A mpo-based sepsis auxiliary detection kit and a preparation method thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a sepsis auxiliary detection kit based on MPO and a preparation method thereof. Three modules of cell sorting, protein separation and MPO detection are integrated. Seven specific antibody coupled negative sorting magnetic beads are used to efficiently obtain high-purity resting neutrophils in peripheral blood. Gradient detergent concentration hypotonic / hypertonic extraction solution is used to realize accurate separation of nucleoplasmic protein by "first breaking cell membrane and then breaking nuclear membrane". Combined with a split double antibody sandwich ELISA system, the nucleoplasmic ratio of MPO in neutrophils is accurately quantified. The application breaks through the limitation of traditional peripheral blood marker total amount detection, effectively eliminates non-infectious systemic inflammatory interference by using an embedded objective diagnostic threshold, significantly improves the specificity of early auxiliary diagnosis of sepsis, and has high clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an MPO-based auxiliary detection kit for sepsis and its preparation method. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulation of the host immune response to infection. Early and accurate diagnosis is crucial for reducing clinical mortality in severe cases. Currently, commonly used immunomarkers for the auxiliary diagnosis of sepsis (such as procalcitonin (PCT), C-reactive protein (CRP), and the traditional myeloperoxidase (MPO) assay) primarily rely on measuring the concentration of free total protein in peripheral serum or plasma. However, this conventional "total quantity assessment" approach has serious limitations in specificity: in the face of non-infectious severe systemic inflammatory responses such as severe trauma, extensive burns, or major surgery, neutrophils in the patient's body also become highly activated and release the aforementioned inflammatory markers. This leads to a high likelihood of false positives in traditional "total quantity detection methods," making it difficult to accurately distinguish between infectious and non-infectious systemic stress. This has become a major clinical challenge hindering the accurate early identification of sepsis.

[0003] To address the aforementioned shortcomings in specificity, assessing the deep functional and pathological changes of neutrophils—specifically, the subcellular redistribution of MPO (metagranulation) from the cytoplasm to the nucleus under abnormally overactivated conditions—has significant diagnostic potential by eliminating background interference. However, existing laboratory peripheral blood cell processing and subcellular separation techniques typically rely on erythrocyte lysis buffers or density gradient centrifugation. These routine procedures are time-consuming and involve significant mechanical and chemical stimulation, easily inducing premature activation and degranulation of fragile neutrophils in vitro, thus disrupting their true in vivo physiological distribution. Furthermore, conventional whole-cell lysis reagents cannot meet the requirements for precise nucleoplasmic separation with low cross-contamination, and the industry has lacked a quantitative threshold for translating this pathological process into a standardized clinical tool. Therefore, there is an urgent need to develop a reagent kit that enables in-situ non-destructive sorting of live cells, precise stepwise extraction of nucleoplasmic proteins, and provides a clear diagnostic ratio quantification standard to fill the technological gap in clinical use of MPO subcellular distribution characteristics to aid in the prediction and diagnosis of sepsis. Summary of the Invention

[0004] To address the above issues, this invention provides an MPO-based auxiliary diagnostic kit for sepsis and its preparation method. The kit uses negative magnetic beads to non-destructively separate neutrophils and extract nuclear and plasma proteins stepwise. Then, ELISA is used to quantitatively determine the MPO concentration to calculate the nucleocytoplasmic ratio. This approach aims to overcome the shortcomings of traditional biomarker "total detection," which is prone to false positives. By using the MPO nucleocytoplasmic ratio as a quantitative indicator, it provides a highly specific auxiliary diagnostic tool for sepsis.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides an MPO-based auxiliary detection kit for sepsis. The outer packaging of the detection kit is a paper box, and the inside of the paper box contains a pre-coated 96-well plate and a detection module.

[0006] Furthermore, the pre-wrapped 96-hole plate is located at the top of the paper packaging box, and there are two of them.

[0007] Furthermore, the detection module is located at the bottom of the paper packaging box.

[0008] Furthermore, the detection module includes a cell sorting module, a protein separation module, and an MPO detection module.

[0009] Furthermore, the cell sorting module includes a sorting magnetic bead solution and a sorting buffer solution.

[0010] Furthermore, the protein separation module includes cytoplasmic protein extract, nuclear protein extract, and protease inhibitor concentrate.

[0011] Furthermore, the MPO detection module includes MPO standard, enzyme-labeled signal amplification solution, colorimetric solution, washing solution, and stop solution.

[0012] Furthermore, the sorting buffer is a PBS (phosphate buffer) containing 2 mM EDTA (ethylenediaminetetraacetic acid) and 0.5% BSA (bovine serum albumin) at pH 7.4.

[0013] Furthermore, the cytoplasmic protein extraction solution is a pH 7.9 buffer containing 10 mM KCl (potassium chloride), 1.5 mM MgCl2 (magnesium chloride), 0.1 mM EDTA, 0.5 mM DTT (dithiothreitol), 0.1% NP-40 (ethyl phenyl polyethylene glycol), and 10 mM HEPES (tetrahydroxyethylpiperazine ethanesulfonic acid).

[0014] Furthermore, the nuclear protein extraction solution is a pH 7.9 buffer containing 400 mM NaCl (sodium chloride), 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol (v / v), 0.5 mM DTT, 1% NP-40 (v / v), and 20 mM HEPES buffer.

[0015] Furthermore, the solvent of the protease inhibitor concentrate is DMSO (dimethyl sulfoxide), and the concentration factor is 100× (100 times).

[0016] Furthermore, the protease inhibitor concentrate contains 100 mM AEBSF (4-2-aminoethylbenzenesulfonyl fluoride hydrochloride), 80 μM Aprotinin, 5 mM Bestatin, 1.5 mM E-64 (cysteine ​​protease inhibitor), 2 mM Leupeptin, and 1 mM Pepstatin (pepsin inhibitor).

[0017] Furthermore, the MPO standard is a 40 ng / mL human MPO recombinant protein solution.

[0018] Furthermore, the enzyme-labeled signal amplification solution includes reagent A and reagent B.

[0019] Furthermore, reagent A is PBS buffer, and reagent A also contains 1% BSA and 0.5 μg / mL biotin-labeled anti-human MPO monoclonal antibody by mass / volume fraction.

[0020] Furthermore, reagent B is PBS buffer, and reagent B also contains 1% BSA, 0.2 μg / mL horseradish peroxidase-labeled streptavidin, and 0.05% Tween 20.

[0021] Furthermore, the colorimetric solution is a TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution.

[0022] Furthermore, the washing solution is a pH 7.4 PBS buffer concentrated 20 times and containing 1% Tween 20 by mass / volume.

[0023] Furthermore, the terminating solution is a 2 M sulfuric acid solution.

[0024] Furthermore, the sorting magnetic bead solution comprises the following raw materials: carboxylated magnetic bead suspension, antibody composition, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), blocking solution, and preservation solution.

[0025] Furthermore, the mass-to-volume ratio of the carboxylated magnetic bead suspension, antibody composition, EDC, NHS, blocking solution, and preservation solution is 1 mL: 1 mg: 3 mg: 5 mg: 1 mL: 1 mL.

[0026] Furthermore, the concentration of the carboxyl magnetic bead suspension is 10 mg / mL, wherein the size of the carboxyl magnetic beads is 200 nm.

[0027] Furthermore, the antibody composition comprises anti-CD2 monoclonal antibody, anti-CD3 monoclonal antibody, anti-CD14 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD56 monoclonal antibody, anti-CD235a monoclonal antibody and anti-CD9 monoclonal antibody in a mass ratio of 1:1:1:1:1:1:1:1.

[0028] Furthermore, the sealing solution is a 1 M ethanolamine solution.

[0029] Furthermore, the preservation solution is PBS buffer, and the preservation solution also contains 0.1% BSA and 0.05% NaN3 (sodium azide) by mass / volume fraction.

[0030] Furthermore, the preparation method of the sorting magnetic bead liquid is as follows: S1: Centrifuge the carboxyl magnetic bead suspension and discard the supernatant to obtain magnetic beads. After resuspending the magnetic beads, add EDC and NHS to them, mix well, and incubate for 30 min. The carboxyl groups on the surface of the magnetic beads are activated into semi-stable NHS esters to obtain activated ester magnetic beads. S2: Prepare an antibody solution from the antibody composition, place the activated ester magnetic beads on a magnetic rack, remove the liquid, add the antibody solution to resuspend, and incubate for 2 h. The primary amino groups on the surface of the antibody undergo an amidation reaction with the NHS activated esters on the surface of the magnetic beads to obtain the coupled magnetic bead solution. S3: Perform magnetic separation on the coupled magnetic bead solution to obtain coupled magnetic beads. Add blocking solution to resuspend and incubate. Ethanolamine will completely quench (block) the remaining NHS activated ester groups on the surface of the magnetic beads that have not participated in antibody coupling, eliminating non-specific adsorption during subsequent whole blood sorting. After blocking, centrifuge and discard the supernatant to obtain a precipitate. Add preservation solution to the precipitate for resuspending, washing, and dispersion to obtain the sorted magnetic bead solution.

[0031] Furthermore, the pre-coated 96-well plate is prepared by coating the bottom of a blank 96-well plate with an anti-human MPO monoclonal antibody.

[0032] This invention provides a method for preparing an MPO-based auxiliary detection kit for sepsis, the specific steps of which are as follows: Step 1: Dispense the sorting magnetic bead solution and sorting buffer solution into 2.5 mL / tube and 50 mL / bottle respectively to obtain the cell sorting module; Step 2: The cytoplasmic protein extract, nuclear protein extract, and protease inhibitor concentrate were packaged into 5 mL / tube, 2.5 mL / tube, and 100 μL / tube, respectively, to obtain the protein separation module; Step 3: Dispense the MPO standard, reagent A, reagent B, colorimetric solution, washing solution and stop solution into 200 μL / tube (2 tubes), 12 mL / bottle, 22 mL / bottle, 22 mL / bottle, 50 mL / bottle and 22 mL / bottle respectively to obtain the MPO detection module; Step 4: Fix and embed the cell sorting module, protein separation module and MPO detection module in the bottom of the paper packaging box, and place two sealed pre-coated 96-well plates on the top of the paper packaging box, then seal it to obtain the MPO-based sepsis auxiliary detection kit.

[0033] The beneficial effects achieved by this invention are as follows: The sepsis auxiliary detection kit based on MPO provided by this invention overcomes the technical limitations of traditional sepsis immunomarkers, which mainly rely on peripheral blood "total free concentration" for assessment. It establishes the "subcellular spatial redistribution (nucleocytoplasmic ratio)" of MPO within neutrophils as an auxiliary diagnostic indicator. Traditional total MPO detection is prone to false positives in the face of severe non-infectious systemic stress such as serious trauma or major surgery. This invention, however, quantitatively measures the MPO concentration in the nucleus and cytoplasm separately, and incorporates an objectively quantified judgment threshold. With its built-in internal reference, it can effectively eliminate background interference from differences in basal immune activation and non-specific systemic inflammation, significantly improving the specificity of early auxiliary differential diagnosis of sepsis.

[0034] This invention develops and integrates a non-destructive acquisition and low-cross-contamination separation system specifically designed for fragile, live neutrophils. In the cell acquisition stage, conventional centrifugation and chemical lysis methods, which easily induce premature cell activation and degranulation, are abandoned. Instead, a negative sorting system with a specific target combination is employed to precisely remove contaminating cells under gentle conditions without mechanical shearing or strong chemical stimulation, preserving the true natural resting state of neutrophils to the greatest extent possible. In the protein extraction stage, stepwise lysis—first breaking the cell membrane, then the nuclear membrane—is achieved through precise control of the osmotic pressure of the step-by-step extraction solution and the strength of the detergent. Combined with the physical retention effect of a microporous membrane, this effectively overcomes the cross-contamination problem between cytoplasm and nuclear components caused by traditional single-stage whole-cell lysis, ensuring the authenticity and reliability of MPO nucleocytoplasmic ratio data. This invention's kit has a compact structure and standardized operating procedures, providing a convenient and accurate new translational tool for rapid clinical testing of sepsis. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the structural composition of a sepsis auxiliary detection kit based on MPO according to the present invention; Figure 2 This is a schematic diagram illustrating the detection principle of an MPO-based auxiliary detection kit for sepsis according to the present invention. Figure 3This is a standard curve for a sepsis auxiliary detection kit based on MPO according to the present invention; Figure 4 This is a ROC curve for specificity evaluation of an MPO-based sepsis auxiliary detection kit of the present invention.

[0036] The components include: 1. Paper packaging box; 2. Pre-coated 96-well plate; 3. Cell sorting module; 4. Protein separation module; 5. MPO detection module; 31. Sorting magnetic bead solution; 32. Sorting buffer; 41. Nuclear protein extract; 42. Cytoplasmic protein extract; 43. Protease inhibitor concentrate; 51. MPO standard; 52. Reagent A; 53. Reagent B; 54. Colorimetric solution; 55. Stop solution; 56. Washing solution. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0039] The structure of the sepsis auxiliary detection kit based on MPO described in this application is as follows: Figure 1 As shown; the schematic diagram of the detection kit of this application is as follows. Figure 2 As shown; the CAS number of AEBSF used is 30827-99-7; the CAS number of Aprotinin used is 9087-70-1; the CAS number of Bestatin used is 58970-76-6; the CAS number of E-64 used is 66701-25-5; the CAS number of Leupeptin used is 103476-89-7; the CAS number of Pepstatin used is 26305-03-3; the product number of the carboxylated magnetic bead suspension used is ST401, 50 mL specification, 200 nm.

[0040] Example 1: Preparation of pre-coated 96-well plates: A commercially available blank 96-well polystyrene ELISA plate was used. The anti-human MPO capture antibody (anti-human MPO monoclonal antibody) was diluted to 2 μg / mL using coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6). 100 μL of the solution was added to each well, and the plate was blocked and incubated overnight (16 h) at 4°C. The next day, the plate was drained, and the plates were washed three times with PBS containing 0.05% Tween-20. Then, 200 μL of blocking buffer containing 1% BSA was added to each well, and the plates were incubated at 37°C for 2 h. After washing and patting dry, the plates were dried in a vacuum oven, sealed, and stored for later use to obtain the pre-coated 96-well plates.

[0041] Example 2: Preparation of the cell sorting module: Sorting buffer preparation: Accurately weigh EDTA and BSA with a purity ≥99%, dissolve them in 0.01 M PBS buffer, adjust the pH to 7.4, and make up to a final concentration of 2 mM for EDTA and 0.5% for BSA. Dispense into 50 mL / bottles.

[0042] Preparation of magnetic bead sorting solution: Raw material preparation: Prepare 10 mL of carboxylated magnetic bead suspension with a concentration of 10 mg / mL and a particle size of 200 nm; mix anti-CD2 monoclonal antibody, anti-CD3 monoclonal antibody, anti-CD14 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD56 monoclonal antibody, anti-CD235a monoclonal antibody and anti-CD9 monoclonal antibody in a mass ratio of 1:1:1:1:1:1, totaling 10 mg; weigh 30 mg of EDC and 50 mg of NHS; prepare 10 mL of 1 M ethanolamine solution; prepare 10 mL of PBS buffer at pH 7.4 containing 0.1% BSA and 0.05% NaN3.

[0043] Preparation method: Place 10 mL of carboxyl magnetic bead suspension in a centrifuge tube, centrifuge, and discard the supernatant. Add 4 mL of MES buffer to the magnetic bead precipitate for resuspending, then add 30 mg EDC and 50 mg NHS, mix well, and incubate at room temperature for 30 min. During this process, the carboxyl groups on the surface of the magnetic beads are activated into semi-stable NHS esters, yielding activated ester magnetic beads; The above 10 mg antibody composition was dissolved in 4 mL of pH 7.4 PBS to prepare an antibody solution. A centrifuge tube containing activated ester magnetic beads was placed on a magnetic rack. After removing the reaction liquid, the magnetic beads were quickly resuspended in the antibody solution. The tube was incubated at room temperature for 2 hours with continuous inversion to allow the primary amino groups on the antibody surface to undergo an amidation reaction with the NHS activated esters on the magnetic beads, yielding a coupled magnetic bead solution. Centrifuge tubes were placed on a magnetic rack to perform magnetic separation of the coupled magnetic bead solution. The supernatant was removed to obtain the coupled magnetic beads. 10 mL of 1 M ethanolamine solution was added to the precipitate for resuspension, and the mixture was incubated at room temperature for 1 hour by inversion. After sealing, magnetic separation was performed, and the supernatant was discarded. 10 mL of preservation solution was added to the precipitate for resuspension, washing, and thorough dispersion to obtain the final separated magnetic bead solution. The solution was dispensed in 2.5 mL tubes.

[0044] Example 3: Preparation of the protein separation module: Preparation of cytoplasmic protein extract: Using deionized water as the solvent, dissolve the raw materials, adjust the pH to 7.9, and after bringing the volume to a final concentration, the final concentrations of each component are: 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, and 0.1% (v / v) NP-40. Dispense into 5 mL tubes. Preparation of nuclear protein extract: Using deionized water as the solvent, dissolve the raw materials, adjust the pH to 7.9, and after bringing the volume to a final concentration, the components are: 20 mM HEPES, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol (v / v), 0.5 mM DTT, and 1% NP-40 (v / v). Dispense into 2.5 mL tubes. Preparation of protease inhibitor concentrate: A 100× inhibitor concentrate was prepared using DMSO as the solvent. The concentrations of each active ingredient were: 100 mM AEBSF, 80 μM Aprotinin, 5 mM Bestatin, 1.5 mM E-64, 2 mM eupeptin, and 1 mM Pepstatin. The concentrate was dispensed individually in 100 μL tubes.

[0045] Example 4: Configuration of the MPO detection module: MPO standard: Prepare a human MPO recombinant protein solution with a concentration of 40 ng / mL, and dispense it in 200 μL tubes.

[0046] Reagent A: Using PBS buffer at pH 7.4 as the solvent, add BSA and biotin-labeled anti-human MPO monoclonal antibody to make the final concentrations as follows: 1% BSA and 0.5 μg / mL labeled antibody, dispensed in 12 mL / vial. Reagent B: Using PBS buffer at pH 7.4 as solvent, add BSA, horseradish peroxidase-labeled streptavidin, and Tween 20 to make the final concentrations as follows: 1% BSA, 0.2 μg / mL horseradish peroxidase-labeled streptavidin, and 0.05% Tween 20, dispensed in 22 mL / bottles. TMB chromogenic solution is a commercially available reagent, dispensed in 22 mL / bottles; 20× wash buffer (PBS containing 1% Tween-20 at pH 7.4) is dispensed in 50 mL / bottles; stop solution (2 M sulfuric acid aqueous solution) is dispensed in 22 mL / bottles.

[0047] Example 5: This example provides a method for preparing an MPO-based sepsis auxiliary detection kit. The specific steps are as follows: Take one tube of sorting magnetic bead solution, one bottle of sorting buffer, one tube of cytoplasmic protein extract, one tube of nuclear protein extract, one tube of protease inhibitor concentrate, two tubes of MPO standard, one bottle of reagent A, one bottle of reagent B, one bottle of colorimetric solution, one bottle of washing solution, one bottle of stop solution, and two pre-coated 96-well plates prepared in Examples 1-4 respectively, place them in a paper packaging box, fix and seal them to obtain the MPO-based sepsis auxiliary detection kit, and store it at 2-8℃.

[0048] Detection method using the kit: The biological samples were tested using the detection kit prepared in Example 5. The specific steps are as follows: M1: Sample Collection and Cell Sorting: Collect approximately 1.5 mL of venous whole blood from the subject and place it in an EDTA anticoagulant tube. Transfer 1 mL of whole blood sample to a flow cytometry tube. Add 50 μL of sorting magnetic beads to each 1 mL of whole blood sample (i.e., 50 μL). Gently pipette to mix the magnetic beads and incubate at room temperature for 5 min to allow the magnetic beads to bind to antibodies and label non-neutrophils. Add 1 mL of sorting buffer and mix thoroughly to obtain a diluted sample. Place the flow cytometry tube on a magnetic rack and allow it to stand at room temperature for 10 min to adsorb. The non-neutrophils labeled with magnetic beads will be adsorbed to the tube wall under the action of the magnetic field, while the unlabeled neutrophils will remain in the liquid. Keep the flow cytometry tube on the magnetic rack and carefully pipette the clear cell suspension from the tube into a new flow cytometry tube, avoiding contact with the adsorbate on the tube wall. Centrifuge at 400×g for 5 min, discard the supernatant to purify, and collect the neutrophil pellet at the bottom of the tube.

[0049] M2: Cytoplasmic protein extraction: Immediately before use, add 100× protease inhibitor concentrate to the cytoplasmic protein extraction solution at a volume ratio of 1:99 to prepare the cytoplasmic working solution. Count the neutrophil pellet and take 1×10⁻⁶. 6 One cell was placed in a 1.5 mL centrifuge tube, and 50 μL of pre-chilled cytoplasmic working solution was added immediately. The cells were mixed by gently tapping the tube wall with a finger and incubated on ice for 10 min. Then, the cells were centrifuged at 4 °C and 500 × g for 5 min. The supernatant was collected, which is the cytoplasmic protein extract (cytoplasmic protein extract), and the precipitate (nuclear precipitate) was retained. Nuclear protein extraction: 100× protease inhibitor concentrate was added to the nuclear protein extraction solution at a volume ratio of 1:99 to prepare the nuclear working solution. 500 μL of pre-chilled PBS containing 0.1% NP-40 was added to the precipitate obtained in the cytoplasmic protein extraction step. The precipitate was gently resuspended by pipetting to wash it and remove residual cytoplasmic protein contamination. The precipitate was then centrifuged at 500×g for 5 min at 4 °C. The supernatant was discarded to obtain the purified precipitate. 25 μL of nuclear working solution was added to the purified precipitate, and the mixture was vortexed for 15 s and incubated on ice for 15 min (vortexing once every 5 min). The precipitate was then centrifuged through a centrifuge column with a filter membrane to separate the nucleoprotein from the insoluble chromatin fragments. The mixture was then centrifuged at 15000×g for 10 min at 4 °C. The centrifuge column was discarded, and the liquid collected in the tube was the nuclear component protein extract (nuclear protein extract).

[0050] The cytoplasmic protein extract and the nuclear protein extract were diluted 25 times with sorting buffer, and then diluted 40 times, resulting in a final dilution of 1000 times, to obtain the cytoplasmic and nuclear test samples.

[0051] M3: Quantitative detection of MPO: MPO standard was diluted to prepare a series of standard solutions of 4000, 2000, 1000, 500, 250, 125, 62.5 and 0 pg / mL. The diluted standard solutions, cytoplasmic test samples and nuclear test samples were added to pre-coated 96-well plates at 100 μL / well, with 2 replicates, and incubated at 37℃ for 1 h. Discard the liquid in the wells, wash the plate 3 times with 1× washing buffer (20× washing buffer diluted 20 times with deionized water), add 100 μL of reagent A to each well to detect the antibody, incubate at 37℃ for 1 h, wash the plate 3 times, add 100 μL of reagent B to each well for enzyme-linked amplification, incubate at 37℃ for 30 min, and wash the plate 5 times. After discarding the liquid in each well, add 100 μL of chromogenic solution to each well and incubate at room temperature in the dark for 15 min. Then add 50 μL of stop solution to each well. Measure the absorbance (OD value) of each replicate well using a microplate reader at dual wavelengths of 450 nm and 570 nm. The calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm. The calibrated OD values ​​of the two replicates are recorded as OD1 and OD2, respectively. The average OD = (OD1 + OD2) / 2, and the corrected OD = average OD - average OD of the zero-concentration standard solution. The OD detection results of the standard solutions are shown in Table 1. Plot a standard curve using a binomial formula with the standard concentration as the x-axis and the corresponding OD value as the y-axis. The curve fit R is required to be... 2 ≥0.99, see standard curve Figure 3 .

[0052] M4: Sample Detection Results and Processing: Substitute the OD values ​​of the cytoplasmic and nuclear test samples into the standard curve, calculate the cytoplasmic MPO concentration and nuclear MPO concentration respectively, and calculate the total cytoplasmic MPO = cytoplasmic MPO concentration × cytoplasmic test sample volume × 1000 (dilution factor), and the total nuclear MPO = nuclear MPO concentration × nuclear test sample volume × 1000 (dilution factor). Calculate the nucleocytoplasmic ratio R = total nuclear MPO / total cytoplasmic MPO.

[0053] Raw data processing: Ten healthy controls (Normal, N group) and ten sepsis patients (S group) were included in each group. One mL of peripheral venous blood was collected from each group, and the specimens were tested strictly according to the aforementioned experimental procedures. Corrected OD values ​​were obtained, and subsequent statistical analysis was performed. Sample test results are shown in Table 2, and the results of the difference analysis are shown in Table 3. Based on the sample test results, the cutoff value was determined using the midpoint method, and the significance and suggestions for the R-value range are given. The results are shown in Table 4.

[0054] Reagent kit specificity validation: Additionally, 30 samples from healthy controls (Group N) and 30 samples from sepsis patients (Group S) were collected from clinical sources. The kit prepared in Example 5 was used for testing, and ROC curves were plotted. Results are shown below. Figure 4 .

[0055] Table 1 Standard Curve OD Values

[0056] Table 2. Test results for groups N and S

[0057] Table 3. Difference Analysis between Group N and Group S

[0058] Table 4 Evaluation of R-value index

[0059] Table 1 and Figure 3 The results show that the standard curve is: y = -7 × 10 -8 x 2 +0.0008x-0.0396, R 2 =0.9994, which meets the testing requirements.

[0060] The results in Tables 2 and 3 show that: cytoplasmic MPO content: group N was significantly higher than group S (P < 0.001), indicating a statistically significant difference; nuclear MPO content: group N was significantly lower than group S (P < 0.001), indicating a statistically significant difference; R value: group S had a significantly higher R value than group N (P < 0.001), indicating a highly statistically significant difference.

[0061] Regarding the cutoff value calculation, the R values ​​of the two groups are arranged from smallest to largest: Group N (control): 0.082, 0.106, 0.126, 0.131, 0.135, 0.143, 0.154, 0.167, 0.167, 0.182; Group S (sepsis): 0.248, 0.282, 0.286, 0.298, 0.304, 0.307, 0.308, 0.311, 0.338, 0.428; The two sets of data do not overlap. The maximum value of group N is 0.182, and the minimum value of group S is 0.248. This means the cutoff is within the range of 0.182 to 0.248. The median (midpoint) of the values ​​at the boundary between the two groups is usually taken. The formula is: (maximum value of group N + minimum value of group S) / 2 = (0.182 + 0.248) / 2 = 0.215. Therefore, 0.215 is used as the R-value to distinguish between normal individuals and patients with sepsis.

[0062] Table 4 shows that, using R=0.215 as the interpretation standard, the results of biological sample testing were used for clinical auxiliary evaluation, and reasonable suggestions were given based on the results.

[0063] Figure 4 The results showed that the detection kit prepared in this invention has good specificity and anti-interference performance.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0065] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A sepsis auxiliary detection kit based on MPO, wherein the outer packaging of the detection kit is a paper box, characterized in that, The paper packaging box contains a pre-coated 96-well plate and a detection module; The detection module includes a cell sorting module, a protein separation module, and an MPO detection module; The cell sorting module includes a sorting magnetic bead solution and a sorting buffer solution; The protein separation module includes cytoplasmic protein extract, nuclear protein extract, and protease inhibitor concentrate; The MPO detection module includes MPO standards, enzyme-labeled signal amplification solution, colorimetric solution, washing solution, and stop solution.

2. The sepsis auxiliary detection kit based on MPO according to claim 1, characterized in that, The sorting magnetic bead liquid comprises the following raw materials in the following mass-volume ratio: Carboxylated magnetic bead suspension: antibody composition: EDC: NHS: blocking solution: preservation solution = 1 mL: 1 mg: 3 mg: 5 mg: 1 mL: 1 mL; The preparation method of the sorting magnetic bead liquid is as follows: S1: Centrifuge the carboxyl magnetic bead suspension and discard the supernatant to obtain magnetic beads. After resuspending the magnetic beads, add EDC and NHS to react and obtain activated ester magnetic beads. S2: Prepare an antibody solution by taking the antibody composition, remove the liquid from the activated ester magnetic beads, add the antibody solution to resuspend and incubate to obtain the coupled magnetic bead solution; S3: After magnetic separation of the coupled magnetic bead solution, add blocking solution to resuspend, incubate, centrifuge and discard supernatant to obtain precipitate, add preservation solution to precipitate to resuspend, wash and disperse to obtain sorted magnetic bead solution.

3. A sepsis auxiliary detection kit based on MPO according to claim 2, characterized in that, The concentration of the carboxyl magnetic bead suspension is 10 mg / mL, wherein the size of the carboxyl magnetic beads is 200 nm; The antibody composition consists of anti-CD2 monoclonal antibody, anti-CD3 monoclonal antibody, anti-CD14 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD56 monoclonal antibody, anti-CD235a monoclonal antibody and anti-CD9 monoclonal antibody in a mass ratio of 1:1:1:1:1:1:1:

1. The sealing solution is a 1 M ethanolamine solution; The preservation solution is a PBS buffer containing 0.1% BSA and 0.05% NaN3 by mass / volume.

4. A sepsis auxiliary detection kit based on MPO according to claim 1, characterized in that, The sorting buffer is a PBS buffer containing 2 mM EDTA and 0.5% BSA at pH 7.

4.

5. A sepsis auxiliary detection kit based on MPO according to claim 1, characterized in that, The cytoplasmic protein extraction solution is a pH 7.9, 10 mM HEPES buffer containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.1% NP-40 (v / v). The nuclear protein extraction solution was a pH 7.9 buffer containing 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol (v / v), 0.5 mM DTT, 1% NP-40 (v / v), and 20 mM HEPES buffer. The protease inhibitor concentrate contains 100 mM AEBSF, 80 μM Aprotinin, 5 mM Bestatin, 1.5 mM E-64, 2 mM Leupeptin and 1 mM Pepstatin.

6. A sepsis auxiliary detection kit based on MPO according to claim 1, characterized in that, The MPO standard was a 40 ng / mL human MPO recombinant protein solution; The enzyme-labeled signal amplification solution includes reagent A and reagent B; The colorimetric solution is TMB colorimetric solution; The washing solution is a PBS buffer at pH 7.4, concentrated 20 times and containing 1% Tween 20 by weight / volume. The terminating solution is a 2 M sulfuric acid solution.

7. A sepsis auxiliary detection kit based on MPO according to claim 6, characterized in that, Reagent A is a PBS buffer containing 1% BSA and 0.5 μg / mL biotin-labeled anti-human MPO monoclonal antibody. Reagent B is a PBS buffer containing 1% BSA, 0.2 μg / mL horseradish peroxidase-labeled streptavidin, and 0.05% Tween 20.

8. A method for preparing an MPO-based sepsis auxiliary detection kit according to any one of claims 1-7, characterized in that, The specific steps are as follows: Step 1: Dispense the sorting magnetic bead solution and sorting buffer solution to obtain the cell sorting module; Step 2: The cytoplasmic protein extract, nuclear protein extract, and protease inhibitor concentrate are packaged to obtain a protein separation module; Step 3: Dispense the MPO standard, enzyme-labeled signal amplification solution, colorimetric solution, washing solution, and stop solution into separate containers to obtain the MPO detection module; Step 4: Place the cell sorting module, protein separation module, and MPO detection module at the bottom of the paper packaging box, place the pre-coated 96-well plate at the top of the paper packaging box, and seal it to obtain the MPO-based sepsis auxiliary detection kit.

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