AU2021101912A4 - Use of vitamin d-binding protein (vdbp) as biomarker in diagnosis of depression - Google Patents

Abstract

The present disclosure discloses use of vitamin D-binding protein (VDBP) as a biomarker in the diagnosis of depression, and specifically relates to use of a reagent for detecting a VDBP content in the preparation of products such as a reagent for diagnosing depression. In the present disclosure, it can be seen from the differential expression of VDBP in the plasma of patients with schizophrenia, bipolar mania, and depression and non-psychiatric controls that the expression of VDBP is specifically increased only in the plasma of depressive patients. The protein alone can be used as a biomarker to diagnose depression by detecting a content of the biomarker in peripheral blood, which can significantly improve the accuracy of differential diagnosis of depressive people from healthy people or patients with other mental disorders such as schizophrenia and bipolar mania. Therefore, the detection of a VDBP content has high clinical applicability in the diagnosis of depression, and provides a new way for the rapid and effective diagnosis of depression. 2/2 Depression vs Non-psychiatric control Depression vs Schizophrenia 100- .------------ 100- ------ 80- 80 60- .160 40- 40 20- 20 0- -- 0 I I I | | | o 20 40 60 80 100 0 20 40 60 80 100 100-specificity 100-specificity Depression vs Bipolar mania 100- - -- 80 60- 40 0- - I I I | | | 0 20 40 60 80 100 100-specificity FIG.2

Description

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P/00/001 Regulation 3.2

AUSTRALIA

Patents Act 1990

COMPLETE SPECIFICATION STANDARDPATENT

Invention title: USE OF VITAMIN D-BINDING PROTEIN (VDBP) AS BIOMARKER IN DIAGNOSIS OF DEPRESSION

The following statement is a full description of this invention, including the best method of performing it known to us:

USE OF VITAMIN D-BINDING PROTEIN (VDBP) AS BIOMARKER IN DIAGNOSIS OF DEPRESSION TECHNICAL FIELD

The present disclosure belongs to the field of biological sciences, and relates to the rapid and convenient specific differential diagnosis of mental disorders, such as depression, and specifically to use of vitamin D-binding protein (VDBP) as a biomarker in the diagnosis of depression.

BACKGROUND

Depression is a type of affective disorder mainly characterized by a depressive mood. The World Health Organization (WHO) reports that depression will become the world's second-largest disease in 2020 and will become the leading cause of death and disability by 2030. The current clinical diagnosis of depression and other mental disorders mainly refers to "Chinese Classification and Diagnostic Criteria for Mental Disorders" (3rd Edition) (CCMD-3) and criteria of "InternationalClassification ofDiseases" (10th Edition) (ICD-10), "Diagnostic and Statistical Manual of Mental Disorders" (4th Edition) (DSM-IV) in the United States, but the method itself has critical shortcomings: strong subjectivity, poor diagnosis consistency, and high misdiagnosis rate. Therefore, in order to achieve early diagnosisand effective intervention for depression, the identification of effective and objective biomarkers is of great significance to the majority of patients with depression. Although a variety of objective biomarkers have been discovered through decades of research, no reliable biomarkers have been determined for the diagnosis of depression. The biggest problem is that most biomarkers may be used to accurately distinguish patients with depression from healthy people, but cannot be used to effectively distinguish among depression, schizophrenia, bipolar mania, and the like at the same time. Therefore, how to specifically diagnose depression and thus provide effective treatment has become a urgent matter in the current clinical diagnosis and treatment of depression. VDBP, also known as GC globulin, is a polymorphic single-chain glycoprotein (about 52 kDa to 59 kDa), which was first discovered by professor Hirschfeld in 1959. Because GC exhibits a high degree of genetic diversity, which leads to polymorphisms in the VDBP protein structure, VDBP exhibits diverse functions (Lisowska-Myjak B, J6fwiak-Kisielewska

A, Lukaszkiewicz J, Skarzyiska E: Vitamin D-binding protein as a biomarker to confirm specific clinical diagnoses. Expert review of molecular diagnostics 2019: 1-8). VDBP in peripheral blood is mainly synthesized by hepatocytes and expressed in a variety of tissues, including liver, kidney, gonad, fat, and neutrophils. VDBP has three protein domains, where, a first domain involves in vitamin D-binding, and the other two domains exhibit a variety of other functions, such as clearing actin, activating macrophages, enhancing neutrophil chemotaxis, activating osteoclasts, and other immuno-inflammatory response processes (Delanghe JR, Speeckaert R, Speeckaert MM: Behind the scenes of vitamin D binding protein: more than vitamin D binding. Best Pract Res Clin Endocrinol Metab 2015, 29: 773-786). Vitamin D has the function of protecting neurons. As the main carrier of vitamin D, an abnormal level of VDBP will inevitably cause the function of vitamin D in the brain. In addition, as an important inflammation-associated protein, VDBP participates in a variety of immunoregulation processes in the body, and a very important pathogenesis hypothesis for depression is the neuroinflammation hypothesis (Liu CH, Zhang GZ, Li B, Li M, Woelfer M, Walter M, Wang L: Role of inflammation in depression relapse. J Neuroinflammation 2019, 16: 90). In 2009, professor Jirikowski et al. first discovered the expression of mRNA and protein of VDBP in the brain tissue (hypothalamus) of rodents, and proposed that the protein may be transported via axoplasm and finally released like neurohormones in the hypothalamic-neurohypophysial system (Jirikowski GF, Kaunzner UW, Dief AEE, Caldwell JD: Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus. Histochemistry and cell biology 2009, 131: 365-370). It is almost impossible to detect VDBP in the brain of living patients, but peripheral plasma is an effective approach to detect levels of VDBP. Moreover, there is currently no research about using VDBP as a differential diagnosis biomarker for distinguishing depression from other mental disorders (such as schizophrenia and bipolar mania).

SUMMARY

Purpose of the present disclosure: In order to solve the defects and deficiencies of the clinical diagnosis methods for mental disorders such as depression in the prior art, the present disclosure provides new use of VDBP in the diagnosis of mental disorders, mainly depression, namely, use of VDBP as a biomarker in the diagnosis of mental disorders such as depression, and specifically, use of a reagent for detecting a VDBP content in the preparation of a reagent for diagnosing mental disorders such as depression. In the present disclosure, it is determined whether a subject suffers from depression by detecting a VDBP content in plasma of the subject, which can realize the differential diagnosis of depression from other mental disorders such as schizophrenia and bipolar mania and provides a new way for the rapid and effective diagnosis of depression. Technical solution: In order to achieve the above purpose, the present disclosure provides use of the VDBP as a biomarker in the preparation of a reagent for diagnosing depression. The present disclosure also provides use of a reagent for detecting a VDBP content in the preparation of a reagent for diagnosing depression. The reagent for diagnosing depression may include a reagent for diagnosing depression from healthy people or patients with schizophrenia and bipolar mania including schizophrenia and bipolar mania. Further, the reagent for detecting VDBP may be used in the preparation of a reagent product or tool for diagnosing depression. The reagent may include a reagent for detecting a VDBP content by enzyme-linked immunosorbent assay (ELISA), and may mainly include a specific polyclonal antibody (pAb) for VDBP, which is used to detect a content of the protein. Preferably, the reagent for detecting a VDBP content by ELISA may be an ELISA kit for VDBP content. Preferably, the reagent for detecting a VDBP content by ELISA may be an ELISA kit including Human Vitamin D BP Quantikine ELISA Kit (DVDBPOB; R&D Systems, Minneapolis, MN, USA), human VDBP ELISA kit (RGB-60486H; RIGORBIO SCIENCE DEVELOPMENT Co., LTD., Beijing, China), or human VDBP ELISA kit (MEXN-H2746; Meixuan Biotechnology Co.,Ltd., Shanghai, China), etc. The present disclosure may preferably adopt Human Vitamin D BP Quantikine ELISA Kit; DVDBPOB; R&D Systems, Minneapolis, MN, USA. The specific antibody for VDBP may be a pAb produced by injecting purified human VDBP or an antigen fragment thereof into an animal body, or an antibody produced by immunizing an animal with cells expressing human VDBP or an antigen fragment thereof. The kit for diagnosing mental disorders such as depression in the present disclosure may include an antibody that can specifically bind to VDBP and an auxiliary reagent that can be used to detect a VDBP content. The kit can use VDBP alone as an indicator to achieve the early screening of depression and the differential diagnosis of depressive people from healthy people or patients with other mental disorders (schizophrenia and bipolar mania).

The components of the kit can be packaged in the form of an aqueous medium or a lyophilized product. Appropriate containers in the kit may usually include at least a vial, a test tube, etc., which can hold a component that can be appropriately divided into equal parts. When more than one component is present in the kit, the kit may usually also include a second, a third, or other additional containers, which hold the additional components separately. However, different combinations of components can be held in one vial. The kit of the present disclosure will usually also include a container for holding reactants, which is sealed for commercial sale. Such a container may include an injection-molded or blow-molded plastic container that can hold desired vials. Based on the differential expression of VDBP in the plasma of patients with depression, schizophrenia, and bipolar mania, and non-psychiatric controls (VDBP shows specific high expression in patients with depression) discovered by the applicants, the present disclosure uses this protein as a peripheral molecule marker so that the quantitative detection of a level of the protein in plasma can serve as a specific diagnostic indicator for depression. In addition, the applicants also evaluate the differential diagnosis performance of plasma VDBP alone for depression from schizophrenia and bipolar mania by comparing the plasma VDBP concentrations in different study groups. Beneficial effects: Compared with the prior art, the present disclosure has the following advantages: The present disclosure finds for the first time the use of VDBP as a biomarker for specifically diagnosing depression, which provides a brand-new way for the detection and differential diagnosis of depression. The present disclosure also provides the use of a reagent for detecting a VDBP content in the preparation of a reagent for diagnosing depression, where, VDBP alone is used as an indicator and a VDBP content is detected to distinguish a patient with depression from non-psychiatric healthy people and patients with schizophrenia and bipolar mania, with AUC values under the ROC curve of 0.750, 0.734, and 0.648, respectively. In addition, the protein alone is used as a peripheral blood biomarker to diagnose depression by detecting a content of the protein, which can achieve high accuracy for the differential diagnosis of patients with depression from healthy people or patients with other mental disorders (schizophrenia and bipolar mania). Therefore, VDBP has high clinical applicability in the diagnosis of depression. The present disclosure also provides a kit for diagnosing depression, including a reagent that can be used to detect a VDBP content, a specific pAb for VDBP, etc. With the peripheral plasma of a test subject, the kit can achieve convenient and rapid differential diagnosis of patients with major depression from healthy people, and can also be used to differentially diagnose depression in healthy people or patients with other mental disorders (schizophrenia and bipolar mania).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the quantitative results of VDBP in peripheral plasma of patients with depression, schizophrenia, and bipolar mania, and non-psychiatric controls; and FIG. 2 is a schematic diagram illustrating the comparison results of depression patients with non-psychiatric controls and patients with schizophrenia and bipolar mania using receiver operating characteristic (ROC) curve analysis.

DETAILED DESCRIPTION

The present disclosure can be better understood according to the following examples. Those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present disclosure, and should not and will not limit the present disclosure described in detail in the claims. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified. Experimental methods in the examples which are not specified with specific conditions are generally conducted according to conventional conditions or conditions recommended by manufacturers. Example Plasma samples were collected from 72 depression patients, 42 schizophrenia patients, 39 mania patients, and 60 non-psychiatric controls who had never taken antipsychotic drugs, mood stabilizers, benzodiazepines, and the like (2 mL of fasting venous blood was collected and centrifuged at 200 g for 10 min, and 1 mL of the upper plasma was retained). Specifically, the depression, schizophrenia, and bipolar mania were diagnosed and confirmed by three-level psychiatric specialists with rich clinical experience (chief/associate chief physician, attending physician, and senior resident) according to the diagnostic criteria in "Diagnostic and Statistical Manual of Mental Disorders" (4th Edition) (DSM-V). The above psychiatric patients were recruited from Zhongda Hospital Affiliated to Southeast University, Second Affiliated Hospital of Xinxiang Medical College, and Wutaishan Hospital of Yangzhou. The samples of healthy controls came from the Physical Examination Center of Zhongda Hospital Affiliated to Southeast University and Second Affiliated Hospital of Xinxiang Medical

College. The following tests and evaluations were conducted: plasma VDBP concentration determination; and rating scales to assess the severity of conditions in patients of each group, including 24-item Hamilton Depression Rating Scale (HAMD-24), Self-Rating Depression Scale (SDS), Beck Hopelessness Scale (BHS), Brief Psychiatric Rating Scale (BRPS), and Young Mania Rating Scale (YMRS). Test results were shown in Table 1. The plasma VDBP concentration was detected with an ELISA kit with detailed information as follows: Human Vitamin D BP Quantikine ELISA Kit; DVDBPOB; R&D Systems, Minneapolis, MN, USA. The kits had a detection range of 6.3 ng/mL to 100 ng/mL. This kit provided the necessary but not all reagents or materials needed to detect a VDBP concentration: 1 96-well microtiter plate (coated with recombinant human VDBP-specific antibody), 2 tubes of human VDBP standard, 1 vial of 12 ml test diluent RD1-38, 1 vial of 21 ml human VDBP conjugated substrate solution, 1 vial of 21 ml calibration diluent RD5P, 1 vial of 21 ml washing solution (25-fold dilution), 1 vial of 12 ml chromogenic solution A, 1 vial of 12 ml chromogenic solution A, and 1 vial of 6 ml stop solution. In addition, the following additional reagents and materials were required: microplate reader, pipettor, pipette, graduated cylinder, absorbent paper, distilled water or deionized water, data analysis and plotting software, etc. All required items were prepared before the start of the test: 1) Preparation of washing solution: 20 mL of a washing solution was diluted with deionized water to 500 mL for later use. 2) A substrate solution: 15 min before use, the chromogenic solutions A and B were mixed in equal volumes. 3) Dilution of calibration diluent RD5P: 20 mL of a RD5P stock solution was diluted with ml of deionized water to obtain 100 mL of a diluted calibration diluent RD5P for later use. 4) Preparation of VDBP standard: The diluted calibration diluent RD5P was added to 1 vial of VDBP standard to obtain standards with the following concentration gradient: 100 ng/mL, 50 ng/mL, 25 ng/mL, and 6.25 ng/mL. Experimental operations: 1) The 96-well microtiter plate (coated with recombinant human VDBP-specific antibody) provided in the kit was used to determine the number of plate wells required for this test, and replicates should be set for each sample, standard, and blank. In addition, according to specific needs, a conventional immune antibody preparation method can also be used to produce an antibody, that is, purified human VDBP or an antigen fragment thereof can be injected into an animal to produce a specific antibody or cells expressing human VDBP or an antigen fragment thereof are used to immunize an animal to produce an antibody, and the antibody can be coated on a clean microtiter plate to obtain a new kit. 2) 50 1 of the test diluent RD1-38 was added to each well. 3) Addition of samples: After the VDBP standard was serially diluted, 50 L of each of 100 ng/mL, 50 ng/mL, 25 ng/mL, and 6.25 ng/mL standards was added to the wells of the microtiter plate, and 1 blank well was set for comparison (the blank well was not added with a sample and an ELISA reagent, and the other steps were the same). 50 L of a to-be-tested plasma sample diluted with the calibration diluent RD5P was added to the remaining wells (it should be ensured that a diluted plasma VDBP concentration was in the middle of the concentration range shown in the standard, and a dilution factor was recorded for each sample to facilitate the calculation after the experiment, thus ensuring the detection accuracy of the kit). The plate was then incubated on a shaker for 2 h at room temperature and a shaker speed of 500 ±50 rpm. 4) The liquid in the wells was completely removed, and each well was washed 4 times with the diluted washing solution, with about 400 1 of washing solution each time. After the washing was completed, the plate was upended on absorbent paper and pat-dried. 5) 200 1 of human VDBP conjugated substrate solution was added to each well, and then the plate was incubated on a shaker for 1 h at room temperature. 6) The washing in step 4 was repeated. 7) 200 1 of the substrate solution was added to each well, and then the plate was incubated for 30 min in the dark at room temperature. 8) 50 1 of the stop solution was added to each well to stop the reaction (the color immediately changed from blue to yellow). 9) Taking the blank well as zero, an optical density (OD value) was determined at 450 nm with a microplate reader within 15 min after the stop solution was added. The ELISACalc software was used to plot a standard curve from OD values determined for the VDBP standards and to obtain a mathematical formula for the curve, and then an OD value of each plasma sample was input into the standard curve formula obtained by the software to calculate a VDBP content in the current sample. Based on a dilution factor of each sample recorded before the experiment, an undiluted concentration was calculated as a final plasma VDBP content. The demographic and clinical characteristics of the subjects were shown in Table 1; the plasma VDBP concentration results of patients with different mental disorders and non-psychiatric controls were shown in FIG. 1 and Table 1; and the diagnosis and diagnostic performance analysis of plasma VDBP were shown in FIG. 2. It can be seen from FIG. 1 that the plasma VDBP level was significantly increased only in patients with depression, and there is no statistically-significant difference among other populations, indicating that the present disclosure can effectively and specifically diagnose depression by detecting a VDBP content. FIG. 2 shows that the area under the ROC curve (AUC) of plasma VDBP in the diagnosis of depression from healthy people, schizophrenia patients, and bipolar mania patients were 0.750 (95% IC: 0.669-0.831), 0.734 (95% IC: 0.643-825), and 0.648 (95% IC: 0.549-0.748), respectively. It is generally considered that, if 0.5 < AUC < 1, the classifier has predictive value; and the greater the AUC, the greater the classification performance. Therefore, VDBP shows high differential diagnosis performance for depression in populations, indicating that the present disclosure can accurately achieve the differential diagnosis of depression in healthy people or patients with other mental disorders (schizophrenia and bipolar mania), and the present disclosure can achieve the convenient, rapid, and effective detection by an ELISA kit. In addition, some other ELISA kits that can detect a VDBP content can achieve a consistent effect with the kit used in the above example, such as human VDBP ELISA kit (RGB-60486H) and human VDBP ELISA kit (MEXN-H2746).

Table 1 Basic clinical information and VDBP detection results of subjects

MDD (n=74) HC (n=60) SZ (n=42) BD-I (n=39) p value

Age (years) 31.54 (11.99) 34.35 (10.76) 32.50 (9.23) 30.46 (9.58) 0.255a

Gender (female) 43 (58.11%) 36(60.00%) 20(47.62%) 15 (38.45%) 0.124b

BMI index 22.59 (3.50) 22.96 (3.25) 21.21 (2.89) 24.43 (2.99) 0.149c

Smoking 13 (17.57%) 13(21.67%) 5 (11.90%) 7(17.59%) 0.655b

Onset of age (years) 27.53 (11.37) - 30.36 (9.10) 27.79 (8.66) 0.136a

Course of disease (months) 49.46 (72.95) - 27.73 (27.92) 32.74 (38.23) 0.754a

Psychiatric history 26(35.14%) 2(3.33%) 6(14.29%) 3(7.69%) 0.000b

HAMD-24 score 35.12 (1.85) 1.95 (2.33) -0.000

SDS score 70.22 (11.43) 34.62 (6.50) -0.000

BHS score 10.93 (4.84) 6.12(1.86) - 0.0001

BRPS score - - 48.69 (3.50) -

YMRS score - - - 31.18 (5.96)

VDBP (pg/ml) 289.35 (77.96) 219.72 (70.39) 225.32 (55.82) 247.26 (45.16) 0.000°

Notes: The data are expressed as an average value (standard deviation) or the number of cases (percentage, %). MDD: depression; HC: non-psychiatric healthy people; SZ: schizophrenia; BD-I: bipolar mania a Kruskal-Wallis H test; b Chi-square test; c One-way analysis of variance (one-way ANOVA); d Mann-Whitney U test

Claims (5)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. Use of vitamin D-binding protein (VDBP) as a plasma biomarker in the preparation of a reagent for diagnosing unipolar depression in adults.

2. Use of a reagent for detecting a VDBP content in plasma in the preparation of a reagent for diagnosing unipolar depression.

3. The use according to claim 1 or 2, wherein, the reagent for diagnosing unipolar depression comprises a reagent for diagnosing depression from healthy people or patients with schizophrenia and bipolar mania; wherein, the reagent for detecting a VDBP content in plasma is used in the preparation of a reagent product or tool for diagnosing depression.

4. The use according to claim 2, wherein, the reagent comprises a reagent for detecting a VDBP content by enzyme-linked immunosorbent assay (ELISA), and the reagent comprises a specific polyclonal antibody (pAb) for VDBP; wherein, the reagent for detecting a VDBP content by ELISA is an ELISA kit for VDBP content; wherein, the reagent for detecting a VDBP content by ELISA is an ELISA kit; wherein, the specific antibody for VDBP is a pAb produced by injecting purified human VDBP or an antigen fragment thereof into an animal body, or an antibody produced by immunizing an animal with cells expressing human VDBP or an antigen fragment thereof.

5. Use of the VDBP according to claim 1 as a plasma biomarker in the preparation of a kit for diagnosing depression in adults, wherein, the kit comprises an antibody that can specifically bind to VDBP and an auxiliary reagent that can be used to detect a VDBP content; the kit can use VDBP alone as an indicator to achieve the early screening of depression and the differential diagnosis of depressive people from healthy people or patients with other mental disorders, such as schizophrenia and bipolar mania.