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Enzyme-linked immunosorbent assay kit for detecting fluoroquinolones and its application

An enzyme-linked immunosorbent reagent and a technology of fluoroquinolones, which are applied in the field of enzyme-linked immunosorbent immunoassay kits for detecting fluoroquinolones, can solve the problems of high capital and personnel investment costs, achieve simple pretreatment process, convenient and easy inspection methods, high precision effect

Active Publication Date: 2018-01-30
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have the advantages of high sensitivity and accurate results, but the cost of capital and personnel is high

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting fluoroquinolones and its application
  • Enzyme-linked immunosorbent assay kit for detecting fluoroquinolones and its application
  • Enzyme-linked immunosorbent assay kit for detecting fluoroquinolones and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 kit components

[0029] 1. Preparation of fluoroquinolone haptens

[0030] Dissolve 1.0 g of norfloxacin in 20 ml of pyridine, add 0.29 g of carboxymethylhydroxylamine, react at 50°C for 12 hours, evaporate the solvent to dryness under reduced pressure, and recrystallize from ethanol-water to obtain a white powder product with a yield of 58%.

[0031] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the signal peak of the carboxyl group was enhanced, and the signal peak of the methylene group increased at about 4.7ppm, indicating that the hapten was successfully synthesized.

[0032] 2. Antigen preparation

[0033] Preparation of immunogens—Fluoroquinolone haptens were conjugated with bovine serum albumin (BSA) to obtain immunogens.

[0034] Take 10mg of hapten, dissolve in 1mL DMF, take 30mg of EDC and NHS, fully dissolve with 0.2ml of water, add to the above DMF, ...

Embodiment 2

[0046] Embodiment 2 detects the formation of the enzyme-linked immunosorbent assay kit of fluoroquinolones

[0047] An enzyme-linked immunosorbent assay kit for detecting fluoroquinolones was constructed to include the following components:

[0048] (1) ELISA plates coated with fluoroquinolone-conjugated antigens;

[0049] (2) 6 bottles of standard solution of fluoroquinolones, the concentrations are 0μg / L, 0.1μg / L, 0.4μg / L, 1.6μg / L, 6.4μg / L, 25.6μg / L;

[0050] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0051] (4) Antibodies specific to fluoroquinolones;

[0052] (5) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0053] (6) The stop solution is 2mol / L sulfuric acid;

[0054] (7) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, the percentage ...

Embodiment 3

[0057] The detection of fluoroquinolones in the sample of embodiment 3 honey, chicken, pork, fish, shrimp, milk, milk powder and eggs

[0058] 1. Sample pretreatment

[0059] (1) Animal tissue samples

[0060] Homogenize the tissue sample with a homogenizer; weigh 2.0±0.05g of the homogenized tissue sample into a 50ml polystyrene centrifuge tube; add 8ml of the sample extract, oscillate with an oscillator for 5min, mix well, 3000g at room temperature (20- Centrifuge at 25°C / 68-77°F) for 10min; pipette 2ml of the upper organic phase into a 10ml clean and dry glass test tube, and dry it in a water bath at 50-60°C (122-140°F) under nitrogen flow; add 1ml of n-hexane and vortex Vortex with vortex for 2 minutes, then add 1ml reconstituted working solution, vortex for 30s with vortex, mix well, centrifuge at 3000g room temperature (20-25℃ / 68-77℉) for 5min; remove the upper organic phase, and take 50ml of the lower aqueous phase for analysis.

[0061] (2) Honey tissue samples

[...

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Abstract

The invention provides an enzyme-linked immunoassay kit for detecting a fluoroquinolone medicine. The enzyme-linked immunoassay kit comprises an enzyme-labelled plate coated with a coating antigen, a standard solution of the fluoroquinolone medicine, an enzyme-labelled bivalent antibody, a specific antibody of the fluoroquinolone medicine, a developing solution of a substrate, a stop solution, a washing solution, a combination solution and a diluent of a honey sample; the coating antigen is a coupled antigen of the fluoroquinolone medicine; and the enzyme-labelled bivalent antibody is an enzyme-labelled anti-goat and anti-rat antibody. The invention further discloses a method for detecting the fluoroquinolone medicine by using the enzyme-linked immunoassay kit. The method comprises the following steps: pre-processing a sample at first, then, detecting by using the kit, and finally, analyzing a detection result. The enzyme-linked immunoassay kit provided by the invention can be used for detecting the content of the fluoroquinolone medicine in honey, chicken, pork, fish, shrimp, milk, milk powder and egg samples, is simple and convenient to operate, low in cost and high in sensitivity and can be used for monitoring on site and applied to screening of a lot of samples.

Description

technical field [0001] The invention relates to enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoimmunoassay kit for detecting fluoroquinolones, which can qualitatively and quantitatively detect fluorine in honey, chicken, pork, fish, shrimp, milk, milk powder and egg samples Residues of quinolone drugs. Background technique [0002] Fluoroquinolones have become clinically commonly used antibacterial drugs because of their broad antibacterial spectrum and strong antibacterial activity. However, due to the characteristics of the drug itself and the existence of irrational drug use and drug abuse, it has caused a series of adverse reactions. Common skin allergies and photosensitivity reactions, central nervous system reactions, circulatory system reactions, digestive system reactions, urinary system reactions, respiratory system reactions, cartilage toxicity, liver toxicity, reproductive toxicity, Achilles tendinitis and local irritation ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/544
CPCG01N33/53G01N33/544
Inventor 冯才伟徐念琴冯静崔海峰冯才茂贾芳芳杨昌松杨烁
Owner BEIJING KWINBON BIOTECH
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