CN113252896B - Kit for predicting acute respiratory distress syndrome prognosis by using ND1 in alveolar lavage fluid - Google Patents

Kit for predicting acute respiratory distress syndrome prognosis by using ND1 in alveolar lavage fluid Download PDF

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CN113252896B
CN113252896B CN202110524995.1A CN202110524995A CN113252896B CN 113252896 B CN113252896 B CN 113252896B CN 202110524995 A CN202110524995 A CN 202110524995A CN 113252896 B CN113252896 B CN 113252896B
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lavage fluid
alveolar lavage
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respiratory distress
distress syndrome
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CN113252896A (en
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王浩
汪涛
曾妮
袁智城
文富强
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West China Hospital of Sichuan University
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Abstract

The invention belongs to the field of acute respiratory distress syndrome detection. The invention provides a kit for predicting acute respiratory distress syndrome prognosis by using ND1 in alveolar lavage fluid, and application of a reagent for detecting ND1 protein in the alveolar lavage fluid in preparation of an acute respiratory distress syndrome prognosis evaluation kit. Experiments have shown that the higher the content of ND1 in alveolar lavage fluid, the higher the risk of death in patients with acute respiratory distress syndrome. The rapid prognosis evaluation of the acute respiratory distress syndrome is realized by detecting the content of the ND1 protein in the alveolar lavage fluid, and the application prospect is good.

Description

Kit for predicting acute respiratory distress syndrome prognosis by using ND1 in alveolar lavage fluid
Technical Field
The invention belongs to the field of acute respiratory distress syndrome prognosis evaluation.
Background
Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by refractory hypoxemia, caused by intrapulmonary and/or extrapulmonary causes, that is of great interest for high mortality.
The prognosis of the ARDS patient is evaluated in time, so that high-risk patients can be discovered and identified as soon as possible, and the method is very important for reducing the death risk of the patients. Acute pathophysiological and Chronic Health (APACHE II) scores can be used for prognosis evaluation of ARDS, including Acute physiological scores, age scores, and Chronic Health scores. The APACHE II score has the defects of more evaluation items, time consumption, complexity and certain subjectivity.
Biomarkers are an emerging means of assessing the prognosis of ARDS. Several biomarkers in plasma have been reported so far, such as: procalcitonin, sRAGE, Ang-2, vWF, SP-D, TNFR-1, IL-6, IL-8, etc., but the markers in plasma tend not to be specific for lung lesions. Meanwhile, patients with ARDS need mechanical ventilation by trachea cannula, bronchofiberscope examination is a routine examination for evaluating respiratory tract and lung lesions in the treatment process, and the retained alveolar lavage fluid specimen in the examination can be examined by microorganism culture, cell staining and the like. Alveolar lavage fluid is from the lung, can reflect the characteristics of lung lesions more accurately, and is superior to a blood specimen (Garci-Larden MI, et al. biomakers for the access respiratory syndrome: how to make the diagnosis more accurate than Ann Transl Med.2017; 5: 283.) when the lung lesions of patients are evaluated. Previous studies found that IL-8 in alveolar lavage fluid more accurately predicted the occurrence of ARDS than plasma (Donnelly SC, et al. Interleukin-8 and maintenance of additive respiratory breakdown syndrome in aa-risk patent groups. Lancet Lond Engl. 1993; 341: 643-7.). However, currently, the number of studies relating to the role of biomarkers in alveolar lavage fluid in the prognostic assessment of ARDS is much lower than that relating to plasma markers.
ND1(UniProtKB accession number P03886), also known as MT-ND1, is a mitochondrially encoded NADH dehydrogenase. After the cells are damaged, mitochondrial proteins and nucleic acids such as ND1 and the like can be released and are used as damage-related molecules to participate in the development of various diseases. While the level of ND1 RNA in Plasma can be used as a marker for the diagnosis of non-small cell lung cancer (Wang L, et al. plasmid RNA sequencing of extracellular RNAs temporal biologicals for non-small cell lung cancer. Clin biochem.2020 Sep; 83: 65-73.), the mitochondrial DNA damage pattern in Plasma ND1 can be used to assess transfusion-related acute lung injury (Lee YL, et al. Blood transfer products DNA damagees-associated molecular tissues: a particulate impact of transfer-related amino fusion Surg. J. (Oct.) (20142): 286-9). However, no objective marker for prognosis evaluation of ARDS is found, which is the ND1 protein, especially ND1 protein in alveolar lavage fluid.
Disclosure of Invention
The invention aims to provide another novel ARDS prognostic marker, and the novel ARDS prognostic marker is used for preparing an ARDS prognostic evaluation kit.
The technical scheme of the invention comprises the following steps:
use of a reagent for detecting the ND1 protein content in alveolar lavage fluid in preparation of an ARDS prognostic evaluation kit.
As the application, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is an enzyme-linked immunosorbent assay (ELISA) reagent.
As the application, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is a reagent for a liquid chromatography method or a colloidal gold detection reagent.
As the application, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is a liquid chip detection reagent.
An ARDS prognostic evaluation kit, which comprises a reagent for detecting the ND1 protein content in alveolar lavage fluid.
As the prognosis evaluation kit, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is an enzyme-linked immunosorbent assay reagent.
As the prognostic evaluation kit, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is a reagent for liquid chromatography or a colloidal gold detection reagent.
As the prognosis evaluation kit, the reagent for detecting the ND1 protein content in the alveolar lavage fluid is a liquid chip detection reagent.
The experiment shows that the prognosis of the ARDS patient is significantly different from the content of ND1 protein in the alveolar lavage fluid of the ARDS patient, and the ND1 protein content in the alveolar lavage fluid of the ARDS critical patient (easy to die within 28 days) is much higher than that of the non-critical patient. According to the discovery, the kit provided by the invention can be used for rapidly evaluating the prognosis of the ARDS patient, so as to identify the critically ill patient.
The core idea of the invention is to evaluate ARDS patient prognosis by the concentration of ND1 protein in alveolar lavage fluid, rather than by any technical means. Therefore, the ND1 protein can be detected by using a conventional protein detection method such as liquid chromatography (such as high performance liquid chromatography and ultra-high performance liquid chromatography), liquid chip Luminex, enzyme-linked immunosorbent assay and colloidal gold, and the technology for further evaluating the prognosis of the ARDS patient is in the scope of the invention.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: the content of ND1 protein in alveolar lavage fluid of different groups of patients;
FIG. 2: ROC curve predicting ARDS patient death within 28 days by ND1 protein content in alveolar lavage fluid, APACH II and oxygenation index;
FIG. 3: patient 28-day survival curves were grouped according to the best predictive cutoff for ND1 in alveolar lavage fluid.
Detailed Description
Example 1 detection and survival assay of ND1 protein in alveolar lavage fluid and plasma from ARDS patients
1. Object to be inspected
Patients with ARDS were recruited 51, 20 after 28 days of follow-up, and 31 after 28 days of survival. The subject population was informed and consented to prior to the experiment.
2. Method of producing a composite material
2.1ND1 protein detection
Collecting alveolar lavage fluid (BALF) of a detected object, and performing ELISA detection on ND1 protein in the alveolar lavage fluid, wherein the detection method specifically comprises the following steps:
(1) adding 100 mu L of standard substance or sample into a 96-well plate (coated with ND1 antibody), sealing the membrane, and incubating for 2 hours at 37-degree shaking;
(2) washing the plate for 3 times by using 300 mu L of plate washing liquid;
(3) adding 100 mu L biotin-labeled secondary antibody into each hole, sealing the membrane, and then incubating for 1 hour at 37 ℃ by shaking;
(4) washing the plate for 3 times by using 300 mu L of plate washing liquid;
(5) adding 50 μ L of color development solution into each well, shaking at room temperature, and mixing for 20 min;
(6) adding 50 mu L of stop solution into each hole, shaking at room temperature and uniformly mixing;
(7) plates were read within 30min (Bio-Rad).
2.2PaO 2 And FiO 2 Detection of
Extracting arterial blood of patient via radial artery, immediately sending the sample into blood gas analyzer (Roche) for blood gas analysis and detection to obtain arterial blood oxygen partial pressure (PaO) 2 ) And simultaneously recording the oxygen supply concentration (FiO) of the patient when the patient takes the arterial blood sample 2 ) Calculating to obtain oxygenation index (PaO) according to the detection result 2 /FiO 2 )。
2.3APACHE II score
The APACHE II score of the subject is obtained according to the method specified by Knaus et al (Knaus WA, et al. APACHE II: a specificity of diagnosis system. Crit Care Med.1985, 13: 818-829.). Briefly, the APACHII score primarily includes an acute physiological score, an age score, and a chronic health score.
2.4 group comparison, ROC analysis and survival analysis
Analyzing the difference of ND1 content in alveolar lavage fluid between the viable group and the non-viable group by using a t test; performing ROC analysis according to the ND1 level and the 28-day survival condition of the patient, and analyzing the value of APACH II score and oxygenation index in ARDS prognosis evaluation by using an ROC curve; then, ARDS patients were divided into high ND1 and low ND1 level groups based on the best predictive cutoff determined by ROC analysis, and the survival rates of the two groups over 28 days were compared.
2.5 Single-factor COX proportional Risk model analysis and Multi-factor COX model analysis
Aiming at the influence of ND1 content, APACH II score and oxygenation index on the death of a patient, SPSS software is used for carrying out single-factor COX proportional risk model analysis; and then, bringing the indexes into a multi-factor COX proportional risk model in SPSS software for further analysis, and further determining the value of each index in patient prognosis evaluation.
3. Results
3.1 results of comparisons between groups, ROC analysis and survival
(1) The ND1 protein content in BALF of death group patients is obviously increased
Compared with the survival patients, the BALF of the death patients has a significant increase in the content of ND1 protein within 28 days (as shown in fig. 1);
(2) ROC curve for predicting death within 28 days by ND1 protein content in BLAF
The ROC curve analysis shows that the area under the curve (AUC) of death of the patients predicted by the content of ND1 protein in BALF within 28 days is 0.7226 (as shown in figure 2), and is remarkably superior to APACH II score (AUC 0.6266) and oxygenation index (AUC 0.5266), which indicates that the ND1 protein level in BALF can more accurately predict survival of the ARDS patients within 28 days and is superior to the APACH II score and oxygenation index of the traditional indexes.
(3) Survival assay
According to the ROC curve, the optimal prediction critical value of ND1 in BALF for prognosis evaluation is 0.5095ng/mL, and ARDS patients are divided into a high ND1 level group and a low ND1 level group, as shown in figure 3, the mortality rate of the low ND1 level group is 0%, and the mortality rate of the high ND1 level group is obviously increased.
3.2 results of one-factor COX proportional Risk model analysis and Multi-factor COX model analysis
A risk ratio HR greater than 1 indicates that the predictive factor will increase risk, and less than 1 indicates that the predictive factor will decrease risk.
As shown in table 1, the amount of ND1 polypeptide released in BALF was found to correlate with the prognosis of ARDS patients using a one-way COX proportional risk model analysis, while the APACH II score also correlated with the prognosis, but the oxygenation index was not correlated with the patient's prognosis. Further analysis of the multifactorial COX model revealed that the BALF ND1 and APACHEII scores correlated with prognosis, and that for each 1ng/mL increase in BALF ND1, the patient's risk of death increased 1.2-fold, with a predictive power 1.1-fold higher than the APACH II score.
TABLE 1 COX proportional Risk model analysis results
Figure BDA0003064765150000051
The results show that the prognosis of the ARDS patient can be effectively evaluated by using the concentration of ND1 protein in the alveolar lavage fluid, and the high-risk patients can be distinguished in time.
Example 2 kit of the invention
1. Compositions of the kits of the invention
The composition of the kit is shown in table 2.
TABLE 2 kit composition
Figure BDA0003064765150000052
Figure BDA0003064765150000061
The antibody specific to ND1 was ND1 antibody (3H3) from Santa Cruz, cat #: sc-293243.
2. Kit using method
(1) Adding 100 mu L of standard substance or sample into a 96-well plate (coated with ND-1 antibody), sealing the membrane, and incubating for 2 hours at 37-degree shaking;
(2) washing the plate for 3 times by using 300 mu L of plate washing liquid;
(3) adding 100 mu L biotin-labeled secondary antibody into each hole, sealing the membrane, and then incubating for 1 hour at 37 ℃ by shaking;
(4) washing the plate for 3 times by using 300 mu L of plate washing liquid;
(5) adding 50 mu L of color development liquid into each hole, shaking at 37 ℃ and uniformly mixing for 20 min;
(6) adding 50 mu L of stop solution into each hole, shaking at room temperature and uniformly mixing;
(7) plates were read within 30min (Bio-Rad).
When the value of ND1 protein detected in alveolar lavage fluid was 0.5095ng/mL (i.e., cut-off value) or less, the risk of death in patients with ARDS was considered to be low, whereas when 0.5095ng/mL or more, the risk of death in patients with ARDS was considered to be critically ill and high.
In conclusion, the kit can realize rapid judgment of ARDS prognosis by quantitatively detecting the ND1 protein, and provides effective early warning for critically ill patients.

Claims (5)

1. The application of the reagent for detecting the ND1 protein content in alveolar lavage fluid in the preparation of the acute respiratory distress syndrome prognosis evaluation kit is characterized in that when the detection value of the ND1 protein in the alveolar lavage fluid is 0.5095ng/mL or less, the death risk of patients with acute respiratory distress syndrome is considered to be low.
2. The use of claim 1, wherein the reagent for detecting the ND1 protein content in alveolar lavage fluid is an ELISA detection reagent.
3. The use according to claim 1, wherein the reagent for detecting the ND1 protein content in alveolar lavage fluid is a reagent for liquid chromatography.
4. The use according to claim 1, wherein the reagent for detecting the ND1 protein content in alveolar lavage fluid is a colloidal gold detection reagent.
5. The use according to claim 1, wherein the reagent for detecting the ND1 protein content in alveolar lavage fluid is a reagent for liquid chip assay.
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