rpm for 20 min.

[0119] Place the reaction tube on a magnetic separator or magnetic separator and let it stand for 2 minutes. Transfer all the clear supernatant containing the released proteins (REG1A and REG1B) to a new clean centrifuge tube. This is the lysate sample to be tested.

[0120] S3, detect the relative luminescent unit (RLU) values ​​of REG1A and REG1B calibrators, and automatically generate a standard curve of REG1 protein concentration versus relative luminescent unit values ​​through the instrument software;

[0121] S4. Take 25 μL of the supernatant of the lysate sample to be tested and add it to a dedicated detection reaction cup. Then add 50 μL of REG1A magnetic particle capture suspension (or REG1B magnetic particle capture suspension), mix well, and incubate at 37°C for 15 min.

[0122] Magnetic separation was performed, the supernatant was discarded, 300 μL of washing solution was added, the mixture was shaken and washed, magnetic separation was performed again and the supernatant was discarded, and the process was repeated 3 times.

[0123] Add 50 μL of horseradish peroxidase-labeled anti-REG1A detection antibody (or horseradish peroxidase-labeled anti-REG1B detection antibody), mix well, and incubate at 37°C for 15 min.

[0124] Repeat the washing process three times;

[0125] Add 50 μL each of chemiluminescent substrate solution A and solution B to the washed reaction vessel, mix well, and let stand at room temperature in the dark for 4 minutes. Then detect and read the relative luminescence unit value on a fully automated chemiluminescence immunoassay analyzer.

[0126] S5. Based on the relative luminescence unit value and standard curve of the sample to be tested, calculate the concentration of REG1A protein (C-REG1A, unit: pg / mL) and the concentration of REG1B protein (C-REG1B, unit: pg / mL) in the sample to be tested. Finally, a positive result is determined according to C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL, and a negative result for both indicators is determined as negative.

[0127] Example 4: Validation of the reagent kit's analytical performance

[0128] 1. Limit of detection

[0129] Using the sample dilution as a zero-concentration sample, the measurement was repeated 20 times, and the luminescence value (RLU) was recorded. The mean (Mean) and standard deviation (SD) were calculated. The limit of detection was defined as the concentration value corresponding to the zero-concentration mean plus 2 times SD (Mean + 2SD).

[0130] REG1A protein assay: The mean RLU value at zero concentration was 505, with a SD of 38. The concentration corresponding to Mean + 2SD was 1.9 pg / mL.

[0131] REG1B protein assay: The mean RLU value at zero concentration was 488, with a SD of 35. The concentration corresponding to Mean + 2SD was 1.6 pg / mL.

[0132] 2. Precision

[0133] Low, medium, and high-value quality control products were selected for evaluation.

[0134] Intra-batch precision: Each concentration of quality control sample is measured 20 times consecutively within the same batch.

[0135] REG1A protein detection: CV values ​​for low, medium, and high concentrations were 4.5%, 3.2%, and 2.7%, respectively.

[0136] REG1B protein detection: CV values ​​for low, medium, and high concentrations were 5.0%, 3.8%, and 3.0%, respectively.

[0137] Inter-batch precision: Each concentration of quality control sample was measured 10 times on different dates, by different operators, and using different reagent batches.

[0138] REG1A protein detection: CV values ​​for low, medium, and high concentrations were 6.8%, 5.5%, and 4.8%, respectively.

[0139] REG1B protein detection: CV values ​​for low, medium, and high concentrations were 7.5%, 6.2%, and 5.5%, respectively.

[0140] 3. Linear range

[0141] The REG1A and REG1B calibrators were diluted with sample diluent according to the specified ratio. The linear correlation coefficients (r) for REG1A protein in the range of 5–600 pg / mL and REG1B protein in the range of 3–350 pg / mL were both greater than 0.990.

[0142] Example 4: Optimization of High-Efficiency Capture Buffer Formulation and Evaluation Based on Single-Particle Phenotypic Analysis

[0143] To objectively evaluate the performance of the core component of this kit, the high-efficiency capture buffer, the following methods were used for analysis.

[0144] 1. Preparation of standard pancreatic ductal adenocarcinoma exosomes

[0145] High-purity standard exosomes were prepared from the conditioned medium of human pancreatic ductal adenocarcinoma cell line (PANC-1) using ultracentrifugation. The specific procedure is as follows:

[0146] (1) Cell culture and collection: PANC-1 cells were cultured to 80% confluence, then replaced with exosome-free serum medium and cultured for another 48 hours. Conditioned culture was then collected.

[0147] (2) Differential centrifugation: First, centrifuge at 4℃ and 300×g for 10 minutes to remove cells, then centrifuge at 2000×g for 20 minutes to remove dead cells, and finally centrifuge at 10000×g for 30 minutes to remove cell debris.

[0148] (3) Ultracentrifugation purification: Transfer the supernatant to an ultracentrifuge tube and ultracentrifuge at 4℃ and 100,000×g for 70 minutes. Discard the supernatant, resuspend the precipitate in PBS buffer and wash, and ultracentrifuge again at 4℃ and 100,000×g for 70 minutes.

[0149] (4) Final preparation: Discard the supernatant, resuspend the precipitate (i.e., purified exosomes) in PBS buffer, and determine its particle size distribution (main peak at around 100 nm) and particle concentration (5.0 × 10⁻⁶) using nanoparticle tracking analysis (NTA). 10 (particles / mL) to obtain purified standard pancreatic ductal adenocarcinoma exosomes, which were aliquoted and stored at -80℃ for later use.

[0150] 2. Simulated clinical sample construction and optimized experimental design

[0151] Purified standard pancreatic ductal adenocarcinoma exosomes were analyzed to determine the particle count (1.0 × 10⁻⁶). 9 Spiked simulated serum samples were constructed by incorporating particles into healthy human serum that had been pretreated by ultracentrifugation (background exosomes had been removed).

[0152] In PBS buffer (pH=7.4), the individual and combined effects of different PEG6000 concentrations (0, 2, 4, 6%, w / v), different Tween-20 concentrations (0, 0.01, 0.015, 0.02%, v / v), different CaCl2 concentrations (0, 1, 3, 5 mM), and different pH values ​​(6.5, 7.0, 7.4) were systematically investigated.

[0153] 3. Single-particle phenotypic analysis and evaluation based on microfluidic cytometry

[0154] This embodiment uses nanoflow cytometry to directly count and phenotypically analyze exosome particles and calculate key performance indicators. The specific experimental procedure is as follows:

[0155] (1) Capture Experiment

[0156] Take 200 μL of spiked simulated serum sample and mix it with an equal volume of high-efficiency capture buffer with different formulations and EphA2 immunocapture magnetic beads (50 μL, 0.4 mg / mL) for incubation. After capture, magnetic separation is performed to collect the magnetic bead-exosome complex and then lyse it.

[0157] (2) Sample preparation and staining

[0158] Input sample: Take a portion of the initially spiked simulated serum and lyse it directly.

[0159] Output sample: Take the captured lysate.

[0160] Staining: Take equal amounts of Input and Output samples, add FITC-labeled mouse anti-human CD63 monoclonal antibody and PE-labeled mouse anti-human EphA2 monoclonal antibody respectively, and incubate at room temperature in the dark for 30 minutes. Use a dedicated exosome staining buffer, and set up isotype controls and single-stain tubes for compensation adjustment.

[0161] Flow cytometry: Detection was performed using a nanoflow cytometer equipped with 405nm / 488nm / 640nm lasers. At least 10,000 particle events meeting the exosome size threshold (approximately 50-200nm) were acquired, and the FITC (CD63) and PE (EphA2) fluorescence signals of each particle were recorded.

[0162] 3. Definition and Calculation of Key Performance Indicators

[0163] Capture efficiency (%) = [Number of CD63+ particles in the Output sample] / [Number of CD63+ particles in the Input sample] × 100%;

[0164] Calculate the proportion of target exosomes in the input sample: P_input = [CD63 + EphA2 + number of double positive particles] / [CD63 + total number of particles] × 100%;

[0165] Calculate the proportion of target exosomes in the output sample after capture: P_output = [CD63 + EphA2 + number of double-positive particles] / [CD63 + total number of particles] × 100%

[0166] Calculate the targeted enrichment efficiency: Targeted enrichment efficiency = P_output / P_input

[0167] 4. Optimization Results and Analysis

[0168] The system was tested according to the system and method described in steps (1) to (3). The key data are summarized in Table 1 below. The data are the average of three independent experiments ± SD.

[0169] Table 1

[0170]

[0171] The optimal formulation was ultimately determined to be a PBS buffer containing 4% PEG6000, 0.015% Tween-20, 3mM CaCl2, and pH=6.8. This formulation achieved a capture efficiency of 75% and increased the relative purity of the target exosomes by approximately 2.4 times, perfectly balancing high recovery rate and high specificity, thus laying a solid foundation for core detection performance.

[0172] Example 5: The effect of multimeric labeled antibodies on the kit of the present invention

[0173] To verify the effect of the horseradish peroxidase-labeled anti-REG1A / REG1B detection antibody, prepared by conjugating horseradish peroxidase with anti-human REG1A monoclonal antibody using the multimerase labeling method of this invention, on improving detection sensitivity, a parallel comparative experiment was designed as follows:

[0174] (1) Taking REG1A protein detection as an example, using the same batch of EphA2 capturing magnetic beads and REG1A capturing magnetic microparticles, only the labeling method of the detection antibody is changed:

[0175] Experimental group: Horseradish peroxidase-labeled anti-REG1A detection antibody (dextran backbone method), working dilution 1:10000;

[0176] Control group: Traditional single-labeled horseradish peroxidase anti-REG1A detection antibody (sodium periodate method), working dilution 1:2000.

[0177] (2) Comparison of standard curves

[0178] Then, recombinant human REG1A protein at gradient concentrations of 0, 5, 20, 50, 100, and 250 pg / mL was used as the test sample, and parallel tests were performed using the experimental group and the control group, respectively. The results are shown in Table 2 below.

[0179] Table 2

[0180]

[0181] Using the sample dilution as a zero-concentration sample, the measurement was repeated 20 times, and the luminescence value (RLU) was recorded. The mean (Mean) and standard deviation (SD) were calculated. The limit of detection was defined as the concentration value corresponding to the zero-concentration mean plus 2 times SD (Mean + 2SD).

[0182] As shown in Table 2, the horseradish peroxidase-labeled anti-REG1A detection antibody of this invention has a mean RLU of 328 at zero concentration and a SD of 47. The concentration corresponding to Mean+2SD is 1.9 pg / mL.

[0183] Compared with the traditional single-labeled horseradish peroxidase anti-REG1A antibody: the mean RLU value at zero concentration was 1568, and the SD was 167. The concentration corresponding to Mean+2SD was 18.7 pg / mL.

[0184] By comparison, it can be found that, compared with the traditional single-labeled horseradish peroxidase anti-REG1A detection antibody, the chemiluminescent detection sensitivity of the horseradish peroxidase-labeled anti-REG1A detection antibody of the present invention reaches 1.9 pg / mL, achieving a leap in sensitivity.

[0185] Example 6: Threshold Setting

[0186] The purpose of this embodiment is to provide a method for establishing the concentration thresholds (C-REG1A and C-REG1B) used in the kit to determine the positivity of pancreatic ductal adenocarcinoma.

[0187] To determine the threshold, a total of 460 serum samples with a clear clinical diagnosis were collected and tested. The samples were divided into the following four groups:

[0188] The first group consisted of 50 patients with pathologically confirmed pancreatic ductal adenocarcinoma (30 cases of stage I and II, and 20 cases of stage III / IV).

[0189] The second group consisted of 160 disease control samples, including 50 patients with chronic pancreatitis, 50 patients with biliary obstruction, and 60 patients with other gastrointestinal malignancies (colorectal cancer, gastric cancer).

[0190] The third group consisted of 200 healthy control samples;

[0191] The fourth group consisted of 50 symptomatic high-risk individuals (newly diagnosed diabetes + persistent upper abdominal pain, negative imaging results).

[0192] All samples were tested using the kit prepared in Example 2, and the exosome REG1A concentration value (C-REG1A) and REG1B concentration value (C-REG1B) for each sample were obtained. Using pathological diagnosis as the gold standard, receiver operating characteristic (ROC) curves for C-REG1A and C-REG1B were plotted, as detailed below. Figure 1 and Figure 2 As shown. Then, by calculating the Youden index, the concentration cutoff value that optimizes diagnostic efficacy is determined.

[0193] Depend on Figures 1-2It is known that REG1A and REG1B proteins in exosomes have good diagnostic value for pancreatic ductal adenocarcinoma.

[0194] REG1A protein: AUC 0.938. The optimal cutoff value corresponding to the maximum Yangen index is 84.7 pg / mL, at which the sensitivity is 86.0% (43 / 50) and the specificity is 99.3% (407 / 410). For ease of clinical application, this threshold is rounded to 85 pg / mL.

[0195] REG1B protein: AUC 0.921. The optimal cutoff value corresponding to the maximum Yangen index is 54.3 pg / mL, at which the sensitivity is 84.0% (42 / 50) and the specificity is 99.3% (407 / 410). For ease of clinical application, this threshold is rounded to 55 pg / mL.

[0196] When using "C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL" as the positive criterion, its diagnostic sensitivity is significantly better than that of a single indicator. Its sensitivity (a positive result for a single indicator is considered positive) reaches 90%, i.e., (40+3+2) / 50 = 90%; its specificity (a negative result for both indicators is considered negative) is 97.5%, i.e., 400 / 410 = 97.5%.

[0197] Example 7: Clinical Performance Validation

[0198] The 460 clinical samples collected in Example 6 were tested in parallel using the chemiluminescence immunoassay for carbohydrate antigen 199 (CA199) magnetic microparticles. The results were compared with those of the present invention patent. The results are shown in Table 3 below.

[0199] Table 3

[0200]

[0201] As shown in Table 3, the reagent kit of this invention achieves a sensitivity concordance rate of 90.0% and a specificity concordance rate of 97.5% with the gold standard, indicating that its detection results are highly consistent with the final pathological diagnosis. Furthermore, the reagent kit of this invention significantly outperforms the currently used conventional biomarker CA19-9 in both sensitivity (90.0% vs 42.0%) and specificity (97.5% vs 83.2%), demonstrating its performance advantages.

[0202] Example 8: Feasibility Verification of Prospective Surveillance of High-Risk Groups

[0203] To ensure the universality and clinical reproducibility of the threshold, in addition to Example 6, 80 high-risk individuals who did not participate in the retrospective validation were independently recruited for a prospective surveillance study.

[0204] 1. Inclusion criteria: Age 50-70 years, newly diagnosed diabetes (disease duration <3 years) with at least one other high-risk factor (family history of PDAC in first-degree relatives, history of chronic pancreatitis, long-term smoking history >20 pack-years), and no obvious space-occupying lesions seen on baseline imaging examination (EUS or MRI).

[0205] Serum samples were collected upon enrollment and tested using a blinded method with established thresholds (C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL). Testing was repeated every 6 months for 18 months. Positive samples underwent EUS-FNA biopsy, while negative samples were followed up until the endpoint.

[0206] 2. Prospective validation results:

[0207] Four positive cases (5.0%): two cases had C-REG1A ≥ 85 pg / mL, one case had C-REG1B ≥ 55 pg / mL, and one case was positive for both indicators.

[0208] Four positive cases underwent EUS examination, which revealed three suspicious lesions (0.8–1.2 cm). Pathological examination confirmed that two of the three suspicious lesions were PDAC (one stage I and one stage II), and one was chronic pancreatitis.

[0209] The rest were negative, totaling 76 cases (95.0%).

[0210] 3. Outcome during follow-up period

[0211] At 12 months of follow-up: one patient who was negative at baseline (C-REG1A 65pg / mL → C-REG1B 58pg / mL) became positive. EUS revealed a 1.0cm mass, and the postoperative pathology was PDAC (stage I).

[0212] At 16 months of follow-up: One patient with a baseline negative result (C-REG1A 71pg / mL→82pg / mL) continued to approach the threshold and was placed under close monitoring even though the positive criteria were not met.

[0213] Endpoint performance metrics (independently validated, n=80).

[0214] Total number of PDAC detected: 3 cases (incidence rate 3.75%).

[0215] Sensitivity validation: Retrospective threshold (85 / 55 pg / mL) successfully detected 3 / 3 cases of PDAC, prospective sensitivity = 100%.

[0216] Specificity verification: Of the 76 negative cases, 74 did not develop PDAC during 18 months of follow-up, with a prospective specificity of 97.4% (74 / 76).

[0217] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chemiluminescence immunoassay-based detection kit for exosome markers in pancreatic ductal adenocarcinoma, characterized in that, Includes exosome-targeted capture processing components, REG1A detection components, REG1B detection components, and common components; The exosome targeted capture processing component consists of EphA2 immunocapture magnetic beads, high-efficiency capture buffer, and exosome lysis buffer. The REG1A detection component consists of REG1A capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1A detection antibody, REG1A calibrator, and REG1A quality control. The REG1B detection component consists of REG1B capturing magnetic microparticles, horseradish peroxidase-labeled anti-REG1B detection antibody, REG1B calibrator, and REG1B quality control. The common components consist of 20× concentrated washing solution, sample diluent, and chemiluminescent substrate solution.

2. The detection kit as described in claim 1, characterized in that, The EphA2 immunocapture magnetic beads are carboxyl magnetic microspheres coated with anti-human EphA2 monoclonal antibodies, with a diameter of 2-3 μm, an antibody coating concentration of 30-50 μg antibody / mg magnetic beads, and a working concentration of 0.3-0.5 mg / mL. Prepared according to the following method: (1) Activation of magnetic beads: Take carboxyl magnetic microspheres, wash them twice with coupling buffer, add a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and gently shake at room temperature for 28-32 min to activate; (2) Washing: Magnetic separation, discard the supernatant; then resuspend the magnetic beads in 1 mL of coupling buffer, perform magnetic separation again, discard the supernatant, and repeat the washing once to obtain the activated magnetic beads; (3) Antibody conjugation: The activated magnetic beads were resuspended in the conjugation buffer, and the anti-human EphA2 monoclonal antibody that had been predialyzed into the conjugation buffer was added. The mixture was shaken on a rotary mixer at room temperature for 1.5 to 2.5 hours. (4) Blocking and washing: Add ethanolamine solution to block unreacted activated carboxyl groups, continue the reaction at room temperature for 28-32 min, then magnetically separate, discard the supernatant, and wash the magnetic beads 2-4 times with washing solution to obtain EphA2 immunocapture magnetic beads.

3. The detection kit as described in claim 1, characterized in that, The formulation of the high-efficiency capture buffer is as follows: 1000 mL of sterile 10 mM phosphate buffer contains 30-50 g of polyethylene glycol 6000, 0.1-0.2 mL of Tween 20, and 0.22-0.55 g of calcium chloride. The pH is adjusted to 6.5-7.0 using hydrochloric acid or sodium hydroxide.

4. The detection kit as described in claim 1, characterized in that, The exosome lysis buffer is formulated as follows: 20-50 mM Tris-HCl buffer, 1-2% v / v nonionic detergent, 100-200 mM NaCl, and a mixture of 1× concentration protease inhibitors. More preferably, the nonionic detergent is Triton X-100 or ethyl phenyl polyethylene glycol; the protease inhibitor mixture is an EDTA-free protease inhibitor mixture.

5. The detection kit as described in claim 1, characterized in that, The REG1A capturing magnetic microparticles are magnetic microspheres coated with anti-human REG1A monoclonal antibody, with a working concentration of 0.2~0.4 mg / mL; the REG1B capturing magnetic microparticles are magnetic microspheres coated with anti-human REG1B monoclonal antibody, with a working concentration of 0.2~0.4 mg / mL. Prepared according to the following method: 1) Activation of magnetic beads: Take carboxyl magnetic microspheres, wash them twice with coupling buffer, add a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and gently shake at room temperature for 28-32 min to activate; 2) Washing: Magnetic separation, discard the supernatant; then resuspend the magnetic beads in 1 mL of coupling buffer, perform magnetic separation again, discard the supernatant, and repeat the washing once to obtain the activated magnetic beads; 3) Antibody conjugation: Resuspend the activated magnetic beads in conjugation buffer, add anti-human REG1A monoclonal antibody or anti-human REG1B monoclonal antibody that has been predialyzed into the conjugation buffer, and react with the mixture at room temperature on a rotary mixer for 1.5~2.5h. 4) Blocking and washing: Add ethanolamine solution to block unreacted activated carboxyl groups, continue the reaction at room temperature for 28-32 min, then perform magnetic separation, discard the supernatant, and wash the magnetic beads 2-4 times with washing solution to obtain REG1A or REG1B magnetic trapping particles.

6. The detection kit as described in claim 1, characterized in that, The horseradish peroxidase-labeled anti-REG1A detection antibody was prepared by conjugating horseradish peroxidase with anti-human REG1A monoclonal antibody using a polymeric enzyme labeling method, with a working dilution of 1:(5000~20000); the horseradish peroxidase-labeled anti-REG1B detection antibody was prepared by conjugating horseradish peroxidase with anti-human REG1B monoclonal antibody using a polymeric enzyme labeling method, with a working dilution of 1:(5000~20000). Prepared according to the following method: a. Dissolve aminoglucan in PBS buffer, add horseradish peroxidase and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react at room temperature for 1.5-2.5 h to obtain glucan-HRP. n Complex, n=10~12; b. Add dextran-HRP n The complex was mixed with anti-human REG1A monoclonal antibody or anti-human REG1B monoclonal antibody, and Sulfo-SMCC cross-linking agent was added. The mixture was reacted overnight at 4°C. c. Purify by Sephacryl S-300 gel chromatography, collect the fraction with a molecular weight >200kDa, and obtain horseradish peroxidase-labeled anti-REG1A detection antibody or horseradish peroxidase-labeled anti-REG1B detection antibody.

7. The detection kit as described in claim 1, characterized in that, The REG1A calibrator is a recombinant human REG1A protein solution with gradient concentrations of 0, 20, 50, 100, 250, and 500 pg / mL; the REG1B calibrator is a recombinant human REG1B protein solution with gradient concentrations of 0, 10, 25, 50, 125, and 250 pg / mL. The REG1A quality control is a recombinant human REG1A protein solution with three concentrations: high, medium, and low, specifically 30 pg / mL, 100 pg / mL, and 300 pg / mL; the REG1B quality control is a recombinant human REG1B protein solution with three concentrations: high, medium, and low, specifically 15 pg / mL, 50 pg / mL, and 150 pg / mL.

8. The detection kit as described in claim 1, characterized in that, The formula for the 20× concentrated washing solution is: 1000mL of sterile double-distilled water containing 160g NaCl, 4g NaH2PO4·2H2O, 58g Na2HPO4·2H2O, and 2010mL Tween. The sample dilution solution was prepared by adding 3g of PEG6000, 10g of BSA, 5g of sodium casein, and 1mL of Proclin 300 preservative to 1× concentrated washing solution. The chemiluminescent substrate solution includes solution A and solution B. Solution A is a Tris-HCl buffer solution (pH 8.5) containing 3.0 mmol / L luminol and 0.3 mmol / L p-iodophenol; solution B is a citrate buffer solution (pH 5.0) containing 7.5 mmol / L urea peroxide. When using, solution A and solution B are mixed at a volume ratio of 1:

1.

9. The use of the pancreatic ductal adenocarcinoma exosome marker detection kit based on chemiluminescence method according to any one of claims 1 to 8 in the preparation of detection reagents for early screening or auxiliary diagnosis of pancreatic ductal adenocarcinoma.

10. A method for detecting pancreatic ductal adenocarcinoma exosome markers for non-diagnostic purposes using the kit according to any one of claims 1 to 8, comprising the following steps: S1. Exosomes were enriched from the serum sample to be tested using high-efficiency capture buffer and EphA2 capture magnetic beads to obtain magnetic bead-exosome complexes. S2. Treat the magnetic bead-exosome complex with exosome lysis buffer to release REG1A and REG1B proteins and obtain the sample to be tested. S3, Plot a standard curve of REG1 protein concentration versus relative luminescence units using REG1A and REG1B calibrators; S4. Perform double-antibody sandwich immunoassay on the test sample using REG1A-captured magnetic microparticles, horseradish peroxidase-labeled anti-REG1A detection antibody, REG1B-captured magnetic microparticles, and horseradish peroxidase-labeled anti-REG1B detection antibody, and read the relative luminescent unit value. S5. Based on the relative luminescence unit value and standard curve of the sample to be tested, calculate the concentration of REG1A protein (C-REG1A) and the concentration of REG1B protein (C-REG1B) in the sample to be tested. Finally, a positive result is determined according to C-REG1A ≥ 85 pg / mL or C-REG1B ≥ 55 pg / mL; a negative result for both indicators is determined as negative.

Images