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Method for quantum dot marked indirect competition fluoroimmunoassay detection for dexamethason

A technology of dexamethasone 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: 2012-09-05
JIANGNAN UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

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 certain period of time, use a microplate reader to detect a certain Calculate the concentration of the drug to be tested in the sample based on the absorbance value of a specific wavelength based on the known content of the standard, which is cumbersome and time-consuming

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  • Method for quantum dot marked indirect competition fluoroimmunoassay detection for dexamethason
  • Method for quantum dot marked indirect competition fluoroimmunoassay detection for dexamethason
  • Method for quantum dot marked indirect competition fluoroimmunoassay detection for dexamethason

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Embodiment 1

[0028] (1) Green-emitting QD545-labeled anti-dexamethasone polyclonal antibody coated with denatured bovine serum albumin (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, 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 dysprosium chromium (CdTe) in a certain molar ratio (1:1, 1:2, 1:4, 1:6), heat in a water bath at 60-80°C for 15min, and keep it at room temperature for two days to make Package completely.

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

[0034] Mix 5 μL 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC (0.056M) with 5 μL sulfo-N-hydroxysuccinimide sulfo-NHS (0.1M) for 10 seconds, then add to 25 μL d...

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, 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 dexamethasone standard solution to 2g of blank chicken sample to make its concentration 1ng / g, 5ng / g, 10ng / g, 20ng / g, and prepare five samples for each concentration respectively, according to the above Processing methods for post-extraction analysis. The ...

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Abstract

Disclosed is a method of quantum dot-labeled indirect competitive fluorescence immunoassay of dexamethasone, which belongs to the immunoassay method technique field. Quantum dots for labeling antibodies of the invention have the emission spectra of QD545, and the method comprises: directly covering coating antigens in micro-holes of an enzyme label plate, adding dexamethasone standard solution ora 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 compoundbody with a fluorescence enzyme-labeling instrument, and obtaining the concentration of dexamethasone in the sample under test through comparing with the standard solution. The invention can detect the content of dexamethasone in the sample under test without adding chromogenic substance, namely, the concentration of dexamethasone 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 detecting dexamethasone by indirect competitive fluorescent immunoassay labeled with quantum dots, which belongs to the technical field of immunoassay methods. Background technique [0002] Dexamethasone is a synthetic glucocorticoid with a chemical name of 16α-methyl-11β, 17α, 21-trihydroxy-9α-fluoropregna-1,4-diene-3,20dione. It has the functions of anti-allergy, anti-inflammation and affecting sugar metabolism, and is often used to treat inflammatory reactions, immune diseases, ketosis of cattle and toxemia of sheep pregnancy in livestock. Dexamethasone is also often used as a growth promoter to increase the feed intake of livestock to achieve weight gain. However, it has been proved by toxicological tests that the drug has mutagenicity and cumulative toxicity, with an ADI value of 0.000015 mg / kgbw / day. If the drug is randomly added to the feed, the accumulated drug will enter the human body through the food c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/74G01N33/543G01N21/64
Inventor 胥传来袁媛陈伟彭池方李秋生尹丽梅
Owner JIANGNAN UNIV
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