CN102788883B

CN102788883B - Kit for detecting depression

Info

Publication number: CN102788883B
Application number: CN201210313623.5A
Authority: CN
Inventors: 谢鹏; 杨涌涛; 周婵娟; 詹远; 房亮
Original assignee: Chongqing Medical University
Current assignee: SICHUAN CREDIT PHARMA Co.,Ltd.
Priority date: 2012-08-30
Filing date: 2012-08-30
Publication date: 2014-11-05
Anticipated expiration: 2032-08-30
Also published as: CN102788883A

Abstract

The invention relates to the field of biotechnology, in particular to a kit for detecting depression. The kit comprises sealing liquid, an enzyme labeled antibody, an enzyme substrate solution, a stop buffer, an enzyme label plate, PEDF (Pigment Epithelium Derived Factor) protein or a protein fragment containing the PEDF protein, and an antibody in specific binding with the PEDF protein, wherein the PEDF protein has an amino acid sequence of SEQIDNO1 (Sequence Identifier Number). The kit can be applicable to a patient in the early stage of depression, and is helpful for the patient in incubation period to determine the pharmacotherapy time so as to delay the development of the disease. The kit provided by the invention is high in accuracy and precision.

Description

A kind of kit for detection of depression

Technical field

The present invention relates to biological technical field, specifically a kind of kit for detection of depression.

Background technology

Depression is a kind of common mood disorder, and its core symptom is that depressed, retardation of thinking, bulesis go down, and can also occur sleep-disorder, suicidal thought etc. repeatedly.According to WHO statistics, approximately there are 1.21 hundred million patients with depression in the whole world at present, has every year 850.000 people's death to depressed relevant.The heritability of depression is about 30% ~ 40%, wherein may relate to multiple inherent causes.Meanwhile, multiple non-genetic factor (stress, neurodevelopment abnormal etc.) has increased the complicacy of depression, and depression mechanism is not clear and definite yet so far.Can make depressive symptom alleviate by drug therapy, psychotherapy or physical treatment clinically.

At present, can only diagnose in conjunction with SDS by clinical symptoms depression clinically, this diagnostic method is difficult to clearly detect depression in disease latent period and commitment thereof, and its adverse consequences causing is to be difficult to clearly detect depression in disease latent period and commitment thereof.Therefore, be badly in need of now finding a kind of new, that there is diagnostic significance, Diagnosis of Depression material and using method thereof that can accurate quantification, depression is carried out to latent period and commitment is diagnosed, and provide better methods for the treatment of for patient.

Summary of the invention

The object of the invention is provides a kind of kit for detecting depression, can realize the diagnosis to patients with depression in accurate quantification ground.

The technical solution used in the present invention is for achieving the above object: a kind of kit for detection of depression, comprise the composition (being confining liquid, enzyme labelled antibody, enzyme substrate solution, stop buffer, ELISA Plate) that uses in general diagnostic kit and pedf protein or the protein fragments that comprises pedf protein and the antibody (hereinafter to be referred as anti-PEDF antibody) with pedf protein specific binding, wherein the amino acid sequence of pedf protein is SEQ ID NO1.

Above-mentioned antibody is monoclonal antibody or polyclonal antibody.Preferably monoclonal antibody.

Right is wanted pedf protein described in 1 or the protein fragments that comprises pedf protein or is detected the application in the kit of depression with the antibody of pedf protein specific binding in preparation, wherein, normally, in schizophrenia, depression and two-way disturbance of emotion serum, the mean value of pedf protein is respectively 2918.9ug/mL, 3037.8ug/mL, 1624.8ug/mL, 2838ug/mL.

The present invention uses proteomics method to analyze to show in depression the protein group of special variation.Find following result by protein number change and the protein type analyzed in patients with depression blood plasma, schizophrenia, the two-way disturbance of emotion and normal plasma.First, select normal plasma as a control group, separate and graphical analysis by two-dimentional gel, in the blood plasma by patients with depression, separate the protein group who shows character and quantity variance, use MALDI-TOF analyser identification of protein group, proved wherein to observe and identified that the protein of variation is PEDF.The second, using normal blood as Normal group, using the blood of schizophrenia, the two-way disturbance of emotion during as disease control group, the expression of observing PEDF in depressed blood plasma reduces, and in schizophrenia blood plasma, expresses and increases.The 3rd, after having checked that the target protein in blood changes, use suitable immunological method to prepare the kit of diagnosis depression, select for measuring the simple, sensitive of protein existence value and accurate method.Wherein, be depression when patient's blood plasma PEDF concentration is diagnosable during lower than 2485ug/ml, can diagnose patients with depression.

Brief description of the drawings

Fig. 1 represents the preprocessing process of the blood plasma that will be applied to proteome analysis;

Fig. 2 represents that blood plasma silver after two dimensional electrophoresis dyes; A: normal group blood plasma dielectrophoresis figure, B: major depression group blood plasma dielectrophoresis figure, shows and have excessive protein in the marked region of normal plasma, and show in the marked region of depression blood plasma that the quantity of protein reduces;

Fig. 3 represents to use in Fig. 2 marked region that MALDI-TOF analyser obtains protein through the mass spectrum of the peptide of trypsin treatment, and the amino acid sequence of peptide in fragments of peptides, one-level mass spectrogram;

The corresponding second order ms figure of Fig. 4 presentation graphs 3;

The corresponding second order ms figure of Fig. 5 presentation graphs 3;

The corresponding second order ms figure of Fig. 6 presentation graphs 3;

Fig. 7 represents that the expression of PEDF PEDF in Normal group, schizophrenia group, two-way disturbance of emotion group and depression group changes;

Fig. 8 represents PEDF standard solution and the reacted optical density measurements of ELISA.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention, should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.

Embodiment 1, the pre-service of the blood plasma of proteome analysis.

As shown in Figure 1, the plasma sample that will be applied to proteome analysis contrasts with normal plasma, then remove 7 kinds of high-abundance proteins (Agilent), Speed Vac concentrates plasma sample, adopt again TCA method to carry out desalination, purifying to sample after concentrated, be finally precipitated as dry powder (experiment of carrying out again after dissolving in the embodiment below) for subsequent use.

Embodiment 2, the protein group's who changes in the blood plasma being obtained by normal person and patients with depression research.

First two different characteristics that, use protein by method progressively by protein in two dimension separately.In the first step, by protein application point is stimulated and makes protein move (IEF, pH3-10) according to the static charge of protein; In second step, protein molecular weight according to every kind of protein on acrylamide gel moves.To the albumen electrophoresis (protein moves according to pH) that is shifted.Then, on polyacrylamide, protein is carried out to two dimensional electrophoresis (protein moves according to molecular weight).With coomassie brilliant blue R250 dyestuff and argentation by these egg removal white matters dyeing for developing.In computer, use image analysis software PDQuest(Bio-Rad, the U.S.) analyze the protein difference of the case of normal plasma and depression blood plasma, the inventor has confirmed to exist the protein group of real difference, referring to seeing that in Fig. 2, square frame frame goes out part.

Embodiment 3, shows the qualification of the plasma proteins of difference between normal person and patients with depression.

Searched for and identified that the protein (being that in Fig. 2, square frame frame goes out part) with quantity and nature difference, to know the kind of protein, is shown in Fig. 3-Fig. 6 by MALDI-TOF analyser, qualification shows: the PEDF of patients with depression expresses the blood lower than normal person.

Embodiment 4, detects PEDF by WesternBlotting Western blot and expresses.

As Fig. 4, first prepare 12 normal, 12 schizophrenias, 12 two-way disturbances of emotion and 12 patients with depression blood plasma sex change samples.On polyacrylamide gel, protein is carried out to electrophoresis (protein moves according to molecular weight), every clotting glue 100V.Rear employing Western blot is transferred to albumen in SDS-PAGE glue on pvdf membrane, pvdf membrane is carried out to immune response with anti-PEDF antibody, and 4 DEG C are spent the night.Take out afterwards, TBST washes film 3 times, each 10 minutes.Then react with PVDF with two anti-(enzyme labelled antibodies) of HRP mark, under room temperature, hatch 2 hours.Then adopt ECL method to carry out X-exposure to PVDF, and with Quantity-One software analysis gray-scale value.Comparative analysis prompting, with Normal group comparison, in schizophrenia and two disease control groups of the two-way disturbance of emotion, PEDF gray-scale value is large, and prompting protein quantity is many; And PEDF gray-scale value is the reduction change of 1.5 times in depression group, prompting protein quantity is few.

Embodiment 5, antibody preparation.

The method of monoclonal antibody: 1) PEDF gene or protein immune animal, 2) immune spleen cell and myeloma cell's preparation; 3) selection of hybridoma is cultivated, screening and clone; 4) foundation of secrete monoclonal antibody hybridoma cell line; 5) a large amount of preparations of monoclonal antibody.

The method of polyclonal antibody: 1) PEDF gene or protein immune animal, 2) collect immune serum, purifying is for the polyclonal antibody of PEDF.

Embodiment 6, by enzyme linked immunological absorption measurement method (ELISA) diagnosis depression.

Carry out this research and be can using anti-PEDF antibody in normal, schizophrenia and the two-way disturbance of emotion, to differentiate the serum of patients with depression by enzyme linked immunological absorption measurement method (ELISA) in order to find out.In hospital, obtain blood plasma by 50 normal healthy peoples, 12 schizophreniacs, 12 two-way patients with affective disorders and 70 patients with depression.First prepare the 96 hole ELISA kits (Merck Millipore, Billerica, Massachusetts, USA) of the detection PEDF being coated with.Due to PEDF in blood mainly with in conjunction with state exist, use in advance 8mM Urea treatment blood plasma, room temperature cracking 30min.Then in each hole, add patient's blood plasma 100ul of damping fluid dilution (1:100), 37 DEG C are reacted 1 hour, then clean 6 times with Wash buffer.To the anti-PEDF antibody (Merck Millipore, Billerica, Massachusetts, USA) that adds 100ul dilution in each hole, then 37 DEG C of reactions 1 hour.After reaction, each hole is cleaned 6 times with Wash buffer.Then, add wherein the Streptavidin of the peroxidase coupling after 100ul dilution, 37 DEG C are reacted 1 hour.Wash buffer cleans 6 times.Add 100ul TMB/E substrate to develop, after 5-10 minute, add 100ul stop buffer to stop.Reading under microplate reader 450nm wavelength.With pedf protein or the protein fragments production standard liquid that comprises pedf protein, carry out the drafting experiment of standard titration curve.Determine the PEDF quantity (see figure 5) in each unit volume (ml) blood by converting standard titration curve and dilutability to optical density.The result that ELISA measures is, scope in Normal group blood plasma is 1897.6-5409.4ug/mL, scope in two-way disturbance of emotion group blood plasma is 1676.1-6160.5ug/mL, scope in schizophrenia group blood plasma is 2267.6-7836.6ug/mL, the scope 702.5-2461.5ug/mL in depression group blood plasma.These results show with mean value in table 1.As the measurement mean value of PEDF, normal person is 2918.9ug/mL, and two-way patients with affective disorders is 2838ug/mL, and schizophreniac is 3037.8ug/mL, and patients with depression is 1624.8ug/mL.Here significantly having shown in the blood plasma of patients with depression exists PEDF to reduce.Show 95.5% diagnosis sensitivity according to ROC tracing analysis.In the situation of psychiatric patient, PEDF has shown 91.7% specificity (in table 1).Wherein, be depression when patient's blood plasma PEDF concentration is diagnosable during lower than 2485ug/ml.

Table 1: the mean value of measuring PEDF in Healthy People and patient's blood plasma by ELISA

	Mean P EDF concentration (ug/ml)
		Healthy People	2918.9±816.7
The two-way disturbance of emotion	2838±1219.4
		Schizophrenia	3730±1493.0
Major depression	1624.8±459.7

The present invention relates to the technology for easily diagnosing depression.There is no at present the effective commodity for depression, depression relies on the psychiatrist of hospital to diagnose completely, for patients with depression, and can not be in its early diagnosis depression in the situation that not carrying out regular psychological consultation and there is no remarkable somatization.The present invention can pass through simple blood testing, and can very effectively before progression of disease, diagnose the illness.Particularly, of the present invention have kit spend cheap and process simply, especially, for accepting the patients with depression of medical science test or referring physician, only need adjust the ELISA method of use 96 orifice plates and just can test in a large number.Equally, by using immuno-chemical method, accuracy of the present invention and accuracy are good.In a word, the present invention effectively diagnoses depression in its screening and early diagnosis, and contributes to latent period patient to determine the drug therapy time, thereby delays disease progression.

<110> Medical University Of Chongqing

<120> kit for detection of depression

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Claims

1.PEDF albumen or the protein fragments that comprises pedf protein or detect the application in the kit of depression in preparation with the antibody of pedf protein specific binding, described PEDF represents pigment epidermal derived factors, wherein, normally, in schizophrenia, depression and two-way disturbance of emotion serum, the mean value of pedf protein is respectively 2918.9 μ g/mL, 3037.8 μ g/mL, 1624.8 μ g/mL, 2838 μ g/mL; Described kit comprises confining liquid, the anti-PEDF antibody of enzyme mark, enzyme substrate solution, stop buffer and ELISA Plate, pedf protein or the protein fragments that comprises pedf protein, with with the antibody of pedf protein specific binding, wherein the amino acid sequence of pedf protein is SEQ ID NO:1; Described antibody is monoclonal antibody or polyclonal antibody.