Fluorescence polarization immunoassay method for detection of erythromycin

A technology of fluorescence polarization and erythromycin, which is applied in the analysis of materials, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of complicated operation and long time consumption, and achieve the effect of simple operation

Inactive Publication Date: 2015-12-09
CHINA AGRI UNIV
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Problems solved by technology

[0005] Aiming at the disadvantages of complex operation and long time consumption in the existing erythromycin immunoassay technology, the present invention establishes a fluorescence polarization immunoassay method for detecting erythromycin. The whole detection process only needs one-step competition reaction, and the operation is simple and fast

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  • Fluorescence polarization immunoassay method for detection of erythromycin
  • Fluorescence polarization immunoassay method for detection of erythromycin
  • Fluorescence polarization immunoassay method for detection of erythromycin

Examples

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example 1

[0039] Example 1, preparation of fluorescent marker based on hapten erythromycinamine

[0040] Taking ERM-FITC as an example, the synthesis method is as follows:

[0041] Weigh 0.2mg of erythromycin standard substance and 1mg of FITC powder, add to 200μL of DMF and shake until dissolved; add 50μL of triethylamine, and react in the dark at room temperature for 7 days; take 50μL of the reaction solution and separate it with thin layer chromatography (TLC) to obtain pure FITC For comparison, the developer is dichloromethane / methanol (v:v, 1:1); scrape off the silica gel plate to scrape R f = 0.5 yellow band, eluting with methanol, ready for detection. After identification by mass spectrometry, the peak m / z of the ERM-FITC product was 1123.4.

[0042] The reaction steps of other fluorescent markers of erythromycylamine such as DTAF, tetramethylrhodamine sulfonyl chloride, and AF647 are similar to those of FITC, and the fluorescent markers are stored at 4°C.

example 2

[0043] Example 2, Preparation of fluorescent marker based on modified erythromycin hapten

[0044] Step 1: Modification of erythromycin hapten

[0045] will contain 44mg carboxymethylhydroxylamine NaHCO 3Adjust the pH to between 5-6 and add 2 mL of distilled water dropwise to 1 mL of absolute ethanol containing 100 mg of erythromycin raw material; put it in a constant temperature water bath at 50°C and stir for 5 hours; after the reaction, cool naturally to room temperature and add water to quench , adjust the pH of the solution to be acidic; extract twice with dichloromethane, combine the dichloromethane layers, add anhydrous Na 2 SO 4 Shake and stand overnight; take the dichloromethane layer, 40°C rotary evaporation to dryness under reduced pressure, dissolve with 5mL DMF, and store at -20°C.

[0046] Step 2: Synthesis of modified erythromycin fluorescent marker (ERY-CMO-4'-AMF)

[0047] Weigh 200 μL ERY-CMO DMF solution and mix with 200 μL DMF solution containing 0.4 mg...

example 3

[0048] Example 3. Screening of the best fluorescent marker and antibody combination

[0049] Step 1: Combining various synthetic fluorescent markers with three erythromycin monoclonal antibodies (5B2, 6C1 and 6D9), first set the working concentration of each fluorescent marker so that the fluorescence intensity is the background of the buffer solution The concentration of the fluorescent marker corresponding to 10 times the fluorescence intensity (15nM), each antibody with borate buffer solution according to 1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, 1 / 3200, Dilute 1 / 6400, 1 / 12800, 1 / 25600 and 1 / 51200, draw the antibody binding curve, and obtain the maximum change value of signal intensity δmP (δmP=mP max -mP min ), (ERM-FITC, ERM-DTAF and ERY-CMO-4'-AMF excitation wavelength 485nm, emission wavelength 530nm, cutoff value 515nm; ERM-AF647 excitation wavelength 644nm, emission wavelength 685nm, cutoff value 665nm) Among them, ERM- FITC had the greatest signal change value. The expe...

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Abstract

The invention provides a fluorescence polarization immunoassay method for detection of erythromycin. The method includes: subjecting an erythromycin standard substance solution with a known concentration to mixed incubation respectively with a fluorescent marker solution and an erythromycin monoclonal antibody solution to carry out competing reaction, thus obtaining a fluorescence polarization value of the system; taking the measured fluorescence polarization value as the ordinate, and adopting the concentration of a series of sulfamethazine standard substances with known concentration as the abscissa to draw a standard curve; using a to-be-measured sample to replace the erythromycin standard substance in step 1), according to the operation in step 1), determining the fluorescence polarization value of the system after incubation, and according to the standard curve, calculating the erythromycin concentration of the to-be-measured sample. The invention provides the homogeneous and rapid erythromycin fluorescence polarization immunodetection method, which only needs sample adding, has no need of separation and washing operation, and can obtain the detection result within 10min.

Description

technical field [0001] The invention belongs to the field of immune analysis technology and detection of veterinary drug residues, in particular to a fluorescence polarization immunoassay method for detecting erythromycin. Background technique [0002] Erythromycin (ERY) is the first macrolide drug that came out, mainly used to treat livestock and poultry diseases caused by Gram-positive bacteria and mycoplasma, and is also used as a feed additive to improve feed conversion rate , to promote growth. However, this kind of compound metabolizes slowly in the livestock and poultry body, easily remains in the animal body, and can produce side effects on the body, which poses a certain threat to people's health. Therefore, all countries in the world have made requirements for the residue limit of erythromycin in animal foods. Since 2006, the European Union has banned all antibiotics from being used as feed additives to promote growth. EC1181 / 2002 stipulates that the maximum resi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/577
Inventor 王战辉沈建忠张素霞张会艳温凯江海洋史为民
Owner CHINA AGRI UNIV
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