CN111351945A - Application of vitamin D binding protein as marker in diagnosis of mental disease depression - Google Patents
Application of vitamin D binding protein as marker in diagnosis of mental disease depression Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/30—Psychoses; Psychiatry
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/30—Psychoses; Psychiatry
- G01N2800/302—Schizophrenia
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/304—Mood disorders, e.g. bipolar, depression
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The invention discloses application of vitamin D binding protein as a marker in diagnosis of mental disease depression, and particularly relates to application of a reagent for detecting the content of vitamin D binding protein in preparation of a reagent for diagnosing mental disease depression. The present invention demonstrates that differential expression of vitamin D binding protein in the plasma of schizophrenic, bipolar mania, depressive, and non-psychotic controls, is specifically elevated only in the plasma of depressive patients; the protein is independently used as a peripheral blood biomarker to detect the content of the protein for diagnosing the depression, so that the differential diagnosis accuracy of the depression and healthy people or other mental diseases such as schizophrenia and bipolar mania can be obviously improved. Therefore, the content detection of the vitamin D binding protein has high clinical application value in the diagnosis of depression, and provides a brand new way for the rapid and effective diagnosis of mental diseases depression.
Description
Technical Field
The invention belongs to the field of biological science, relates to rapid, convenient and specific differential diagnosis of mental diseases such as depression, and particularly relates to application of vitamin D binding protein as a marker in diagnosis of mental diseases such as depression.
Background
Depression, also known as melancholia, is an affective disorder characterized primarily by depressed mood. WHO reported that depression will be the second largest disease in the world in 2020 and will be the leading cause of death and disability by 2030. Currently, in clinical diagnosis of mental diseases such as depression, reference is mainly made to the criteria of Chinese mental disorder classification and diagnosis standard (CCMD-3), American International Classification of diseases tenth (ICD-10), mental disease diagnosis and statistics manual (fourth edition) DSM-IV and mental disease diagnosis and statistics manual (fifth edition) DSM-V, but the method itself has serious disadvantages: the subjectivity is strong, the diagnosis consistency is poor and the misdiagnosis rate is high. Therefore, development of effective objective biomarker detection for early diagnosis and early intervention of various diseases is of great significance to a large number of patients.
Based on several decades of research, reliable biomarkers for diagnosing depression have not been identified, although a variety of objective biomarkers have been discovered. The biggest problem is that most biomarkers can accurately identify depression and healthy people, but the effective identification of mental diseases such as depression, schizophrenia, bipolar mania and the like cannot be realized at the same time. Therefore, it is a very urgent matter in the clinical depression diagnosis and treatment at present to specifically diagnose depression and provide effective treatment.
Vitamin D binding protein (VDB), also known as GC globulin, is a polymorphic single-chain glycoprotein (-52-59 kDa), first discovered in 1959 by professor Hirschfeld. Since GC shows high genetic diversity, resulting in polymorphism of VDB protein structure, VDB shows diverse functions (Lisowska-Myjak B,
-KisielewskaA,
J,
vitamin D-binding protein as a biomarker toconfirm specific diagnostic reagents Expert review of molecular diagnostics2019: 1-8). VDB is synthesized primarily by hepatic parenchymal cells in peripheral blood and is expressed in a variety of tissues, including liver, kidney, gonads, fat and neutrophils. VDB has three protein domains, and two other domains, in addition to the first domain involved in vitamin D binding, exhibit various other functions, such as actin elimination, macrophage activation, neutrophil chemotaxis enhancement, osteoclast activation, and the like (Delanghe JR, Speeckaert R, Speec)kaert MM, Behindthe scenes of vitamin D binding protein more than one vitamin D binding BestPract Res Clin Endocrinol Metab 2015,29: 773-. Vitamin D has the function of protecting neurons, and as the main carrier of vitamin D, abnormal levels of VDB inevitably cause the function of vitamin D in the brain. Furthermore, as an important inflammation-related protein, VDB is involved in a variety of immunomodulatory processes in vivo, and a very important pathogenesis hypothesis in depression is the neuroinflammatory hypothesis (Liu CH, Zhang GZ, Li B, Li M, Woeler M, Walter M, Wang L: Role of inflammation in depression relief. J Neuroinflammation 2019,16: 90). In 2009, professor Jirikowski et al found expression of VDB mRNA and protein for the first time in rodent brain tissue (hypothalamus) and suggested that transplasmatic transport and terminal release in the hypothalamic-neurohypophyseal system may be in a neurohormone-like manner (Jirikowski GF, Kanzner UW, Dief AEE, Caldwell JD: Distribution of vitamin D binding protein expressing nerves in the rat hypothalamus. histochemistry and cell biology 2009,131:365 370). Since the detection of VDB in the brain is almost impossible in living bodies and peripheral plasma is easily available, no research on using VDB as a biomarker for differential diagnosis between depression and other mental diseases (such as schizophrenia, bipolar mania and the like) exists at present.
The invention content is as follows:
the purpose of the invention is as follows: in order to solve the defects and deficiencies of clinical diagnosis means of mental diseases such as depression in the prior art, the invention provides a novel application of VDB protein (vitamin D binding protein) in the diagnosis of mental diseases mainly depression, namely the application of vitamin D binding protein as a marker in the diagnosis of mental diseases such as depression, in particular to the application of a reagent for detecting the content of vitamin D binding protein in the preparation of a reagent for diagnosing mental diseases such as depression. The invention judges whether the subject has the mental disease depression by detecting the content of the VDB protein in the plasma of the subject, can realize the differential diagnosis of other mental diseases such as schizophrenia and bipolar mania, and provides a brand new way for the rapid and effective diagnosis of the mental disease depression.
The technical scheme is as follows: in order to achieve the above objects, the use of vitamin D binding protein according to the present invention as a marker for the preparation of a reagent for diagnosing mental illness depression.
The invention discloses application of a reagent for detecting the content of vitamin D binding protein in preparing a reagent for diagnosing mental disease depression.
Wherein the agent for diagnosing depressive disorder includes an agent for diagnosing depressive disorder from a healthy person or a psychiatric disease including schizophrenia and bipolar mania.
Further, the application of the reagent for detecting the vitamin D binding protein in preparing a depression diagnosis product reagent product or a reagent tool.
The reagent comprises a reagent for detecting the content of the vitamin D binding protein by using an enzyme-linked immunosorbent assay, and mainly comprises a specific polyclonal antibody of the vitamin D binding protein, and is used for detecting the content of the protein.
Preferably, the reagent for detecting the content of the vitamin D binding protein by using an enzyme-linked immunosorbent assay kit is the vitamin D binding protein content enzyme-linked immunosorbent assay kit.
Preferably, the ELISA Kit used as the reagent for detecting the content of the Vitamin D binding protein by using the ELISA method comprises a Human Vitamin D BP Quantikine ELISA Kit (DVDBP 0B; R & D Systems, Minneapolis, MN, USA), a Human Vitamin D Binding Protein (VDBP) ELISA Kit (RGB-60486H; Riegbock technology development Co., Ltd., Beijing, China), a Human Vitamin D Binding Protein (VDBP) ELISA Kit (MEXN-H2746; Meixuan Biotechnology Co., Ltd., Shanghai, China), and the like. Preferably, a Human Vitamin D BPQuantikine ELISA Kit is used; DVDBP 0B; r & D Systems, Minneapolis, MN, USA.
Wherein the antibody specific for vitamin D binding protein is a polyclonal antibody produced by injecting purified human vitamin D binding protein or an antigenic fragment thereof into an animal; or by immunizing an animal with cells expressing human vitamin D binding protein or an antigenic fragment thereof.
The kit for diagnosing and diagnosing the mental diseases comprises an antibody which can be specifically combined with the vitamin D binding protein and other auxiliary reagents which can be used for detecting the content of the vitamin D binding protein.
The kit can adopt vitamin D binding protein alone as an index to complete early screening of depression and differential diagnosis of healthy people or other mental diseases (schizophrenia and bipolar mania).
The components of the test kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in a kit typically include at least one vial, test tube, or the like, in which one component may be placed and suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.
The invention is based on the differential expression (the specificity and high expression of the VDB protein in the blood plasma of depression, schizophrenia, bipolar mania and non-mental disease control persons) discovered by the applicant, and the quantitative detection of the blood plasma level of the VDB protein by taking the VDB protein as a peripheral molecule marker can be used as a specific diagnostic index of depression. In addition, applicants have evaluated the diagnostic efficacy of plasma VDB protein alone in the identification of depression from schizophrenia and bipolar mania by comparing plasma VDB concentrations between different study groups.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the invention discovers the application of the VDB protein as a biomarker for specifically diagnosing the depression for the first time, and provides a brand-new way for detecting, identifying and diagnosing the depression. The application of the reagent for detecting the content of the vitamin D binding protein in the preparation of the reagent for diagnosing the mental disease depression is utilized, wherein the VDB protein is independently adopted as an index to detect the content of the vitamin D binding protein, the depression patient and non-mental disease healthy population, schizophrenia and biphase patient are analyzed, and the area values AUC under an ROC curve are respectively 0.750, 0.734 and 0.648; in addition, the protein is used as a peripheral blood biomarker alone to detect the content of the protein for diagnosing depression, so that higher differential diagnosis accuracy of depression and healthy people or other mental diseases (schizophrenia and bipolar mania) can be provided. Therefore, the vitamin D binding protein has higher clinical application value in the diagnosis of depression.
The invention also provides a kit for diagnosing the mental disease depression, which comprises a reagent for detecting the content of the vitamin D binding protein, a specific polyclonal antibody of the vitamin D binding protein and the like, utilizes the peripheral blood plasma of a person to be detected to conveniently and quickly carry out differential diagnosis on the severe depression and healthy people, and can be used for differential diagnosis on the depression from the healthy people or other mental diseases (schizophrenia and bipolar mania).
Drawings
FIG. 1 is a graph showing the results of quantification of VDB protein in peripheral plasma of patients with depression, schizophrenia, bipolar mania and non-psychotic controls;
figure 2 is a graphical representation of the results of a comparison of depression patients with non-psychotic controls, schizophrenia, bipolar mania patients using a subject work profile analysis.
Detailed Description
The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
Examples
Plasma samples of 72 depression patients, 42 schizophrenia, 39 mania patients and 60 non-psychotic controls who had never taken antipsychotics, mood stabilizers, benzodiazepines and the like were collected (2 mL of fasting venous blood was obtained, centrifuged at 200g for 10 minutes, and 1mL of upper plasma was retained). Specifically, the diagnosis of depression, schizophrenia and bipolar mania was diagnosed and confirmed by three-level mentally specialized physicians (chief/assistant chief physicians, treating physicians and high-age hospitalization physicians) with abundant clinical experience according to the diagnostic criteria in handbook of mental illness diagnosis and statistics (fourth edition) (DSM-V), in which patients with mental illness were recruited from the middle-to-large hospital affiliated with southeast university, the second subsidiary hospital of the new county medical school and the fifth mountain hospital of the Yangzhou city, and samples of healthy controls were from the middle-to-large hospital affiliated with southeast university and the second subsidiary hospital physical examination center of the new county medical school, respectively.
The tests and evaluations that were made included: plasma VDB protein concentration determination; scales to assess the severity of the disease in each group of patients individually include the Hamilton Depression Scale (24-item Hamilton Depression Rating Scale, HAMD-24), the Self-Rating Depression Self-Rating Scale (SDS), the Beck despair Scale (BHS), the Brief psychosis Scale (BRPS), the Young Mania Rating Scale (YMRS), and the results are shown in Table 1.
Plasma VDB protein concentrations were measured using an ELISA kit using the following detailed information: HumanVitamin D BP Quantikine ELISA Kit; DVDBP 0B; r & D Systems, Minneapolis, MN, USA. The detection range of the kit is 6.3-100 ng/mL.
The kit provides the necessary but not all reagents or materials required to detect the concentration of VDB protein: 1 piece of 96-well enzyme label plate (coated with recombinant human VDB specific antibody), 2 tubes of human VDB protein standard, 1 bottle of 12ml test diluent RD1-38, 1 bottle of 21ml human VDB conjugate binding substrate solution, 1 bottle of 21ml calibration diluent RD5P, 1 bottle of 21ml washing solution (25-fold dilution), 1 bottle of 12ml developing solution A and 1 bottle of 6ml stop solution. In addition, the additional reagents and materials required are as follows: enzyme-linked immunosorbent assay instrument, pipettor, pipette, graduated cylinder, absorbent paper, distilled water or deionized water, data analysis and drawing software, etc.
All required items are prepared before the detection starts:
1) preparing a washing solution: adding 20mL of washing liquid into deionized water, and diluting to 500mL of washing liquid for later use;
2) substrate solution: mixing equal volumes of color developing solutions A and B15 minutes before the start of use;
3) diluting the calibration diluent RD5P, adding 20mL of RD5P stock solution into 80mL of deionized water, and mixing to obtain 100mL of diluted calibration diluent RD5P for later use;
4) VDB standard configuration: diluted calibration diluent RD5P was added to a 1-tube VDB protein standard to construct a concentration gradient standard with the following concentrations: 100ng/mL, 50ng/mL, 25ng/mL, 6.25 ng/mL.
The experimental procedure was as follows:
1) determining the hole number of an enzyme label plate required by the detection by using a 96-hole enzyme label plate (coated with a recombinant human VDB specific antibody) provided in the kit, wherein each sample, standard and blank are provided with multiple holes; in addition, according to the self-requirement, the purified human vitamin D binding protein or the antigen fragment thereof can be injected into the animal body by adopting the conventional immune antibody preparation method to generate specific antibodies or the cells expressing the human vitamin D binding protein or the antigen fragment thereof are used for immunizing the animal to generate the antibodies, and the antibodies are coated in a clean enzyme label plate to prepare a novel kit.
2) Adding 50 mul of test diluent RD1-38 into each well;
3) adding a sample: after diluting the VDB protein standard in a multiple proportion, 50 mu L of each standard substance of 100ng/mL, 50ng/mL, 25ng/mL and 6.25ng/mL is added into an ELISA plate hole in sequence, and 1-hole blank comparison is set (the blank comparison hole is not added with a sample and an ELISA reagent, and the operation of other steps is the same). Adding 50 mu L of the plasma sample to be detected diluted by using the calibration diluent RD5P into the other holes (the diluted plasma VDB concentration needs to be ensured to be in the middle of the concentration interval shown by the standard substance, and the dilution factor of each sample is recorded, so that the calculation is convenient after the experiment is finished, and the detection accuracy of the kit is ensured); incubating on a shaker for 2 hours at room temperature; the shaker speed was set at 500 + -50 rpm;
4) completely pouring out liquid in the holes, repeatedly washing each hole for 4 times by using diluted washing liquid, adding about 400 mu l of the washing liquid into each hole, and after complete washing, placing the plate upside down on absorbent paper and patting the plate dry;
5) adding 200 mul of human VDB conjugated substrate solution into each hole, and incubating for 1 hour on a shaking table at room temperature;
6) repeating operation 4 for washing;
7) adding 200 mul of substrate solution into each hole, and incubating for 30 minutes at room temperature in a dark place;
8) stop the reaction by adding 50. mu.l of stop solution to each well (color immediately changed from blue to yellow);
9) the blank well was set to zero, and the optical density (OD value) was measured at 450nm with an enzyme-labeled detector within 15 minutes after the addition of the stop solution. And (3) drawing a standard curve and a mathematical formula of the curve by using ELISACalc software according to the OD value measured by the standard protein of the VDB, sequentially inputting the OD value of each plasma sample into the standard curve formula obtained by the software to calculate the content of the VDB in the current sample, and calculating the concentration of the VDB in the undiluted state to be used as the final content of the VDB in the plasma based on the dilution multiple of each sample recorded before the experiment starts.
The demographic and clinical characteristics of the subjects are shown in table 1, the results of the concentration of VDB protein in plasma of different mental disease patients and non-mental disease controls are shown in table 1 and table 1, and the analysis of the diagnosis and diagnostic efficacy of the plasma VDB is shown in table 2.
As can be seen in FIG. 1, the plasma levels of VDB were significantly increased only in depression patients, while there were no statistically significant differences among other populations, indicating that the present invention can effectively and specifically diagnose the psychotic depression using the assay for vitamin D binding protein content.
FIG. 2 shows that the area under the ROC curve (AUC) for plasma VDB protein in the diagnosis of depression and healthy population, schizophrenia, bipolar mania was 0.750 (95% IC: 0.669-0.831), 0.734 (95% IC: 0.643-825), 0.648 (95% IC: 0.549-0.748) in order. It is generally considered that 0.5< AUC <1, the classifier is of predictive value, and the classification efficacy is greater when the AUC is greater. Therefore, the VDB has higher identifying efficacy in diagnosing depression and other people, which shows that the invention can accurately identify and diagnose the depression from healthy people or other mental diseases (schizophrenia, bipolar mania), and the invention is convenient, rapid and effective to detect by an enzyme-linked immunosorbent assay kit. In addition, the effect of some other ELISA kits capable of detecting the content of VDB is consistent with the kit effect adopted in the above examples, such as human Vitamin D Binding Protein (VDBP) ELISA kit (RGB-60486H), human Vitamin D Binding Protein (VDBP) ELISA kit (MEXN-H2746), and the like.
TABLE 1 basic clinical information of the Subjects and VDB test results
Note: data are expressed using mean (standard deviation) or number of cases (percentage%).
MDD: depression; HC for healthy people with non-mental diseases; SZ: schizophrenia; BD-I biphase maniaaKruskal-Wallis H test;bchecking a chi square;canalyzing the single-factor variance;dMann-Whitney U test.
Claims (10)
1. Use of vitamin D binding protein as a marker in the preparation of a reagent for diagnosing mental illness depression.
2. The application of the reagent for detecting the content of the vitamin D binding protein in the preparation of the reagent for diagnosing the mental disease depression.
3. The use according to claim 1 or 2, wherein the agent for diagnosing depressive disorders comprises an agent for diagnosing depressive disorders from a healthy human or from psychiatric disorders including schizophrenia and bipolar mania.
4. The use according to claim 2, wherein the reagent for detecting the content of vitamin D binding protein is preferably used in the preparation of a depression diagnostic reagent product or tool.
5. The use according to claim 2, wherein the reagents comprise reagents for detecting the content of vitamin D binding protein by enzyme-linked immunosorbent assay, said reagents comprising mainly polyclonal antibodies specific for vitamin D binding protein.
6. The use of claim 5, wherein the reagent for detecting the content of vitamin D binding protein by using an enzyme-linked immunosorbent assay kit is a vitamin D binding protein content enzyme-linked immunosorbent assay kit.
7. The use of claim 5 or 6, wherein the reagent for detecting the content of Vitamin D binding protein by ELISA is ELISA Kit comprising Human Vitamin D BP Quantikine ELISA Kit (DVDBP 0B; R & D Systems, Minneapolis, MN, USA), Human Vitamin D Binding Protein (VDBP) ELISA Kit (RGB-60486H; Ruegbock technology development Co., Ltd., Beijing, China), or Human Vitamin D Binding Protein (VDBP) ELISA Kit (MEXN-H2746; Meixuan Biotechnology Co., Ltd., Shanghai, China), etc.
8. The use of claim 5, wherein the antibody specific for vitamin D binding protein is a polyclonal antibody produced by injecting purified human vitamin D binding protein or an antigenic fragment thereof into an animal; or by immunizing an animal with cells expressing human vitamin D binding protein or an antigenic fragment thereof.
9. A kit for diagnosing a psychiatric disorder comprising an antibody capable of specifically binding to vitamin D binding protein and an auxiliary reagent capable of detecting the amount of vitamin D binding protein.
10. The kit according to claim 9, wherein said kit can be used for early screening of depression and differential diagnosis of healthy persons or other mental disorders such as schizophrenia, bipolar mania using vitamin D binding protein alone as an index.
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| WO2021185124A1 (en) * | 2020-03-18 | 2021-09-23 | 东南大学 | Use of vitamin d binding protein as marker in diagnosis of mental illness depression |
| CN113444786A (en) * | 2021-06-29 | 2021-09-28 | 中国人民解放军军事科学院军事医学研究院 | Application of EDA-A2 in serum as auxiliary diagnostic marker for mood disorder diseases |
| CN114410773A (en) * | 2022-01-27 | 2022-04-29 | 宁波大学 | Marker combination for predicting or diagnosing depression recurrence and application thereof |
| CN114438191A (en) * | 2022-01-27 | 2022-05-06 | 宁波大学 | Application of hypoxia inducible factor 1alpha as marker in depression recurrence diagnosis |
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| CN114438191B (en) * | 2022-01-27 | 2024-04-30 | 宁波大学 | Application of hypoxia inducible factor 1 alpha as marker in diagnosis of depression recurrence |
| CN114410773B (en) * | 2022-01-27 | 2024-05-03 | 宁波大学 | Marker combination for predicting or diagnosing depression recurrence and application thereof |
| CN115097143A (en) * | 2022-07-11 | 2022-09-23 | 东南大学 | Application of vitamin D binding protein in total exosomes of peripheral blood plasma in diagnosing depression |
| CN115166260A (en) * | 2022-07-11 | 2022-10-11 | 东南大学 | Application of vitamin D binding protein in plasma brain cell source exosome in diagnosing depression |
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| WO2021185124A1 (en) | 2021-09-23 |
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