Immunoassay kit for predicting the occurrence of mental and psychological symptoms after stress and application thereof

Abstract

The present application relates to a biomarker for predicting mental and psychological symptoms such as anxiety and depression after stress and a detection kit thereof. The present application provides a group of biomarkers for predicting the occurrence of mental and psychological symptoms such as anxiety and depression after stress in potential patients, which are selected from one or more of catalase, dopamine-beta-hydroxylase, neuropilin 1 or an amino acid sequence having 80% homology therewith. The present application also provides a detection kit and a detection method for detecting these proteins in blood samples; and the application of the above antigens and kits; and provides a reference concentration threshold of high risk of anxiety and depression after stress of these proteins. The immunodetection kit made therefrom can quickly, simply and accurately detect the concentration of biomarkers in blood, and is suitable for large-scale popularization and application.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202311409525.6, filed on December 6, 2021, entitled "Immunoimmunoassay kit for predicting the occurrence of post-stress mental and psychological symptoms and its application". Technical Field

[0002] This invention relates to the detection and identification of biomarkers generated in individuals suffering from post-stress anxiety and depression, and provides a metric for distinguishing high-risk individuals in predicting potential cases of anxiety and depression. Specifically, it refers to a set of biomarkers that can predict the occurrence of post-stress anxiety and depression, as well as detection kits and methods for detecting these biomarkers in blood samples, and provides reference concentration thresholds for high-risk occurrence of these biomarkers, enabling reliable detection and identification of biomarkers for the diagnosis and prevention of post-stress anxiety and depression. Background Technology

[0003] In recent years, the incidence of stressful events such as public health emergencies, various major accidents, and natural and man-made disasters has been gradually increasing. Worldwide, over 70% of people will experience at least one traumatic event in their lifetime. Those who experience such events, as well as rescuers such as medical personnel, often experience anxiety, depression, and other mental health symptoms after the stressful event, with some individuals even developing serious mental illnesses. Post-traumatic stress disorder (PTSD) is one of the more serious mental illnesses. PTSD refers to a delayed-onset and persistent mental disorder that occurs after an individual experiences, witnesses, or encounters one or more events involving actual or threatened death, serious injury, or a threat to physical integrity, either themselves or others. Clinically, it manifests as traumatic memory intrusion, avoidance of trauma-related stimuli, negative cognitive alterations, increased arousal, and excessive behavior. Depending on the cultural background and economic development level of different countries and regions, the lifetime prevalence of PTSD is approximately 1.3%–12.2%, and the annual prevalence is approximately 0.2%–3.8%. In today's society, post-stress mental illnesses, including PTSD, have become a major concern. These illnesses have a long course and are prone to relapse. Patients often exhibit behaviors such as self-harm, suicide, and substance abuse, which seriously affect their quality of life and impose a severe psychological, physiological, and economic burden on individuals, families, and society.

[0004] Among post-stress mental illnesses, PTSD is the most severe and receives the most attention. The widely accepted clinical diagnostic criteria for PTSD are the American Psychiatric Association's DSM-5 and the World Health Organization's ICD-11. In DSM-5, the diagnostic criteria for PTSD consist of eight items, divided into four symptom clusters and one subtype. The four symptom clusters are traumatic memory intrusion, avoidance of trauma-related stimuli, negative cognitive and emotional changes, increased arousal, and hyperactive behavior. In ICD-11, the diagnosis of PTSD is simplified to three symptom clusters, totaling six symptoms, including recurrent retrieval of the traumatic event, avoidance of the traumatic stimulus, and hypervigilance. Although the diagnostic criteria for PTSD differ slightly, its treatment primarily includes psychological intervention, pharmacology, and other innovative therapies. Psychological intervention, as the first-line treatment recommended by most guidelines, mainly includes extended exposure therapy, cognitive processing therapy, cognitive behavioral therapy, eye movement desensitization, and reprocessing therapy. Drug therapy is generally used as a second-line treatment, including selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, monoamine oxidase, sympatholytic agents, anticonvulsants, benzodiazepines, and other drugs.

[0005] Although PTSD has a high incidence rate in individuals who have experienced one or more severe traumas, not all individuals who have experienced trauma will develop PTSD. The actual lifetime prevalence of PTSD is far lower than theoretically estimated, indicating significant individual variability in its incidence. Studies on PTSD incidence based on different racial groups have found a significantly lower incidence in Caucasians than in other racial groups, while twin studies have also found a significantly higher incidence in identical twins than in fraternal twins after a traumatic event, suggesting that genetic factors significantly influence the incidence of PTSD. Currently, the diagnosis of PTSD mainly relies on the patient's medical history, assessment of mental status, duration of symptoms, clinical psychiatric scale tests, and patient self-report, lacking the detection of objective and effective biomarkers for PTSD. Considering that most traumatic events are unavoidable, predicting risk factors for PTSD after trauma becomes particularly important. However, there are few studies on the screening of objective, effective, and specific biomarkers for PTSD, and there is still a lack of objective indicators for the early warning diagnosis and treatment efficacy evaluation of PTSD. The detection of blood biomarkers is considered the most promising and convenient method for PTSD diagnosis due to its convenience and minimally invasive nature. Studies have reported that decreased blood cortisol levels after trauma may be associated with the development of PTSD. Imbalances in certain neurotransmitters in the blood, as well as levels of adrenocorticotropic hormone (ACTH), glucocorticoids (GC), adrenaline, and catecholamines, are also considered biomarkers related to PTSD. Other biomarkers potentially associated with PTSD include cholecystokinin (CCK), nitric oxide synthase (NOS), neuropeptide Y (NPY), and p11 protein. However, the specificity and sensitivity of these biomarkers have not been well validated. Furthermore, specific biomarkers for post-stress mental disorders other than PTSD are rarely reported. Therefore, there is an urgent need to discover and develop specific biomarkers for post-stress anxiety, depression, and other mental health symptoms.

[0006] Therefore, in order to effectively predict the occurrence of mental and psychological symptoms such as anxiety and depression after stress, it is necessary to identify novel biomarkers and develop test kits that can specifically detect these biomarkers. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a set of biomarkers that can predict the occurrence of post-stress anxiety, depression, and other mental and psychological symptoms, as well as detection kits and methods for detecting these biomarkers in blood samples. Detection kits based on this set of biomarkers can be used to detect the concentration of these biomarkers in the blood of potential stress patients with high sensitivity, high specificity, and high stability. The concentration thresholds provided by the present invention indicate a high risk of developing mental and psychological symptoms such as anxiety and depression, thus providing doctors with a basis for accurately predicting that stress patients will suffer from mental illnesses such as anxiety, depression, and PTSD, enabling timely treatment to prevent the occurrence of such diseases and alleviate patient suffering and burden. The immunoassay kits made from these biomarkers can quickly, simply, and accurately detect the concentration of biomarkers in blood, making them suitable for large-scale application.

[0008] To achieve the above objectives, in a first aspect of the present invention, an immunoassay kit for predicting the occurrence of post-stress anxiety, depression, and other mental and psychological symptoms is provided. The immunoassay kit comprises reagents for quantitatively detecting the expression levels of biomarkers, wherein the biomarkers are selected from one or more of the following: butyrylcholinesterase (BCHE), catalase (CAT), dopamine beta-hydroxylase (DBH), insulin-like growth factor binding protein 2 (IGFBP2), neuropilin 1 (NRP1), an amino acid sequence having at least 80% homology with the amino acid sequence of BCHE, an amino acid sequence having at least 80% homology with the amino acid sequence of CAT, an amino acid sequence having at least 80% homology with the amino acid sequence of DBH, an amino acid sequence having at least 80% homology with the amino acid sequence of IGFBP2, and an amino acid sequence having at least 80% homology with the amino acid sequence of NRP1.

[0009] Preferably, the aforementioned post-stress anxiety and depression symptoms include post-stress anxiety, post-stress depression, post-traumatic stress disorder, post-stroke anxiety, post-stroke depression, post-traumatic brain injury anxiety, post-traumatic brain injury depression, and other post-stress-related mental illnesses.

[0010] In a second aspect of the invention, an application is provided in the preparation of a reagent for detecting and predicting the occurrence of post-stress psychosomatic symptoms. The reagent is used to quantitatively detect the expression level of the biomarker. The biomarker is selected from one or more of the following: butyrylcholinesterase, catalase, dopamine-β-hydroxylase, insulin-like growth factor binding protein 2, neurociliacin 1, an amino acid sequence having at least 80% homology with the amino acid sequence of butyrylcholinesterase, an amino acid sequence having at least 80% homology with the amino acid sequence of catalase, an amino acid sequence having at least 80% homology with the amino acid sequence of dopamine-β-hydroxylase, an amino acid sequence having at least 80% homology with the amino acid sequence of insulin-like growth factor binding protein 2, and an amino acid sequence having at least 80% homology with the amino acid sequence of neurociliacin 1.

[0011] In a third aspect of the invention, a detection method for an immunoassay kit is provided to detect the levels of the aforementioned biomarker proteins in whole blood, central nervous system tissue, serum, plasma, cerebrospinal fluid, saliva, sweat, tears, urine, oral samples, or combinations thereof. Preferably, the bodily fluid is blood. The detection of the aforementioned proteins can be performed by conventional enzyme-linked immunosorbent assay (ELISA), preferably using a sandwich ELISA method.

[0012] Preferably, the immunoassay kit further includes a carrier on which an antigen for detecting the aforementioned biomarker protein is coated.

[0013] Preferably, the immunoassay kit further includes a primary antibody, which is a primary antibody used to detect the aforementioned biomarker protein.

[0014] Preferably, the immunoassay kit further includes a secondary antibody labeled with the primary antibody of the aforementioned biomarker protein. The label used may be horseradish peroxidase, alkaline phosphatase, the fluorescent molecule FITC (or other fluorescent labels), or chemiluminescence detection. The detection method may be a colorimetric method, a fluorescence method, chemiluminescence, or electrochemiluminescence for qualitative and quantitative analysis.

[0015] More preferably, the secondary antibody against the primary antibody of the aforementioned biomarker protein is a horseradish peroxidase-labeled secondary antibody against the primary antibody of the aforementioned protein.

[0016] In a fourth aspect of the invention, an immunoassay kit for the aforementioned biomarker protein is provided for the detection of the aforementioned biomarker protein in human blood. By comparing the measured level of the aforementioned biomarker protein in a subject (e.g., a patient who has experienced a traumatic event) with the measured level of the aforementioned biomarker protein using quantitative mass spectrometry, a high-risk concentration threshold for the aforementioned protein is determined.

[0017] The beneficial effects of this invention are as follows: This invention provides a set of biomarkers that can predict the occurrence of post-stress anxiety, depression, and other mental and psychological symptoms, including butyrylcholinesterase (BCHE), catalase (CAT), dopamine beta-hydroxylase (DBH), insulin-like growth factor binding protein 2 (IGFBP2), and neuropilin 1. 1. One or more of the following amino acid sequences are used: NRP1, an amino acid sequence with at least 80% homology to BCHE, an amino acid sequence with at least 80% homology to CAT, an amino acid sequence with at least 80% homology to DBH, an amino acid sequence with at least 80% homology to IGFBP2, and an amino acid sequence with at least 80% homology to NRP1. After a stressful event, the concentration of these biomarker proteins in the blood can be detected with high specificity, high sensitivity, and high specificity. This provides doctors with a basis for accurately predicting whether stressful patients will suffer from mental illnesses such as anxiety, depression, and PTSD, enabling timely treatment to prevent the occurrence of such diseases and alleviate patient suffering and burden. Immunoassay kits made from these sequences can quickly, simply, and accurately detect the concentration of biomarkers in the blood, making them suitable for large-scale application. Attached Figure Description

[0018] Figure 1 A comparison of serum BCHE protein levels between patients with and without post-traumatic anxiety and depression symptoms.

[0019] Figure 2 A comparison of serum CAT protein levels between patients with and without post-traumatic anxiety and depression symptoms.

[0020] Figure 3 A comparison of serum DBH protein levels between patients with and without post-traumatic anxiety and depression symptoms.

[0021] Figure 4 A comparison of serum IGFBP2 protein levels between patients with and without post-traumatic anxiety and depression symptoms.

[0022] Figure 5 A comparison of serum NRP1 protein levels between patients with and without post-traumatic anxiety and depression symptoms. Detailed Implementation

[0023] To better understand the technical content of this invention, the following embodiments are provided for detailed explanation.

[0024] Protein biomarkers Biomarkers used to predict the occurrence of post-stress anxiety, depression, and other mental health symptoms include butyrylcholinesterase (BCHE), catalase (CAT), dopamine beta-hydroxylase (DBH), insulin-like growth factor binding protein 2 (IGFBP2), and neuropilin 1 (NRP1).

[0025] BCHE is a broadly substrate-specific esterase that contributes to the inactivation of the neurotransmitter acetylcholine. BCHE can degrade neurotoxic organophosphates. For example... Figure 1 As shown, the present invention identified that, compared with the levels in patients who experienced post-traumatic anxiety and depression, the levels of BCHE protein in whole blood, plasma, and serum of patients who experienced post-traumatic anxiety and depression were significantly lower (p<0.05).

[0026] CAT is present in almost all aerobic organisms and serves to protect cells from the toxic effects of hydrogen peroxide. CAT can promote cell growth, including T cells, B cells, myeloid leukemia cells, melanoma cells, mast cell tumor cells, and normal and transformed fibroblasts. For example... Figure 2 As shown, the present invention identified that, compared with the levels in patients who experienced post-traumatic anxiety and depression, the levels of CAT protein in whole blood, plasma, and serum of patients who experienced post-traumatic anxiety and depression were significantly lower (p<0.05).

[0027] DBH protein is expressed in neurosecreting vesicles and chromaffin granules of the adrenal medulla, catalyzing the conversion of dopamine to norepinephrine, a hormone and a major neurotransmitter of the sympathetic nervous system. DBH exists in soluble and membrane-bound forms, depending on the absence or presence of the signal peptide, respectively. Mutations in this gene lead to dopamine-β-hydroxy acid deficiency in human patients, characterized by autonomic and cardiovascular dysfunction, including hypotension and ptosis. This invention identified significantly lower levels of DBH protein in whole blood, plasma, and serum of patients with post-traumatic anxiety and depression compared to those without such symptoms (p<0.05).

[0028] IGFBP2 inhibits IGF-mediated growth and development rates. IGFBP2 prolongs the half-life of IGF and has been shown to inhibit or stimulate the growth-promoting effect of IGF on cell cultures. IGFBP2 can alter the interaction between IGF and its cell surface receptors. This invention identified significantly lower levels of IGFBP2 protein in whole blood, plasma, and serum of patients with post-traumatic anxiety and depression compared to patients without such symptoms (p<0.05).

[0029] NRP1 is a cell surface receptor involved in cardiovascular development, angiogenesis, neural circuit formation, and extra-neural organogenesis. NRP1 mediates the chemical repulsion activity of serotonins. NRP1 recognizes the C-terminal motif R / KXXR / K on its ligands, leading to internalization and vascular leakage. It binds to serotonin 3A, the PLGF-2 isoform of PGF, and the VEGF165 isoform of VEGFA and VEGFB. Co-expression of NRP1 with KDR leads to increased binding of VEGF165 to KDR and increased chemotaxis. NRP1 regulates VEGF-induced angiogenesis. NRP1 binding to VEGFA initiates signaling pathways required for motor neuron axonal guidance and cell body migration, including the caudal migration of facial motor neurons from rhomboid 4 to rhomboid 6 during embryonic development (via similarity). NRP1 regulates mitochondrial iron transport through interaction with ABCB8 / MITOSUR. NRP1 binds to VEGF-165 and may inhibit its binding to cells. NRP1 can induce apoptosis by isolating VEGF-165. NRP1 can also bind to various members of the serotonin family. NRP1 expression has a negative impact on angiogenesis and vessel integrity. NRP1 acts as a host factor for human coronavirus SARS-CoV-2 infection. NRP1 recognizes and binds to the RRAR motif on the S1 of the SARS-CoV-2 spike protein using C-terminal rules to enhance SARS-CoV-2 infection. This invention identified significantly lower levels of NRP1 protein in whole blood, plasma, and serum of patients with post-traumatic anxiety and depression compared to those without such symptoms (p<0.05).

[0030] Reagent test kit In this invention, five biomarkers (BCHE, CAT, DBH, IGFBP2, and NRP1) for the occurrence of post-stress anxiety and depression in serum were detected using five different enzyme-linked immunosorbent assay (ELISA) kits. Sandwich ELISA was preferred for the assays.

[0031] 1. Establishment of the sandwich ELISA method 1) Take several Costar strips coated with primary antibodies against biomarkers for the occurrence of post-stress anxiety, depression and other mental and psychological symptoms, according to the number of test samples.

[0032] 2) Add 100 μL of sample diluent per well.

[0033] 3) Add standard samples, blank controls and diluted serum samples, 50 μL / well, incubate at 37°C for 2 hours on a shaker at 200 rpm.

[0034] 4) Wash with PBST, 400 μL / well, 5 times, then blot dry.

[0035] 5) Add another horseradish peroxidase-labeled primary antibody of the corresponding biomarker, 200 μL / well, incubate at 37°C for 2 hours, shake at 200 rpm.

[0036] 6) Wash with PBST, 400 μL / well, 5 times, then blot dry.

[0037] 7) TMB color development: Mix equal volumes of TMB solution A and solution B, 100 μL / well, incubate at 37°C for 20 min.

[0038] 8) Stop solution, 50 μL / well, read the value using a microplate reader at 450 nm.

[0039] 2. Kit components: This kit consists of the following parts: 1). One Costar ELISA plate containing the primary antibody for detecting the occurrence of post-stress anxiety, depression, and other mental and psychological symptoms of this invention. 2) One tube (1 ml) each of PTSD biomarker standard samples. 3) One bottle (100ml) of sample diluent 4). One tube (20ml) each of horseradish peroxidase-labeled anti-biomarker antibodies. 5) One bottle (100ml) of PBST (10*10) 6) One bottle (60ml) of TMB developer solution A and one bottle (60ml) of TMB developer solution B. 7) One bottle of stop solution (60ml) 3. Identification of biomarkers for post-stress anxiety, depression, and other mental health symptoms. 1) Patients: Eleven outpatients who had experienced traumatic brain injury were selected. Six months after their injury, these patients were assessed by two psychiatrists according to the DSM-5 diagnostic criteria for anxiety and depression. Eight patients did not exhibit anxiety and depression symptoms and were not diagnosed with PTSD, while three patients did exhibit anxiety and depression symptoms and were diagnosed with PTSD. Demographic data of the eleven patients are shown in Table 1. 2) Blood Sample Collection: Blood samples were collected via venipuncture into vacuum tubes containing heparin anticoagulant. 10 ml of blood was collected from each patient. After centrifugation at 3000 rpm for 5 minutes, the blood samples were separated into serum and plasma. All whole blood, serum, and plasma were aliquoted and stored at -80°C for subsequent testing.

[0040] 3) Protein content identification in serum samples: The protein content of serum samples was determined using a 4D-Label-free succinylation quantitative proteomics method. Label-free proteomics is a novel protein quantification technique that does not rely on isotope labeling. This technique analyzes enzymatically digested peptides using liquid chromatography-mass spectrometry (LC-MS / MS), eliminating the need for expensive stable isotope labels as internal tags. It only requires analyzing the mass spectrometry data generated during large-scale protein identification and comparing the signal intensity of corresponding peptides in different samples to perform relative quantification of the corresponding proteins. After removing serum samples from -80°C, an appropriate amount of sample was weighed into a mortar pre-cooled with liquid nitrogen and thoroughly ground into powder. After adding lysis buffer to the powder, an equal amount of protein from each sample was used for enzymatic digestion. The peptides obtained from the digestion were separated using a NanoElute ultra-high performance liquid chromatography system, injected into a Capillary ion source for ionization, and then analyzed by timsTOF Pro mass spectrometry. The raw files obtained from mass spectrometry detection are analyzed using database search software. After quality control of peptide and protein levels based on the search results, protein annotation is performed, followed by protein quantification analysis.

[0041] 4) Results: Protein levels were compared between patients with and without anxiety and depression symptoms who had experienced traumatic brain injury. A group of differentially expressed proteins were identified in patients with anxiety and depression symptoms, as shown in Table 2. Figure 1 The results showed that compared with patients without anxiety and depression symptoms, patients with anxiety and depression symptoms had significantly lower serum BCHE protein levels (p<0.05). Figure 2 The study showed that, compared with patients without anxiety and depression symptoms, patients with anxiety and depression symptoms had significantly lower serum CAT protein levels (p<0.05). Figure 3 The results showed that compared with patients without anxiety and depression symptoms, patients with anxiety and depression symptoms had significantly lower serum DBH protein levels (p<0.05). Figure 4 The study showed that, compared with patients without anxiety and depression symptoms, patients with anxiety and depression symptoms had significantly lower serum IGFBP2 protein levels (p<0.05). Figure 5 The results showed that, compared with patients without anxiety and depression symptoms, patients with anxiety and depression symptoms had significantly lower serum NRP1 protein levels (p<0.05).

[0042] 5) Summary: Five proteins were identified that significantly differentiated patients with and without anxiety-depressive symptoms after stress. These included (1) butyrylcholinesterase (BCHE) (SwissProt P06276), which, according to DIA mass spectrometry results, was reduced by approximately 44% in serum samples from patients with anxiety-depressive symptoms compared to those without. (2) catalase (CAT) (SwissProt P04040), which, according to DIA mass spectrometry results, was reduced by approximately 47% in serum samples from patients with anxiety-depressive symptoms compared to those without. (3) dopamine-β-hydroxylase (DBH) (SwissProt P09172), which, according to DIA mass spectrometry results, was reduced by approximately 52% in serum samples from patients with anxiety-depressive symptoms compared to those without. (4) Insulin-like growth factor binding protein 2 (IGFBP2) (SwissProt P18065): According to DIA mass spectrometry results, IGFBP2 was reduced by approximately 75% in serum samples from patients with anxiety and depression symptoms compared to patients without such symptoms. (5) Neurociliacin 1 (NRP1) (SwissProt O14786): According to DIA mass spectrometry results, NRP1 was reduced by approximately 23% in serum samples from patients with anxiety and depression symptoms compared to patients without such symptoms.

[0043] 4. Application of immunoassay kits for biomarkers of post-stress anxiety, depression, and other mental health symptoms. 1) Subjects: Sixteen healthy subjects and 15 outpatients who had experienced traumatic events were recruited for the application of the kit. The demographic data of these subjects are shown in Table 3: 2) Blood Sample Collection: Blood samples were collected via venipuncture into vacuum tubes containing heparin anticoagulant. 10 ml of blood was collected from each patient. After centrifugation at 3000 rpm for 5 minutes, the blood samples were separated into serum and plasma. All whole blood, serum, and plasma were aliquoted and stored at -80°C for subsequent testing.

[0044] 3) Detection of protein content of biomarkers for post-stress anxiety and depression in serum samples: The serum samples were tested using the kit of this invention and the protein content of butyrylcholinesterase, catalase, dopamine-β-hydroxylase, insulin-like growth factor binding protein 2, and neurocilia 1 were detected by ELISA.

[0045] 4) Results: The serum test results of the 31 subjects are shown in Table 4: 5) Data Analysis: Based on the results in Table 4, the mean, standard deviation, and 95% CI of biomarker protein levels for post-traumatic anxiety, depression, and other mental health symptoms were calculated for both healthy controls and subjects who had experienced traumatic events. The results are shown in Table 5. 6) Summary: The expression levels of biomarkers for post-stress anxiety, depression, and other mental health symptoms in the serum of healthy controls and subjects who experienced traumatic events were as follows: BCHE: 2748±158.4 ng / ml (healthy controls) and 3064±237.8 ng / ml (trauma group); CAT: 59.69±4.083 pg / ml (healthy controls) and 67.24±3.718 pg / ml (trauma group); DBH: 24.15±5.577 ng / ml (healthy controls) and 25.29±7.149 ng / ml (trauma group); IGFBP2: 142.2±19.85 ng / ml (healthy controls) and 276.6±34.95 ng / ml (trauma group); NRP1: 265.2±11.31 ng / ml (healthy controls) and 239.2±17.66 ng / ml (trauma group).

[0046] 5. Determination of high-risk thresholds for biomarkers of post-stress anxiety, depression, and other mental health symptoms. 1) Based on the decrease in the expression levels of BCHE, CAT, DBH, IGFBP2, and NRP1 in patients with post-stress anxiety and depression symptoms compared to those without, as determined by quantitative proteomics, and combined with the expression levels of these proteins in the serum of subjects who experienced traumatic events as determined by this kit, the high-risk cutoff thresholds for the above biomarkers were calculated using the following formula: The cutoff calculation formula is: cutoff = mean expression level in the trauma group determined by ELISA × (1 - rate of decrease in expression in patients with anxiety and depression symptoms determined by quantitative mass spectrometry) Based on the above formula and the average expression levels in Table 4 for the trauma group, the high-risk thresholds for biomarkers of each post-stress anxiety, depression, and other mental health symptoms were calculated to be less than the following expression levels: BCHE: 3064×(1-44%)=1716 ng / ml; CAT: 67.24×(1-47%)=35.64 pg / ml; DBH: 25.29×(1-52%)=12.14 ng / ml; IGFBP2: 276.6×(1-75%)=69.15 ng / ml; NRP1: 239.2×(1-23%)=184.2 ng / ml.

[0047] 2) By using the cutoff value, the risk of developing stress in the tested patients is determined. Those below this cutoff value are considered to be at high risk. If any protein test shows a high risk, it is considered that there is a high risk of developing mental illnesses such as anxiety, depression, and PTSD.

[0048] This invention utilizes label-free proteomics quantification technology to compare protein levels in blood samples from patients with and without post-traumatic brain injury (PTB) anxiety and depression, identifying a group of biomarkers that can effectively predict the risk of post-traumatic stress disorder (PTSD) and other mental health symptoms: BCHE, CAT, DBH, IGFBP2, and NRP1. Based on the identified biomarkers for PTSD and other mental health symptoms, this invention prepares an immunoassay kit for detecting expression levels in blood samples and establishes a detection method. The blood sandwich ELISA of this invention, based on immunobiochemistry principles, provides a highly sensitive, specific, and stable antigen-antibody detection method. By repeatedly screening and validating the prepared kit using serum from healthy controls and patients who have experienced traumatic events, a biomarker detection kit with high sensitivity, specificity, and stability for PTSD and other mental health symptoms was obtained. Furthermore, by combining the analysis of mass spectrometry quantification data and ELISA results, concentration thresholds for predicting the high risk of mental illnesses such as anxiety, depression, and PTSD in the target population are provided. The blood ELISA test kit of this invention is faster and simpler to operate; it greatly improves the accuracy and precision of the test results; it is inexpensive, easy to promote, and suitable for the market. The ELISA test method, optimized with various parameters and experimental procedures, can be used as a tool for large-scale screening of early post-stress mental illness.

[0049] In summary, this invention provides a set of biomarkers that can predict the occurrence of post-stress anxiety, depression, and other mental health symptoms, as well as detection kits and methods for detecting these biomarkers in blood samples. Potential patients experiencing stressful events can use the kits of this invention to detect the concentration of these proteins in the blood with high specificity, sensitivity, and specificity. Based on the high-risk threshold range provided by this invention, doctors can accurately predict whether stress patients will suffer from anxiety, depression, PTSD, or other mental illnesses, enabling timely treatment to prevent such diseases and alleviate patient suffering and burden. The immunoassay kits made from these biomarkers can quickly, simply, and accurately detect the concentration of biomarkers in the blood, making them suitable for large-scale application.

[0050] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, this specification should be considered illustrative rather than restrictive.

Claims

1. An immunoassay kit for predicting the occurrence of post-stress psychological symptoms, characterized in that, The immunoassay kit contains reagents for quantitatively detecting the expression levels of biomarkers, wherein the biomarkers are selected from one or more of the following: catalase, dopamine-β-hydroxylase, neurocilia 1, amino acid sequences having at least 80% homology with butyrylcholinesterase, amino acid sequences having at least 80% homology with catalase, amino acid sequences having at least 80% homology with dopamine-β-hydroxylase, and amino acid sequences having at least 80% homology with neurocilia 1.

2. The immunoassay kit according to claim 1, characterized in that, The immunoassay kit also includes a carrier, on which the reagent is coated.

3. The immunoassay kit according to claim 1, characterized in that, The reagents include primary antibodies against the biomarkers.

4. The immunoassay kit according to claim 3, characterized in that, The reagents include secondary antibodies containing primary antibodies labeled with the biomarkers described above.

5. The application of a biomarker in the preparation of a reagent for detecting and predicting the occurrence of post-stress psychological symptoms, characterized in that, The reagent is used to quantitatively detect the expression level of the biomarker. The biomarker is selected from one or more of the following: catalase, dopamine-β-hydroxylase, neurocilia 1, an amino acid sequence having at least 80% homology with butyrylcholinesterase, an amino acid sequence having at least 80% homology with catalase, an amino acid sequence having at least 80% homology with dopamine-β-hydroxylase, and an amino acid sequence having at least 80% homology with neurocilia 1.

6. The application according to claim 5, characterized in that, The mental and psychological symptoms mentioned include one or more of the following: post-stress anxiety, post-stress depression, post-traumatic stress disorder, post-stroke anxiety, post-stroke depression, post-traumatic brain injury anxiety, or post-traumatic brain injury depression.

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