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Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay)

A olaquindox, direct technique, applied in the field of time-resolved fluorescence immunoassay, achieves the effect of high average recovery, small coefficient of variation and short detection time

Inactive Publication Date: 2015-04-29
ZHEJIANG GONGSHANG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, enzyme-linked immunosorbent assay (ELISA) is the most widely used and most maturely developed. There are many reports about ELISA detection, but there is no time-resolved fluorescent immunoassay (TRFIA) detection of olaquindox at home and abroad. Therefore, it is of great significance to develop a detection method for time-resolved fluorescent immunoassay (TRFIA) of olaquindox with independent intellectual property rights.

Method used

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  • Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay)
  • Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay)

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Test feed samples as follows:

[0028] (1) Preparation of coating agent (OLA-OVA) and immunogen (OLA-BSA):

[0029] A. First, accurately add 2.106g of olaquindox and 1.6g of succinic anhydride to a three-necked round-bottomed flask, then add 80mL of pyridine, reflux at 115°C for 4 hours, then evaporate pyridine under reduced pressure, and add 60mL of ice-distilled water to the remaining mixture, 2mol L -1 Adjust the pH to 2.0-3.0 with HCl, and place it overnight at 4°C. Suction filtration under reduced pressure and wash with ice distilled water for 3 times, then drained, the light yellow powdery substance is OLA-HS;

[0030] B. Next, weigh 14.528mg OLA-HS and dissolve it in 0.8mL DMF, add 4.603mg NHS and 8.253mg DCC, stir at room temperature for 10h in the dark, then centrifuge at 2000r / min for 10min, the supernatant after centrifugation is liquid A. Weigh 20mg OVA (or BSA) and dissolve in 5mL 0.01mol L -1 In phosphate buffered saline (PBS, pH7.4), this is solution B...

Embodiment 2

[0054] To test a pond water sample, proceed as follows:

[0055] (1) The preparatory work of detection is the same as (1)~(4) of embodiment 1

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Abstract

The invention discloses a method for detecting olaquindox by a directly-competing TRFIA (Time Resolved Fluorescence Immunoassay). The method comprises the following steps of: respectively adding standard olaquindox solution or treated sample solution and europium-labeled monoclonal antibodies into each micropore of an antigen precoating strip, mixing to be uniform, incubating for 1 hour at the temperature of 37 DEG C, and washing the micropores for 3-5 times by using washing liquid; adding 200muL of enhancement solution into each micropore, and carrying out light-proof oscillating incubation for 10 minutes at the temperature of 37 DEG C; determining the fluorescence intensity value cps by using a time resolution meter; drawing a standard curve; calculating out the corresponding concentration of the olaquindox from the standard curve according to the cpsx / cps0 value of each sample, multiplying by the corresponding dilution ratio and calculating out the actual concentration of the olaquindox in the sample. The method disclosed by the invention has the advantages that the operation is simple and convenient, the sensitivity is high, the stability is good and the lowest detection limit can reach 0.83ng.mL<-1>.

Description

technical field [0001] The invention relates to a detection method of olaquindox, which belongs to the technical field of time-resolved fluorescence immunoassay in biotechnology, and specifically relates to a detection method for directly competing with TRFIA of olaquindox drug residues in feed, water and animal-derived food. Background technique [0002] Olaquindox (OLA) is an antibacterial and growth-promoting agent, which was widely used in aquaculture and was once called "aquatic lean meat extract". The toxicity and side effects of olaquindox should not be underestimated, and there are obvious genotoxicity and cumulative toxicity. Therefore, strict usage regulations and residue limit standards have been formulated successively at home and abroad. For example, the United States and the European Union prohibit the use of olaquindox, and Japan stipulates that the maximum residue limit (MRL) of olaquindox in animal tissues and viscera is 300 μg kg -1 In 2001, the Ministry o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/542
CPCG01N21/6408G01N33/53G01N33/543
Inventor 金仁耀桑永玉
Owner ZHEJIANG GONGSHANG UNIVERSITY
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