Reagent prediction method for treatment prognosis effect of acute respiratory distress syndrome

Abstract

The invention provides an acute respiratory distress syndrome treatment prognosis effect reagent prediction method, which comprises: adding 100 [mu] L of a standard substance or a sample into a 96-well plate (coated with an NF1 antibody), sealing a film, shaking at 37 DEG C, and incubating for 2 h; washing the plate for three times by 300 microliters of plate washing liquid; adding 100 [mu] L of biotin-labeled secondary antibody into each hole, sealing a film, shaking at 37 DEG C, and incubating for 1 hour; washing the plate for three times by 300 microliters of plate washing liquid; adding 50 [mu] L of a color developing solution into each hole, and shaking and uniformly mixing at room temperature for 20 min; adding 50 microliters of stop solution into each hole, and shaking and uniformly mixing at room temperature; according to the present invention, the plate (Bio-Rad) is read within 30 min, and when the detection value of the NF1 protein in the pulmonary alveolar lavage fluid is less than 0.62 ng / mL (cut-off value), the death risk of the ARDS patient is low, and when the detection value of the NF1 protein in the pulmonary alveolar lavage fluid is more than 0.62 ng / mL, the ARDS patient is the critical patient, and the death risk is high;

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a reagent prediction method for the prognostic effect of acute respiratory distress syndrome treatment. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a clinical syndrome caused by intrapulmonary and / or extrapulmonary causes, with refractory hypoxemia as its prominent feature. It has attracted much attention due to its high mortality rate. Timely and accurate evaluation of the status of ARDS patients, the differences before and after diagnosis and treatment, and the prognosis progress, so as to detect and identify high-risk patients as early as possible, is crucial to reducing the risk of death in patients. The Acute Physiology and Chronic Health Evaluation II (APACHEII) score can be used for ARDS prognosis evaluation, which includes acute physiology score, age score and chronic health score. The disadvantage of the APACHEII score is that it involves many evaluation items, is time-consuming and cumbersome, and has a certain degree of subjectivity.

[0003] Biomarkers are an emerging means of evaluating the prognosis of ARDS. Several biomarkers in plasma have been reported, such as procalcitonin, sRAGE, Ang-2, vWF, SP-D, TNFR-1, IL-6, IL-8, etc., but markers in plasma are often not specific for lung lesions. At the same time, since ARDS patients need mechanical ventilation through endotracheal intubation, fiberoptic bronchoscopy is a routine examination for evaluating respiratory and lung lesions during treatment. The alveolar lavage fluid specimens collected during the examination can be used for microbial culture, cell staining and other examinations. Alveolar lavage fluid comes from the lungs and can more accurately reflect the characteristics of lung lesions. It is better than blood specimens in evaluating patients' lung lesions. However, at present, the number of studies on the role of biomarkers in alveolar lavage fluid in the prognosis assessment of ARDS is much lower than that on plasma markers.

[0004] NF1 (PO00712 in UniProt), reduced NF1 expression leads to cancer resistance to EGFR inhibition Activating mutations of the EGFR receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib. However, resistance eventually emerges, usually due to a second EGFR mutation, most commonly T790M. Through patient testing and evaluation, it was found that changes in NF1 expression in bronchoalveolar lavage fluid can be used to evaluate the prognostic effect of acute lung injury and acute respiratory distress syndrome. Summary of the invention

[0005] The main purpose of the present invention is to provide a reagent prediction method for the prognosis effect of acute respiratory distress syndrome treatment, which realizes the prediction of the prognosis effect of acute respiratory distress syndrome treatment. The present invention provides an important detection means for early diagnosis, intervention treatment and prognosis monitoring of acute respiratory distress syndrome.

[0006] To achieve the above object, the present invention proposes a method for predicting the prognostic effect of a reagent for the treatment of acute respiratory distress syndrome, the method comprising:

[0007] Add 100 μL of standard or sample to the 96-well plate (coated with NF1 antibody), seal the plate, and incubate at 37°C for 2 hours with shaking;

[0008] Wash the plate three times with 300 μL of plate washing buffer;

[0009] Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37 degrees with shaking for 1 hour;

[0010] Wash the plate three times with 300 μL of plate washing buffer;

[0011] Add 50 μL of color development solution to each well and shake to mix at room temperature for 20 min;

[0012] Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0013] The plate was read within 30 min (Bio-Rad). When the detection value of NF1 protein in the alveolar lavage fluid was below 0.62 ng / mL (i.e., the cut-off value), the ARDS patient was considered to have a low risk of death. When it was above 0.62 ng / mL, the ARDS patient was considered to be a critically ill patient with a high risk of death.

[0014] Furthermore, the detection method in the sample includes an immunological method, a fluorescent method, a flow cytometry, a latex turbidimetry, a biochemical method, an enzymatic method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method, or a photoelectric conversion method.

[0015] Furthermore, the reagent is a reagent that can be used to detect the expression level of the protein marker NF1, including a kit, or a test paper, or a chip.

[0016] The present invention provides a reagent prediction method for the prognosis effect of acute respiratory distress syndrome treatment. In the experiment, it was found that the prognosis of ARDS patients was significantly different from the expression level of NF1 protein in their alveolar lavage fluid. The expression level of NF1 protein in the alveolar lavage fluid of ARDS critically ill patients (who are prone to die within 28 days) is much higher than that of non-critically ill patients. Based on this finding, the reagent of the present invention can quickly evaluate the prognosis of ARDS patients and then identify critically ill patients among them. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0019] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0020] Unless otherwise specified, the reagents, biological materials, etc. used in the following examples can be obtained from commercial sources.

[0021] Example 1: NF1 protein expression level in bronchoalveolar lavage fluid of ARDS patients and survival analysis detection object:

[0022] 41 ARDS patients were recruited, 12 of whom died after 28 days of follow-up, and 29 survived after 28 days. All subjects were informed and agreed before the experiment.

[0023] Sample collection and testing

[0024] The patients' bronchoalveolar lavage fluid (BALF) was collected and ELISA was performed to detect the NF1 protein in the bronchoalveolar lavage fluid.

[0025] (1) Add 100 μL of standard or sample to a 96-well plate (coated with NF1 antibody), seal the plate, and incubate at 37°C for 2 h with shaking.

[0026] (2) Wash the plate three times with 300 μL of washing buffer;

[0027] (3) Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37°C with shaking for 1 hour;

[0028] (4) Wash the plate three times with 300 μL of washing buffer;

[0029] (5) Add 50 μL of color development solution to each well and shake at room temperature for 20 min;

[0030] (6) Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0031] (7) Read the plate within 30 min (Bio-Rad).

[0032] Protein expression level

[0033] According to the experimental results, the NF1 protein expression level of the sample was recorded.

[0034] Survival analysis

[0035] According to the prognosis of the patients, they were divided into the death group and the survival group. Comparing the NF protein expression levels in the death group and the survival group, we can see a significant difference. We can set the threshold to 0.62ng / mL. When it is greater than this value, the risk of death is high; when it is lower than this value, the prognosis is good and the survival rate is high.

[0036] Summarize

[0037] Through the expression level of NF1 protein, we can distinguish the prognosis of patients, which has clinical and industrial value. In the future, we can also explore potential mechanism research by studying the mechanism of NF1 and provide new ideas for potential drug target research.

[0038] Example 2 Kit of the present invention

[0039] 1. Composition of the kit of the present invention

[0040] The composition of the kit is shown in Table 1.

[0041] Table 1 Kit composition

[0042]

[0043] The NF1-specific antibody is the NF1 antibody (D-2) produced by Santa Cruz, with the catalog number: sc-74444.

[0044] 2. How to use the kit

[0045] (1) Add 100 μL of standard or sample to a 96-well plate (coated with NF-1 antibody), seal the plate, and incubate at 37°C for 2 h with shaking.

[0046] (2) Wash the plate three times with 300 μL of washing buffer;

[0047] (3) Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37°C with shaking for 1 hour;

[0048] (4) Wash the plate three times with 300 μL of washing buffer;

[0049] (5) Add 50 μL of colorimetric solution to each well and shake at 37°C for 20 min;

[0050] Add 50 μL of stop solution to each well and shake at room temperature to mix;

[0051] (7) Read the plate within 30 minutes (Bio-Rad). When the detection value of NF1 protein in the bronchoalveolar lavage fluid is below 0.62 ng / mL (i.e., the cut-off value), the ARDS patient can be considered to have a low risk of death, while when it is above 0.62 ng / mL, the ARDS patient can be considered to be a critically ill patient with a high risk of death.

[0052] In summary, the kit of the present invention can realize rapid judgment of ARDS prognosis by quantitatively detecting the expression level of NF1 protein, and provide effective early warning for critically ill patients.

[0053] Of course, the detection method in the sample includes immunoassay, fluorescence method, flow fluorescence method, latex turbidimetry, biochemical method, enzyme method, hybridization method, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, chromatography, chemiluminescence method, magnetoelectric method, or photoelectric conversion method. The reagent is a reagent that can be used to detect the expression level of the protein marker NF1, including a kit, a test paper, or a chip.

[0054] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent transformations made using the contents of the present invention's specification and embodiments, or direct / indirect applications in other related technical fields, within the concept of the present invention, are included in the patent protection scope of the present invention.

Claims

1. A method for predicting the prognosis of acute respiratory distress syndrome treatment, characterized in that: The reagent prediction method for the acute respiratory distress syndrome treatment prognosis effect comprises: Add 100 μL of standard or sample to the 96-well plate (coated with NF1 antibody), seal the plate, and incubate at 37°C for 2 hours with shaking; Wash the plate three times with 300 μL of plate washing buffer; Add 100 μL of biotin-labeled secondary antibody to each well, seal the membrane and incubate at 37 degrees with shaking for 1 hour; Wash the plate three times with 300 μL of plate washing buffer; Add 50 μL of color development solution to each well and shake to mix at room temperature for 20 min; Add 50 μL of stop solution to each well and shake at room temperature to mix; The plate was read within 30 min (Bio-Rad). When the detection value of NF1 protein in the alveolar lavage fluid was below 0.62 ng / mL (i.e., the cut-off value), the ARDS patient was considered to have a low risk of death. When it was above 0.62 ng / mL, the ARDS patient was considered to be a critically ill patient with a high risk of death.

2. The method for predicting the prognosis of acute respiratory distress syndrome according to claim 1, characterized in that: The detection method in the sample includes an immunological method, a fluorescent method, a flow fluorescence method, a latex turbidimetry method, a biochemical method, an enzymatic method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method, or a photoelectric conversion method.

3. The method for predicting the prognosis of acute respiratory distress syndrome according to claim 1, characterized in that: The reagent is a reagent that can be used to detect the expression level of the protein marker NF1, including a kit, a test paper, or a chip.

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