Use of biomarkers in products for in vitro aid in the diagnosis of sepsis complicated by ards

Abstract

The application relates to the field of biological medicine, discloses application of a biomarker in an in-vitro auxiliary diagnosis product of sepsis complicated with ARDS, the biomarker is PDLIM1 in peripheral blood; further includes a detection kit and a use method thereof. The application first proposes that the PDLIM1 protein can be used as a biomarker to detect sepsis complicated with ARDS, compared with detection by means of a "combined judgment" method, the detection product can help medical staff to perform rapid detection, and in a ROC curve diagram, the AUC of PDLIM1 is greater than 0.8, the detection result is reliable, and the operation mode is relatively simple, so that the detection time is compressed, and the medical starting time is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a biomarker in the preparation of products for in vitro auxiliary diagnosis of sepsis complicated with ARDS. Background Technology

[0002] Sepsis complicated by acute respiratory distress syndrome (ARDS) is one of the most common and serious complications in critical care medicine. When severe systemic infection (sepsis) triggers an excessive and uncontrolled systemic inflammatory response, the lungs often become the primary target organ, leading to acute, diffuse lung injury, i.e., ARDS. Sepsis is the leading cause of ARDS, accounting for approximately 40%-50% of all ARDS cases. Diagnosing sepsis complicated by ARDS is crucial, and rapid diagnosis of this symptom remains a challenge in clinical practice.

[0003] In current technology, the diagnosis of sepsis complicated by acute respiratory distress syndrome (ARDS) in hospitals mainly relies on a set of internationally accepted clinical standards for comprehensive judgment, such as the APACHE 2 scoring system and the SOFA scoring system. These standards are typically combined with clinical manifestations, chest imaging, and blood oxygenation parameters for confirmation. Doctors need to integrate all these standards to make a judgment. Therefore, from the onset of respiratory distress in a sepsis patient to the completion of imaging, blood gas analysis, and meeting all diagnostic criteria, it may take one to two days.

[0004] Therefore, providing a more accurate, simple, and rapid means of diagnosing sepsis complicated by acute respiratory distress syndrome is an urgent problem that needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a biomarker in the preparation of products for the in vitro auxiliary diagnosis of sepsis complicated with ARDS, so as to solve the technical problem of the lack of products in the prior art for rapidly, simply and accurately determining whether a patient has sepsis complicated with ARDS.

[0006] To solve the above-mentioned technical problems, the present invention specifically provides the following technical solution: This invention provides the application of a biomarker in the preparation of products for in vitro auxiliary diagnosis of sepsis complicated with ARDS, wherein the biomarker is PDLIM1.

[0007] As a preferred embodiment of the present invention, the product assists in diagnosis by detecting the concentration of PDLIM1 in the sample. When the concentration is greater than or equal to 5 ng / mL, it indicates that the sample originated from a patient with sepsis complicated by ARDS.

[0008] As a preferred embodiment of the present invention, the sample is peripheral blood, serum, plasma, tissue homogenate, or cell supernatant.

[0009] This invention provides an in vitro diagnostic kit for assisting in the diagnosis of sepsis complicated with ARDS. The kit contains an anti-PDLIM1 antibody for specifically binding to and detecting the PDLIM1 protein in a sample.

[0010] As a preferred embodiment of the present invention, the kit includes a sealing film, an enzyme-labeled coated plate, standards, standard diluent, chromogenic reagent A, chromogenic reagent B, stop solution, enzyme-labeled reagent, and concentrated washing solution.

[0011] As a preferred embodiment of the present invention, the standard is a recombinant human PDLIM1 protein solution; The standard diluent is a PBS buffer containing 1% bovine serum albumin; The color developer A solution is a hydrogen peroxide solution; The colorimetric reagent B solution is a 3,3',5,5'-tetramethylbenzidine substrate solution; The terminating solution is a 2 mol / L sulfuric acid solution; The enzyme-labeled reagent is a streptavidin-horseradish peroxidase conjugate; The concentrated washing solution contains 0.05% Tween-20 PBS buffer.

[0012] As a preferred embodiment of the present invention, the concentration of the recombinant human PDLIM1 protein is 20 ng / mL.

[0013] This invention provides a method for in vitro detection of PDLIM1 concentration, comprising the following steps: S100. React the test sample and standard with the anti-PDLIM1 antibody in the kit, respectively. S200, detects the signal of the complex formed by the antibody binding to the PDLIM1 protein; S300. Calculate the concentration of PDLIM1 protein in the sample to be tested based on the standard curve of standard concentration versus signal value.

[0014] In a preferred embodiment of the present invention, the reaction in step S100 is carried out in an enzyme-labeled plate; the detection in step S200 is a colorimetric detection, and the signal is the absorbance value at a wavelength of 450 nm.

[0015] In a preferred embodiment of the present invention, the standard curve is characterized by the following linear regression equation: y = 0.02559 + 0.31895*x, r 2 =0.99942; Where y is absorbance and x is the concentration of PDLIM1, and the effective detection range of x is 0.125 ng / mL to 10 ng / mL.

[0016] Compared with the prior art, the present invention has the following advantages: This invention is the first to propose the application of PDLIM1 protein as a biomarker in the detection of sepsis complicated with ARDS. By detecting whether the content of PDLIM1 protein in human serum is greater than 5 ng / ml, it is possible to quickly determine whether a patient has sepsis complicated with acute respiratory distress syndrome. It can be used as an auxiliary detection method or a pre-detection method for other detection methods, and can provide a new product for the detection of sepsis complicated with acute respiratory distress syndrome. This invention provides a test kit that helps medical staff determine whether a patient has sepsis-induced acute respiratory distress syndrome. Compared to the "combined judgment" method, this test product can help medical staff perform rapid testing, and the operation method is relatively simple, thereby reducing the testing time and medical initiation time. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0018] Figure 1 This invention provides a statistical chart showing the correlation between biomarkers and sepsis, and sepsis complicated with ARDS. Figure 2 A schematic diagram of the standard dilution process in the method for establishing a linear regression equation provided by this invention; Figure 3 This invention provides a standard curve for detecting the concentrations of human PDZ and LlM domain protein 1; Figure 4 ROC curves of APACHE2 score, PDLIM1 index and SOFA score are provided for this invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This study combines a series of techniques, including protein extraction, enzyme digestion, liquid chromatography-mass spectrometry (LC-MS), database retrieval analysis, and bioinformatics analysis, to investigate blood proteins in blood samples from patients with sepsis complicated by acute respiratory distress syndrome (ARDS). The results are as follows: Figure 1 As shown, the relative concentration of serum PDLIM1 protein was significantly correlated with sepsis complicated by acute respiratory distress syndrome (ARDS). Patients with serum PDLIM1 protein levels less than 5 ng / mL were diagnosed with sepsis, while those with levels greater than 5 ng / mL were diagnosed with ARDS. (Diagnosis was aided by detecting PDLIM1 expression levels in samples and comparing the detected values ​​with preset thresholds.) This indicator is an auxiliary diagnostic indicator and needs to be combined with other clinical indicators for comprehensive judgment.

[0021] This invention is the first to propose the application of biomarkers in the preparation of products for in vitro auxiliary diagnosis of sepsis complicated with ARDS. This product can detect the content of PDLIM1 protein in human serum, helping medical staff to determine whether a patient has sepsis complicated with acute respiratory distress syndrome. Compared with the detection method of "joint judgment", this detection product can help medical staff to perform rapid detection, and the operation method is relatively simple, thereby reducing the detection time and medical initiation time.

[0022] This can be derived from the patient's peripheral blood, including human serum and plasma. Studies have shown that both human serum and plasma contain PDLIM1 protein, and the relative concentration of PDLIM1 protein is significantly correlated with the incidence of ARDS in sepsis.

[0023] Furthermore, PDLIM1 protein was found in human tissues, cell supernatants, and related fluid samples, and the relative concentration of PDLIM1 protein was significantly correlated with the incidence of sepsis complicated with ARDS.

[0024] It is evident that human serum, plasma, tissue, cell supernatant, and related liquids can all be used as test samples, and the collection method for the test samples is simple.

[0025] The full name of the PDLIM1 protein is PDZ and LIM domain protein 1. In existing studies, PDLIM1 is a key "brake" protein that regulates T cell exhaustion. Its antibody has shown strong anti-tumor effects in animal models, but it has not revealed the relationship between PDLIM1 and sepsis complicated with ARDS.

[0026] This invention proposes that PDLIM1, as a protein 1 of the PDZ and LIM domains, is known to be a key molecule regulating T cell exhaustion, and the core pathology of sepsis complicated by ARDS is systemic excessive inflammatory response leading to lung injury. It is hypothesized that PDLIM1 promotes ARDS by regulating T cell dysfunction, exacerbating pulmonary inflammatory infiltration and barrier disruption, and that its concentration changes are directly related to the intensity of the inflammatory response and the degree of lung injury.

[0027] This invention reveals for the first time the relationship between PDLIM1 and sepsis complicated with ARDS, provides correlation information data, and offers a specific product that can be used to detect sepsis complicated with ARDS in patients.

[0028] Specifically, this product is a test kit.

[0029] This test kit contains an anti-PDLIM1 antibody and is used for the in vitro quantitative detection of PDZ and LIM domain protein 1 in human serum, plasma, tissue, cell supernatant, and related liquid samples. Shelf life is 6 months; store at 2-8°C.

[0030] The test kit includes a sealing film, an enzyme-labeled coated plate, standards, standard diluent, chromogenic reagent A, chromogenic reagent B, stop solution, enzyme-labeled reagent, and concentrated washing buffer.

[0031] The standard used was recombinant human PDLIM1 protein (concentration 20 ng / mL).

[0032] The standard diluent, colorimetric reagent A solution, colorimetric reagent B solution, stop solution, enzyme-labeled reagent, and concentrated washing solution can all be selected from a wide range.

[0033] Specifically, the standard diluent was selected using PBS buffer (pH 7.4) containing 1% bovine serum albumin (BSA).

[0034] The color developer A solution is selected using hydrogen peroxide (H2O2) solution.

[0035] The colorimetric reagent B solution uses 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution.

[0036] The stop solution is a 2 mol / L sulfuric acid (H2SO4) solution.

[0037] The enzyme-labeled reagent used was streptavidin-horseradish peroxidase (HRP) conjugate.

[0038] For concentrated washing, PBS buffer (pH 7.4) containing 0.05% Tween-20 was selected.

[0039] The usage instructions for this test kit are as follows: S100. Add the sample to be tested, standard, standard diluent and enzyme-labeled reagent to the sample well of the enzyme-labeled plate, and gently shake to mix to obtain the first solvent. S200: After sealing the plate with the sealing film, incubate at 37°C for 30 minutes. After incubation, remove the sealing film, discard the first solvent, and add diluted washing solution to clean the wells of the sample to be tested. S300. Add color developer A and color developer B to the sample well in sequence, shake to mix, develop color at 37°C in the dark for 10 minutes, add stop solution to stop the reaction, so that the solution in the sample changes from blue to yellow. S400. Within 15 minutes, the absorbance of the solution in the sample to be tested is measured at a wavelength of 450 nm using an ELISA reader, and the result is substituted into the linear regression equation to obtain the concentration of PDLIM1 in the sample to be tested.

[0040] In this invention, the sample processing and requirements are as follows: 1. Serum Collect blood samples using pyrogen-free and endotoxin-free collection tubes or centrifuge tubes, and leave them at room temperature for 2 hours or overnight at 4°C to allow serum to separate (it is best to tilt the test tubes or centrifuge tubes to increase the cross-sectional area of ​​the liquid surface, which will allow more serum to separate).

[0041] Centrifuge at 2500-3500×g for 20 minutes at 4℃, and carefully collect the supernatant. It is recommended to aliquot the serum and store it at -20℃ or -80℃, avoiding repeated freeze-thaw cycles, and avoiding the use of hemolyzed and hyperlipidemic samples.

[0042] 2. Plasma Collect blood samples using collection tubes or centrifuge tubes containing anticoagulants. Centrifuge the samples at 4°C and 3000×g for 15 minutes within 30 minutes of collection, and collect the supernatant, which is the plasma.

[0043] Aliquot the supernatant into multiple portions and store at -20°C or -80°C. Avoid repeated freeze-thaw cycles and avoid using samples with hemolysis or high blood lipids.

[0044] 3. Cell supernatant Centrifuge the collected cell supernatant sample at 3000×g for 20 minutes to remove cell debris and impurities, and collect the supernatant. Perform experiments as soon as possible, or aliquot and store at -20°C or -80°C, avoiding repeated freeze-thaw cycles.

[0045] 4. Cell lysate Collect cell suspension samples, centrifuge at 1000×g for 10 minutes and discard the culture medium, wash 3 times with pre-cooled PBS; add an appropriate amount of pre-cooled PBS (protease inhibitors can be added) to resuspend the cells. Repeated freeze-thaw disruption: Freeze the sample at -20 or -80°C for 30 minutes, thaw at 37°C, and repeat the freeze-thaw process three times until the cells are completely disrupted; Ultrasonic disruption: Perform ultrasonic disruption in an ice bath, disrupting for 2 seconds with a 3-second interval, repeating this process three times until the cells are completely disrupted; Collect the sample and centrifuge at 10000×g for 10 minutes at 4℃. Take the supernatant and store it at 20°C or -80°C, avoiding repeated freeze-thaw cycles.

[0046] 5. Tissue homogenate After the tissue is processed, add pre-cooled 0.01 MPa H7.4 PBS (protease inhibitors can be added) for homogenization. It is recommended that the tissue weight to PBS volume ratio be 1:9, that is, 1g of tissue to 9mL of PBS. Homogenization should be carried out on ice or in an ice bath. Collect the homogenate and centrifuge at 10000×g at 4℃ for 5 to 10 minutes. Take the supernatant and store it at -20°C or -80°C to avoid repeated freeze-thaw cycles.

[0047] 6. Other liquid biological samples such as urine and saliva Centrifuge at 3000×g for 10 minutes, take the supernatant for testing or aliquot and store at -20°C or -80°C, avoiding repeated freeze-thaw cycles.

[0048] The processing of the samples to be tested is relatively simple, and the process for medical staff is also relatively simple. The time span for detecting concentration values ​​is between 2 and 6 hours. Once the test results are available, they can help determine whether the patient has sepsis complicated by ARDS. The testing time is short, the testing methods are diverse, and it is easy to operate.

[0049] The present invention further provides embodiments to further illustrate the detection kit: Example 1 describes the configuration of a 96T test kit, and Example 2 describes the configuration of a 48T test kit.

[0050] The compositions of Examples 1 and 2 are shown in Table 1: Table 1 In Examples 1 and 2, the enzyme-labeled coated plates are detachable. If the plates are not used up after opening, they should be placed in an aluminum foil bag, sealed, and stored at 2-8°C.

[0051] The following example, Example 1, illustrates how to use this test kit: The reagents and equipment are prepared as follows: Example 1, an ELISA reader for detecting absorbance at a wavelength of 450 nm, 1-2 mL pipettes, 100 mL and 1 L graduated cylinders, absorbent paper, a 37°C incubator, distilled water, data analysis and plotting software, drawings, and test tubes for standard and sample dilution.

[0052] I. Establishing a standard curve: The present invention further provides a method for establishing this linear regression equation, comprising the following steps: 1. Dilution of standards: such as Figure 2 As shown, prepare 6 small test tubes and number them sequentially. First, add 150 μL of standard diluent to each test tube. Then, add 150 μL of the original concentration standard to one of the numbered test tubes and mix thoroughly. Next, add 150 μL of the standard to the second test tube and mix thoroughly. Then, add 150 μL of the standard to the third test tube and mix thoroughly. Finally, add 150 μL of the standard to the fourth test tube and mix thoroughly. Add 150 μL of the solution to the fifth test tube and mix thoroughly. The sixth test tube serves as standard 0. The concentrations after dilution are: 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0 ng / mL. Set up standard wells on the enzyme-labeled plate and add 50 μL of different concentrations of standard to each well sequentially (it is recommended to run two parallel wells for each concentration).

[0053] 2. Sample Addition: Set up blank wells (blank control wells do not contain sample, enzyme-labeled reagent, or biotin-labeled anti-PDLIM1 antibody; all other steps are the same) and sample wells. Add 40 μL of sample to the sample wells on the enzyme-labeled plate, followed by 10 μL of biotin-labeled anti-PDLIM1 antibody. Add the sample to the bottom of the well, avoiding contact with the well walls, and gently shake to mix. (Note: Biotin-labeled antibody is already pre-added to the standard; no additional antibody is needed.) 3. Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells.

[0054] 4. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0055] 5. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing solution, let stand for 30 seconds and then discard. Repeat this process 5 times, then pat dry.

[0056] 6. Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0057] 7. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0058] 8. Measurement: Zero the instrument using the blank well, and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0059] 9. Detection range: 0.125ng / mL-10ng / mL.

[0060] 10. Sensitivity: ≤0.025ng / mL.

[0061] With the concentration of the standard as the x-axis and the OD value as the y-axis, a standard curve is obtained through four-parameter fitting. The OD value of the sample is then substituted into the curve equation to calculate the concentration of the sample.

[0062] Standard curve such as Figure 3 As shown, in Figure 3 In the equation, the linear regression equation is y = 0.02559 + 0.31895*x, r 2 =0.99942, where y is the absorbance and x is the PDLIM1 concentration.

[0063] The threshold for PDLIM1 content is 5 ng / ml. When the relative concentration of PDLIM1 in the sample is greater than 5 ng / ml, the patient can be identified as a patient with sepsis complicated by ARDS; when it is less than 5 ng / ml, the patient is identified as a patient with simple sepsis.

[0064] The value of x ranges from 0.125 ng / mL to 10 ng / mL. When the sample concentration exceeds 10 ng / mL or is lower than 0.125 ng / mL, it is not statistically significant.

[0065] How to use: 1. Sample Processing: Taking plasma samples as an example, peripheral blood is collected from the patient using collection tubes containing anticoagulant. After collection, the sample is centrifuged at 3000×g for 15 minutes at 4°C within 30 minutes, and the supernatant, i.e., plasma, is collected. The supernatant is then aliquoted into multiple portions. 2. Sample addition: First, add 40 μL of sample to the wells of the enzyme-labeled plate, then add 10 μL of biotin-labeled anti-PDLIM1 antibody. Add the sample to the bottom of the wells, avoiding contact with the well walls as much as possible. Gently shake to mix. Add 50 μL of enzyme-labeled reagent to each well. Seal the plate with sealing film and incubate at 37°C for 30 minutes.

[0066] 3. Solution preparation: Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside.

[0067] 4. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds and then discard. Repeat this 5 times, pat dry, add 50 μL of color developer A to each well first, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0068] 5. Termination: Add 50 μL of stop solution to each well to terminate the reaction. TMB is converted to blue under the catalysis of HRP enzyme, and then to yellow under the action of acid.

[0069] 6. Measurement: Within 15 minutes, zero the instrument using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution. Substitute the absorbance value y into the linear regression equation y = 0.02559 + 0.31895*x to calculate the value of x.

[0070] In this embodiment, purified human PDZ and LIM domain protein 1 (PDLIM1) antibodies were coated onto microplates to prepare solid-phase detection antibodies. PDZ and LIM domain protein 1 (PDLIM1) standards and samples were then added sequentially to the microplates. Subsequently, biochemical detection antibodies and streptavidin-labeled enzyme-labeled reagents were added sequentially to form antibody-antigen-antibody-enzyme complexes. After thorough washing, the substrate TMB was added for color development.

[0071] TMB is converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The intensity of the color is positively correlated with the presence of PDZ and LIM domain protein 1 (PDLIM1) in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader, and the concentration of PDLIM1 in the sample is calculated using a standard curve. The PDLIM1 concentration is then compared with 5 ng / ml. If the concentration is greater than 5 ng / ml, the patient is considered to have a high probability of sepsis-induced acute respiratory distress syndrome.

[0072] In the actual testing, the kit was used for detection and APACHE2 score assessment. The SOFA score and APACHE2 score of the experimental group subjects are shown in Table 1 as baseline data.

[0073] Table 1 It displays the basic clinical and physiological data of the study subjects (sepsis group and sepsis complicated with ARDS group) at the time of enrollment, including but not limited to age, sex, underlying diseases (such as diabetes, COPD), sepsis severity score (such as SOFA score), initial blood oxygen saturation, and baseline lung imaging, etc., to exclude confounding factors, ensure the comparability of the two groups of study subjects, and support the reliability of the experimental results.

[0074] Detection Example 1: Figure 1 The operation and methods for generating the publicly available PDLIM1 relative concentration statistics chart are as follows: Sample processing: Serum samples should be placed at room temperature for 2 hours or overnight at 4°C, then centrifuged at 4°C and 2500-3500×g for 20 minutes to collect the supernatant; plasma samples should be centrifuged at 4°C and 3000×g for 15 minutes within 30 minutes of collection to collect the supernatant.

[0075] Detection procedure: Add sample according to the kit instructions, incubate at 37℃ for 30 minutes, wash, develop color, terminate the reaction, and measure absorbance at 450nm wavelength using an ELISA reader within 15 minutes. Substitute the absorbance into the regression equation to calculate the PDLIM1 concentration.

[0076] Data presentation (graphpad): The relative concentration of serum PDLIM1 (ng / mL) is plotted on the vertical axis, and the two groups of patients are plotted on the horizontal axis. The concentration distribution of each group is statistically analyzed, and the differences between the two groups are displayed intuitively.

[0077] The results are as follows Figure 1 As shown, from Figure 1 It can be seen that PDLIM1 concentration is significantly associated with sepsis complicated by ARDS.

[0078] Detection Example 2: Furthermore, this invention provides ROC curves (Receiving Operating Characteristic curves) for APACHE2 score, PDLIM1 index, and SOFA score, and the detection method is as follows: ROC curve related operations and methods Patient selection (based on the latest guidelines and Berlin definition): 44 patients with simple sepsis and 46 patients with sepsis-related ARDS were included (all met the criteria, and baseline information such as age and underlying diseases was recorded; confounding factors were excluded to ensure comparability between the two groups). Exclusion criteria: Patients with a recent history of acute myocardial infarction, pregnancy, organ transplantation, chronic viral infection (hepatitis virus, human immunodeficiency virus (HIV), autoimmune disease, and / or receiving immunosuppressant therapy) were excluded.

[0079] Timing of testing: Peripheral blood samples were collected from patients upon enrollment, and after immediate processing, PDLIM1 concentration was detected using a kit. APACHE2 score and SOFA score were recorded simultaneously.

[0080] Data recording: PDLIM1 concentration, APACHE2 score, and SOFA score were recorded for each group. ROC curves were plotted using the statistical software Graphpad, and AUC values ​​were calculated to assess the diagnostic value, with "whether it is sepsis complicated with ARDS" as the outcome variable.

[0081] The results are as follows Figure 4 As shown, the ROC curve can evaluate the diagnostic value of different indicators for "sepsis complicated with ARDS". The key is the area under the curve (AUC) - the closer the AUC is to 1, the better the diagnostic performance.

[0082] As shown in the figure, the APACHE2 score (red, AUC=0.9248) has an AUC close to 1, which is the best in terms of diagnostic performance. It can accurately identify patients with sepsis complicated by ARDS (high true positive rate) and rarely misdiagnose non-patients as patients (low false positive rate).

[0083] PDLIM1 (blue, AUC=0.8128) has an AUC>0.8, indicating good diagnostic performance and demonstrating its reliable ability as a biomarker to differentiate between sepsis and ARDS.

[0084] The SOFA score (green, AUC=0.7681) has an AUC>0.7, indicating acceptable diagnostic performance, but its discriminative ability is weaker than the first two indicators.

[0085] The application of the biomarker in this embodiment in the preparation of a product for in vitro auxiliary diagnosis of sepsis complicated with ARDS enables rapid detection of PDLIM1 concentration in the peripheral blood of patients. This kit can help medical staff determine whether a patient has sepsis complicated with acute respiratory distress syndrome. Compared with the detection method of "combined judgment", this detection product is simpler to use, thereby reducing detection time and medical initiation time.

[0086] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. The application of biomarkers in in vitro auxiliary diagnosis of sepsis complicated with ARDS, characterized in that, The biomarker is PDLIM1.

2. The application according to claim 1, characterized in that, The product assists in diagnosis by detecting the concentration of PDLIM1 in the sample. When the concentration is greater than or equal to 5 ng / mL, it indicates that the sample originated from a patient with sepsis complicated by ARDS.

3. The application according to claim 1 or 2, characterized in that, The samples are peripheral blood, serum, plasma, tissue homogenate, or cell supernatant.

4. An in vitro diagnostic kit for assisting in the diagnosis of sepsis complicated with ARDS, characterized in that, The kit contains an anti-PDLIM1 antibody for specifically binding to and detecting the PDLIM1 protein in a sample.

5. A detection kit according to claim 4, characterized in that, The kit includes a sealing film, an enzyme-labeled coated plate, standards, standard diluent, chromogenic reagent A, chromogenic reagent B, stop solution, enzyme-labeled reagent, and concentrated washing solution.

6. The detection kit according to claim 5, characterized in that, The standard was a recombinant human PDLIM1 protein solution; The standard diluent is a PBS buffer containing 1% bovine serum albumin; The color developer A solution is a hydrogen peroxide solution; The colorimetric reagent B solution is a 3,3',5,5'-tetramethylbenzidine substrate solution; The terminating solution is a 2 mol / L sulfuric acid solution; The enzyme-labeled reagent is a streptavidin-horseradish peroxidase conjugate; The concentrated washing solution contains 0.05% Tween-20 PBS buffer.

7. A test kit according to claim 6, characterized in that, The concentration of the recombinant human PDLIM1 protein was 20 ng / mL.

8. A method for in vitro detection of PDLIM1 concentration according to any one of claims 4-7, characterized in that, Includes the following steps: S100. React the test sample and standard with the anti-PDLIM1 antibody in the kit, respectively. S200, detects the signal of the complex formed by the antibody binding to the PDLIM1 protein; S300. Calculate the concentration of PDLIM1 protein in the sample to be tested based on the standard curve of standard concentration versus signal value.

9. A method according to any one of claims 8, characterized in that, The reaction in step S100 is carried out in an enzyme-labeled plate; the detection in step S200 is a colorimetric detection, and the signal is the absorbance value at a wavelength of 450 nm.

10. A method according to any one of claims 9, characterized in that, The standard curve is characterized by the following linear regression equation: y = 0.02559 + 0.31895*x, r 2 =0.99942; Where y is absorbance and x is the concentration of PDLIM1, and the effective detection range of x is 0.125 ng / mL to 10 ng / mL.

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