Method for quantum dot mark indirect competition fluoroimmunoassay detection for glucocorticosteroid residual

A technology of glucocorticoid and fluorescence immunity, which is applied in the field of immunoassay, can solve the problems of cumbersome operation and time-consuming, and achieve the effect of simple operation, strong fluorescence intensity and long fluorescence stability time

Inactive Publication Date: 2008-11-19
JIANGNAN UNIV
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  • Abstract
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Problems solved by technology

This type of method is to coat the enzyme-labeled plate with the original coating, add drugs and anti-drug antibodies, then add the enzyme-labeled secondary antibody, that is, the detection antibody, and finally add the substrate for color development. After a certai

Method used

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  • Method for quantum dot mark indirect competition fluoroimmunoassay detection for glucocorticosteroid residual
  • Method for quantum dot mark indirect competition fluoroimmunoassay detection for glucocorticosteroid residual

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Embodiment 1

[0028] (1) Coating three kinds of quantum dots QD545, QD590, QD650 with denatured bovine serum albumin (dBSA) labeled anti-dexamethasone, anti-betamethasone, anti-diflumetasone polyclonal antibodies

[0029] (1) BSA denaturation:

[0030] Dissolve 16.5mg BSA in 10mL double distilled water, add 0.42mg NaBH under stirring 4 , react at room temperature for 1h, heat at 60-80°C for 20min to decompose excess NaBH 4 , BSA is denatured, the disulfide bond is opened into -SH, and the concentration of the final dBSA aqueous solution is 5×10 -5 M.

[0031] (2) Denatured BSA-wrapped quantum dots (dBSA-QDs):

[0032] Mix dBSA and quantum dot chromium dysprosium (CdTe) in a molar ratio (1:1), heat in a water bath at 60-80°C for 15 minutes, and keep at room temperature for two days to completely wrap.

[0033] (3) Denatured BSA-coated quantum dot-coupled antibody

[0034] Mix 5 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC (0.056M) with 5 μL of sulfo-N-hydroxysuccinimide sulfo-...

Embodiment 2

[0041] Embodiment 2 adds recovery experiment:

[0042] (1) Extraction and purification of samples: add 10 mL of acetonitrile / water (7:3) mixed solution to 2 g of chicken meat samples, vortex for 1 min, ultrasonically sonicate for 30 min, centrifuge at 2000×g for 10 min, absorb 2.5 mL of the supernatant in a clean Add 4mL of n-hexane and 1mL of dichloromethane to degrease in a glass tube, vortex and mix for 1min to separate the three phases, draw 1mL of the intermediate phase (corresponding to 0.2g sample) into a clean test tube; at 50°C, slowly N 2 Blow dry, and dissolve the residue with 0.2mL standard diluent (phosphate buffered saline PBST containing Tween-20 containing 10% methanol), and use it as a sample for analysis.

[0043] (2) Add standard substance (dexamethasone, three kinds of mixtures of betamethasone and diflumetasone) liquid in the chicken sample of 2g blank, make its concentration be 10ng / g, each concentration prepares five parts of samples respectively, accord...

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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 [0001] The invention relates to a method for indirect competitive fluorescent immunodetection of multiple residues of glucocorticoids labeled with quantum dots, which belongs to the technical field of immunodetection methods. Background technique [0002] Glucocorticoids have anti-allergic, anti-inflammatory and glucose metabolism effects, and are often used to treat inflammatory reactions, immune diseases, ketosis in cattle, and pregnancy toxemia in sheep. Dexamethasone, betamethasone, and diflumethasone are also often used as growth promoters to increase the feed intake of livestock to achieve weight gain. However, it has been proved by toxicological tests that such drugs are highly mutagenic and cumulatively toxic. If such drugs are randomly added to the feed, the accumulated drugs will enter the human body through the food chain, causing great harm. For this reason, many countries include such drugs in the list of prohibited drugs. The commonly used det...

Claims

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Application Information

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IPC IPC(8): G01N33/74G01N33/543G01N21/64
Inventor 胥传来袁媛陈伟彭池方李灼坤李哲徐丽广
Owner JIANGNAN UNIV
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