CN114137214A - Immune detection kit and application for predicting the occurrence of mental and psychological symptoms after stress - Google Patents
Immune detection kit and application for predicting the occurrence of mental and psychological symptoms after stressDownload PDFInfo
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Abstract
Description
Technical Field
The present invention relates to the detection and identification of biomarkers produced in subjects suffering from psychophysiological symptoms such as anxiety and depression after stress and provides a metric for predicting high risk of developing in subjects potentially suffering from anxiety and depression. In particular to a group of biomarkers for predicting the occurrence of psychological symptoms such as anxiety and depression after stress, a detection kit and a detection method for detecting the biomarkers in a blood sample, and provides a reference concentration threshold value of the markers with high occurrence risk, so that the reliable detection and identification of the biomarkers for assisting the diagnosis and prevention of the psychological symptoms such as anxiety and depression after stress are possible.
Background
In recent years, the incidence rate of stressful events such as public health emergencies, various major accidents, natural and artificial disasters gradually increases, more than 70% of people worldwide experience at least one traumatic event in a lifetime, and psychological symptoms such as anxiety and depression often appear after the stressful event for rescuers such as event experience persons and medical staff involved in rescue, and some individuals even develop serious mental diseases. Among them, post-traumatic stress disorder (PTSD) is one of the more serious mental diseases. Post-traumatic stress disorder refers to a mental disorder that is delayed in the appearance and persistence of an individual after the individual experiences, witnesses, or encounters one or more physical deaths involving themselves or others, or is threatened by death, or is severely injured, or is threatened by physical integrity. Clinically manifested as invasion of wound memory, avoidance of wound-related stimuli, negative changes in cognition, increased arousal and behavioral hyperactivity. According to the cultural background and the economic development level of different national regions, the lifetime prevalence rate of PTSD is about 1.3% -12.2%, and the annual prevalence rate is about 0.2% -3.8%. In today's society, post-stress mental illness, including PTSD, has become a disease of great concern, especially during the current COVID-19 pandemic. The disease has a long course and is easy to relapse, the patient often has behaviors of self-disability, suicide, drug abuse and the like, the life quality of the patient is seriously influenced, and the serious psychological, physiological and economic burden is caused to the individual, the family and the society.
PTSD is the most serious and most interesting disease in the post-stress psychiatric category, and the clinical diagnostic criteria of which are currently widely accepted are DSM-5 of the American society for mental medicine and ICD-11 of the world health organization. In DSM-5, the diagnostic criteria for PTSD have 8 large entries, divided into 4 symptom clusters and a subtype, where the 4 large symptom clusters are invasive in wound memory, avoidance of wound-related stimuli, negative changes in cognition and mood, increased arousal and behavioral hyperactivity. Whereas in ICD-11, the diagnosis of PTSD is simplified to 3 symptom groups for 6 categories of symptoms, including constant reexperiencing to traumatic events, avoidance of traumatic stimuli, and a high degree of vigilance. Although the diagnostic criteria for PTSD are slightly different, the treatment modalities include mainly psychological interventions, drug therapies and some other innovative therapies. Psychological intervention is recommended as the first-line treatment in most guidelines, and mainly includes prolonged exposure therapy, cognitive processing therapy, cognitive behavioral therapy, ocular desensitization, and reprocessing therapy. While drug therapy is generally used as a second line therapy, including selective 5-hydroxytryptamine reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, monoamine oxidases, sympatholytic agents, anticonvulsants, benzodiazepines, and the like.
Although the incidence of PTSD is high in people who experience one or more severe wounds, not all individuals who experience a wound become PTSD patients, and the actual lifetime prevalence of PTSD is far below the theoretical presumptive value, suggesting that there is significant individual variability in the incidence of PTSD. PTSD incidence rate research based on different ethnic groups discovers that the incidence rate of white race is obviously lower than that of other races, and the twin-based research also discovers that the incidence rate of homozygotic twins is obviously higher than that of heterozygotic twins after suffering from traumatic events, and suggests that genetic factors obviously influence the incidence rate of PTSD. At present, PTSD is mainly diagnosed based on the medical history of a patient, mental state judgment, symptom duration, clinical mental scale detection and self-description of the patient, and the detection of objective and effective biomarkers of PTSD is lacked. Predicting risk factors for the occurrence of PTSD after a trauma event becomes particularly important given that the occurrence of most traumatic events is unavoidable. However, PTSD is less subject to screening and research on objective, effective and specific biological markers, and currently, there is still no objective index for PTSD early warning diagnosis and therapeutic efficacy evaluation. The detection of biomarkers in blood is considered to be the most potential and convenient way to facilitate PTSD diagnosis due to its advantages of convenience, minimal invasiveness, and the like. Studies have reported that a decrease in cortisol levels in blood following trauma may be associated with the development of PTSD, and imbalance in partial neurotransmitters in blood, levels of adrenocorticotropic hormone (ACTH), Glucocorticoid (GC), epinephrine, and catecholamine, and the like are also considered as markers associated with PTSD. In addition, other biomarkers that may be associated with PTSD include cholecystokinin (CCK), Nitric Oxide Synthase (NOS), neuropeptide y (npy), and p11 protein, among others. However, the specificity and sensitivity of these markers have not been well validated. Meanwhile, specific markers of mental diseases after stress other than PTSD are more rarely reported, and therefore, it is urgently required to discover and develop and utilize specific biomarkers for mental symptoms such as anxiety and depression after stress.
Therefore, in order to effectively predict the occurrence of psychophysiological symptoms such as anxiety and depression after stress, it is necessary to identify completely novel biomarkers and develop a detection kit capable of specifically detecting the biomarkers.
Disclosure of Invention
In order to solve the above problems, the present invention provides a set of biomarkers for predicting the occurrence of psychological symptoms such as anxiety and depression after stress, and a detection kit and a detection method for detecting the biomarkers in a blood sample, wherein the detection kit based on the biomarkers can be used for detecting the concentration of the biomarkers in the blood of a potential stress patient with high sensitivity, high specificity and high stability, and according to the concentration threshold provided by the invention, the high occurrence risk of the psychological symptoms such as anxiety and depression is prompted, so that the basis can be provided for doctors to accurately predict the occurrence of mental diseases such as anxiety, depression and PTSD of the stress patient, and further to take treatment in time to prevent the occurrence of the diseases and relieve the pain and burden of the patient in time, and the immunoassay kit prepared by the biomarkers can quickly, simply and accurately detect the concentration of the biomarkers in the blood, is suitable for large-scale popularization and application.
In order to achieve the above objects, according to a first aspect of the present invention, there is provided an immunoassay kit for predicting the occurrence of psychophysiological symptoms such as anxiety and depression after stress, the immunoassay kit comprising a reagent for quantitatively detecting the expression level of a biomarker selected from the group consisting of: butyrylcholinesterase (BCHE), Catalase (CATase, CAT), Dopamine-Beta-Hydroxylase (DBH), Insulin-Like Growth Factor Binding Protein 2(Insulin Like Growth Factor Binding Protein 2, IGFBP2), Neuropilin 1(Neuropilin1, 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 NRP 1.
Preferably, the psychosocial symptoms such as anxiety and depression after stress include anxiety after stress, depression after stress, post traumatic stress disorder, anxiety after stroke, depression after stroke, anxiety after brain trauma, depression after brain trauma and other stress-related mental diseases.
In a second aspect of the present invention, there is provided a use of a biomarker for preparing an agent for quantitatively measuring the expression level of the biomarker for predicting the occurrence of psychophysiological symptoms after stress, the biomarker being selected from the group consisting of: butyrylcholinesterase, catalase, dopamine-beta-hydroxylase, insulin-like growth factor binding protein 2, neuropilin1, 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-beta-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 neuropilin 1.
In a third aspect of the invention, there is provided a method of immunoassay kit for detecting the level of the above biomarker protein in whole blood, central nervous system tissue, serum, plasma, cerebrospinal fluid, saliva, sweat, tears, urine, oral sample, or a combination thereof. Preferably, the body fluid is blood. The method for detecting the above-mentioned protein can be carried out by a conventional Enzyme-linked immunosorbent assay (ELISA), preferably by using a sandwich ELISA method.
Preferably, the immunoassay kit further comprises a carrier, and the antigen for detecting the biomarker protein is coated on the carrier.
Preferably, the immunoassay kit further comprises a primary antibody, wherein the primary antibody is used for detecting the biomarker protein.
Preferably, the immunoassay kit further comprises a labeled secondary antibody of the biomarker protein primary antibody. The label can be horseradish peroxidase, alkaline phosphatase, fluorescent molecule FITC (or other fluorescent labels), and chemiluminescence detection. The detection method can be qualitative and quantitative analysis by a color development method, a fluorescence method, chemiluminescence and an electrochemiluminescence method.
More preferably, the labeled secondary antibody against the primary antibody against the biomarker protein is a secondary antibody against the primary antibody against the protein labeled by horseradish peroxidase.
In a fourth aspect of the invention, there is provided the use of an immunoassay kit for the above biomarker protein in the detection of the above biomarker protein in human blood to determine a high risk concentration threshold for the above protein by comparing the measured level of the above biomarker protein in a subject (e.g., a patient who has experienced a traumatic event) with the measured level of the above biomarker protein by quantitative mass spectrometry techniques.
The invention has the beneficial effects that: the biomarker capable of predicting the occurrence of psychological symptoms such as anxiety and depression after stress is one or more of Butyrylcholinesterase (BCHE), Catalase (Catalase, CAT), Dopamine-Beta-Hydroxylase (DBH), Insulin-Like Growth Factor Binding Protein 2 (IGFBP 2), Neuropilin 1(Neuropilin1, 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 FBP2, and an amino acid sequence having at least 80% homology with the amino acid sequence of NRP1, and potential patients after stress events, the concentration of the biomarker protein can be detected in blood with high specificity, high sensitivity and high specificity, so that the method can provide a basis for doctors to accurately predict stress patients suffering from anxiety, depression, PTSD and other mental diseases, and then timely take treatment to prevent the occurrence of the diseases and timely relieve the pain and burden of the patients.
Drawings
FIG. 1 is a graph comparing the protein levels of BCHE in serum of patients with post-traumatic anxious depressive symptoms and patients with post-traumatic anxious depressive symptoms.
FIG. 2 is a graph showing the comparison of the protein level of CAT in serum of a patient having no anxiety and depression symptoms after trauma and a patient having anxiety and depression symptoms after trauma.
FIG. 3 is a graph showing a comparison of the protein level of DBH in serum of a patient having no anxiety-depressive symptom after trauma and a patient having anxiety-depressive symptom after trauma.
FIG. 4 is a graph comparing the protein levels of IGFBP2 in the serum of patients with post-traumatic anxiety-free depressive symptoms and patients with post-traumatic anxiety-depressive symptoms.
FIG. 5 is a graph comparing the protein level of NRP1 in serum of patients with post-traumatic anxiety-free depressive symptoms and patients with post-traumatic anxiety-depressed symptoms.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following examples are given in detail.
Protein biomarkers
Biomarkers for predicting the occurrence of psychophysiological symptoms such as anxiety and depression after stress are Butyrylcholinesterase (BCHE), Catalase (Catalase, CAT), Dopamine-Beta-Hydroxylase (DBH), Insulin-Like Growth Factor Binding Protein 2 (IGFBP 2), Neuropilin 1(Neuropilin1, NRP 1).
BCHE is an esterase with broad substrate specificity that contributes to the inactivation of the neurotransmitter acetylcholine. BCHE degrades neurotoxic organophosphates. As shown in figure 1, the present invention identifies that levels of BCHE protein in whole blood, plasma, serum of patients experiencing symptoms of anxiety depression after trauma are significantly reduced (p <0.05) compared to levels in patients experiencing symptoms of anxiety depression after trauma.
CAT is present in almost all organisms that breathe aerobically and serves to protect cells from the toxic effects of hydrogen peroxide. CAT promotes cell growth, including T cells, B cells, myeloid leukemia cells, melanoma cells, mast cell tumor cells, and normal and transformed fibroblasts. As shown in figure 2, the present invention identifies that CAT protein levels in whole blood, plasma, serum of patients experiencing symptoms of anxiety depression after trauma are significantly reduced (p <0.05) compared to levels in patients experiencing symptoms of anxiety depression after trauma.
DBH protein is expressed in neurosecretory vesicles and chromaffin granules of the adrenal medulla, catalyzing the conversion of dopamine to norepinephrine, which is both a hormone and the 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. This gene mutation results in dopamine- β -hydroxy acid deficiency in human patients characterized by defects in autonomic and cardiovascular function, including hypotension and ptosis. The present invention identifies that the levels of DBH protein in whole blood, plasma, serum of patients experiencing symptoms of anxiety depression after trauma are significantly reduced (p <0.05) compared to the levels in patients experiencing symptoms of anxiety depression after trauma.
IGFBP2 inhibits IGF-mediated growth and development rates. IGFBP2 extends the half-life of IGFs and has been shown to inhibit or stimulate the growth promoting effects of IGFs on cell culture. IGFBP2 alters IGF interaction with its cell surface receptors. The present invention identifies that IGFBP2 protein levels in whole blood, plasma, serum of patients experiencing symptoms of anxiety depression after trauma are significantly reduced (p <0.05) compared to levels in patients experiencing symptoms of anxiety depression after trauma.
NRP1 is a cell surface receptor involved in the development of the cardiovascular system, angiogenesis, formation of neural circuits, and organogenesis outside the nervous system. NRP1 mediates the chemorepulsive activity of semaphorin. NRP1 recognizes the motif R/KXXR/K on its ligand as a rule of C-terminus, leading to cellular internalization and vascular leakage. It binds to semaphorin 3A, the PLGF-2 subtype of PGF, the VEGF165 subtypes VEGFA and VEGFB. The co-expression of NRP1 with KDR results in increased binding of VEGF165 to KDR and increased chemotaxis. NRP1 modulates VEGF-induced angiogenesis. NRP1 in combination with VEGFA initiated the signaling pathway required for motor neuron axon guidance and cell body migration, including facial motor neuron migration (by similarity) from diamond 4 to the caudal side of diamond 6 during embryonic development. NRP1 regulates mitochondrial iron transport by interacting with ABCB 8/mitosus. NRP1 binds VEGF-165 and may inhibit its binding to cells. NRP1 can induce apoptosis by sequestering VEGF-165. NRP1 may also bind to various members of the semaphorin family. NRP1 expression has a negative impact on blood vessel number and integrity. NRP1 acts as a host factor for infection by human coronavirus SARS-CoV-2. NRP1 recognizes and binds with C-terminal rules to the motif RRAR on SARS-CoV-2 spinous process protein S1 to enhance infection with SARS-CoV-2. The present invention identifies that NRP1 protein levels in whole blood, plasma, serum of patients experiencing symptoms of anxiety depression after trauma are significantly reduced (p <0.05) compared to levels in patients experiencing symptoms of anxiety depression after trauma.
Reagent kit
In the invention, five enzyme-linked immunoassay kits are used for detecting 5 serum biomarkers (BCHE, CAT, DBH, IGFBP2, NRP1) of psychopsychological symptoms such as anxiety and depression after stress. The assay is performed by a sandwich ELISA method.
1. Establishment of Sandwich ELISA method
1) According to the number of the detection samples, a plurality of costar bars are measured, wherein the costar bars are coated with the biomarker for resisting psychopsychological symptoms such as anxiety and depression after stress.
2) 100ul of sample diluent was added per well.
3) The standard, blank and diluted serum samples were added at 50 ul/well for 2 hours at 37 degrees, and shaker 200 rpm.
4) PBST wash, 400 ul/well, 5 times, pat dry.
5) Add another horseradish peroxidase labeled biomarker primary antibody, 200 ul/well, incubate at 37 ℃ for 2 hours, shaker 200 rpm.
6) PBST wash, 400 ul/well, 5 times, pat dry.
7) TMB color development, wherein TMB solution A and B solution are mixed in equal volume, 100 ul/hole, and 37 ℃ for 20 min.
8) Stop solution, 50 ul/well stop, read 450nm with microplate reader.
2. The kit comprises the following components:
the kit consists of the following parts:
1) coating costar enzyme plates for detecting biomarker-antibody for resisting psychology symptoms such as anxiety and depression after stress
2) One tube (1ml) of PTSD biomarker standard sample each
3) Sample diluent a bottle (100ml)
4) Tubes of horseradish peroxidase-labeled anti-biomarker antibody (20ml)
5) 10 PBST one bottle (100ml)
6) One bottle (60ml) of TMB color developing solution A and one bottle (60ml) of B solution B
7) One bottle of stop solution (60ml)
3. Identification of biomarkers for psychosocial symptoms such as anxiety and depression after stress
1) And the patient: 11 outpatients who underwent brain trauma were evaluated 6 months after brain trauma for the presence of anxious depressive symptoms by 2 psychiatrists according to the diagnostic criteria of DSM-5, 8 of which were not anxious depressive symptoms and were not diagnosed as PTSD and 3 of which were anxious depressive symptoms and were diagnosed as PTSD. The demographic data for 11 patients are shown in table one:
table one: demographic data of 11 outpatient brain trauma patients
| Brain trauma patient numbering | Sex | Age (age) | Diagnosis of |
| PT-071 | Female with a view to preventing the formation of wrinkles | 25 | Anxious depression, non-PTSD |
| PT-072 | Female with a view to preventing the formation of wrinkles | 34 | Anxious depression, non-PTSD |
| PT-073 | Female with a view to preventing the formation of wrinkles | 30 | Anxious depression, non-PTSD |
| PT-076 | Female with a view to preventing the formation of wrinkles | 24 | Anxious depression, non-PTSD |
| PT-078 | Female with a view to preventing the formation of wrinkles | 44 | Anxious depression, non-PTSD |
| PT-079 | Male sex | 55 | Anxious depression, non-PTSD |
| PT-083 | Male sex | 29 | Anxious depression, non-PTSD |
| PT-087 | Female with a view to preventing the formation of wrinkles | 50 | Anxious depression, non-PTSD |
| PT-077 | Female with a view to preventing the formation of wrinkles | 49 | With anxiety, depression, PTSD |
| PT-082 | Female with a view to preventing the formation of wrinkles | 25 | With anxiety, depression, PTSD |
| PT-095 | Female with a view to preventing the formation of wrinkles | 29 | With anxiety, depression, PTSD |
2) And collecting a blood sample: blood samples were collected by venipuncture in heparin-containing anticoagulation vacuum tubes, and 10ml blood samples were collected from each patient. The blood sample was centrifuged at 3000rpm for 5 minutes and separated into serum and plasma. All whole blood, serum and plasma were dispensed and stored in a-80 ℃ freezer for subsequent assays.
3) Identifying the protein content in the serum sample: the serum sample adopts a 4D-Label-free succinylation quantitative proteomics method to carry out protein content determination. The technology analyzes the proteolysis peptide section by liquid chromatography-mass spectrometry, does not need an expensive stable isotope Label as an internal Label, only needs to analyze mass spectrum data generated during large-scale protein identification, and compares the signal intensity of corresponding peptide sections in different samples, so that the corresponding protein can be relatively quantified. Taking out a serum sample from-80 ℃, weighing a proper amount of sample into a mortar precooled by liquid nitrogen, adding liquid nitrogen, and fully grinding the sample into powder. After adding lysis buffer to the powder, equal amounts of protein were taken from each sample for enzymatic hydrolysis. The peptide fragments obtained by enzymolysis are separated by a NanoElute ultra-performance liquid phase system, injected into a Capillary ion source for ionization and then analyzed by a timeTOF Pro mass spectrum. And (3) carrying out database search by adopting analysis software based on raw files obtained by mass spectrum detection, carrying out quality control on peptide fragments and protein levels based on search results, carrying out protein annotation, and then carrying out quantitative analysis on the protein.
4) And the result is as follows: comparing the protein content of patients with anxiety and depression symptoms and patients without anxiety and depression symptoms who have undergone brain trauma, a group of proteins differentially expressed in patients with anxiety and depression symptoms was identified, and the results are shown in table two:
table two: protein specific for patient with anxiety depression symptoms
Figure 1 shows that the protein levels of BCHE are significantly reduced in serum of patients with symptoms of anxious depression (p <0.05) compared to patients without symptoms of anxious depression. Figure 2 shows that the protein levels of CAT in serum were significantly reduced (p <0.05) in patients with symptoms of anxious depression compared to patients without symptoms of anxious depression. Figure 3 shows that the protein level of DBH in serum was significantly reduced (p <0.05) in patients with anxiety-depressive symptoms compared to patients without anxiety-depressive symptoms. Figure 4 shows that the protein levels of IGFBP2 are significantly reduced in the serum of patients with symptoms of anxious depression (p <0.05) compared to patients without symptoms of anxious depression. Figure 5 shows that NRP1 protein levels in serum were significantly reduced in patients with symptoms of anxious depression (p <0.05) compared to patients without symptoms of anxious depression.
5) And summarizing: 5 proteins were found which significantly distinguish patients with anxiety and depression symptoms after stress from patients without anxiety and depression symptoms. Including (1) butyrylcholinesterase BCHE (SwissProt P06276), according to the results of DIA mass spectrometry, BCHE was reduced by about 44% in serum samples from patients with anxiety depression symptoms compared to patients without anxiety depression symptoms. (2) The catalase CAT (SwissProt P04040), according to the results of DIA mass spectrometry, reduced CAT by about 47% in serum samples from patients with anxiety depression symptoms compared to patients without anxiety depression symptoms. (3) Dopamine-beta-hydroxylase DBH (SwissProt P09172), as a result of DIA mass spectrometry, reduces DBH by about 52% in serum samples from patients with anxiety depression symptoms compared to patients without anxiety depression symptoms. (4) Insulin-like growth factor binding protein 2IGFBP2(SwissProt P18065), as a result of DIA mass spectrometry, reduced IGFBP2 in serum samples from patients with anxiety-depressive symptoms by about 75% compared to patients without anxiety-depressive symptoms. (5) Neuropilin 1NRP1(SwissProt O14786), as a result of DIA mass spectrometry, reduced NRP1 in serum samples from patients with anxiety-depressive symptoms by about 23% compared to patients without anxiety-depressive symptoms.
4. Application of biomarker immunoassay kit for psychopsychological symptoms such as anxiety and depression after stress
1) And a subject: the use of the kit to recruit 16 healthy subjects and 15 outpatients who had experienced a traumatic event for the detection of the kit, the demographics of these subjects are as shown in table three:
table three: demographic data of the subject
2) And collecting a blood sample: blood samples were collected by venipuncture in heparin-containing anticoagulation vacuum tubes, and 10ml blood samples were collected from each patient. The blood sample was centrifuged at 3000rpm for 5 minutes and separated into serum and plasma. All whole blood, serum and plasma were dispensed and stored in a-80 ℃ freezer for subsequent assays.
3) And protein content of biomarker for psychosocial symptoms such as anxiety and depression after stress detection in serum sample: the serum sample adopts the kit of the invention, and the protein contents of butyrylcholinesterase, catalase, dopamine-beta-hydroxylase, insulin-like growth factor binding protein 2 and neuropilin1 are respectively detected by an ELISA method.
4) And the result is as follows: the results of the serum test for 31 subjects are shown in table four:
table four: ELISA detection result of biomarker of psychopsychological symptoms such as anxiety and depression after stress of subject
5) And data analysis: according to the results in the table four, the average value, the standard deviation and the 95% CI interval of the content of the biomarker protein of the psychosocial symptoms such as anxiety and depression after stress of healthy control subjects and subjects who have suffered from traumatic events are respectively calculated, and the results are shown in the table five:
table five: analysis data table of marker detection results of psychopsychological symptoms such as anxiety and depression after stress of subjects
6) And summarizing: the expression levels of the markers of psychophysiological symptoms such as anxiety and depression after stress in the serum of healthy control subjects and subjects who have suffered from traumatic events are respectively as follows: BCHE: 2748 ± 158.4ng/ml (healthy control) and 3064 ± 237.8ng/ml (wound group); CAT: 59.69 + -4.083 pg/ml (healthy control) and 67.24 + -3.718 pg/ml (wound group); DBH: 24.15 + -5.577 ng/ml (healthy control) and 25.29 + -7.149 ng/ml (wound group); IGFBP 2: 142.2. + -. 19.85ng/ml (healthy control) and 276.6. + -. 34.95ng/ml (wound group); NRP 1: 265.2 + -11.31 ng/ml (healthy control) and 239.2 + -17.66 ng/ml (wound group).
5. Determination of high risk threshold of biomarkers for psychophysiological symptoms such as anxiety and depression after stress
1) The reduction rate of expression levels of BCHE, CAT, DBH, IGFBP2 and NRP1 of a patient with anxiety and depression symptoms after stress, which is determined by a quantitative proteomics group, compared with a patient without anxiety and depression symptoms, and the expression level of the protein in the serum of a subject suffering from a traumatic event determined by the kit are combined to calculate the high risk cutoff threshold of the marker, and the calculation formula is as follows:
the Cutoff calculation formula is as follows: the mean expression level of the wound group determined by ELISA × (1-the rate of decrease in expression of patients with anxiety and depression symptoms determined by quantitative mass spectrometry)
The high risk threshold of the biomarker for each psychophysiological symptom such as anxiety and depression after stress is calculated according to the formula and the average expression of the trauma group in the fourth table and is respectively less than the following expression:
BCHE：3064×(1-44％)＝1716ng/ml；
CAT：67.24×(1-47％)＝35.64pg/ml；
DBH：25.29×(1-52％)＝12.14ng/ml；
IGFBP2：276.6×(1-75％)＝69.15ng/ml；
NRP1：239.2×(1-23％)＝184.2ng/ml。
2) and determining the risk of the detected stress patient according to the cutoff value, wherein the risk is considered to be high when the cutoff value is lower than the cutoff value, and the risk of the mental diseases such as anxiety, depression and PTSD is considered to be high when only one protein is detected to be high.
The invention obtains a group of biomarkers which can effectively predict the occurrence risk of psychopsychological symptoms such as anxiety and depression after stress by screening through a non-standard (Label-free) proteome quantitative technology and comparing the content of protein in blood samples of patients who have anxiety and depression after brain trauma and patients without anxiety and depression: BCHE, CAT, DBH, IGFBP2, and NRP 1. The invention prepares an immunoassay kit for detecting expression level in a blood sample according to the identified biomarkers of psychological symptoms such as anxiety and depression after stress and establishes a detection method. The blood sandwich enzyme-labeled immunoassay (sandwich ELISA) provided by the invention is based on the immune biochemical principle, and provides an antigen-antibody detection method with high sensitivity, high specificity and high stability. The prepared kit is repeatedly screened and verified by using the serum of healthy controls and patients who have suffered traumatic events, so that the biomarker detection kit for psychopsychological symptoms such as anxiety and depression after stress with high sensitivity, high specificity and high stability is obtained. And by combining the analysis mass spectrum quantitative data and the ELISA result, the biomarker concentration threshold value for predicting high occurrence risk of mental diseases such as anxiety, depression, PTSD and the like in the target population is given. The blood ELISA detection kit is quicker and simpler to operate; the accuracy and the precision of the detection result are greatly improved; the cost is low, the popularization is easy, and the method is suitable for the market. The ELISA detection method can be used as a large-scale screening tool for early screening of mental diseases after stress through optimization of various parameters and experimental processes.
In conclusion, the invention provides a group of biomarkers capable of predicting the occurrence of psychological symptoms such as anxiety and depression after stress and a detection kit and a detection method for detecting the biomarkers in a blood sample, and potential patients can use the kit to detect the concentrations of the proteins in the blood with high specificity, high sensitivity and high specificity after experiencing a stress event, and can provide basis for doctors to accurately predict the anxiety, depression, PTSD and other mental diseases of the stress patients according to the high risk threshold range provided by the invention, so that the treatment can be timely taken to prevent the occurrence of the diseases and relieve the pain and burden of the patients, and the immunodetection kit prepared from the biomarkers can quickly, simply and accurately detect the concentrations of the biomarkers in the blood, and is suitable for large-scale popularization and application.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The description is thus to be regarded as illustrative instead of limiting.
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