Method for quantum dot mark indirect competition fluoroimmunoassay detection for diflunisal

A technology of diflumetasone and fluorescence immunoassay, which is applied in the field of immunoassay, can solve the problems of accumulation toxicity, cumbersome operation, and time-consuming, and achieve the effects of long fluorescence stability time, strong fluorescence intensity, and simple operation

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

It has anti-allergic and anti-inflammatory effects, and it has cumulative toxicity. Many countries have included this drug in the list of banned drugs, especially beef products and milk.
The commonly used detection methods for diflumetasone residues in the world today are: thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS), etc., but these instrumental analysis methods not only require expensive instruments and equipment, but also have relatively high requirements for inspection materials, and require complex sample pretreatment before they can be carried out.
Research on the analysis methods of glucocorticoids has already been carried out abroad. The commonly used method is enzyme-linked immunoassay (ELISA) or immunoenzyme quantitative analysis (IEMA). Plate, add drug and anti-drug antibody, then add enzyme-labeled secondary antibody, that is, detection antibody, and finally add substrate for color development, after a certain period of time, use a microplate reader to detect the absorbance value of a specific wavelength, according to the known standard content Calculating the concentration of the drug to be tested in the sample is cumbersome and time-consuming

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  • Method for quantum dot mark indirect competition fluoroimmunoassay detection for diflunisal
  • Method for quantum dot mark indirect competition fluoroimmunoassay detection for diflunisal

Examples

Experimental program
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Embodiment 1

[0028] (1) Green-emitting QD650-labeled anti-diflumetasone polyclonal antibody coated with denatured BSA (dBSA)

[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 dots in a certain molar ratio (1:1-1:6), 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] After mixing 5 μL EDC (0.056M) and 5 μL sulfo-NHS (0.1M) for 10 s, add to 25 μL dBSA-QDs (2×10 -5 In M), the denatured BSA-wrapped quantum dots were activated for 15 min, and 9.6 μL of antibody (Ab, 5×10 -5 M). In th...

Embodiment 2

[0041] Embodiment 2 adds recovery experiment:

[0042] (1) Extraction and purification of samples: Add 10 mL of acetonitrile / water (volume ratio 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, and absorb 2.5 mL of the supernatant In a clean glass tube, add 4 mL of n-hexane and 1 mL of dichloromethane to degrease, vortex and mix for 1 min to separate the three phases, draw 1 mL of the intermediate phase (corresponding to 0.2 g 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 diflumethasone standard solution to 2g of blank chicken sample to make its concentration 1ng / g, 5ng / g, 10ng / g, 20ng / g, prepare five samples for each concentration respectively, according to the above Processing methods f...

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Abstract

Disclosed is a method of quantum dot-labeled indirect competitive fluorescence immunoassay of diflucortolone, which belongs to the immunoassay method technique field. Quantum dots for labeling antibodies of the invention have the emission spectra of QD650, and the method comprises: directly covering coating antigens in micro-holes of an enzyme label plate, adding diflucortolone 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 diflucortolone in the sample under test through comparing with the standard solution. The invention can detect the content of diflucortolone in the sample under test without adding chromogenic substance, namely, the concentration of diflucortolone in the sample under test can be detected indirectly 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 quantitatively detecting diflumetasone with a quantum dot-labeled diflumetasone antibody, and in particular discloses a quantum dot-labeled indirect competitive immunoassay method, which belongs to the technical field of immunoassay methods. Background technique [0002] Flumethasone (Flumethasone, FLM) is a synthetic glucocorticoid, chemical name is 6α, 9α-diflupregna-16α-methyl-11β, 17α, 21-trihydroxy-1,4-diene-3 , 20 diketones. It has anti-allergic and anti-inflammatory effects, and it has cumulative toxicity. Many countries have included this drug in the list of banned drugs, especially beef products and milk. The commonly used detection methods for diflumetasone residues in the world today are: thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS), etc., but these instrumental analysis methods not only require exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/543G01N21/64
Inventor 胥传来袁媛陈伟彭池方许定花边爱马伟
Owner JIANGNAN UNIV
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