Blood marker for assisting in diagnosing influenza

Abstract

The invention belongs to the field of biological medicine industry, and discloses a blood marker for assisting in diagnosing influenza. The inventor verifies that the LRG1 is a peripheral blood marker of influenza for the first time and can be used for auxiliary diagnosis of influenza and increase of the detection rate of influenza virus by being matched with multiple times of detection of influenza virus. A test result shows that the peripheral blood LRG1 content 5ng / ml is a CUTOFF value, 46 patients and 4 false negative patients are successfully detected for 50 influenza patients, and the sensitivity is 92%; the number of normal persons is 60, the number of the normal persons is 12, and the specificity is 80%. After influenza treatment, LRG1 and Plt are obviously reduced compared with those before influenza treatment; 0.001, which is equivalent to the level of normal people, Pgt; and 0.05%.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical industry, and specifically relates to the application of LRG1 quantitative reagent in the preparation of influenza detection reagent. Background Art

[0002] Influenza, or the flu, is a Class C infectious disease caused by influenza viruses. It is primarily caused by influenza A and B viruses, with influenza A being the most common in China. After infection, the virus binds to cells on the surface of the respiratory tract, enters the cells, and replicates, causing systemic symptoms such as fever, headache, and myalgia. The antigenic variation of influenza viruses makes them highly contagious, making them universally susceptible, and the same person can be infected multiple times. Because influenza viruses are adapted to cold, humid climates, they are more common in winter and spring.

[0003] Flu symptoms include high fever, fatigue, and headache, while respiratory symptoms are milder. The incubation period is typically 1-7 days. Flu is primarily spread through droplets, including sneezing and coughing, and is most contagious during the first two to three days of illness. Furthermore, the entire population is susceptible to flu, especially high-risk groups such as children, the elderly, and those with underlying health conditions.

[0004] Common treatments for influenza include general therapy and antiviral therapy. Oseltamivir and Mabalosac are both effective specific medications for the treatment of influenza. Specific treatment should be sought promptly at a hospital and followed by a doctor's advice. The prognosis for influenza is typically 4-7 days, though a few patients may experience persistent coughing for several weeks.

[0005] 1. Influenza Prevention: Numerous studies are underway to understand and research the spread and prevention of influenza. Research from the China Centers for Disease Control and Prevention has found that climate variables and latitude are the factors most strongly correlated with influenza seasonality. Global studies have shown that influenza vaccination is the most effective way to prevent influenza infection and its severe complications.

[0006] 2. Clinical manifestations of influenza: fever, sore throat, cough, body aches, or runny nose 3. Diagnosis: This usually involves history taking, physical examination, and imaging studies as necessary to confirm the diagnosis and severity.

[0007] 3.1. Medical history collection: The doctor will ask the patient in detail about symptoms, duration, inducing factors, etc.

[0008] 3.2. Physical examination: red throat, fever, and rapid heart rate 3.3. Imaging examinations: Imaging methods such as X-ray, ultrasound, CT or MRI can help identify pneumonia.

[0009] 3.4. Influenza virus antigen detection is the basis for diagnosis 4. Prevention and treatment: Wear a mask, disinfect the environment, treat symptoms, and administer oseltamivir and mabaloxavir tablets. 5. Relationship between influenza and peripheral blood proteins Influenza virus infection causes related symptoms in patients. Any symptom has a material basis, resulting in changes in gene expression and proteins.

[0010] Proteins are the executors of gene functions. Research on protein structure, localization, and protein-protein interactions will provide a direct basis for elucidating the essence of life phenomena. Almost all physiological and pathological processes, as well as the effects of drugs and environmental factors, rely on proteins and cause changes in the proteome. Before any disease manifests any noticeable symptoms, certain proteins must have changed. Therefore, finding key proteins and marker proteins for various diseases is of great significance for disease diagnosis, pathological research, and drug screening. Leucine-rich repeat family proteins include eight leucine-rich repeat sequences (LRR). Studies have shown that LRR proteins are transmembrane proteins, which mainly participate in normal physiological activities as cell adhesion molecules or ligand-binding proteins.

[0011] Leucine-rich α2-glycoprotein 1 (LRG1) is a member of the leucine-rich repeat family and belongs to the LRR protein family. Previous studies have shown that LRG1 is involved in important physiological and pathological processes in the body, such as protein interactions, signal transduction, and cell adhesion.

[0012] Numerous proteins containing leucine-rich repeats have been identified within the nervous system. Reduced levels of LRG1 have been detected in cerebrospinal fluid (CSF) in several neurodegenerative diseases, and expression of LRG1 has been observed in autopsied human cerebral cortices. Previous studies, including proteomic analysis of CSF samples from healthy individuals and patients with idiopathic normal pressure hydrocephalus (INPH), have revealed that LRG1 expression is specifically elevated in the CSF of INPH patients. This suggests that LRG1 has potential clinical diagnostic value for INPH. Abnormal LRG1 expression has been associated with the development of various tumors, including gliomas, lung cancer, colon cancer, epithelial ovarian cancer, and hepatocellular carcinoma.

[0013] During research into retinopathy caused by vascular remodeling, LRG1 was found to promote endothelial cell mitosis and angiogenesis. This hypothesis was further confirmed in an in vitro human vascular endothelial cell (HUVEC) tube formation assay. Administration of recombinant human LRG1 resulted in a significant increase in tube formation and branching, while anti-LRG1 antibodies significantly blocked tube formation. Other studies have shown that the angiogenic activity of LRG1 is not limited to the eye and may play a crucial role in other major biological processes, including tumorigenesis and immune responses. LRG1's regulatory effect on the TGF-β1 signaling pathway provides strong evidence for the function of LRG1. LRG1 is a promising therapeutic target for controlling ocular angiogenic disorders and may also play a significant role in other diseases such as cancer and atherosclerosis.

[0014] The function of LRG1 may be related to its LRR structure. LRG is believed to regulate other proteins, such as cytochrome c, by binding to them, thereby limiting their function. Glioma, originating from glial cells, is the most common tumor of the central nervous system, accounting for 30% of brain and central nervous system tumors and 80% of all malignant brain tumors. Related experiments have shown that LRG1 plays a key role in promoting the proliferation, migration, and invasion of glioma cells by mediating the transforming growth factor β1 (TGF-β1) signaling pathway. Furthermore, LRG1 can promote endothelial cell proliferation and angiogenesis through the TGF-β signaling pathway, suggesting that LRG1 may be involved in the development and progression of astrocytic malignancies. Previous studies have shown that inhibiting LRG1 expression inhibits glioma cell growth in vitro and delays glioma development in xenograft mouse models, providing evidence for a role for LRG1 in promoting glioma pathology. LRG1 is associated with atherosclerosis and microcirculatory impairment and may serve as a marker of vascular damage in different vascular beds. LRG1 has a potential role in early identification of vascular disease risk, but the pathological basis and mechanisms underlying these associations require further exploration. Modulating LRG1 activity may be a potential therapeutic approach in vascular diseases, particularly with minimally invasive surgery and endovascular revascularization. LRG1 expression is upregulated after myocardial infarction, inhibiting post-MI cardiac remodeling. As a novel cardiovascular protective factor, LRG1 may be a therapeutic target for cardiovascular disease. Genetic deletion of LRG1 exacerbates post-MI cardiac dysfunction and increases myocardial fibrosis. In LRG1-deficient mice, impaired Smad1 / 5 / 8 signaling leads to reduced capillary density at the infarct margin. Genetic ablation of LRG1 exacerbates this post-MI capillary density reduction in a cardiac remodeling model. Furthermore, our studies have shown that LRG1 levels are elevated in the blood of patients with cardiorespiratory arrest after cardiopulmonary and cerebral resuscitation, potentially related to brain and vascular damage caused by cardiorespiratory arrest. This is consistent with the mechanism of increased LRG1 in the blood of patients with cerebral infarction and myocardial infarction.

[0015] On the other hand, LRG1, as an angiogenic factor, may have promising prospects for the treatment of vascular diseases by regulating the smad1 / 5 / 8 pathway, but its specific regulatory mechanism is still unclear.

[0016] The inventor's prior Chinese patent application CN117110626A disclosed that the average LRG1 level in the blood of patients with carotid artery disease is higher than that of normal subjects, and that as the disease worsens, the shedding of plaque in carotid vascular disease increases more significantly. As a peripheral blood marker for dangerous carotid artery disease, it has high sensitivity and specificity and can be used in combination with ultrasound results to diagnose carotid artery disease. CN115856325A disclosed that the level of LRG1 in peripheral blood is closely related to lower limb artery damage. By detecting the level of LRG1 in peripheral blood, it is possible to effectively predict lower limb artery damage, particularly lower limb arteriosclerosis or plaques, lower limb arterial stenosis, thrombosis or occlusion, and in particular, to effectively predict lower limb arteriosclerosis or plaques, lower limb arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia. CN110261617A disclosed that LRG1 can be used as a peripheral blood marker for cerebral hemorrhage.

[0017] In the prior art, no studies have shown that LRG1 is associated with influenza. Summary of the Invention

[0018] The object of the present invention is to overcome at least one deficiency of the prior art and provide a use of an LRG1 quantitative reagent in the preparation of an influenza detection reagent.

[0019] The technical solution adopted by the present invention is: The first aspect of the present invention provides: Application of LRG1 quantitative reagent in the preparation of influenza diagnostic or auxiliary diagnostic reagents.

[0020] In some examples, the LRG1 quantitative reagent is an ELISA reagent, an immunofluorescence reagent, or a mass spectrometry reagent.

[0021] In some examples, the sample detected by the LRG1 quantitative reagent is peripheral blood.

[0022] The second aspect of the present invention provides: An influenza diagnosis or auxiliary diagnosis system, comprising: LRG1 quantification device, used to quantify the amount of LRG1 in a sample; An analytical device that determines influenza risk based on the level of LRG1; The result output device is used to output the analysis result of the analysis device.

[0023] In some examples, the LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

[0024] In some instances, high levels of LRG1 indicate a high risk of influenza.

[0025] In some embodiments, a high level of LRG1 means a level of LRG1 greater than 5 ng / mL in a peripheral blood sample.

[0026] In some examples, the sample is peripheral blood.

[0027] The beneficial effects of the present invention are: The inventors' research has confirmed for the first time that LRG1 is a peripheral blood marker for influenza. Combined with multiple tests for influenza virus, it can be used as an auxiliary diagnostic marker for influenza and improve the detection rate of influenza virus.

[0028] The test results showed that, using a peripheral blood LRG1 level of 5 ng / ml as the cutoff value, 46 of 50 influenza patients were successfully detected, with 4 false negatives, for a sensitivity of 92%. Among 60 healthy controls, 12 were false positives, for a specificity of 80%. LRG1 levels were significantly reduced after influenza treatment compared to pre-treatment levels (P<0.001), and comparable to levels in healthy controls (P>0.05). DETAILED DESCRIPTION

[0029] The first aspect of the present invention provides: Application of LRG1 quantitative reagent in the preparation of influenza diagnostic or auxiliary diagnostic reagents.

[0030] The LRG1 quantitative reagent can be any conventional protein quantitative reagent, without special requirements. In some embodiments, the LRG1 quantitative reagent is an ELISA reagent, an immunofluorescence reagent, or a mass spectrometry reagent. The LRG1 quantitative reagent is preferably an ELISA reagent, which is easy to operate and can produce results that meet the requirements.

[0031] In some embodiments, the sample used for the LRG1 quantitative reagent is peripheral blood, which is easy to obtain and easily accepted by people.

[0032] The second aspect of the present invention provides: An influenza diagnosis or auxiliary diagnosis system, comprising: LRG1 quantification device, used to quantify the amount of LRG1 in a sample; An analytical device that determines influenza risk based on the level of LRG1; The result output device is used to output the analysis result of the analysis device.

[0033] There is no particular requirement for the type of LRG1 quantification device, as long as it can effectively quantify the amount of LRG1. In some embodiments, the LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

[0034] In some instances, high levels of LRG1 indicate a high risk of influenza.

[0035] In some embodiments, a high level of LRG1 means a level of LRG1 greater than 5 ng / mL in a peripheral blood sample.

[0036] In some examples, the sample is peripheral blood.

[0037] Screening and collection of clinical samples: Fifty patients with influenza infection were enrolled over the past five years. Gender and age were recorded. All patients underwent blood pressure, lipid profiles, blood sugar, liver function, renal function, coagulation tests, and electrocardiograms (ECGs). All cases underwent ultrasound, CT, and neuroelectrophysiological confirmation. Sixty healthy controls, matched for age and gender, were also selected. Sample collection: 5 mL of venous blood was collected from patients who met the inclusion criteria. The blood was allowed to stand for 15 minutes and then centrifuged at 3000 rpm for 15 minutes. Serum was extracted and placed in sterile cryovials and stored at -80°C for research purposes.

[0038] Study of blood samples from case patients Separate the serum. The inspection method is as follows: Separate the serum and cells using a high-speed centrifuge.

[0039] Main equipment, materials, and reagents: homogenizer, high-speed centrifuge, 3kDa ultrafiltration centrifuge tubes, ICP-MS. 18.2 MΩ / cm ultrapure water was used throughout the experiment. Sample pretreatment methods: Fully automatic biochemical analyzer (one each of Beckman DXC800 and AU400), 1 / 10,000 electronic analytical balance (Shimadzu), microplate reader, Roche electrochemiluminescence automated immunoassay analyzer (Elecsys 2010), specific protein analyzer (imported), water purification machine (MEDICA60), electric constant temperature water bath (DK-600), low-temperature high-speed centrifuge (Sigma), ultra-low temperature freezer (Thermo Fisher Scientific), low-temperature freezer (HFC350, Germany). The above equipment is fully capable of implementing this project.

[0040] Statistical methods: The mean values ​​of each group were compared with T test using SPSS statistical software, and P < 0.05 was considered statistically significant.

[0041] result: Normal values ​​are defined as the mean plus two standard deviations. The upper limit of normal for the Huizhou-produced LRG1 test kit (LRG1 Huizhou) is 5 ng / ml. In a test of 50 influenza patients using the Huizhou-produced reagent, the cutoff value was 5 ng / ml. Results exceeded the cutoff value in 46 patients, with 4 false negatives, for a sensitivity of 92%. In a test of 60 normal individuals, 12 exceeded the cutoff value, resulting in a false positive rate of 20% and a specificity of 80%.

[0042] The results of changes in peripheral blood LRG1 in influenza are shown in Table 1 .

[0043] Table 1: Changes in peripheral blood LRG1 in influenza patients (X±SD, unit ng / ml) Number of cases LRG1 / ng / ml Normal people 60 3.2±1.1 influenza 50 15.2±3.2 The influenza rate was significantly higher than that of normal people, P < 0.001.

[0044] Sensitivity and specificity of LRG1 in diagnosing influenza Sensitivity = number of true positives / (number of true positives + number of false negatives) * 100%, the percentage of patients correctly diagnosed Specificity = number of true negatives / (number of true negatives + number of false positives)*100%, the percentage of correctly identified non-patients False positive rate = number of false positives / number of gold standard negatives False negative rate = number of false negatives / number of gold standard positives The normal upper limit of the LRG1 detection kit produced in Huizhou is 5ng / ml. 50 cases of influenza were tested with Huizhou-produced reagents, with a CUTOFF value of 5ng / ml. 46 cases exceeded the CUTOFF value, and there were 4 false negatives. 60 normal people were tested, 12 of whom exceeded the CUTOFF value, and the false positive rate was 20%.

[0045] The changes of LRG1 before and after influenza treatment are shown in Table 2 .

[0046] Table 2: Changes in LRG1 before and after influenza treatment Number of cases LRG1 / ng / ml Normal people 60 3.2±1.1 Before flu treatment 50 15.2±3.2 After flu treatment 32 4.3±2.1 After influenza treatment, LRG1 was significantly reduced compared with that before treatment (P<0.001), which was equivalent to the level of normal people (P>0.05).

[0047] The above test results show that the average LRG1 level in the blood of influenza patients is higher than that of normal people. LRG1 has high sensitivity and specificity as a peripheral blood marker for influenza.

[0048] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

[0049] References: [1] Tessa Gordon 1, Nasim Amirjani, David CEdwards, K Ming Chan,Briefpost-surgical electrical stimulation accelerates axon regeneration and musclereinnervationwithout affecting the functional measures in carpal tunnelsyndrome patients. Trial Exp Neurol. 2010 May;223(1):192-202. [2] Proteomic analysis of hippocampus in micefollowing long-termexposure to low levels of copper. Qian Sun, Ming Ying, Quan Ma, Zhijun Huang,Liangyu Zou, Jianjun Liu, Zhixiong Zhuang, Xifei Yang.Toxicol. Res.,2016,4,28 [3]Serumproteomic analysis reveals potential serum biomarkers foroccupational medicamentosa-like dermatitis caused by trichloroethylene PeiwuHuang a,d,1,Xiaohu Ren a,1, Zhijun Huang c,1, Xifei Yang a, Wenxu Hong a,Yanfang Zhang b, Hang Zhang a,d, Wei Liu a, Haiyan Huang a, Xinfeng Huang a,Desheng Wua,Linqing Yang a, Haiyan Tang b, Li Zhou a, Xuan Li a, Jianjun Liu。Toxicology Letters 2014,6,21(sci 3.75) [4] 1. Ditochondrial proteomic alterations caused by long-term low –dose copper exposure in mouse cortex. Xuemei Lin, Gang Wei, Zhijun Huang,etc. Toxicology Letter, 2016 [5] Detection of coagulation factor VIIR353Q genotype in patients with intracerebral hemorrhage in the Han ethnic group in South China. Chinese Journal of Arteriosclerosis, 2007, 6. [6]. Research progress on the application of gene chips in the diagnosis and treatment of cardiovascular diseases, China Medical Engineering, 2005, 10. [7] Application of DNA molecular genetic markers RFLP, STR and SNP in coagulation genotype analysis. Chinese Journal of Misdiagnosis Medicine, 2006, 4.

Claims

1. Application of LRG1 quantitative reagent in the preparation of influenza diagnostic or auxiliary diagnostic reagents.

2. The use according to claim 1, characterized in that The LRG1 quantitative reagents include ELISA reagents, immunofluorescence reagents, and mass spectrometry reagents.

3. The use according to claim 1, characterized in that The sample for LRG1 quantitative reagent detection is peripheral blood.

4. An influenza diagnosis or auxiliary diagnosis system, comprising: LRG1 quantification device, used to quantify the amount of LRG1 in a sample; An analytical device that determines influenza risk based on the level of LRG1; The result output device is used to output the analysis result of the analysis device.

5. The influenza diagnostic system according to claim 4, characterized in that The LRG1 quantitative device is an ELISA detection device, an immunoblotting device, an immunofluorescence device or a mass spectrometry device.

6. The influenza diagnostic system according to claim 4, characterized in that: High levels of LRG1 indicate a high risk of influenza.

7. The influenza diagnostic system according to claim 6, characterized in that: High LRG1 levels refer to levels higher than 5 ng / mL in peripheral blood samples.

8. The influenza diagnostic system according to claim 4, characterized in that: The sample is peripheral blood.