Products for early diagnosis of sepsis and use thereof

Abstract

The present application relates to the field of immunoassay technology, discloses a product for early diagnosis of sepsis and its application, the composition of the product for early diagnosis of sepsis includes detecting SAA1 protein and PDL1 + The product for detecting the content of exosome in sample is based on antigen-antibody specific immune binding to detect SAA1 protein and PDL1 + The product for detecting the content of exosome in sample, the detection method adopted by the detection product is selected from one of fluorescence immunoassay, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, chemiluminescence immunoassay, electrochemical immunoassay and nanopore-based immunosensing detection method, the product for early diagnosis of sepsis improves the accuracy of early diagnosis of sepsis and disease condition assessment in a double-index cooperative manner, effectively solves the technical bottleneck that the existing sepsis detection method is insufficient in sensitivity, poor in specificity and difficult to meet the requirements of early precise diagnosis and dynamic disease condition assessment.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to products and their applications for the early diagnosis of sepsis. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which can rapidly progress to severe sepsis, septic shock, and multiple organ dysfunction. It is one of the leading causes of death in critically ill patients. Early and accurate diagnosis and assessment of sepsis are crucial for improving treatment success rates and patient prognosis. Currently, clinical diagnosis of sepsis mainly relies on clinical symptoms, inflammatory marker detection, and etiological examination. However, traditional inflammatory markers such as C-reactive protein and procalcitonin have insufficient sensitivity and poor specificity. Single inflammatory marker detection is easily affected by non-infectious factors, while etiological examination is time-consuming and cannot meet the needs of early diagnosis. More accurate and efficient detection methods are needed.

[0003] Serum amyloid A1 (SAA1) is an acute-phase reactive protein whose serum concentration can rise sharply within hours during infection and inflammation, and the magnitude of the increase is positively correlated with the severity of sepsis. It is a potentially highly sensitive biomarker for the early diagnosis of sepsis. Programmed death-ligand 1-positive exosomes (PDL1) + Exosomes are extracellular vesicles with PDL1 protein on their outer membrane. They can participate in the immune dysregulation process of sepsis by mediating immunosuppression. Their serum levels are closely related to the immune status of sepsis patients and are an important indicator for assessing the condition of sepsis.

[0004] However, current technologies for detecting sepsis mostly employ single inflammatory marker detection schemes, which are neither accurate nor efficient. Summary of the Invention

[0005] The main objective of this invention is to provide products and their applications for the early diagnosis of sepsis, and to solve the problems of insufficient sensitivity and poor specificity of sepsis detection in the prior art, which make it difficult to meet the needs of early and accurate diagnosis and disease assessment.

[0006] According to a first aspect of the present invention, a product for early diagnosis of sepsis is provided, the product comprising the detection of SAA1 protein and PDL1. + The product containing the content of exosomes in the sample; Detection of SAA1 protein and PDL1 + The product for detecting the content of exosomes in samples is based on antigen-antibody specific immunobinding assay for SAA1 protein and PDL1. + Products for detecting the content of exosomes in samples; The detection method used in the detection product provided by this invention is selected from one of the following: fluorescence immunochromatography, enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, chemiluminescence immunoassay, electrochemical immunoassay, and nanopore-based immunosensing detection. The capture antibody for detecting SAA1 protein is 15-7F-5A, and the detection antibody is 13-5H-F11. The detection antibody for PDL1... + The capture antibody for exosomes is CD9 antibody, and the detection antibody is PDL1 antibody.

[0007] Compared to single-indicator detection, combined dual-indicator immunoassay can improve diagnostic accuracy and clinical guidance value, achieving high sensitivity and high specificity for early diagnosis. This involves detecting SAA1 protein and simultaneously detecting PDL1 in the sample. + Exosomes have high detection specificity and no cross-interference, which can ensure accurate and reliable test results and help in the clinical diagnosis of sepsis.

[0008] Combined detection of SAA1 protein and PDL1 + Products containing exosomes in samples can be used for the early diagnosis of sepsis, effectively shortening the detection cycle and achieving rapid detection while addressing the issues of insufficient sensitivity and specificity, thus providing an efficient detection tool for the early diagnosis and disease assessment of sepsis.

[0009] In some implementations, SAA1 protein and PDL1 are detected. + The product containing exosomes in the sample can be obtained by detecting SAA1 protein and PDL1 using any known method in the art based on antigen-antibody specific immunobinding. + Products for detecting the content of exosomes in samples.

[0010] In some embodiments, the detection product can be suitable for detecting SAA1 protein and PDL1 in a sample by at least one of the following methods. + Reagents, test strips, kits, chips and / or instruments for exosome content: fluorescence immunochromatography, enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, chemiluminescence immunoassay, electrochemical immunoassay or nanopore-based immunosensing detection.

[0011] In some implementations, fluorescence immunochromatography can be time-resolved fluorescence immunochromatography.

[0012] In some implementations, the sample is serum.

[0013] According to a second aspect of the present invention, a time-resolved fluorescence immunochromatographic test strip for early diagnosis of sepsis is provided, comprising a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the PVC base plate is located at the bottom, and the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are sequentially laid on the PVC base plate along the sample flow direction, wherein the sample pad partially overlaps the conjugate pad, and the conjugate pad and the absorbent pad partially overlap both ends of the nitrocellulose membrane; Along the sample flow direction, the nitrocellulose membrane is sequentially provided with a first detection line coated with exosome capture antibody CD9 antibody, a second detection line coated with anti-SAA1 protein capture antibody 15-7F-5A, and a quality control line coated with goat anti-mouse antibody.

[0014] Time-resolved fluorescence immunochromatography (TRFIA) combines the speed and convenience of immunochromatography with the high sensitivity of time-resolved fluorescence. Using lanthanide-labeled fluorescent microspheres as tracers, it employs time-gated detection technology to eliminate background fluorescence interference, resulting in high detection sensitivity. Furthermore, it is simple to operate, requires no large instruments, and is suitable for rapid point-of-care testing. This invention combines time-resolved fluorescence immunochromatography with the use of SAA1 protein and PDL1... + This test combines exosome detection with a dual-indicator approach, providing a time-resolved fluorescence immunochromatographic strip. The detection limit for SAA1 protein is down to the ng / mL level, and for PDL1... + The detection limit for exosomes can reach 10. 7 With particle / mL level, this test strip boasts high sensitivity and specificity, is easy to operate, and allows for rapid detection. It enables simultaneous detection of two indicators of sepsis, providing an efficient tool for the early diagnosis of sepsis.

[0015] In time-resolved fluorescent immunochromatographic test strips, time-resolved fluorescent microsphere-labeled antibody probes can bind to antigens, namely SAA1 and PDL1. + The binding of exosomes and the binding of fluorescent microspheres to antibodies ensure that the corresponding antibodies can remain stable in the detection system, thus guaranteeing the signal stability and response specificity of subsequent fluorescence detection.

[0016] The time-resolved fluorescence immunochromatographic test strip for early diagnosis of sepsis provided by this invention is mainly based on SAA1 protein and PDL1. + The exosome dual-marker immunoassay uses the high-affinity and high-specificity capture antibody 15-7F-5A and the detection antibody 13-5H-F11 to detect the serum amyloid A1 (SAA1) content in the sample, and the capture antibody CD9 and the detection antibody PDL1 to detect PDL1 in the sample. + The level of exosomes can reduce the levels of SAA1 protein and PDL1 in the serum of sepsis patients. +Sensitive and specific detection of exosomes can aid in the clinical diagnosis of sepsis.

[0017] Based on SAA1 protein and PDL1 + The standard curve of exosomes and the fluorescence intensity of the first and second detection lines obtained using the time-resolved fluorescence immunochromatographic test strip can be used to calculate the concentration of SAA1 protein and PDL1 in the sample. + The content of exosomes can be used to achieve early diagnosis of sepsis. Among them, PDL1 + The standard curve of exosomes is based on PDL1 + The exosome count was plotted on the x-axis, and the fluorescence intensity of the first detection line (T1 line) was plotted on the y-axis. The standard curve for SAA1 protein was plotted with SAA1 protein concentration on the x-axis and the fluorescence intensity of the second detection line (T2 line) on the y-axis. When the control line (C line) shows no fluorescence signal, it indicates chromatography failure or antibody inactivation, thus the test result is deemed invalid. This control line, coated with goat anti-mouse antibody, effectively monitors the validity of the test strip and the standardization of the testing procedure.

[0018] In some implementations, PDL1 is drawn. + PDL1 used in the standard curve process of exosomes + Exosomes were prepared using BGC-823 cells as the cell source via a combination of differential gradient centrifugation and ultracentrifugation. The specific preparation method included collecting cell culture supernatant, low-speed centrifugation for impurity removal, 0.22 μm filtration, concentration using 100 kDa concentration tubes, and enrichment of exosomes by 120,000 g ultracentrifugation. The PDL1 exosomes prepared using this method had a particle size distribution of 30-150 nm, consistent with typical exosome characteristics, and exhibited stable PDL1 protein expression, meeting the requirements for subsequent PDL1... + The need to plot exosome standard curves.

[0019] In some embodiments, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad overlap by 2 mm between adjacent portions.

[0020] In some embodiments, anti-SAA1 protein capture antibody 15-7F-5A and anti-SAA1 protein detection antibody 13-5H-F11 can be prepared using a hybridoma cell fusion method. Specifically, this may include steps such as prokaryotic expression and purification of SAA1 protein, immunization with Balb / c mice, hybridoma cell fusion, positive cell screening and subcloning, ascites preparation and antibody purification. The resulting antibodies have high affinity and high specificity, and their EC50 binding to SAA1 protein is highly specific. 50 It reaches the ng / mL level and has high detection sensitivity.

[0021] In some embodiments, the concentration ratio of exosome capture antibody CD9 antibody, anti-SAA1 protein capture antibody 15-7F-5A, and goat anti-mouse antibody is 1:1:1.

[0022] In some embodiments, the coating concentrations of exosome capture antibody CD9, anti-SAA1 protein capture antibody 15-7F-5A, and goat anti-mouse monoclonal antibody are all 2.0 mg / mL. This improves detection repeatability and ensures no competitive binding interference among the three, guaranteeing efficient capture of exosome capture antibody CD9 and stable fluorescence signal response from anti-SAA1 protein capture antibody 15-7F-5A.

[0023] According to a third aspect of the present invention, a method for preparing a time-resolved fluorescence immunochromatographic test strip is provided, comprising the following steps: S1. Sample pad and binding pad pretreatment: The glass cellulose membrane is impregnated with the sample pad pretreatment solution and dried to obtain the pretreated sample pad. The glass cellulose membrane was impregnated with a conjugate pretreatment solution and dried to obtain the pretreated conjugate. S2. Nitrocellulose membrane treatment: Fix the nitrocellulose membrane onto a PVC (polyvinyl chloride) substrate. Draw the first detection line, the second detection line, and the quality control line on the nitrocellulose membrane along the sample flow direction using exosome capture antibody CD9 antibody, anti-SAA1 protein capture antibody 15-7F-5A, and goat anti-mouse antibody, respectively. Then dry. S3. Assembly of the test strip: The pretreated sample pad and conjugate pad are overlapped sequentially at the front end of the nitrocellulose membrane, and the absorbent pad is overlapped at the rear end of the nitrocellulose membrane to complete the assembly of the test strip.

[0024] In some embodiments, the sample pad pretreatment solution comprises 2.4428 g / L Tris, 20 g / L BSA, 1.0 g / L sodium caseinate, 50 g / L trehalose, 20 g / L sucrose, 1 mL / L Tween-20, 1 mL / L ProClin300, and the balance ddH2O.

[0025] In some embodiments, the components of the conjugation pad pretreatment solution include 50 mM Na2HPO4·12H2O, 0.5% (w / v) PVA, 0.5% (w / v) BSA and 1% (v / v) Tritium-X100, and the pH of the conjugation pad pretreatment solution is 7.4.

[0026] The sample pads and binding pads pretreated in step S1 can effectively reduce non-specific binding and improve the specificity and stability of the detection.

[0027] In some implementations, after assembly, the test strips can be cut into 3mm×60mm sizes, packaged into a card case, and stored in a dehumidifying cabinet.

[0028] In some embodiments, a first detection line, a second detection line, and a control line are sequentially drawn with a coating amount of 2 μg / cm. The distance between the first and second detection lines is 3 mm, and the distance between the second detection line and the control line is also 3 mm. The antibody concentration coating the first, second, and control lines is 2.0 mg / mL, and the lines are drawn using a gold sputtering apparatus at a speed of 1 μL / cm. Under these coating concentration and streaking parameters, the antibody coating amount for the first, second, and control lines is 2 μg / cm. This coating amount ensures the signal intensity and resolution of the detection bands, improving detection accuracy.

[0029] According to a fourth aspect of the present invention, a kit for early diagnosis of sepsis is provided, comprising a time-resolved fluorescence immunochromatographic test strip, a reaction buffer, and a mixture of fluorescent microsphere-labeled probes provided by the present invention; The reaction buffer consists of 50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1% (v / v) ProClin 300, 0.01% (v / v) Tween-20 and 1.5% (w / v) BSA, and the pH of the reaction buffer is 7.8. The fluorescent microsphere-labeled probe mixture was prepared by mixing time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe and time-resolved fluorescent microsphere-labeled PDL1 antibody probe in equal concentrations and volumes.

[0030] In some embodiments, the concentrations of the time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe and the time-resolved fluorescent microsphere-labeled PDL1 antibody probe are 1 mg / mL.

[0031] Tris-base is the basic raw material for the reaction buffer. Together with NaCl, it maintains the stable pH and ionic strength of the system. At this concentration, ProClin 300, Tween-20, and BSA work synergistically to improve detection specificity and reduce background interference.

[0032] In some embodiments, the preparation of time-resolved fluorescent microsphere-labeled probes includes the following steps: Time-resolved fluorescent microspheres were activated with an EDC / NHS cross-linking agent combination, coupled with antibodies, blocked, washed, and resuspended in microsphere preservation solution to obtain antibody probes labeled with time-resolved fluorescent microspheres at a final concentration of 0.5-1.5 mg / mL.

[0033] The EDC / NHS crosslinking agent combination is a mixture of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), which can activate the carboxyl functional groups on the surface of time-resolved fluorescent microspheres, thereby achieving covalent coupling between the antibody and the fluorescent microspheres.

[0034] In some implementations, the molar ratio of EDC to NHS in the EDC / NHS crosslinker combination is 1:(15~16).

[0035] In some implementations, the molar ratio of EDC to NHS is 1:15.7.

[0036] In some embodiments, the mass ratio of time-resolved fluorescent microspheres to antibody is (8~12):1.

[0037] In some embodiments, the mass ratio of time-resolved fluorescent microspheres to antibodies is 10:1. This coupling ratio ensures efficient binding of the fluorescent microspheres to the antibodies, resulting in a probe with strong fluorescence signal and good specificity.

[0038] In some embodiments, the microsphere preservation solution comprises 25 mM Tris, 150 mM NaCl, 0.05% (v / v) Tween-20, 1% (w / v) BSA, 5% (w / v) trehalose, and 0.1% (v / v) ProClin 300. The pH of this microsphere preservation solution is 7.2, which can effectively maintain the stability of the fluorescent microsphere labeled probe. It can be stored at 4°C in the dark for long-term use.

[0039] In some implementations, the method of using the kit may include the following steps: (1) Take serum as the test sample, and dilute the test sample with the reaction buffer at a volume ratio of 1:50 to obtain the diluted sample; (2) Take 100 μL of reaction buffer, 2 μL of diluted sample and 2 μL of fluorescent microsphere labeled probe mixture, incubate at room temperature for 5 min to obtain the test solution; (3) Add 35 μL of the test solution to the sample pad of the test strip. After reacting at room temperature for 15 min, use a time-resolved fluorescence immunoassay analyzer to detect the fluorescence intensity of each band. Obtain the SAA1 protein and PDL1 in the test sample according to the standard curve. + The content of exosomes can be used to achieve early diagnosis of sepsis.

[0040] According to a fifth aspect of the invention, the use of time-resolved fluorescence immunochromatographic test strips or kits for early diagnosis of sepsis in the preparation of detection products for the diagnosis or assessment of sepsis is provided.

[0041] With SAA1 protein and PDL1 + Exosomes were used as a dual detection marker. The optimal antibody pair for SAA1 protein was screened, and a time-resolved fluorescence immunochromatographic strip for dual detection was constructed to detect SAA1 protein and PDL1 in the serum of sepsis patients. + Simultaneous detection of two exosome indicators offers high sensitivity and specificity, is simple to operate, and is rapid, providing an efficient tool for the early diagnosis or disease assessment of sepsis. Specifically, it involves the simultaneous detection of SAA1 protein concentration and PDL1... + The content of exosomes and their relative levels / ratios are used to assess and determine sepsis subtypes. SAA1 protein concentration reflects inflammatory burden, and PDL1... + Exosomes reflect immune paralysis, PDL1 + The higher the exosome content, the more severe the immunosuppression. When the SAA1 protein concentration increases, and PDL1 also increases... + A significant increase in exosome levels indicates immunosuppressive sepsis; when SAA1 protein concentration increases, along with PDL1... + When exosome levels are not elevated or are only slightly elevated, it is diagnosed as non-immunosuppressive sepsis.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows: Using the anti-SAA1 protein monoclonal antibodies 13-5H-F11 and 15-7F-5A, which have high affinity and high specificity for binding to SAA1 protein, and combining them with CD9 / PDL1 antibody pairing, a dual-indicator specific recognition method was constructed to simultaneously detect SAA1 protein and PDL1 in the serum of sepsis patients. + This dual-indicator exosome detection system enables early diagnosis and disease assessment of sepsis using two indicators, offering higher accuracy. Furthermore, by incorporating time-resolved fluorescence immunochromatography (RTI) with lanthanide-labeled fluorescent microspheres as tracers, it eliminates background fluorescence interference from serum samples, improving detection sensitivity. The detection limit for SAA1 protein reaches the ng / mL level, and for PDL1… + The detection limit for exosomes can reach 10. 7 Particles per mL are measured, making the operation simple and the detection rapid. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the test strip structure according to an embodiment of the present invention; Figure 2 The graph shows the affinity test results of the anti-SAA1 protein monoclonal antibody of this invention. Figure 3 For PDL1 + Transmission electron microscopy image of exosomes; Figure 4 For PDL1+ Particle size analysis diagram of exosomes; Figure 5 For PDL1 + Exosome biomarker detection diagram; Figure 6 The image shows the detection results of SAA1 at different concentrations under a 365nm UV lamp using time-resolved fluorescence immunochromatography. Figure 7 The standard curve for time-resolved fluorescence immunochromatography of SAA1; Figure 8 PDL1 at different concentrations under a 365nm UV lamp + Image showing the results of time-resolved fluorescence immunochromatography of exosomes; Figure 9 For PDL1 + Standard curve for time-resolved fluorescence immunochromatography of exosomes; Figure 10 The image shows the detection results of time-resolved fluorescence immunochromatography in the sample of Experiment Example 1; Figure 11 Principal component analysis yields a scatter plot. Figure 12 For SAA1 and PDL1 + ROC curve of combined exosome-based dual-indicator detection of sepsis; Figure 13 ROC curve for combined detection of two indicators in the immunosuppressed subgroup of sepsis; Figure 14 ROC curve for combined detection of two indicators in the non-immunosuppressed subgroup of sepsis; Figure 15 ROC curves for combined detection of sepsis immunosuppressed and non-immunosuppressed subgroups. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all commercially available.

[0045] The experimental materials used in the following experiments are as follows: Cell lines: SP2 / 0 cells (myeloma cells) and BGC-823 cells (human gastric cancer cell line), derived from laboratory cell banks; Laboratory animals: 6-8 week old male Balb / c mice, purchased from Guangdong Provincial Animal Experiment Center; Main reagents: SAA1 gene prokaryotic expression vector pColdII, BL21(DE3) competent cells, SP2 / 0 myeloma cells, Protein G affinity chromatography column, BCA protein quantification kit, time-resolved fluorescent microspheres (1% w / v), EDC, NHS, goat anti-mouse monoclonal antibody, CD9 antibody, PDL1 antibody, and all buffers were commercially available analytical grade. Main instruments: ultra-high speed centrifuge, refrigerated centrifuge, ultrasonic cell disruptor, time-resolved fluorescence immunoassay analyzer, streak sprayer, programmable strip cutter, enzyme-linked immunosorbent assay (ELISA) reader, and four-dimensional rotator.

[0046] Preparation: I. Preparation and screening of monoclonal antibodies against SAA1 protein: Monoclonal antibodies against SAA1 protein were prepared using a hybridoma cell fusion method, and the optimal detection / capture antibody pair 13-5H-F11 / 15-7F-5A was screened to obtain the best results. 1. Prokaryotic expression and purification of SAA1 protein The cDNA sequence of the human SAA1 gene (NCBI gene ID: 6288) was found on the Genbank website. After codon optimization, it was cloned into the prokaryotic expression vector pColdII, transformed and plated, and the clones were picked and the plasmids were extracted. The recombinant plasmid was transformed into BL21(DE3) competent cells containing the pTf16 molecular chaperone plasmid, and heat-shocked at 42℃ for 90s. Single colonies were picked and cultured in LB medium at 37℃ and 220rpm until the OD600nm reached 0.6-0.8. The LB medium contained 100μg / mL ampicillin and 35μg / mL chloramphenicol. Then, 0.1mM IPTG was added and induced at 16℃ for 16h. After centrifugation at 4℃ and 8000rpm for 6 minutes, the bacterial cells were collected, sonicated, and the supernatant was purified by nickel column chromatography. The supernatant was then replaced with PBS buffer by dialysis and quantified by BCA (diquinoline carboxylic acid) to obtain SAA1 protein for later use.

[0047] 2. Animal immunization Primary immunization: The purified SAA1 protein was emulsified with Freund's complete adjuvant in a 1:1 volume ratio and injected subcutaneously and intradermally at multiple points in the neck and back of Balb / c mice, 50 μg / mouse. Two weeks later, booster immunization was performed by emulsifying Freund's incomplete adjuvant with SAA1 protein in a 1:1 ratio, 30 μg / animal, for a total of 3 immunizations; Seven days after the second booster immunization, tail blood was collected and serum titers were measured using indirect ELISA. The results are shown in Table 1 below: Table 1

[0048] SAA1-1 mice with the highest titer were selected for cell fusion.

[0049] 3. Cell fusion and positive cell screening Sprint immunization: 100 μg of SAA1 protein was dissolved in physiological saline and injected into mice intraperitoneally. Blood was collected from the eyeballs 72 h later. Mice were euthanized by cervical dislocation. The cells were soaked in 75% alcohol for 5 min. The spleen was taken, ground to obtain spleen cell suspension. The red blood cells were lysed and centrifuged and washed. Spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 8:1, and cell fusion was mediated by PEG. After fusion, the cells were resuspended in complete medium containing 2×HAT and seeded into 96-well feeder cell plates and cultured at 37°C and 5% CO2. On day 5, the medium was partially changed (1×HAT), and on day 10, the medium was completely changed (1×HT). 3-4 days after the medium change, positive hybridoma cells were screened using indirect ELISA. Wells with an OD450 value ≥ 10 times that of the negative control wells were selected as positive wells.

[0050] 4. Positive cell subcloning Positive hybridoma cells were subcloned using a three-round limiting dilution method. After each round of subcloning, the cell supernatant was detected by indirect ELISA. Monoclonal hybridoma cell lines were screened and finally five cell lines that could stably secrete monoclonal antibodies against SAA1 protein were obtained.

[0051] 5. Ascites preparation and antibody purification Eight-week-old Balb / c mice were pre-sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. Seven days later, each mouse was injected intraperitoneally with 1 mL of 1×10⁻⁶ liquid paraffin. 6 Hybridoma cell suspension was collected at a concentration of 10 cells / mL. Ascites fluid was collected after 7-10 days. The ascites fluid was filtered through a 0.45 μm filter and loaded onto a Protein G affinity chromatography column. The column was equilibrated with PBS, eluted with 0.1 M glycine buffer (pH 2.2), neutralized with Tris-HCl (pH 9.0), dialyzed to 0.01 M PBS, and antibody concentration was determined by NanoDrop. The antibody was stored at -20°C for later use.

[0052] 6. Antibody affinity detection and pairing screening Indirect ELISA detection of EC5 antibodies against 5 strains 50 Filter out EC 50 High-affinity antibodies at the ng / mL level, such as Figure 2As shown, compared to the negative control (NC), the absorbance of the detection antibody 13-5H-F11 and the capture antibody 15-7F-5A was higher than that of other antibodies (13-10B-4D, 6-10B-7F, and 3-8E-12G). A sandwich ELISA method was used for antibody pairing screening. Different antibodies were used as capture antibodies to coat the ELISA plate, and different antibodies were used as detection antibodies and conjugated with fluorescent microspheres to detect SAA1 protein standards. The optimal pairing combination was selected based on fluorescence intensity. The detection antibody 13-5H-F11 and the capture antibody 15-7F-5A showed the strongest and best specificity.

[0053] 2. PDL1 + Preparation and identification of exosomes PDL1 was prepared using BGC-823 cells as the cell source. + Exosomes were identified and processed.

[0054] 1. BGC-823 cell culture BGC-823 cells were cultured in RPMI-1640 medium containing 10% (v / v) fetal bovine serum at 37°C and 5% CO2 until 80% confluence. The medium was then replaced with exosome-free serum medium and cultured for another 48 hours. The cell culture supernatant was then collected.

[0055] 2. PDL1 + Extraction of exosomes Centrifuge the cell culture supernatant at 4℃ and 3000g for 10 minutes to remove cell debris, and transfer the supernatant to a new container; Use a 0.22μm filter to filter the supernatant, remove impurities, and transfer the supernatant to a new container; Centrifuge at 3000g for 5 min using a 100kD concentration tube to concentrate to 240mL. Transfer to an ultracentrifuge tube and centrifuge at 120000g for 2 h. Discard the supernatant and retain the precipitate at the bottom of the tube. The exosomes were precipitated by repeatedly pipetting with 500 μL of PBS for 2 min, resuspended, quantified by BCA, and stored at -80℃ for later use.

[0056] 3. PDL1 + Identification of exosomes Transmission electron microscopy (TEM) observation: A suspension of exosomes was dropped onto a copper mesh, and the morphology was observed under a TEM. Typical cup-shaped exosome morphology was visible, such as... Figure 3 As shown; Nanoparticle size analysis: Nanoparticle tracking and analysis showed that the exosome particle size distribution was 30-150 nm, consistent with the typical particle size characteristics of exosomes. Figure 4As shown, the horizontal axis corresponds to particle size (Size), with the unit being nanometers; the vertical axis corresponds to particle concentration, with the unit being particles / mL. The exosome marker proteins PDL1, CD9, and TSG101 were detected, with the Golgi apparatus marker GM130 used as a negative control. Results showed that PDL1, CD9, and TSG101 were all positive, while GM130 was negative. Figure 5 As shown, the extracted exosomes were confirmed to be PDL1. + exosomes.

[0057] III. Preparation of Time-Resolved Fluorescent Microsphere Labeled Probes Preparation of time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe (time-resolved fluorescent microspheres-13-5H): (1) Microsphere pretreatment: Take 50 μL of 1% (w / v) time-resolved fluorescent microspheres, add 1 mL of activation buffer (10 mM MMES, 0.05% ProClin 300, pH 6.2), sonicate for 10 min, centrifuge at 15℃ and 20000g for 10 min, discard the supernatant, and repeat the washing once. (2) Microsphere activation: Resuspend the microspheres in 1 mL of activation buffer, disperse by sonication, add 3.5 μL of EDC (10 mg / mL) and 33 μL of NHS (10 mg / mL) in sequence, vortex to mix, rotate at 37°C in the dark for 30 min; centrifuge at 15°C and 20000g for 10 min, discard the supernatant, and wash twice with 1.5 mL of activation buffer; (3) Antibody conjugation: Add 750 μL of conjugation buffer (10 mM MES, 0.05% ProClin 300, pH 6.2) to resuspend the microspheres and sonicate for 10 min; take 50 μg of the prepared 13-5H-F11 antibody and dissolve it in 250 μL of conjugation buffer, vortex to mix, add the microsphere suspension, and rotate at 37°C in the dark for 2 h. (4) Blocking and washing: Add 500 μL of microsphere blocking solution, which consists of 5 mM H3BO3, 11.2 mM Na2B4O7, 0.05% (v / v) Tween-20, 1% (w / v) BSA, and 0.24% (v / v) ethanolamine. The pH of the microsphere blocking solution is 9.0. Rotate at 37℃ in the dark for 1 h. Centrifuge at 15℃ and 20000g for 10 min, discard the supernatant, and wash twice with 1.5 mL of microsphere washing solution, which consists of 50 mM Tris, 0.5% (w / v) BSA, 0.05% (v / v) Tween-20, and 0.03% (v / v) ProClin 300, with a pH of 8.0. (5) Preservation: Add 500 μL of microsphere preservation solution (25 mM Tris, 150 mM NaCl, 0.05 v / v% Tween-20, 1% w / v BSA, 5% trehalose, 0.1 v / v% ProClin 300, pH 7.2), sonicate to obtain time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe with a final concentration of 1 mg / mL, and store at 4°C protected from light.

[0058] Preparation of time-resolved fluorescent microsphere-labeled PDL1 antibody probe (time-resolved fluorescent microsphere-PDL1): The difference between the preparation method of the time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe and the method of preparation method is that in the antibody conjugation step (3), 50 μg of the prepared 13-5H-F11 antibody is replaced with 50 μg of commercially available PDL1 antibody to obtain a time-resolved fluorescent microsphere-labeled PDL1 antibody probe with a final concentration of 1 mg / mL, which is stored at 4°C in the dark.

[0059] Example 1 Preparation of time-resolved fluorescence immunochromatographic test strips S1. Pretreatment of sample pad and conjugate pad (1) Sample pad pretreatment: Cut the glass cellulose membrane into 300mm×14mm pieces, add sample pad pretreatment solution to soak, apply evenly with a roller, dry at 37℃ for 6 hours, and seal and store in a moisture-proof cabinet. The sample pad pretreatment solution consisted of 2.4428 g / L Tris, 20 g / L BSA, 1.0 g / L sodium caseinate, 50 g / L trehalose, 20 g / L sucrose, 1 mL / L Tween-20, 1 mL / L ProClin 300, and the balance ddH2O. (2) Pretreatment of the conjugate pad: Cut the glass cellulose membrane into 300mm×11mm pieces, add conjugate pad pretreatment solution to soak, apply evenly with a roller, dry at 37℃ for 6 hours, and seal and store in a moisture-proof cabinet. The pretreatment solution for the conjugated pad consists of 50 mM Na2HPO4·12H2O, 0.5% (w / v) PVA, 0.5% (w / v) BSA and 1% (v / v) Tritium-X100, with a pH of 7.4.

[0060] S2. Treatment of Nitrocellulose Membrane Nitrocellulose membranes were fixed onto a PVC soleplate. Using a spectrophotometer, a first detection line (T1 line) coated with exosome capture antibody CD9, a second detection line (T2 line) coated with anti-SAA1 protein capture antibody 15-7F-5A, and a control line (C line) coated with goat anti-mouse antibody were sequentially drawn at a speed of 1 μL / cm. The coating concentrations of exosome capture antibody CD9, anti-SAA1 protein capture antibody 15-7F-5A, and goat anti-mouse antibody were all 2.0 mg / mL. The distance between T1 and T2 lines was 3 mm, and the distance between T2 and C lines was 3 mm. The membranes were dried overnight at 37°C and then sealed for storage.

[0061] S3. Assembly of test strips The pretreated sample pad and the coated conjugate pad are sequentially overlapped at the front end of the nitrocellulose membrane, and the absorbent pad is overlapped at the rear end of the nitrocellulose membrane. Each component overlaps by 2mm to complete the assembly of the test strip. After assembly, the strips are cut into 3mm × 60mm test strips using a programmable strip cutter, sealed in a cartridge, and stored in a dehumidifier.

[0062] S4. Assembly of the reagent kit Assemble the above test strips with reaction buffer (50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1 v / v% ProClin 300, 0.01 v / v Tween-20, 1.5% w / v BSA, pH 7.8) and fluorescent microsphere-labeled probe mixture into a kit, and store it in a sealed container. The fluorescent microsphere-labeled probe mixture was prepared by mixing equal volumes of the time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody 13-5H-F11 probe with a concentration of 1 mg / mL and the time-resolved fluorescent microsphere-labeled PDL1 antibody probe with a concentration of 1 mg / mL.

[0063] Example 2: How to use the kit Includes the following steps: (1) Take serum as the test sample, and dilute the test sample with the reaction buffer at a volume ratio of 1:50 to obtain the diluted sample; (2) Take 100 μL of reaction buffer, 2 μL of diluted sample and 2 μL of fluorescent microsphere labeled probe mixture, incubate at room temperature for 5 min to obtain the test solution; (3) Take 35 μL of the test solution and add it to the sample pad of the test strip. After reacting at room temperature for 15 min, use a time-resolved fluorescence immunoassay analyzer to detect the fluorescence intensity of each band. According to the standard curve, obtain the content of SAA1 protein and PDL1+ exosomes in the test sample, and thus realize the early diagnosis of sepsis.

[0064] The plotting of the standard curve may include the following steps: 1. Construction of the SAA1 protein standard curve (1) Standard processing: The SAA1 protein standard was serially diluted with reaction buffer to 0 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL and 80 ng / mL to obtain diluted samples; (2) Incubation: Take 100 μL of diluted sample and 2 μL of probe mixture, incubate at room temperature for 5 min to obtain the test solution; The reaction buffer consisted of 50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1% (v / v) ProClin 300, 0.01% (v / v) Tween-20, and 1.5% (w / v) BSA, with a pH of 7.8. The probe mixture was the prepared time-resolved fluorescent microspheres-PDL1. + The antibody probe and time-resolved fluorescent microsphere-13-5H-F11 probe were mixed in equal concentrations and volumes. (3) Detection: Add 35 μL of the test solution to the sample pad of the test strip, react at room temperature for 15 min, and detect the fluorescence intensity at T2 using a time-resolved fluorescence immunoassay analyzer. The detection results of SAA1 at different concentrations under a 365 nm UV lamp are as follows: Figure 6 As shown; Record the T2 line fluorescence intensity; plot a standard curve with SAA1 protein concentration (ng / mL) on the x-axis and T2 line fluorescence intensity on the y-axis, as shown below. Figure 7 As shown, the linear equation is obtained: Y = 4726X + 52658, R 2 >0.9957, the detection limit of SAA1 protein was calculated to be 2.18 ng / ml.

[0065] 2. PDL1 + Plotting the exosome standard curve (1) Standard treatment: Prepared PDL1 + Exosomes were serially diluted with reaction buffer to 3 × 10⁻⁶. 7 Particles / mL, 6×10 7 Particles / mL, 12×10 7 Particles / mL, 24×10 7 Particles / mL, 48×10 7 Particles / mL, 97×10 7 Particles / mL, 194×10 7 Particles / mL, to obtain diluted samples; (2) Incubation: Take 100 μL of diluted sample and 2 μL of probe mixture, incubate at room temperature for 5 min to obtain the test solution; The reaction buffer consisted of 50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1% (v / v) ProClin 300, 0.01% (v / v) Tween-20, and 1.5% (w / v) BSA, with a pH of 7.8. The probe mixture was the prepared time-resolved fluorescent microspheres-PDL1. + The antibody probe and time-resolved fluorescent microsphere-13-5H-F11 probe were mixed in equal volumes. (3) Detection: Add 35 μL of the test solution to the sample pad of the test strip, react at room temperature for 15 min, and detect the fluorescence intensity of T1 using a time-resolved fluorescence immunoassay analyzer. Detect PDL1 at different concentrations under a 365 nm UV lamp. + The results of time-resolved fluorescence immunochromatography of exosomes are as follows: Figure 8 As shown; Record the fluorescence intensity of the T1 line, using PDL1 + Exosome particle count (PDL1) + Exosomes Concerntration) (×10 7 Plot a standard curve with particles / mL on the x-axis and fluorescence intensity at the T1 line on the y-axis, as shown below. Figure 9 As shown, the linear equation is obtained: Y = 3416X + 37220, R 2 >0.9982, PDL1 is calculated. + The limit of detection for exosomes is 2.67 × 10⁻⁶. -7 Particles / mL.

[0066] Experimental Example 1 Serum samples were collected from 13 patients with sepsis and 11 healthy individuals according to standard operating procedures. Patients with sepsis were diagnosed using the Sepsis-3 diagnostic criteria.

[0067] Of the 13 patients with sepsis, 8 were immunosuppressed and 5 were non-immunosuppressed. The criteria for distinguishing between immunosuppressed and non-immunosuppressed sepsis are shown in Table 2. Table 2

[0068] The kit prepared in Example 1 of this invention was used to detect SAA1 protein and PDL1 in serum samples. + The determination of exosome content includes the following steps: (1) Sample preparation: Take serum as the test sample, and dilute the test sample with the reaction buffer at a volume ratio of 1:50 to obtain the diluted sample; (2) Incubation: Take 100 μL of reaction buffer, 2 μL of diluted sample and 2 μL of fluorescent microsphere labeled probe mixture, incubate at room temperature for 5 min to obtain the test liquid; (3) Detection: such as Figure 1 As shown in Figure B, 35 μL of the test solution was added to the sample pad of the test strip. After reacting at room temperature for 15 min, the fluorescence intensity of the T1, T2, and C lines was detected using a time-resolved fluorescence immunochromatography analyzer. The detection results of time-resolved fluorescence immunochromatography for each sample are shown in the figure below. Figure 10 As shown; (4) Result determination: If there is no fluorescence signal on the C line, the detection result is invalid; if there is fluorescence signal on the C line, the concentration of SAA1 protein and PDL1 in the serum sample are calculated according to the standard curve. + Exosome content.

[0069] (5) Data statistical methods The concentration of SAA1 protein in serum samples was compared with that of PDL1. + The exosome content data were organized into a standardized matrix, and principal component analysis (PCA) was used to perform dimensionality reduction and cluster visualization of the samples. A scatter plot was drawn with principal component 1 (PC1) and principal component 2 (PC2) as the coordinate axes to visually display the distribution differences and clustering characteristics of the three groups of samples: healthy individuals, immunosuppressed sepsis patients, and non-immunosuppressed patients. The results are as follows: Figure 11 As shown.

[0070] (6) Diagnostic efficacy verification: Diagnostic efficacy was analyzed using ROC curves, with the horizontal axis representing 1 - specificity (100% - Specifity%) and the vertical axis representing sensitivity (Sensitivity%). Figure 12 The diagram shows SAA1 and PDL1. + ROC curve of combined exosome-based dual-indicator detection of sepsis; test samples were serum samples from sepsis patients and healthy individuals undergoing physical examinations; Figure 13 The figure shows the ROC curve of the combined dual-indicator detection of the immunosuppressed subgroup of sepsis. The test samples were serum samples from immunosuppressed sepsis patients and healthy individuals undergoing physical examinations. Figure 14 The figure shows the ROC curve of the combined dual-indicator detection of the non-immunosuppressed subgroup of sepsis. The test samples were serum samples from non-immunosuppressed sepsis patients and healthy individuals undergoing physical examinations. Figure 15 The figure shows the ROC curve of the combined detection of two indicators in the immunosuppressed and non-immunosuppressed sepsis subgroups. The test samples were serum samples from patients with non-immunosuppressed sepsis and patients with immunosuppressed sepsis. SAA1 protein and PDL1 are visible in the curve.+ The overall AUC of the combined detection of exosome indicators was 1.0. The AUC of the combined detection of the two indicators in the non-immunosuppressed subgroup and the immunosuppressed subgroup of sepsis was also 1.0, indicating that the combined detection of the two indicators can accurately distinguish between the two sepsis subtypes, realize the diagnosis and disease assessment of sepsis, and effectively differentiate sepsis subtypes.

[0071] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A kit for early diagnosis of sepsis, characterized in that, The kit detects SAA1 protein and PDL1. + The content of exosomes in the sample; The kit comprises the following components, each individually packaged: time-resolved fluorescence immunochromatographic test strips, reaction buffer, and a mixture of fluorescent microsphere-labeled probes; The time-resolved fluorescence immunochromatographic test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The PVC base plate is located at the bottom, and the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially laid on the PVC base plate along the sample flow direction. The sample pad partially overlaps the conjugate pad, and the conjugate pad and absorbent pad partially overlap the two ends of the nitrocellulose membrane, respectively. Along the sample flow direction, a first detection line coated with exosome-capturing antibody CD9 antibody, a second detection line coated with anti-SAA1 protein-capturing antibody, and a quality control line coated with goat anti-mouse antibody are sequentially arranged on the nitrocellulose membrane. The reaction buffer comprises 50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1% (v / v) ProClin 300, 0.01% (v / v) Tween-20 and 1.5% (w / v) BSA, and the pH of the reaction buffer is 7.

8. The fluorescent microsphere-labeled probe mixture was prepared by mixing time-resolved fluorescent microsphere-labeled anti-SAA1 protein detection antibody probe and time-resolved fluorescent microsphere-labeled PDL1 antibody probe in equal concentrations and volumes.

2. The kit for early diagnosis of sepsis according to claim 1, characterized in that, The method for preparing time-resolved fluorescent microsphere-labeled antibody probes includes the following steps: Time-resolved fluorescent microspheres were activated with an EDC / NHS cross-linking agent combination, coupled with antibodies, blocked, washed, and resuspended in microsphere preservation solution to obtain antibody probes labeled with time-resolved fluorescent microspheres at a final concentration of 0.5-1.5 mg / mL.

3. The kit for early diagnosis of sepsis according to claim 1, characterized in that, The anti-SAA1 protein capture antibody was prepared using a hybridoma cell fusion method.

4. The kit for early diagnosis of sepsis according to claim 1, characterized in that, The preparation method of the time-resolved fluorescence immunochromatographic test strip includes the following steps: S1. Sample pad and binding pad pretreatment: The glass cellulose membrane is impregnated with sample pad pretreatment solution and dried to obtain the pretreated sample pad. The glass cellulose membrane was impregnated with a conjugate pretreatment solution and dried to obtain the pretreated conjugate. S2. Nitrocellulose membrane treatment: Fix the nitrocellulose membrane onto a PVC base plate, and draw the first detection line, second detection line, and quality control line on the nitrocellulose membrane along the sample flow direction using exosome capture antibody CD9 antibody, anti-SAA1 protein capture antibody, and goat anti-mouse antibody, respectively, and then dry it; S3. Assembly of the test strip: The pretreated sample pad and conjugate pad are sequentially overlapped at the front end of the nitrocellulose membrane, and the absorbent pad is overlapped at the rear end of the nitrocellulose membrane to complete the assembly of the test strip.

5. The kit for early diagnosis of sepsis according to claim 4, characterized in that, In step S2, the first detection line, the second detection line, and the quality control line are drawn sequentially with a coating amount of 2 μg / cm. The distance between the first detection line and the second detection line is 3 mm, and the distance between the second detection line and the quality control line is 3 mm.

6. The kit for early diagnosis of sepsis according to any one of claims 1-5, characterized in that, The sample was serum.

7. The use of the kit according to any one of claims 1-6 in the preparation of a detection product for early diagnosis of sepsis or assessment of sepsis subtypes; wherein the sepsis subtypes include immunosuppressive sepsis and non-immunosuppressive sepsis.

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