Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit

A chemiluminescence reagent and furazolidone technology, which is applied to a magnetic particle chemiluminescence kit for detecting furazolidone metabolites and its application field, can solve the problems of poor reagent stability, the influence of reaction time and temperature, and achieves low detection time and fast detection. , the effect of high sensitivity

Inactive Publication Date: 2012-07-04
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction time and temperature have a great influence on the enzyme activity, and the stability of the reagent is poor

Method used

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  • Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of specific components of the kit

[0037] 1. Preparation of luminescent markers

[0038] a) Synthesis of hapten

[0039] The furazolidone metabolite is derivatized to obtain the furazolidone derivative hapten.

[0040] b) Preparation of luminescent markers

[0041] Take 4.5mmol / L ABEI, dissolve it in 4ml distilled water, dissolve 5.0mmol / L N-hydroxysuccinimide in 0.5ml N,N-dimethylformamide, mix the two well and react at room temperature for 3-4h . Take 15 mg of the furazolidone metabolite hapten prepared above, adjust the volume to 1.5 ml with pH 7.4 PBS, then add the above activated ABEI solution, mix well, react overnight at room temperature, and purify it on a G-25 gel column.

[0042] 2. Preparation of fluorescent markers

[0043] a) Preparation of immunogen:

[0044] The furazolidone metabolite derivative hapten and bovine serum albumin are coupled by a diazotization method to obtain an immunogen.

[0045] b) Preparation of monoclonal antibodies to fura...

Embodiment 2

[0057] Example 2 Construction of the kit

[0058] A magnetic particle chemiluminescence detection kit for the detection of furazolidone metabolites was constructed to contain the following components:

[0059] FITC-labeled fluorescent label of furazolidone metabolite monoclonal antibody

[0060] Luminescent label of ABEI-labeled furazolidone metabolite hapten

[0061] Separation reagent of paramagnetic nanobeads coated with goat anti-FITC monoclonal antibody

[0062] Furazolidone metabolite standard solution (0ng / ml, 0.01ng / ml, 0.03ng / ml, 0.09ng / ml, 0.27ng / ml, 0.81ng / ml), the standard dilution is pH7.4, containing 0.05% thimerosal Preservative, 0.1mol / LTRIS buffer. The percentage is the percentage by mass.

[0063] The concentration of the furazolidone metabolite control solution was 0.02ng / ml and 0.5ng / ml respectively, the quality control substance was diluted to pH7.4, containing 0.05% thimerosal preservative, 0.1mol / L TRIS buffer. The percentage is the percentage by mass.

[0064] T...

Embodiment 3

[0065] Example 3 Detection of furazolidone metabolites in actual samples

[0066] 1. Sample pretreatment

[0067] (1) Pre-processing methods for muscle and liver tissue

[0068] Homogenize the sample with a homogenizer; weigh 1.0±0.05g of the homogenized tissue sample (liver sample / meat sample), add 4ml of deionized water, 0.5ml of 1M hydrochloric acid solution and 100μl of derivatization reagent, fully use a shaker Shake for 2min; incubate at 37℃ overnight (about 16h); add 5ml 0.1M dipotassium hydrogen phosphate solution, 0.4ml 1M sodium hydroxide solution and 5ml ethyl acetate respectively, shake vigorously with a shaker for 30s; 3000g above, room temperature (20- 25℃) Centrifuge for 10min; take 2.5ml ethyl acetate phase into 10ml dry glass test tube and blow dry under nitrogen stream in a water bath at 50~60℃; add 1ml n-hexane (or n-heptane), vortex for 30s with a vortexer, Then add 1ml of reconstituted working solution and mix thoroughly with a vortexer for 1min; centrifuge for...

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Abstract

The invention relates to a magnetic particle chemiluminescence kit for detecting frazolidone metabolites. The kit comprises reagents of a luminance marker, a fluorescein marker, a standard substance, a quality control substance and a separation reagent, wherein the luminance marker is a frazolidone metabolite hapten marked by an isoluminol luminance marker; the fluorescein marker is a frazolidone metabolite monoclonal antibody marked by fluorescein or a derivative of the fluorescein; and the separation reagent is a paramagnetic nano micro-bead coated with an anti-goat fluorescein isothiocyanate monoclonal antibody. The invention also relates to a method for detecting the frazolidone metabolites in an animal derived food by adopting the kit. The method is relatively high in sensitivity and specificity to detecting the frazolidone metabolites and relatively high in detection speed.

Description

Technical field [0001] The invention relates to a chemiluminescence detection kit and a test method thereof, in particular to a magnetic particle competition direct chemiluminescence detection kit for detecting furazolidone metabolite residues in samples such as honey, eggs, aquatic products, tissues and the like. technical background [0002] Furazolidone (Frazolidone) is a synthetic broad-spectrum antibacterial drug with stable efficacy. It can inhibit or kill a variety of Gram-positive and negative bacteria, and has a certain effect on certain protozoa and fungi. Due to its high efficiency and low cost, furazolidone is effective It has been widely used in animal husbandry and aquaculture. [0003] However, furazolidone residues in animal-derived foods can be passed to humans through the food chain, and long-term intake can cause various diseases. According to the report of the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) Joint Ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/577G01N33/533
Inventor 何方洋万宇平冯才伟汪善良张丽杰郭旭余厚美徐恩宁
Owner BEIJING KWINBON BIOTECH
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