Application of ALDH7A1 protein in preparation of kit for diagnosing asthma

Abstract

The invention discloses an application of ALDH7A1 protein in preparation of a kit for diagnosing asthma. The invention finds that (1) the ALDH7A1 protein content is negatively correlated with the absolute value and IgE content of serum eosinophils serving as typical characteristics of asthma patients and has significance; 2) the area AUC under an ROC curve for diagnosing and distinguishing an asthma patient and a healthy subject by the ALDH7A1 protein drawn in a training set sample is up to 0.907, which shows that the diagnosis has higher accuracy; and 3) the parameters obtained by training of the training set are used as diagnosis thresholds to predict samples in the verification set, the prediction accuracy is nearly 80%, and the accuracy is high. Therefore, the ALDH7A1 protein has high accuracy in diagnosing and distinguishing healthy subjects and asthma patients, and has the prospect of being developed into a kit for diagnosing asthma.

Description

Technical Field

[0001] The present invention belongs to the field of disease diagnosis, relates to the discovery and application of disease diagnosis markers, and specifically relates to the application of ALDH7A1 protein in the preparation of a kit for diagnosing asthma. Background Art

[0002] Bronchial asthma (asthma) is the most common chronic respiratory disease, characterized by chronic inflammation of the airways. Its pathogenesis is complex and is the result of the combined effects of the body's internal and external environments. Asthma affects more than 300 million people worldwide, with children being significantly more likely than adults. It is caused by a variety of cells and cell components. This chronic inflammation leads to increased airway responsiveness, which makes it easy for reversible expiratory airflow obstruction to occur. Symptoms include recurrent wheezing, shortness of breath, chest tightness or coughing, and symptoms are usually exacerbated in the morning or evening.

[0003] In recent years, diagnostic markers have developed rapidly. Using diagnostic markers to diagnose diseases has not only improved the objectivity of diagnosis, but also has good patient compliance.

[0004] ALDH7A1 protein is a subfamily of the aldehyde dehydrogenase superfamily, located on human chromosome 5q31, with 18 exons, encoding a total of 510 amino acids, and is present in the cytosol, mitochondrial matrix and nucleus of the kidney, ovary and heart. The function of ALDH7A1 protein has not yet been clarified.

[0005] Currently, there is no report that ALDH7A1 protein can be used as a diagnostic marker for the diagnosis of asthma. Summary of the invention

[0006] The purpose of the present invention is to provide the use of ALDH7A1 protein in preparing a kit for diagnosing asthma.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A use of ALDH7A1 protein for preparing a kit for diagnosing asthma.

[0009] Preferably, the kit is a serum detection kit.

[0010] Preferably, the kit contains a detection reagent for ALDH7A1 protein.

[0011] Beneficial effects:

[0012] The present invention proves that ALDH7A1 protein has good diagnostic efficacy for asthma from three aspects:

[0013] First, the present invention proves through correlation analysis between the absolute value of eosinophils, IgE content and ALDH7A1 protein content in the serum of asthma patients and healthy subjects that with the increase of the absolute value of serum eosinophils and IgE content, which are typical characteristics of asthma patients, the content of ALDH7A1 protein decreases, and the correlation is negative and significant, indicating that the decreased content of ALDH7A1 protein can also be used as a typical characteristic of asthma patients.

[0014] Secondly, the present invention draws the ROC curve of ALDH7A1 protein diagnosis and differentiation between asthma patients and healthy subjects in the training set samples, and the AUC is as high as 0.907. Those skilled in the art know that the area under the ROC curve AUC has been generally recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it has no diagnostic significance; when AUC is 0.5-0.7, it indicates a low diagnostic accuracy; when AUC is 0.7-0.9, it indicates a medium diagnostic accuracy; when AUC>0.9, it indicates a high diagnostic accuracy.

[0015] Again, the present invention uses the parameters obtained by training the training set as the diagnostic threshold to predict the samples in the validation set, and the prediction accuracy is 76.9% (30 / 39), nearly 80%, with high accuracy.

[0016] In summary, ALDH7A1 protein diagnosis has a high accuracy in distinguishing healthy subjects from asthma patients, and ALDH7A1 protein has the prospect of being developed into a kit for diagnosing asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of serum ALDH7A1 protein levels between asthma patients and healthy subjects in the training set;

[0018] Figure 2 For the correlation analysis of serum IgE and ALDH7A1 protein levels in asthma patients and healthy subjects in the training set;

[0019] Figure 3 For the correlation analysis between the absolute value of eosinophils and the ALDH7A1 protein content in the serum of asthma patients and healthy subjects in the training set;

[0020] Figure 4 ROC curve for ALDH7A1 protein diagnosis and differentiation between asthma patients and healthy subjects in the training set. DETAILED DESCRIPTION

[0021] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0022] 1. Experimental Sample

[0023] A total of 40 asthma patients (15 males, 25 females, aged 18-65 years) and 43 healthy subjects (18 males, 25 females, aged 18-65 years) were collected. Asthma patients were diagnosed according to the Global Initiative for Asthma (GINA) guidelines, and healthy subjects were healthy physical examination subjects who were of the same period, lived in the same place, were of similar age, and had no direct blood relationship with the asthma patients.

[0024] The age, gender and body mass index of healthy subjects and asthma patients were matched, and there was no significant difference. Healthy subjects and asthma patients were randomly divided into training set samples (22 asthma patients and 22 healthy subjects) and validation set samples (18 asthma patients and 21 healthy subjects).

[0025] Exclusion criteria:

[0026] (1) Patients with a history of allergic rhinitis; (2) Patients with other acute diseases or uncontrolled chronic diseases; (3) Patients with serious primary diseases such as cardiovascular, liver, kidney and hematopoietic system diseases; (4) Patients with mental illness and unable to cooperate; (5) Patients with a clear history of tumors and chemotherapy drugs; (6) Patients with a history of upper respiratory tract infection within the past month; (7) Pregnant or lactating women, or those preparing for pregnancy; (8) Patients participating in other clinical trials within the past month; (9) Patients unwilling to participate in this study.

[0027] 2. Instruments and Reagents

[0028] M200 microplate reader (TECAN, USA), MinSpin high-speed centrifuge (Eppendorf, Germany); human immunoglobulin (IgE) Elisa kit (Wuhan Huamei Bioengineering Co., Ltd.), human ALDH7A1 Elisa kit.

[0029] 3. Experimental Methods

[0030] 1. Collection and storage of serum samples

[0031] Peripheral blood was collected from the patient and placed in a test tube without anticoagulant. After the blood was naturally aggregated at room temperature for 60 minutes and coagulated, it was centrifuged at 3000 rpm for 10 minutes. The upper serum was aspirated into a sterile cryopreservation tube, labeled and stored at -80°C for later use.

[0032] 2. Determination of IgE and ALDH7A1 protein content in serum

[0033] The levels of IgE and ALDH7A1 in serum were determined according to the method steps in the ELISA kit manual.

[0034] 3. Determination of absolute eosinophil count

[0035] SYSMEX SE-9500 fully automatic blood cell analyzer was used for detection.

[0036] 4. Data processing and statistical analysis

[0037] GraphpadPrism 9.0 was used to perform statistical tests on the data. T-test was used between the two groups of samples, and p < 0.05 was considered statistically significant. Logistic regression was performed on the training set to establish a regression equation for the target protein ALDH7A1 content level, and ROC curve analysis was performed, and the optimal cut-off value was obtained based on the ROC curve. In the validation set, the diagnostic accuracy of ALDH7A1 was calculated based on the predicted probability given by SPSS 26.0 software.

[0038] IV. Experimental Results

[0039] 1. Results of determination of absolute eosinophil value, IgE content and ALDH7A1 protein content in the training set

[0040] The results of the determination of the absolute value of eosinophils and the content of IgE are shown in Table 1. The results show that the serum IgE content and the absolute value of eosinophils in peripheral blood of asthma patients in the training set were significantly higher than those of healthy subjects, and the difference was statistically significant (p < 0.05).

[0041] Table 1 Comparison of absolute eosinophil values ​​and IgE content between the asthma group and the healthy subjects in the training set

[0042]

[0043] The results of ALDH7A1 protein content determination are as follows Figure 1 As shown, the results showed that the ALDH7A1 protein content in the serum of asthma patients in the training set was significantly lower than that in healthy subjects, and the difference was statistically significant (p < 0.05).

[0044] Those skilled in the art know that elevated eosinophils and IgE are typical characteristics of asthma patients. We analyzed the correlation between the serum IgE content and the ALDH7A1 protein content in the training set of asthma patients and healthy subjects and found that as the serum IgE content increased, the ALDH7A1 protein content decreased, showing a negative correlation and significant (p < 0.05). Figure 2 We also analyzed the correlation between the absolute value of eosinophils in the serum of asthma patients and healthy subjects and the content of ALDH7A1 protein in the training set and found that as the absolute value of eosinophils in serum increased, the content of ALDH7A1 protein decreased, showing a negative correlation and significant (p < 0.05). Figure 3 shown.

[0045] The above results show that compared with healthy subjects, the ALDH7A1 protein content in the serum of asthma patients is significantly reduced; at the same time, the ALDH7A1 protein content in the serum of asthma patients is negatively correlated with the severity of the disease in asthma patients.

[0046] 2. ROC curve of ALDH7A1 protein diagnosis to distinguish healthy subjects from asthma patients

[0047] The ALDH7A1 protein content (X) in the training set samples was used as the independent variable, and the group (i.e., healthy subjects and asthma patients) was used as the dependent variable. Logistic regression was performed on the ALDH7A1 protein content in the serum samples of asthma patients and healthy subjects, and the logistic regression equation Logit[p] = 16.831-3.091X was obtained. Then, the ALDH7A1 protein content of each serum sample in the training set was substituted into the logistic regression equation to obtain the regression value Logit[p] of each sample. The possible regression value was used as the diagnostic point, and the sensitivity and specificity were calculated. Based on this, the ROC curve was drawn (such as Figure 4 The AUC was 0.907, and the optimal cut-off value for distinguishing healthy subjects from asthma patients according to the ROC curve was 0.667.

[0048] Those skilled in the art know that the area under the ROC curve AUC has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it means no diagnostic significance; when AUC is 0.5-0.7, it means low diagnostic accuracy; when AUC is 0.7-0.9, it means medium diagnostic accuracy; when AUC>0.9, it means high diagnostic accuracy. Therefore, the above results show that ALDH7A1 protein has a high accuracy in distinguishing healthy subjects from asthma patients.

[0049] 3. Verify the accuracy of ALDH7A1 protein in diagnosing asthma

[0050] In the validation set, the ALDH7A1 protein content of each sample was imported into the SPSS software, and the best cut-off value obtained in the training set was used as the diagnostic threshold to predict the serum samples of asthma patients VS healthy subjects in the validation set, and the prediction accuracy was obtained. The results showed that the prediction accuracy was 76.9% (30 / 39).

[0051] The above results further indicate that ALDH7A1 protein has a high accuracy in distinguishing healthy subjects from asthma patients, and ALDH7A1 protein has the prospect of being used in the development of a kit for diagnosing asthma.

[0052] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Use of ALDH7A1 protein for preparing a kit for diagnosing asthma.

2. The use according to claim 1, wherein the kit is a serum detection kit.

3. The use according to claim 1, wherein the kit contains a detection reagent for ALDH7A1 protein.

Images