Biomarker for distinguishing between depression and bipolar disorder and uses thereof

Abstract

The present disclosure relates to a biomarker for distinguishing between depression and bipolar disorder and uses thereof, elucidating a composition for distinguishing between depression and bipolar disorders and a method for providing information for distinguishing between depression and bipolar disorder, using same, wherein the composition is based on a biomarker composition comprising eotaxin, nerve growth factors (NGF), tumor necrosis factor-α (TNF-α), glial cell derived neurotrophic factor (GDNF), interleukin-1β (IL-1β), and serum soluble CD40 Ligand (sCD40L) within a serum sample and includes an agent for measuring the expression level of each of the six biomarker proteins.

Description

Biomarkers for differentiating depression and bipolar disorder and their uses

[0001] The present invention relates to a biomarker for distinguishing between depression and bipolar disorder and its use.

[0002] While physical diseases such as internal or surgical diseases are diagnosed using imaging medical devices such as CT and MRI, or hematological medical devices such as biomarkers and diagnostic kits, the diagnosis of mental illness is currently focused on the clinical symptoms that are apparent on the surface.

[0003] In relation to these major mental illnesses, according to the World Health Report published by WHO in 2000, depression ranks first, schizophrenia third, and bipolar disorder fifth in statistics on YLD (Years Lived With Disability).

[0004] In particular, patients with mood disorders such as depression and bipolar disorder may commit suicide if they are not diagnosed and treated in a timely manner, and according to data from Statistics Korea, 80% of suicides are related to mood disorders.

[0005] Therefore, it is of utmost importance to detect and diagnose major mental illnesses such as depression, bipolar disorder, or schizophrenia at an early stage and to initiate treatment quickly.

[0006] However, clinical diagnostic tests performed to quickly diagnose major mental illnesses such as depression, bipolar disorder, or schizophrenia have certain limitations in terms of diagnostic accuracy and reliability.

[0007] For example, although depression and bipolar disorder are different mental illnesses, they both have symptoms of a 'depressive episode', so in clinical practice, 40% to 50% of bipolar disorder patients are misdiagnosed as having depression.

[0008] Another example is that bipolar disorder patients are sometimes misdiagnosed as schizophrenic due to delusions and hallucinations seen in the 'manic episodes' of bipolar disorder.

[0009] Because of this, when bipolar patients initially diagnosed with depression use antidepressants, the course of the disorder may worsen, either by inducing 'manic and hypomanic episodes' or by entering a rapid cycling type with a poor prognosis.

[0010] Therefore, in order to diagnose major mental illnesses such as depression, bipolar disorder, or schizophrenia more objectively and accurately, it is necessary to go beyond clinical symptom analysis and conduct imaging evaluations based on neurological and EEG tests, as well as hematological evaluations based on biomarkers.

[0011] In this regard, a prior art document for diagnosing treatment-resistant depression using brain MRI images is “Method for diagnosing treatment-resistant depression using brain MRI analysis” (hereinafter referred to as “prior art”), Korean Patent Publication No. 10-0425525, in which the inventor of the present invention participated as an inventor.

[0012] However, in the case of existing technologies for diagnosing major mental illnesses such as depression, bipolar disorder, or schizophrenia, including conventional technologies, biomarkers with a significant level of performance and technologies for utilizing them as indicators for diagnosing major mental illnesses such as depression, bipolar disorder, or schizophrenia have not been presented.

[0013] The present invention was created to solve the above problems, and the purpose of the present invention is to provide a biomarker and techniques for utilizing the same that can produce significant results in diagnosing major mental illnesses such as depression, bipolar disorder, or schizophrenia.

[0014] In order to achieve the above purpose, the biomarker composition for distinguishing between depression and bipolar disorder of the present invention includes Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample.

[0015] Meanwhile, as another embodiment for achieving the above purpose, the composition for distinguishing between depression and bipolar disorder of the present invention includes a preparation for measuring the expression level of each of six proteins of biomarkers for distinguishing between depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample.

[0016] Here, the preparation for measuring the expression level of each of the six proteins of the biomarkers for distinguishing between depression and bipolar disorder provides information necessary for calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression using the following mathematical formula 1.

[0017] [Mathematical Formula 1]

[0018] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}

[0019] (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0020] And as another embodiment for achieving the above purpose, the diagnostic kit for distinguishing between depression and bipolar disorder of the present invention includes the composition for distinguishing between depression and bipolar disorder described above.

[0021] Finally, as another embodiment for achieving the above object, a method for providing information for distinguishing between depression and bipolar disorder includes: a step A of detecting a serum sample as a biological sample from a subject; a step B of measuring the expression level of each of six proteins of biomarkers for distinguishing between depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L), in the serum sample detected through the step A; and a step C of calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression by inputting information on the expression level of each of the six proteins of biomarkers for distinguishing between depression and bipolar disorder measured through the step B into the following mathematical formula 1.

[0022] [Mathematical Formula 1]

[0023] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}

[0024] (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0025] Here, the information providing method for distinguishing between the above depression and bipolar disorder further includes a D step for diagnosing that the closer the estimated value (X) of the probability of being diagnosed with bipolar disorder compared to depression calculated through the C step is to -∞, the higher the likelihood of being diagnosed with depression, and the closer it is to ∞, the higher the likelihood of being diagnosed with bipolar disorder.

[0026]

[0027] According to the present invention, the following effects are achieved.

[0028] First, we present a composition of biomarkers that are significantly useful for differentiating between depression and bipolar disorder.

[0029] Second, a composition that can be used as a diagnostic kit and for providing information for diagnosis can be provided, including a preparation that measures the expression level of each of the six proteins of the biomarker composition.

[0030] Third, by calculating an estimate of the probability of being diagnosed with bipolar disorder versus depression, this information can be used to provide information for distinguishing between depression and bipolar disorder and to objectively diagnose depression and bipolar disorder from a hematological perspective.

[0031]

[0032] Figures 1 and 2 are graphs showing the results of analyzing the expression level of each biomarker in a serum sample of an individual using a composition for distinguishing between depression and bipolar disorder.

[0033] Figures 3 to 8 are graphs showing the ROC curve pattern for confirming the diagnostic ability of each biomarker in a serum sample of an individual subject for depression versus bipolar disorder using a composition for distinguishing between depression and bipolar disorder.

[0034] Figure 9 is a graph showing the results of analyzing the comprehensive expression level of all biomarkers in a serum sample of an individual using a composition for distinguishing between depression and bipolar disorder.

[0035] Figure 10 is a graph showing the ROC curve pattern for confirming the diagnostic ability of bipolar disorder compared to depression for all biomarkers in a serum sample of an individual using a composition for distinguishing between depression and bipolar disorder.

[0036] Figures 11 to 13 are reference diagrams for explaining the method of proceeding with the biomarker expression level measurement step during the process of conducting an experiment related to providing information and diagnosis for differentiating between depression and bipolar disorder using a composition for differentiating between depression and bipolar disorder.

[0037]

[0038] According to one embodiment of the present invention, a biomarker composition for distinguishing between depression and bipolar disorder is provided, characterized in that it comprises Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample.

[0039] According to one embodiment of the present invention, a composition for distinguishing between depression and bipolar disorder is provided, characterized in that it includes a preparation for measuring the expression level of each of six proteins of biomarkers for distinguishing between depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample.

[0040] The preparation that measures the expression level of each of the six proteins of the biomarkers for distinguishing between the above depression and bipolar disorder provides information necessary for calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression using the following mathematical formula 1.

[0041] [Mathematical Formula 1]

[0042] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}

[0043] (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0044] According to one embodiment of the present invention, a diagnostic kit for distinguishing between depression and bipolar disorder is provided, characterized in that it comprises a composition for distinguishing between depression and bipolar disorder.

[0045] According to one embodiment of the present invention, a method for providing information for distinguishing between depression and bipolar disorder is provided, characterized by comprising the following steps:

[0046] Step A: Detecting a serum sample as a biological sample from an individual subject;

[0047] Step B of measuring the expression level of each of six proteins of biomarkers for distinguishing depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in the serum sample detected through Step A; and

[0048] Step C of calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression by inputting information on the expression level of each of the six proteins of the biomarker for distinguishing between depression and bipolar disorder measured through the above step B into the following mathematical formula 1;

[0049] [Mathematical Formula 1]

[0050] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}

[0051] (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0052] According to one embodiment of the present invention, the information providing method for distinguishing between depression and bipolar disorder is characterized by further including a D step of diagnosing that the closer the estimated value (X) of the probability of being diagnosed with bipolar disorder compared to depression calculated through the C step is to -∞, the higher the likelihood of being diagnosed with depression, and the closer it is to ∞, the higher the likelihood of being diagnosed with bipolar disorder.

[0053] In this way, according to the present invention, an estimate of the probability of being diagnosed with bipolar disorder versus depression is calculated using a biomarker composition that is significantly usable for differentiating between depression and bipolar disorder, and based on the information, information is provided for differentiating between depression and bipolar disorder, and objective diagnosis of depression and bipolar disorder from a hematological perspective is made possible.

[0054]

[0055] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different embodiments and is not limited to the embodiments described herein.

[0056]

[0057] 1. Description of biomarkers for distinguishing between depression and bipolar disorder.

[0058] The present invention proposes a biomarker related to the diagnosis of bipolar disorder compared to depression in the blood, and based on this, a method can be utilized to classify, identify, and monitor mental illness types according to the expression pattern of the biomarker.

[0059] Specifically, the biomarker composition for distinguishing between depression and bipolar disorder includes Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in serum samples.

[0060] As biomarkers corresponding to these six proteins, it is possible to obtain concentration information indicating the level of expression of Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in serum samples using a bead-based multiplex immunoassay (Luminex).

[0061] To this end, the composition for distinguishing between depression and bipolar disorder includes a formulation for measuring the expression level of each of six proteins of biomarkers for distinguishing between depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample.

[0062] As described above, the expression levels of each of the six proteins of the biomarker for distinguishing depression and bipolar disorder are analyzed by the bead-based multiplex immunoassay (Luminex). The preparations included herein include beads that form a primary complex in the form of an 'antibody-antigen' and are first bound to the biomarker, as well as antibodies that are combined with the primary complex while equipped with biotin to form a secondary complex, and components such as streptavidin-conjugated phycoerythrin (PE) that is equipped with a fluorescent substance and binds to the biotin of the antibody used to form the secondary complex to form the final complex.

[0063] A preparation that measures the expression level of each of the six proteins of the biomarker for distinguishing between depression and bipolar disorder can provide information necessary for calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression using Equation 1.

[0064] Specifically, mathematical expression 1 is defined as 'X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}'. (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0065] Furthermore, by utilizing a preparation that measures the expression levels of each of the six proteins that are biomarkers for differentiating between depression and bipolar disorder, a diagnostic kit for differentiating between depression and bipolar disorder can also be expanded and implemented.

[0066] 2. Description of information and diagnostic experiments for differentiating between depression and bipolar disorder.

[0067] Below, using proteins selected as biomarkers for distinguishing between depression and bipolar disorder according to the present invention, the process of providing information and diagnosing depression and bipolar disorder is sequentially explained based on an example of an actual experiment.

[0068] (1) Sample detection stage (Stage A)

[0069] First, the clinical trial subjects who were to be subjected to sample detection were selected, and the process of detecting and collecting serum samples as biological samples from the selected patients (subjects) was carried out.

[0070] Specifically, serum samples from 114 patients with depression and 103 patients with bipolar disorder were provided by Seoul National University Hospital. The patients with depression were aged between 19 and 65 years, with a mean age of 42 years, and the patients with bipolar disorder were aged between 19 and 62 years, with a mean age of 34 years.

[0071] Serum samples from 114 patients with depression and 103 patients with bipolar disorder were collected from peripheral blood, stored at room temperature for 1 hour, centrifuged, and the supernatant was collected. These serum samples were stored at -80℃ until use.

[0072] (2) Biomarker expression level measurement step (Step B)

[0073] In this step, the expression levels of each of the six proteins, which are biomarkers for distinguishing depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L), in the serum sample detected through the previously performed sample detection step (Step A) are measured.

[0074] Specifically, serum samples collected after detection from 114 patients with depression and 103 patients with bipolar disorder were used to obtain concentration information indicating the degree of expression by bead-based multiplex immunoassay (Luminex).

[0075] The concentrations of six proteins corresponding to Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) were measured using the Milliplex Map Kit (Luminex kit) from Merck, and analyses were performed in a 96-well plate according to each protocol.

[0076] - Preparation of standard materials

[0077] For each standard material (standard protein, Standard) prepared as shown in Table 1 below, 50 μl of standard material A and 450 μl of standard material B are added to one microcentrifuge tube, mixed well by vortexing, and labeled as ‘Standard material 1’. Then, 100 μl of standard material B is added to seven microcentrifuge tubes, and labeled as “Standard material 2 to Standard material 7” and “Blank”.

[0078] Here, standard material A refers to standard material A: an integrated high-concentration target material produced by combining six standard materials (eotaxin, NGF, TNF-α, GDNF, IL-1β, sCD40L) at specific concentrations.

[0079] In addition, standard material B refers to standard material B: an integrated low-concentration target material produced by combining six standard materials (eotaxin, NGF, TNF-α, GDNF, IL-1β, sCD40L) at specific concentrations, or a biological base material produced using distilled water or a buffer material.

[0080] Dispense 100 μl of the mixture in the tube labeled ‘Standard Material 1’ into the tube labeled ‘Standard Material 2’ and vortex.

[0081] In this manner, as shown in FIG. 11, 100 μl of the mixture in the tube labeled as 'standard material 2' is sequentially dispensed into the tube labeled as 'standard material 3' and vortexed, 100 μl of the mixture in the tube labeled as 'standard material 3' is sequentially dispensed into the tube labeled as 'standard material 4' and vortexed, 100 μl of the mixture in the tube labeled as 'standard material 4' is sequentially dispensed into the tube labeled as 'standard material 5' and vortexed, 100 μl of the mixture in the tube labeled as 'standard material 5' is sequentially dispensed into the tube labeled as 'standard material 6' and vortexed, and 100 μl of the mixture in the tube labeled as 'standard material 6' is sequentially dispensed into the tube labeled as 'standard material 7' and vortexed.

[0082]

[0083] - As shown in Figure 12 for preparing serum sample dilution, dispense 60 μl of standard materials 1 to 7 and blank sequentially through the [Standard material preparation] process described above into column 1 of the dilution plate.

[0084] Next, dispense 45 μl of Assay buffer and 5 μl of serum samples (up to 80 samples) in order from rows 3 to 12 of the dilution plate according to the number of tests.

[0085] - Preparation of capture antibody (magnetic bead conjugated antibody)

[0086] First, vortex each marker tube of the capture antibody and then disperse it using an ultrasonic cleaner for 1 minute.

[0087] Next, add 400 μl of each marker of the capture antibody to the mixing container.

[0088] Next, add Assay buffer to the mixing container until the volume reaches 4.0 ml and vortex.

[0089] - Busy process in progress

[0090] Standard materials 1 to 7 and blank in column 1 of the dilution plate shown in Figure 12 are dispensed in 25 μl each into columns 1 and 2 of the 96-well plate for analysis as shown in Figure 13.

[0091] Dispense 25 μl of the diluted serum samples (up to 80 samples) prepared in the previously described [Serum Sample Dilution Preparation] process sequentially into rows 3 to 12 of a 96-well plate for analysis.

[0092] Next, dispense 25 μl of the Capture antibody solution (4 ml) prepared in the [Capture antibody (Magnetic bead conjugated antibody) preparation] process according to the number of tests into rows 1 to 12 of the 96-well plate for analysis.

[0093] - Stirring process in progress

[0094] First, the 96-well plate for analysis as shown in Fig. 13 is shaken at 800 rpm for 2 hours at room temperature in an orbital shaker, then the 96-well plate for analysis is taken out from the orbital shaker and 25 μl of detection antibody is dispensed.

[0095] Next, shake the 96-well plate for analysis on an orbital shaker at 800 rpm for 1 hour at room temperature, then take the 96-well plate for analysis out of the orbital shaker again and dispense 25 μl of SA-PE solution.

[0096] Next, the 96-well plate for analysis is stirred at 800 rpm for 30 minutes at room temperature on an orbital shaker to complete the reaction.

[0097] - Cleaning and measurement preparation process in progress

[0098] First, turn on the microplate washer and place the 96-well plate for analysis on it for 1 minute to precipitate the results.

[0099] After this, remove the supernatant from the 96-well plate for analysis using a microplate washer, and then take the 96-well plate for analysis out of the microplate washer.

[0100] Next, pipette 200 μl of washing buffer into each well of a 96-well plate for analysis, gently tap the sides of the 96-well plate for analysis for 10 seconds to suspend the resulting reactants, and then place it on a microplate washer for 1 minute to sediment the resulting products.

[0101] Then, remove the supernatant from the wells of the 96-well plate for analysis using the microplate washer, and then take the 96-well plate for analysis out of the microplate washer.

[0102] Again, pipette 200 μl of Washing Buffer into each well of the 96-well plate for analysis, gently tap the sides of the 96-well plate for analysis for 10 seconds to suspend the resulting reactants, then place it on the Microplate washer for 1 minute to sediment the resulting products, and as previously done, remove the supernatant from the 96-well plate for analysis using the Microplate washer, and then take the 96-well plate for analysis out of the Microplate washer.

[0103] Once more, pipette 200 μl of Washing Buffer into each well of the 96-well plate for analysis, gently tap the sides of the 96-well plate for analysis for 10 seconds to suspend the resulting reactants, then place it on a microplate washer for 1 minute to sediment the resulting products, and as previously described, remove the supernatant from the 96-well plate for analysis using a microplate washer to complete the washing.

[0104] After this washing process is completed, pipette 100 μl of sheath fluid into each well of a 96-well plate for analysis, shake the 96-well plate for analysis on an orbital shaker at 800 rpm for 5 minutes at room temperature, and prepare for measurement.

[0105] Finally, the results were calculated by performing analysis on the Luminex 200 System.

[0106] As a result, the concentration values ​​representing the expression levels of each of the six proteins of biomarkers for distinguishing depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in serum samples, are numerically informationized.

[0107] In fact, the protein concentration values ​​of Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) expressed in each of the serum samples collected after detection from 114 patients with depression and 103 patients with bipolar disorder are as shown in Figures 1 and 2.

[0108] As shown in Figures 1 and 2, the expression levels of biomarkers Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) expressed in serum samples of the first experimental group (Depression) of 114 patients with depression and the second experimental group (Bipolar) of 103 patients with bipolar disorder showed differences from each other.

[0109] In addition, we analyzed the diagnostic ability of bipolar disorder compared to depression for each of the six proteins of biomarkers for distinguishing between depression and bipolar disorder, whose expression levels were analyzed using the concentration values ​​in the serum samples of Figures 1 and 2.

[0110] Specifically, the diagnostic ability of the six proteins of the biomarker for distinguishing between depression and bipolar disorder was confirmed through the Receiver Operating Characteristic (ROC) curve, which shows the false positive rate (FPR, 1-specificity, X-axis value) and true positive rate (TPR, sensitivity, Y-axis value) for multiple decision criteria (operating conditions, cutoffs, decision thresholds, positive judgment criteria) based on the diagnostic test results, based on the sensitivity and specificity (1-Specificity) measured during the actual diagnosis process.

[0111] As a result, in the case of Eotaxin as a single biomarker, the ROC curve result was shown as in Figure 3, and the AUC (Area under the ROC curve) showed a value of 0.5925.

[0112] In addition, as a single biomarker, NGF (Nerve growth factors) showed an AUC value of 0.5925 through the results of the ROC curve as shown in Fig. 4, TNF-α (Tumor Necrosis Factor-α) showed an AUC value of 0.5925 through the results of the ROC curve as shown in Fig. 5, and GDNF (Glial cell Derived Neurotrophic Factor) showed an AUC value of 0.6762 through the results of the ROC curve as shown in Fig. 6.

[0113] In addition, as a single biomarker, IL-1β (Interleukin-1β) showed an AUC value of 0.5661 through the results of the ROC curve as shown in Figure 7, and sCD40L (Serum soluble CD40 Ligand) showed an AUC value of 0.7475 through the results of the ROC curve as shown in Figure 8.

[0114] (3) Estimation calculation stage (Stage C)

[0115] In this step, the information on the expression level of each of the six proteins of the biomarker for distinguishing between depression and bipolar disorder, measured through the previously performed biomarker expression level measurement step (Step B), is input into the following mathematical formula 1 to calculate an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression.

[0116] Specifically, mathematical expression 1 is defined as 'X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)}'. (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1))

[0117] Here, the variable values ​​for the concentrations of Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in the serum sample included in Equation 1 can be measured through the previously performed biomarker expression level measurement step (Step B) and provided in the form of information.

[0118] Specifically, the X value as a result value produced through mathematical expression 1 is an estimate of the probability of being diagnosed with bipolar disorder versus depression, and becomes a distinguishing indicator information that can be used to classify depression and bipolar disorder.

[0119] Therefore, in this step, the biomarkers for distinguishing between depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L), can be combined and evaluated for their ability to distinguish between depression and bipolar disorder through regression analysis.

[0120] As a result, when the expression levels of all six proteins of the biomarkers for distinguishing between depression and bipolar disorder in the comprehensive serum samples were compared between the first experimental group (Depression) of 114 patients with depression and the second experimental group (Bipolar) of 103 patients with bipolar disorder, the differences were clearly evident, as shown in Figure 9.

[0121] Above all, the comprehensive diagnostic ability of all six proteins of the biomarker for distinguishing between depression and bipolar disorder is confirmed through the ROC (Receiver Operating Characteristic) curve, which shows the false positive rate (FPR, 1-specificity, X-axis value) and true positive rate (TPR, sensitivity, Y-axis value) for multiple decision criteria (operating condition, cutoff, decision threshold, positive judgment criteria) based on the diagnostic test results. It shows a specificity of 87.3% and a sensitivity of 71.7%, and in particular, it shows an AUC value of 0.8304 as shown in the results of the ROC curve as shown in Figure 10, showing excellent diagnostic performance.

[0122] (4) Estimate result analysis stage (Stage D)

[0123] In this step, the process of diagnosing is carried out such that the closer the estimate (X) of the probability of being diagnosed with bipolar disorder compared to depression calculated through the previously performed estimate calculation step (Step C) is to -∞, the higher the likelihood of being diagnosed with depression, and the closer it is to ∞, the higher the likelihood of being diagnosed with bipolar disorder.

[0124] As a result, by using the proteins selected as biomarkers for distinguishing between depression and bipolar disorder of the present invention and going through the series of processes described above, an indicator is provided for diagnosing the patient's mental illness as either depression or bipolar disorder, thereby proving that the use of hematological biomarkers for mental illness is possible.

[0125] The embodiments disclosed in the present invention are intended to illustrate, not limit, the technical concepts of the present invention. These embodiments do not limit the scope of the technical concepts of the present invention. The scope of protection should be interpreted according to the following claims, and all technical concepts within the scope equivalent thereto should be construed as being included within the scope of the present invention.

[0126]

[0127] First, we present a composition of biomarkers that are significantly useful for differentiating between depression and bipolar disorder.

[0128] Second, a composition that can be used as a diagnostic kit and for providing information for diagnosis can be provided, including a preparation that measures the expression level of each of the six proteins of the biomarker composition.

[0129] Third, by calculating an estimate of the probability of being diagnosed with bipolar disorder versus depression, this information can be used to provide information for distinguishing between depression and bipolar disorder and to objectively diagnose depression and bipolar disorder from a hematological perspective.

Claims

1. Characterized in that it contains Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample. Biomarker composition for differentiating depression and bipolar disorder.

2. A preparation characterized by including a preparation for measuring the expression level of each of six proteins of biomarkers for distinguishing depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in a serum sample. Composition for differentiating between depression and bipolar disorder.

3. In paragraph 2, The preparation for measuring the expression level of each of the six proteins of the biomarkers for distinguishing between the above depression and bipolar disorder is characterized in that it provides information necessary for calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression using the following mathematical formula 1. Composition for differentiating between depression and bipolar disorder. [Mathematical Formula 1] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)} (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1)) 4. Characterized in that it comprises a composition for distinguishing between depression and bipolar disorder according to paragraph 2 or 3. Diagnostic kit for differentiating between depression and bipolar disorder.

5. Step A: Detecting a serum sample as a biological sample from an individual subject; Step B of measuring the expression level of each of six proteins of biomarkers for distinguishing depression and bipolar disorder, including Eotaxin, Nerve growth factors (NGF), Tumor Necrosis Factor-α (TNF-α), Glial cell Derived Neurotrophic Factor (GDNF), Interleukin-1β (IL-1β), and Serum soluble CD40 Ligand (sCD40L) in the serum sample detected through Step A; and It is characterized by including a step C of calculating an estimate (X) of the probability of being diagnosed with bipolar disorder versus depression by inputting information on the expression level of each of the six proteins of the biomarkers for distinguishing between depression and bipolar disorder measured through the above step B into the following mathematical formula 1. How to provide information to help distinguish between depression and bipolar disorder. [Mathematical Formula 1] X=α+{β1×(concentration of eotaxin in serum sample)}+{β2×(concentration of NGF in serum sample)}+{β3×(concentration of TNF-α in serum sample)}+{β4×(concentration of GDNF in serum sample)}+{β5×(concentration of IL-1β in serum sample)}+{β6×(concentration of sCD40L in serum sample)} (X: Estimate of the probability of being diagnosed with bipolar disorder versus depression, α: Intercept value when the estimate (X) is 0 (-10≤α≤10), β1: Regression coefficient representing the weight for the concentration value of eotaxin in the serum sample (-1≤β1 ≤1), β2: Regression coefficient representing the weight for the concentration value of NGF in the serum sample (-1≤β2≤1), β3: Regression coefficient representing the weight for the concentration value of TNF-α in the serum sample (-1≤β3≤1), β4: Regression coefficient representing the weight for the concentration value of GDNF in the serum sample (-1≤β4≤1), β5: Regression coefficient representing the weight for the concentration value of IL-1β in the serum sample (-1≤β5≤1), β6: Regression coefficient representing the weight for the concentration value of sCD40L in the serum sample (-1≤β6≤1)) 6. In paragraph 5, The method of providing information for distinguishing between the above depression and bipolar disorder is as follows: The method further comprises a D step, which diagnoses that the closer the estimate (X) of the probability of being diagnosed with bipolar disorder compared to depression produced through the above C step is to -∞, the higher the probability of being diagnosed with depression, and the closer it is to ∞, the higher the probability of being diagnosed with bipolar disorder. How to provide information to help distinguish between depression and bipolar disorder.

Images