CN101308148A - Method for quantum dot mark indirect competition fluoroimmunoassay detection for glucocorticosteroid residual - Google Patents
Method for quantum dot mark indirect competition fluoroimmunoassay detection for glucocorticosteroid residual Download PDFInfo
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- CN101308148A CN101308148A CNA2008101235244A CN200810123524A CN101308148A CN 101308148 A CN101308148 A CN 101308148A CN A2008101235244 A CNA2008101235244 A CN A2008101235244A CN 200810123524 A CN200810123524 A CN 200810123524A CN 101308148 A CN101308148 A CN 101308148A
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Abstract
Disclosed is a method of quantum dot-labeled indirect competitive fluorescence immunoassay of glucocorticoid multi-residue, which belongs to the immunoassay method technique field. Quantum dots for labeling antibodies of the invention have the emission spectra of QD545, QD590 and QD650, and the method comprises: directly covering coating antigens in micro-holes of an enzyme label plate, adding glucocorticoid standard solution or a sample under test to form an antigen-antibody fluorescence immunity compound body, stimulating and detecting the fluorescence intensity of the formed antigen-antibody fluorescence immunity compound body with a fluorescence enzyme-labeling instrument, and obtaining the concentration of glucocorticoid multi-residue in the sample under test through comparing with the standard solution. The invention can indirectly detect the concentration value of glucocorticoid multi-residue in the sample under test without adding chromogenic substance through the fluorescence intensity of the antigen-antibody immunity compound body, and both the operation and reaction need only one step; and the quantum dots for labeling antibodies of the invention have advantages of stronger emitted fluorescence intensity and long stabilization time of fluorescence compared with the traditional fluorescence.
Description
Technical field
A kind of method of quantum dot-labeled indirect competition fluoroimmunoassay detection for glucocorticosteroid residual belongs to the immunologic detection method technical field.
Background technology
Glucocorticoid has the glycometabolic effect of antiallergy, anti-inflammatory and influence, often is used to treat the ketoacidosis of inflammatory reaction, immunity disease, ox of domestic animal and sheep eclampsia etc.Dexamethasone, betamethasone, diflucortolone also are often used as growth promoter, thus the feed intake that increases domestic animal makes it reach the purpose of weightening finish.Yet, prove that through toxicology test such medicine has to mutability, cumulative toxicity, if with being intended to add this medicine in the feed, the medicine of accumulating enters human body by food chain, will cause huge harm.In the forbidden drugs list is listed this type of medicine by many for this reason countries.The current residual detection method of glucocorticoid commonly used in the world has: thin-layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc., but these instrument analytical methods not only need expensive instrument and equipment, also than higher, need through complicated sample pre-treatment just can carry out the requirement of sample.The external research of having carried out already the glucocorticoid analytical approach, method commonly used is enzyme-linked immunosorbent assay (ELISA) or is called immuno-enzymatic quantitative analysis method (IEMA).These class methods are with the coating antigen coated elisa plate, add medicine and anti-drug antibodies, add ELIAS secondary antibody again, promptly detect antibody, add the substrate colour developing at last, behind the certain hour, detect the absorbance of a certain specific wavelength with microplate reader, according to drug concentrations to be measured in the known standard product cubage sample, this complex operation, time-consuming.
Summary of the invention
The method that the purpose of this invention is to provide quantum dot-labeled indirect competition fluoroimmunoassay detection for glucocorticosteroid residual, this method has high sensitivity, high specific, pin-point accuracy, quantum dot-labeled immunofluorescence assay simple to operate, is used for the fast detecting of glucocorticosteroid residual.
Technical scheme of the present invention: a kind of quantum dot-labeled indirect competition fluorescence immunoassay detects the method for glucocorticoid, with the coating antigen direct coated in the micropore of ELISA Plate, add glucocorticoid standard solution or testing sample and form Ag-Ab electrochemiluminescent immunoassay complex, excite and detect the fluorescence intensity of formed Ag-Ab electrochemiluminescent immunoassay compound with fluorescence microplate reader, by obtaining the concentration of glucocorticosteroid residual in the testing sample with the standard solution contrast;
(1) with three kinds of quantum dot QD545 of denatured bovine serum protein dBSA parcel, QD590, the anti-dexamethasone of QD650 mark, anti-betamethasone, anti-diflucortolone polyclonal antibody
(1) BSA sex change:
16.5mg BSA is dissolved in the 10mL distilled water, stirs adding 0.42mg NaBH down
4, react 1h under the room temperature, 60-80 ℃ of NaBH that heats the 20min decomposing excessive down
4, the BSA sex change, disulfide bond is opened into-SH, and the concentration of controlling final dBSA aqueous solution is 5 * 10
-5M;
(2) sex change BSA parcel quantum dot dBSA-QDs:
DBSA and quantum dot dysprosium chromium CdTe were mixed in 1: 1 in molar ratio, and 60-80 ℃ of water-bath heating 15min kept two days under the room temperature, made parcel fully;
(3) sex change BSA parcel quantum point coupling antibody
With behind 5 μ L 1-ethyls-(3-dimethylaminopropyl) carbodiimide EDC 0.056M and the 5 μ L sulfo-N-hydroxy-succinamide sulfo-NHS 0.1M mixing 10s, be added to 25 μ L dBSA-QDs 2 * 10
-5Among the M, the quantum dot activation 15min of sex change BSA parcel adds a kind of Ab in three kinds of glucocorticoid antibody of 12 μ L, 4.0mg/mL; Reactant molar ratio is controlled to be: dBSA-QDs: Ab: EDC: sulfo-NHS=1: 1: 560: 1000, react 4h under the room temperature, obtain quantum dot-labeled glucocorticoid antibody, the concentration of antibody is 1.0mg/mL in the reactant liquor, and it is stand-by to do 1000 times of antibody diluent dilutions with the pH that contains tween 7.4, the 0.01M phosphate buffer of 0.1% gelatin; Sex change BSA parcel quantum dot carries out coupling with three kinds of polyclonal antibodies respectively;
(2) the bag quilt of antigen
Polyclonal antibody with three kinds of parcel quantum dots carries out coupling with the ovoserum albumin respectively, three kinds of quality such as the white conjugate of polyclonal antibody-ovoserum that obtain are mixed as the coupled body of glucocorticoid as coating antigen, with pH 9.6,0.05M carbonate buffer solution is cushioned liquid as bag, be cushioned liquid with bag the glucocorticoid coating antigen is diluted to 1-20 μ g/mL as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of refrigerator overnight, with containing NaCl 8.5g/L, pH 7.2-7.4, the 10mmol/L phosphate buffer is given a baby a bath on the third day after its birth inferior, adds 0.1% gelatin by 200 μ L/ holes and seals;
(3) formation of Ag-Ab binary electrochemiluminescent immunoassay complex
Add glucocorticoid standard solution or testing sample 50 μ L in the ELISA Plate hole after sealing, the quantum dot-labeled glucocorticoid antibody 50 μ L of dilution, hatch 1h for 37 ℃, antigen and glucocorticoid medicine competition antibody, partial antibody and antigen form Ag-Ab binary immune complex, give a baby a bath on the third day after its birth time with containing NaCl 8.5g/L, pH7.2-7.4,10mmol/L phosphate buffer, remove non-specific adsorption;
(4) quantitative fluorescence detects
Excite and detect the fluorescence intensity of formed Ag-Ab electrochemiluminescent immunoassay compound, excitation wavelength: 430nm with fluorescence microplate reader; Emission wavelength: 545nm, 590nm, 650nm is by obtaining glucocorticosteroid residual dexamethasone in the auspicious product, betamethasone, the concentration of diflucortolone with the standard solution contrast.
Said antibody is meant with three kinds of quantum dots of sex change BSA parcel (545nm, 590nm, 650nm) the anti-dexamethasone of difference mark, anti-betamethasone, the polyclonal antibody of anti-diflucortolone; Quantum dot be meant can emitting fluorescence nano particle, its core is CdTe.
Said bag is to utilize conventional method with dexamethasone, betamethasone, and the coating antigen of diflucortolone (dexamethasone, betamethasone, diflucortolone-OVA conjugate) directly is fixed in the ELISA Plate micropore.
The formation of said antigen-antibody complexes is: add dexamethasone, and betamethasone, diflucortolone, three kinds of antigens are competed corresponding antibody with corresponding medicine, combine with the specificity of antibody by antigen and form Ag-Ab binary immune complex.
The detection of said fluorescence intensity is the fluorescence intensity that excites and detect formed antigen-two layers of sandwich electrochemiluminescent immunoassay compound of detection antibody with fluorescence microplate reader, usually sample Chinese traditional medicine concentration is big more, many more by the medication amount of antibody capture, the antibody that combines with coating antigen is few more, and the fluorescence intensity level that then records is more little.
Above-mentioned medicine to be measured is a dexamethasone, betamethasone, and diflucortolone, corresponding antibody should be the anti-dexamethasone of specificity, anti-betamethasone, the polyclonal antibody of anti-diflucortolone.
Beneficial effect of the present invention:
(1) this law is simple to operate, need not add the content that substance that show color just can detect glucocorticoid to be measured, promptly pass through the fluorescence intensity of Ag-Ab immune complex, the concentration value of glucocorticoid to be measured in the indirect detection sample, no matter operation is still reacted was all lacked for two steps than background technology, only needed can finish once going on foot.
(2) this law quantum dot of being used for labelled antibody is compared with conventional fluorescent, and emitted fluorescence intensity is stronger, and fluorescence is long stabilization time.
Description of drawings
Fig. 1 detects the typical curve of glucocorticoid with quantum dot-labeled indirect competition fluorescent immune method.
Embodiment
(1) with three kinds of quantum dot QD545 of denatured bovine serum protein (dBSA) parcel, QD590, the anti-dexamethasone of QD650 mark, anti-betamethasone, anti-diflucortolone polyclonal antibody
(1) BSA sex change:
16.5mg BSA is dissolved in the 10mL distilled water, stirs adding 0.42mg NaBH down
4, react 1h under the room temperature, 60-80 ℃ of NaBH that heats the 20min decomposing excessive down
4, the BSA sex change, disulfide bond is opened into-SH, and the concentration of final dBSA aqueous solution is 5 * 10
-5M.
(2) sex change BSA parcel quantum dot (dBSA-QDs):
With dBSA and quantum dot dysprosium chromium (CdTe) (1: 1) mixing in molar ratio, 60-80 ℃ of water-bath heating 15min kept two days under the room temperature, made parcel fully.
(3) sex change BSA parcel quantum point coupling antibody
5 μ L 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC (0.056M) with after 5 μ L sulfo-N-hydroxy-succinamide sulfo-NHS (0.1M) mix 10s, are added to 25 μ L dBSA-QDs (2 * 10
-5M) in, the quantum dot of sex change BSA parcel activation 15min, add 12 μ L glucocorticoid antibody (Ab, 4.0mg/mL).Reactant molar ratio is: dBSA-QDs: Ab: EDC: sulfo-NHS=1: 1: 560: 1000.React 4h under the room temperature, obtain quantum dot-labeled glucocorticoid antibody.The concentration of antibody is 1.0mg/mL in the reactant liquor, and is stand-by with 1000 times of antibody diluent (the phosphate buffer pH 7.4, the 0.01M that contain tween of 0.1% gelatin) dilutions.
(2) the bag quilt of antigen
Use dexamethasone, betamethasone, diflucortolone and the coupled body of ovoserum albumin are as coating antigen, (pH9.6,0.05M) is cushioned liquid as bag with carbonate buffer solution, is cushioned liquid with bag the glucocorticoid coating antigen is diluted to 1-20 μ g/mL as coating buffer, adds 100 μ L coating buffers in every hole of ELISA Plate, 4 ℃ of refrigerator overnight, give a baby a bath on the third day after its birth time with phosphate buffer (PBS, 10mmol/L, pH 7.2-7.4, contain NaCl 8.5g/L), add 0.1% gelatin by 200 μ L/ holes and seal.
(3) formation of Ag-Ab binary electrochemiluminescent immunoassay complex
Add dexamethasone in the ELISA Plate hole after sealing, betamethasone, diflucortolone standard solution or testing sample 50 μ L, the quantum dot-labeled glucocorticoid antibody 50 μ L of dilution, hatch 1h for 37 ℃, antigen and glucocorticoid medicine competition antibody, partial antibody and antigen form Ag-Ab binary immune complex, with phosphate buffer (PBS, 10mmol/L, pH 7.2-7.4 contains NaCl 8.5g/L) give a baby a bath on the third day after its birth time, remove non-specific adsorption.
(4) quantitative fluorescence detects
The measuring principle of quantum dot-labeled indirect competition immunofluorescence assay is to realize detection to glucocorticoid by the fluorescence intensity that detection is attached to the Ag-Ab binary immune complex on the ELISA Plate micropore.Can obtain the concentration of glucocorticoid in the sample with the typical curve contrast.Be attached to the quantum dot-labeled detection antibody quantity difference on the ELISA Plate micropore, the fluorescence intensity difference that produces, sample Chinese traditional medicine concentration is big more usually, and is many more by the medication amount of antibody capture, the antibody that combines with coating antigen is few more, and the fluorescence intensity level that then records is more little.The drafting of typical curve, with dexamethasone, betamethasone, the diflucortolone standard solution is configured to 8 kinds of variable concentrations (0.1ng/mL, 0.5ng/mL, 1ng/mL from low to high respectively, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL), under concentration, equivalent is mixed respectively for three kinds of medicines, uses the method identical with sample to be tested, repeats the operation of (two) (three) step, measure the fluorescence intensity of the standard solution correspondence of certain concentration, excitation wavelength: 430nm; Emission wavelength: 545nm, 590nm, 650nm is a horizontal ordinate with glucocorticoid concentration, and fluorescence intensity is an ordinate, and the drawing standard curve is seen Fig. 1.
Embodiment 2 adds the recovery experiment:
(1) extraction and cleaning of sample: waste the mixed solution that adds 10mL acetonitrile/water (7: 3) in the sample at 2g chicken, vortex mixed 1min, ultrasonic echography 30min, the centrifugal 10min of 2000 * g, draw 2.5mL upper strata liquid in clean glass tube, add 4mL normal hexane and the degreasing of 1mL methylene chloride, vortex mixed 1min makes three phase separation, draws the middle phase of 1mL (corresponding to the 0.2g sample) in clean test tube; 50 ℃, N slowly
2Stream dries up, and with 0.2mL standard items dilution (the phosphate buffer PBST that contains Tween-20 that contains 10% methyl alcohol) dissolution residual substance, supplies to analyze as sample.
(2) add standard items (dexamethasone, three kinds of potpourris of betamethasone and diflucortolone) liquid in the chicken sample of 2g blank, making its concentration is 10ng/g, and each concentration prepares five parts in sample respectively, extracts post analysis by above-mentioned pre-treating method.The results are shown in Table 1.As can be seen, it is good in 70.5～74.5 these analytical approach repeatability to detect the recovery of three kinds of medicines in chicken meat sample separately, and relative standard deviation is lower than 4.37.Detect the recovery of three kinds of medicines in chicken meat sample simultaneously 60.12%～67.98%.This analytical approach repeatability is good, and relative standard deviation is lower than 5.57%.
Table 1 adds the mensuration of the recovery
Claims (1)
1. the method for a quantum dot-labeled indirect competition fluoroimmunoassay detection for glucocorticosteroid residual, it is characterized in that: with the coating antigen direct coated in the micropore of ELISA Plate, add glucocorticoid standard solution or testing sample and form Ag-Ab electrochemiluminescent immunoassay complex, excite and detect the fluorescence intensity of formed Ag-Ab electrochemiluminescent immunoassay compound with fluorescence microplate reader, by obtaining the concentration of glucocorticosteroid residual in the testing sample with the standard solution contrast;
(1) with three kinds of quantum dot QD545 of denatured bovine serum protein dBSA parcel, QD590, QD650 is the anti-dexamethasone of mark respectively, anti-betamethasone, anti-diflucortolone polyclonal antibody, these three kinds of polyclonal antibodies promptly are respectively one of following glucocorticoid antibody
(1) BSA sex change:
16.5mg BSA is dissolved in the 10mL distilled water, stirs adding 0.42mg NaBH down
4, react 1h under the room temperature, 60-80 ℃ of NaBH that heats the 20min decomposing excessive down
4, the BSA sex change, disulfide bond is opened into-SH, and the concentration of controlling final dBSA aqueous solution is 5 * 10
-5M;
(2) sex change BSA parcel quantum dot dBSA-QDs:
DBSA and quantum dot dysprosium chromium CdTe were mixed in 1: 1 in molar ratio, and 60-80 ℃ of water-bath heating 15min kept two days under the room temperature, made parcel fully;
(3) sex change BSA parcel quantum point coupling antibody
With behind 5 μ L 1-ethyls-(3-dimethylaminopropyl) carbodiimide EDC 0.056M and the 5 μ L sulfo-N-hydroxy-succinamide sulfo-NHS 0.1M mixing 10s, be added to 25 μ L dBSA-QDs 2 * 10
-5Among the M, the quantum dot activation 15min of sex change BSA parcel adds a kind of Ab, 4.0mg/mL in three kinds of glucocorticoid antibody of 12 μ L; Reactant molar ratio is controlled to be: dBSA-QDs: Ab: EDC: sulfo-NHS=1: 1: 560: 1000, react 4h under the room temperature, obtain quantum dot-labeled glucocorticoid antibody, the concentration of antibody is 1.0mg/mL in the reactant liquor, and it is stand-by to do 1000 times of antibody diluent dilutions with the pH that contains tween 7.4, the 0.01M phosphate buffer of 0.1% gelatin; Sex change BSA parcel quantum dot carries out coupling with three kinds of polyclonal antibodies respectively;
(2) the bag quilt of antigen
Polyclonal antibody with three kinds of parcel quantum dots carries out coupling with the ovoserum albumin respectively, three kinds of quality such as the white conjugate of polyclonal antibody-ovoserum that obtain are mixed as the coupled body of glucocorticoid as coating antigen, with pH 9.6,0.05M carbonate buffer solution is cushioned liquid as bag, be cushioned liquid with bag the glucocorticoid coating antigen is diluted to 1-20 μ g/mL as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of refrigerator overnight, with containing NaCl 8.5g/L, pH 7.2-7.4, the 10mmol/L phosphate buffer is given a baby a bath on the third day after its birth inferior, adds 0.1% gelatin by 200 μ L/ holes and seals;
(3) formation of Ag-Ab binary electrochemiluminescent immunoassay complex
Add glucocorticoid standard solution or testing sample 50 μ L in the ELISA Plate hole after sealing, the quantum dot-labeled glucocorticoid antibody 50 μ L of dilution, hatch 1h for 37 ℃, antigen and glucocorticoid medicine competition antibody, partial antibody and antigen form Ag-Ab binary immune complex, give a baby a bath on the third day after its birth time with containing NaCl 8.5g/L, pH7.2-7.4,10mmol/L phosphate buffer, remove non-specific adsorption;
(4) quantitative fluorescence detects
Excite and detect the fluorescence intensity of formed Ag-Ab electrochemiluminescent immunoassay compound, excitation wavelength: 430nm with fluorescence microplate reader; Emission wavelength: 545nm, 590nm, 650nm is by obtaining glucocorticosteroid residual dexamethasone in the auspicious product, betamethasone, the concentration of diflucortolone with the standard solution contrast.
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Cited By (6)
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| CN101762706B (en) * | 2010-01-05 | 2013-11-06 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
| CN105044367A (en) * | 2015-08-03 | 2015-11-11 | 武汉上成生物科技有限公司 | Colloidal gold immunochromatographic assay test strip for detecting dexamethasone residues in milk |
| CN106814065A (en) * | 2017-01-18 | 2017-06-09 | 山东省食品药品检验研究院 | A kind of hydrocortisone method for quick |
| CN109324183A (en) * | 2018-11-23 | 2019-02-12 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | A kind of glucocorticoid immunochromatography wide spectrum detection card and the preparation method and application thereof |
| CN112394166A (en) * | 2020-11-09 | 2021-02-23 | 广东希格生物科技有限公司 | Application of hexafluorophosphate in preparation of preparation for inhibiting ELISA reaction matrix effect |
| CN113341134A (en) * | 2021-06-01 | 2021-09-03 | 河南中泽生物工程有限公司 | Method for detecting tiamulin by quantum dot fluorescence immunoassay |
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| CN1515909A (en) * | 2003-08-27 | 2004-07-28 | 魏景艳 | Quantum point marker sandwich immunodetection method and its diagnosis kit |
| CN1811449A (en) * | 2006-02-23 | 2006-08-02 | 上海交通大学 | Method for detecting quantum dot mark fast immune chromatographic test paper bar |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101762706B (en) * | 2010-01-05 | 2013-11-06 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
| CN105044367A (en) * | 2015-08-03 | 2015-11-11 | 武汉上成生物科技有限公司 | Colloidal gold immunochromatographic assay test strip for detecting dexamethasone residues in milk |
| CN106814065A (en) * | 2017-01-18 | 2017-06-09 | 山东省食品药品检验研究院 | A kind of hydrocortisone method for quick |
| CN109324183A (en) * | 2018-11-23 | 2019-02-12 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | A kind of glucocorticoid immunochromatography wide spectrum detection card and the preparation method and application thereof |
| CN109324183B (en) * | 2018-11-23 | 2022-03-29 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Glucocorticoid immunochromatography broad-spectrum detection card and preparation method and application thereof |
| CN112394166A (en) * | 2020-11-09 | 2021-02-23 | 广东希格生物科技有限公司 | Application of hexafluorophosphate in preparation of preparation for inhibiting ELISA reaction matrix effect |
| CN113341134A (en) * | 2021-06-01 | 2021-09-03 | 河南中泽生物工程有限公司 | Method for detecting tiamulin by quantum dot fluorescence immunoassay |
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