CN104749354A - Kit for simultaneous detection of four depression markers and preparation method thereof - Google Patents
Kit for simultaneous detection of four depression markers and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of immunodetection, and provides a kit for simultaneous detection of four depression markers and a preparation method thereof, the preparation method is as follows: antibody coating is prepared; enzyme label plates prepared in the first step are respectively poured, closed, washed, air-dried, and stored for standby use; a sample is loaded, the enzyme label plates are put into an incubation oscillator for incubation, then a detection antibody is added; enzyme SA HRP is added into plate holes for continuing incubation; CCD imager is used for filming and imaging, the image IDV value can be calculated by combination of image analysis software, according to the IDV value, a standard curve is drawn, and the sample concentration result is calculated; and softMax regression model and Excel table are used for calculation and statistical analysis of the sample concentration result. The preparation method solves effectively the problem of complicated process, operation disadvantage and low sensitivity.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly detect kit of four kinds of depression marks and preparation method thereof simultaneously.
Background technology
In recent years, increasing evidence demonstrates the effect of inflammatory reaction in depressive illness generation, evolution, a lot of inflammatory factors and corresponding soluble recepter, chemotactic molecule, adhesion molecule, acute phase protein etc. significantly can rise in the peripheral blood, cerebrospinal fluid of patients with depression, and indivedual symptoms such as relation between fatigue, cognitive disorder and sleep-disorder etc. and inflammatory factor of depression is also constantly reported.The biomarker that these depression are relevant may be used for the evaluation of adjuvant clinical diagnosis and prognostic, provides biological evidences by the objective diagnosis for depression.Apply the depression correlation factor of more than 600 part of serum sample to bibliographical information further across us and carried out large sample amount screening verification, tentatively determine Brain Derived Neurotrophic Factor, cortisol, epidermal growth factor, myeloperoxidase, prolactin, phylaxin, soluble tumor necrosis factor α receptor II type as the biomarker of depression auxiliary diagnosis.
Have a lot of traditional individual event elisa technique detecting correlation factor in the market, its degree of ripeness is high, and specificity is good, easy and simple to handle.But when common Enzyme-linked Immunosorbent Assay ELISA detects sample, each detect aperture generally needs 100-200 μ l, sample large usage quantity, more than one of the biomarker of depression in addition, need the combined monitoring of multiple factor to diagnose, if still depend on individual event ELISA under these circumstances to detect, then sample consumption is excessive; The diagnosis each factor being detected separately to auxiliary depression is obviously wasted time and energy, and diagnoses with high costs, and patient is generally difficult to bear.
Radioimmunoassay method RIA technology, it had both had immunoreactive high specific, there is again radiometric high sensitivity, but there is the problem such as radiation and pollution, and the health of testing staff is also constituted a threat to, and, operate complicated relative to ELISA, once inspection can only survey an index, also needs to provide special radioactivity experiment place, invest high, harm is large.
Although protein chip can realize the function that multiple factor detects simultaneously, but because it is that antibody is coated on for the preparation of on the substrate slide of protein-chip, microballoon or film, and substrate slide and film before use will through the pre-service such as aldehyde radical or poly-D-lysine, production cost is caused to increase, and subsequent experimental complex operation,, poor repeatability, is difficult to promote.
Therefore, technical field of immunoassay is badly in need of a kind of, convenient operation simple based on chemiluminescent technique, highly sensitive, can detect kit of four kinds of depression marks and preparation method thereof simultaneously.
Summary of the invention
The invention provides kit simultaneously detecting four kinds of depression marks and preparation method thereof, technical scheme is as follows:
Detect the kit of four kinds of depression marks simultaneously, it is characterized in that, comprising:
Kit and this kit include useful Brain Derived Neurotrophic Factor BDNF, epidermal growth factor EGF, myeloperoxidase MPO and prolactin Prolactin tetra-kinds of antigens and corresponding antibodies with the surface of rectangular array arrangement bag quilt through plasma and wall through ELISA Plate, dilution, a positive serum controls sample, a negative serum control sample, washing agent, biotinylated detection antibody, enzyme SA-HRP, the luminescent solution of coating film treatment.
Detect the preparation method of the kit of four kinds of depression marks simultaneously, it is characterized in that, comprise the steps:
The first step, the ELISA Plate of Dispersal risk bag quilt;
Preparation is that 100000-20000000ng/mL is in the phosphate buffered solution of 0.01-0.1mol/l containing Brain Derived Neurotrophic Factor BDNF, epidermal growth factor EGF, myeloperoxidase MPO and prolactin Prolactin;
Further, extract the above-mentioned phosphate buffered solution containing the four species specificity factors of 10-100nl with full-automatic point sample instrument, point sample is in the hole of the ELISA Plate crossed by plasma treatment;
Further, wrap by 16-24 hour under the ELISA Plate that point sample is good being placed in the temperature of 2-8 DEG C;
Second step, after pouring into a mould respectively, close, wash, drying, then saves stand-by to ELISA Plate in the first step;
First, be in the phosphate buffer of 7.4 ± 0.2 to pH value, add the bovine serum albumin(BSA) BSA of the polyoxyethylene sorbitol mono laurate monoester Tween20 and 3% of 0-0.05% successively, after stirring, pour into a mould in the ELISA Plate in the first step; Or to pH value be 7.4 ± 0.2 phosphate buffer in, add the skimmed milk power of polyoxyethylene sorbitol mono laurate monoester Tween20 and 5-10% of 0-0.05% successively, after stirring, pour into a mould in the ELISA Plate in the first step;
Further, the ELISA Plate of this being poured into a mould is closed under being placed on the temperature of 2-8 DEG C and is placed 16-24 hour or close placement 2 ± 0.5 hours at being placed on 37 DEG C, or room temperature closes 3 ± 0.5 hours;
Further, after the ELISA Plate closed being washed containing the PBS solution of non-ionics Tween20, drying, preserve stand-by under being placed in the temperature of 2-8 DEG C;
3rd step, loading, and ELISA Plate is put into incubation oscillator hatch;
First, the serum sample of many parts of patients, 1 part of negative control sample through the normal person of examination and 1 part of positive control is got;
Sample Dilution is carried out further with the PBS Sample dilution containing 1%BSA, 0.05%Tween that pH value is 7.4 ± 0.2; Again the sample to be tested diluted is added corresponding in the ELISA Plate hole processed in second step;
Further, the ELISA Plate having added solution is put into incubation oscillator, react 1 hour under the room temperature condition of 18-25 DEG C;
4th step, adds and detects antibody and hatch;
With the PBS containing Tween as washing lotion, after ELISA Plate washing reacted in the 3rd step, then add detection antibody respectively in each hole of ELISA Plate; Put into incubation oscillator reaction;
5th step, to add in enzyme SA-HRP and to hatch;
With the PBS containing Tween as washing lotion, 4 times are washed to reacted ELISA Plate in the 4th step, then to after SA-HRP dilutes in kit, add the solution after dilution respectively in each hole of ELISA Plate, put into incubation oscillator and react;
6th step, with CCD imager shooting imaging, and combining image analysis software calculates the IDV value of image, according to IDV value drawing standard curve, and calculates concentration of specimens result;
First, stable Peroxidase Solution and the luminol substrate solution containing reinforcing agent are hybridly prepared into luminescent solution with 1:1;
Further, with containing the PBS of Tween as washing lotion, 4 times are washed to reacted ELISA Plate in the 5th step, in each hole of ELISA Plate, add the luminescent solution prepared, to take pictures imaging with CCD imager in 1-10 minute;
Further, by the image analysis software supporting with CCD imager, the image procossing of shooting is become IDV value;
Further, according to IDV value drawing standard curve, and concentration of specimens result is calculated;
7th step, uses softMax regression model and Excel form to calculate and statistical analysis concentration of specimens result, and through comprehensively analyzing, be 100% positive to the testing result of these many parts of patients serum's samples, namely susceptibility is 100%.
The preparation method simultaneously detecting the kit of four kinds of depression marks as above, wherein, in the first step, with full-automatic point sample instrument point sample, be put by four kinds of antibody points on four intersect edge points of the equidistant rectangle in distance plate hole center in ELISA orifice plate, what form rectangular array catches surface.，
The as above preparation method simultaneously detecting the kit of four kinds of depression marks, wherein, the surface of the ELISA Plate in the first step through plasma and wall through coating film treatment.
The invention has the beneficial effects as follows:
1. present invention achieves and polyfactorially to detect simultaneously, improve detection efficiency, saved testing cost, make patient be easy to accept, and save sample consumption, avoid because sample size deficiency cannot complete the drawback that repeatedly individual event detects.
2. the present invention is for the quantitative detection of the four species specificity factors, and detectability can reach 0.1pg/ml, thus has higher detection sensitivity, the biomarker that the low abundance of detection that can be quantitative and downward are expressed.
3. the range of linearity has been brought up to 4-51log from existing 2-31log by the present invention, thus has the wider range of linearity, the factor detection that the wide range of linearity can be quantitative is in harmonious proportion in downward two kinds of situations, can better assess effectiveness.
4. the present invention takes the lead in rectangular array to apply in the biomarker detection of depression, antibody probe point is put on the rectangular loop summit in ELISA orifice plate, what form rectangular array catches surface, thus obtain the good antibody array of consistance, impelled reaction more even, stable, data redundancy is good.
5. the present invention is by designing and plate treatment technology by rectangular array, and make to have very high consistance between different probe site, the combination of these two kinds of technology makes the detection data of product have very high repeatability, can improve the order of accuarcy of diagnosis.
6. four kinds of depression marks of the present invention are combined in the ELISA Plate of plasma treatment, can strengthen and effective combination of antibody protein, and wall is by coating film treatment, makes it can not associated proteins, thus reduce non-specific binding to the impact of background, improve sensitivity.
Accompanying drawing explanation
The present invention is described in detail below in conjunction with the drawings and specific embodiments:
Fig. 1 is the typical curve that BDMP of the present invention draws according to IDV value.
Fig. 2 is the typical curve that EGF of the present invention draws according to IDV value.
Fig. 3 is the typical curve that MPO of the present invention draws according to IDV value.
Fig. 4 is the typical curve that Prolactin of the present invention draws according to IDV value.
Embodiment
In order to make technological means of the present invention, creation characteristic, reach object and effect is easy to understand, below in conjunction with concrete diagram, set forth the present invention further.
The invention provides the kit simultaneously detecting four kinds of depression marks, comprising:
Kit and this kit include useful Brain Derived Neurotrophic Factor BDNF, epidermal growth factor EGF, myeloperoxidase MPO and prolactin Prolactin tetra-kinds of antigens and corresponding antibodies with the surface of rectangular array arrangement bag quilt through plasma and wall through ELISA Plate, dilution, a positive serum controls sample, a negative serum control sample, washing agent, biotinylated detection antibody, enzyme SA-HRP, the luminescent solution of coating film treatment.
Detect the preparation method of the luminescence reagent box of four kinds of depression marks, concrete steps are as follows simultaneously:
The first step, the ELISA Plate of Dispersal risk bag quilt;
Preparation is that 100000-20000000ng/mL is in the phosphate buffered solution of 0.01-0.1mol/l containing Brain Derived Neurotrophic Factor BDNF, epidermal growth factor EGF, myeloperoxidase MPO and prolactin Prolactin;
Further, extract the above-mentioned phosphate buffered solution containing the four species specificity factors of 10-100nl with full-automatic point sample instrument, point sample is in the hole of the ELISA Plate crossed by plasma treatment;
With full-automatic point sample instrument point sample, be put by four kinds of antibody points on four intersect edge points of the equidistant rectangle in distance plate hole center in ELISA orifice plate, what form rectangular array catches surface;
Further, wrap by 16-24 hour under the ELISA Plate that point sample is good being placed in the temperature of 2-8 DEG C;
Second step, after pouring into a mould respectively, close, wash, drying, then saves stand-by to ELISA Plate in the first step;
First, be in the phosphate buffer of 7.4 ± 0.2 to pH value, add the bovine serum albumin(BSA) BSA of the polyoxyethylene sorbitol mono laurate monoester Tween20 and 3% of 0-0.05% successively, after stirring, pour into a mould in the ELISA Plate in the first step; Or to pH value be 7.4 ± 0.2 phosphate buffer in, add the skimmed milk power of polyoxyethylene sorbitol mono laurate monoester Tween20 and 5-10% of 0-0.05% successively, after stirring, pour into a mould in the ELISA Plate in the first step;
Further, the ELISA Plate of this being poured into a mould is closed under being placed on the temperature of 2-8 DEG C and is placed 16-24 hour or close placement 2 ± 0.5 hours at being placed on 37 DEG C, or room temperature closes 3 ± 0.5 hours;
Further, after the ELISA Plate closed being washed containing the PBS solution of non-ionics Tween20, drying, preserve stand-by under being placed in the temperature of 2-8 DEG C;
3rd step, loading, and ELISA Plate is put into incubation oscillator hatch;
First, the serum sample of 38 parts of patients, 1 part of negative control sample through the normal person of examination and 1 part of positive control is got;
1:3 Sample Dilution is carried out further with the PBS Sample dilution containing 1%BSA, 0.05%Tween that pH value is 7.4 ± 0.2; Again the sample to be tested diluted is added corresponding in the ELISA Plate hole processed in second step; Further, the ELISA Plate having added solution is put into incubation oscillator, react 1 hour under the room temperature condition of 18-25 DEG C;
4th step, adds and detects antibody and hatch;
With the PBS containing Tween as washing lotion, after ELISA Plate washing reacted in the 3rd step, then add detection antibody respectively in each hole of ELISA Plate; Put into incubation oscillator in rotating speed 500rpm, room temperature 18-25 DEG C of reaction 0.5 hour;
5th step, adds SA-HRP and hatches
With the PBS containing 0.05%Tween as washing lotion, 4 times are washed to reacted ELISA Plate in the 4th step, again with 1:10000 to after in kit, SA-HRP dilutes, respectively to the solution added in each hole of ELISA Plate after 50 μ l dilutions, put into incubation oscillator in rotating speed 500rpm, room temperature 18-25 DEG C of reaction 0.5 hour;
6th step, with CCD imager shooting imaging, and combining image analysis software calculates the IDV value of image, according to IDV value drawing standard curve, and calculates concentration of specimens result;
First, stable Peroxidase Solution and the luminol substrate solution containing reinforcing agent are hybridly prepared into luminescent solution with 1:1;
Further, with containing the PBS of 0.05%Tween as washing lotion, 4 times are washed to reacted ELISA Plate in the 5th step, in each hole of ELISA Plate, add the luminescent solution that 50 μ l prepare, to take pictures imaging with CCD imager in 1-10 minute;
Further, by the image analysis software supporting with CCD imager, the image procossing of shooting is become IDV value;
Further, according to IDV value drawing standard curve, Fig. 1 is the typical curve that BDMP of the present invention draws according to IDV value, Fig. 2 is the typical curve that EGF of the present invention draws according to IDV value, Fig. 3 is the typical curve that MPO of the present invention draws according to IDV value, Fig. 4 is the typical curve that Prolactin of the present invention draws according to IDV value, and calculates concentration of specimens result, as table one according to typical curve:
7th step, uses softMax regression model and Excel form to calculate and statistical analysis concentration of specimens result, and through comprehensively analyzing, be 100% positive to the test result of these 38 parts of patients serum's samples, namely susceptibility is 100%.
Present invention achieves and polyfactorially to detect simultaneously, improve detection efficiency, saved testing cost, make patient be easy to accept, and save sample consumption, avoid because sample size deficiency cannot complete the drawback that repeatedly individual event detects.
The present invention is for the quantitative detection of the four species specificity factors, and detectability can reach 0.1pg/ml, thus has higher detection sensitivity, the biomarker that the low abundance of detection that can be quantitative and downward are expressed.
The range of linearity has been brought up to 4-51log from existing 2-31log by the present invention, thus has the wider range of linearity, the factor detection that the wide range of linearity can be quantitative is in harmonious proportion in downward two kinds of situations, can better assess effectiveness.
The present invention takes the lead in rectangular array to apply in the biomarker detection of depression, antibody probe point is put on the rectangular loop summit in ELISA orifice plate, what form rectangular array catches surface, thus obtain the good antibody array of consistance, impelled reaction more even, stable, data redundancy is good.
The present invention is by rectangular array design and plate treatment technology, and make to have very high consistance between different probe site, the combination of these two kinds of technology makes the detection data of product have very high repeatability, can improve the order of accuarcy of diagnosis.
Four kinds of depression marks of the present invention are combined in the ELISA Plate of plasma treatment, can strengthen and effective combination of antibody protein, and wall is by coating film treatment, makes it can not associated proteins, thus reduce non-specific binding to the impact of background, improve sensitivity.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (4)
1. detect the kit of four kinds of depression marks simultaneously, it is characterized in that, comprising:
Kit and this kit include useful Brain Derived Neurotrophic Factor, epidermal growth factor, myeloperoxidase and prolactin four kinds of antigens and corresponding antibodies with the surface of rectangular array bag quilt through plasma and wall through ELISA Plate, dilution, a positive serum controls sample, a negative serum control sample, washing agent, biotinylated detection antibody, enzyme SA-HRP, the luminescent solution of coating film treatment.
2. detect the preparation method of the luminescence reagent box of four kinds of depression marks simultaneously, it is characterized in that, comprise following steps:
The first step, the ELISA Plate of Dispersal risk bag quilt;
Preparation is that 100000-20000000ng/mL is in the phosphate buffered solution of 0.01-0.1mol/l containing Brain Derived Neurotrophic Factor, epidermal growth factor, myeloperoxidase and prolactin;
Further, extract the above-mentioned phosphate buffered solution containing the four species specificity factors of 10-100nl with full-automatic point sample instrument, point sample is in the hole of the ELISA Plate crossed by plasma treatment;
Further, wrap by 16-24 hour under the ELISA Plate that point sample is good being placed in the temperature of 2-8 DEG C;
Second step, after pouring into a mould respectively, close, wash, drying, then saves stand-by to ELISA Plate in this first step;
First, be in the phosphate buffer of 7.4 ± 0.2 to pH value, add the polyoxyethylene sorbitol mono laurate monoester of 0-0.05% and the bovine serum albumin(BSA) of 3% successively, after stirring, pour into a mould in the ELISA Plate in this first step; Or to pH value be 7.4 ± 0.2 phosphate buffer in, add the polyoxyethylene sorbitol mono laurate monoester of 0-0.05% and the skimmed milk power of 5-10% successively, after stirring, pour into a mould in the ELISA Plate in this first step;
Further, the ELISA Plate of this being poured into a mould is closed under being placed on the temperature of 2-8 DEG C and is placed 16-24 hour or close placement 2 ± 0.5 hours at being placed on 37 DEG C, or room temperature closes 3 ± 0.5 hours;
Further, after the ELISA Plate closed being washed containing the phosphate buffered solution of non-ionics, drying, preserve stand-by under being placed in the temperature of 2-8 DEG C;
3rd step, loading, and ELISA Plate is put into incubation oscillator hatch antibody;
First, the serum sample of many parts of patients, 1 part of negative control sample through the normal person of examination and 1 part of positive control is got;
Sample Dilution is carried out further with the phosphate buffered solution Sample dilution containing 1% bovine serum albumin(BSA), 0.05% polyoxyethylene sorbitol mono laurate monoester that pH value is 7.4 ± 0.2; Again the sample to be tested diluted is added corresponding in the ELISA Plate hole processed in this second step;
Further, the ELISA Plate having added solution is put into incubation oscillator, react under the room temperature condition of 18-25 DEG C;
4th step, adds and detects antibody and hatch;
By the phosphate buffered solution containing polyoxyethylene sorbitol mono laurate monoester as washing lotion, then add detection antibody respectively in each hole of ELISA Plate; Put into incubation oscillator reaction;
5th step, to add in kit and to hatch;
By the phosphate buffered solution containing polyoxyethylene sorbitol mono laurate monoester as washing lotion, 4 times are washed to reacted ELISA Plate in the 4th step, after again kit being diluted, add the solution after dilution respectively in each hole of ELISA Plate, put into incubation oscillator and react;
6th step, with CCD imager shooting imaging, and combining image analysis software calculates the IDV value of image, according to IDV value drawing standard curve, and calculates concentration of specimens result;
First, stable Peroxidase Solution and the luminol substrate solution containing reinforcing agent are hybridly prepared into luminescent solution with 1:1; Further, by the phosphate buffered solution containing polyoxyethylene sorbitol mono laurate monoester as washing lotion, 4 times are washed to reacted ELISA Plate in the 5th step, in each hole of ELISA Plate, adds the luminescent solution prepared, to take pictures imaging with CCD imager in 1-10 minute;
Further, by the image analysis software supporting with CCD imager, the image procossing of shooting is become IDV value;
Further, according to IDV value drawing standard curve, and concentration of specimens result is calculated;
7th step, uses softMax regression model and Excel form to calculate and statistical analysis concentration of specimens result, and through comprehensively analyzing, be 100% positive to the test result of these many parts of patients serum's samples, namely susceptibility is 100%.
3. the preparation method simultaneously detecting the kit of four kinds of depression marks according to claim 2, it is characterized in that, with full-automatic point sample instrument point sample in this first step, be put by four kinds of antibody points on four intersect edge points of the equidistant rectangle in distance plate hole center in ELISA Plate, what form rectangular array catches surface.
4. the preparation method of luminescence reagent box simultaneously detecting four kinds of depression marks according to claim 2, is characterized in that, the surface of the ELISA Plate in this first step through plasma and wall through coating film treatment.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105510586A (en) * | 2015-12-22 | 2016-04-20 | 湖北鹊景生物医学有限公司 | Kit for lung cancer diagnosis and use method of kit |
| CN111141863A (en) * | 2018-11-06 | 2020-05-12 | 中国科学院大连化学物理研究所 | Depression diagnosis marker and preparation method thereof |
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| CN101358976A (en) * | 2008-04-28 | 2009-02-04 | 北京华大吉比爱生物技术有限公司 | Micro array-ELISA detecting kit for detecting six tumor markers |
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| CN101358976A (en) * | 2008-04-28 | 2009-02-04 | 北京华大吉比爱生物技术有限公司 | Micro array-ELISA detecting kit for detecting six tumor markers |
| US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105510586A (en) * | 2015-12-22 | 2016-04-20 | 湖北鹊景生物医学有限公司 | Kit for lung cancer diagnosis and use method of kit |
| CN111141863A (en) * | 2018-11-06 | 2020-05-12 | 中国科学院大连化学物理研究所 | Depression diagnosis marker and preparation method thereof |
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