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Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application

An enzyme-linked immunological reagent and quinolone technology, applied in the field of immunological detection, can solve problems such as inability to achieve

Active Publication Date: 2013-09-25
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The current detection method is mainly instrumental analysis method, and the current enzyme-linked immunoassay method can only detect one or several drugs, and it is far from meeting the requirements of simultaneous detection of multiple residues for many types of quinolones

Method used

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  • Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application
  • Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application
  • Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application

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Embodiment 1

[0024] Embodiment 1: the preparation of the solution of the present invention

[0025] The quinolones drug standard solution, enzyme-labeled goat anti-mouse antibody solution, quinolones drug antibody solution, substrate chromogenic solution and washing solution formula involved in the kit of the present invention have a great influence on the sensitivity of the kit detection of the present invention; wherein each solution The main ingredients and their preparation methods are:

[0026] 1. Standard solution of quinolones: the pure quinolones were formulated with 0.05mmol / L PBS containing 10% methanol and pH=7.4 in a conventional method to prepare concentrations of 0ng / mL, 0.2ng / mL, and 0.6ng / mL respectively. mL, 1.8ng / mL, 5.4ng / mL and 16.2ng / mL quinolone standard solutions, the percentages are volume percentages.

[0027] 2. Enzyme-labeled goat anti-mouse antibody solution: Enzyme-labeled goat anti-mouse antibody is horseradish peroxidase-goat anti-mouse IgG stock solution, w...

Embodiment 2

[0035] Embodiment 2: the coating of microtiter plate of the present invention

[0036] In the present invention, the coated ELISA plate is coated by placing the QNS-OVA conjugate in a set coating solution at a set concentration and a set time in a thermostat at 37°C.

[0037] The coating liquid used in the present invention is a sodium carbonate-sodium bicarbonate buffer solution with pH=9.6. In the present invention, the QNS-OVA coated in the microporous plate can be well combined on the plastic surface of the microporous plate in an alkaline environment, and can withstand repeated washing of the plate, and the concentration of the coated antigen used is 5.0 μg / mL .

[0038] The coated microplate can be blocked with a blocking solution. The inert protein in the blocking solution is preferably OVA, and NaN needs to be added 3 Prevent spoilage.

[0039] The enzyme-linked immunoassay kit of the present invention has the characteristics of high sensitivity, simplicity and spee...

Embodiment 3

[0040] Example 3: Preparation of derivatives, immunogens, coating agents and monoclonal antibodies

[0041] (1) Synthesis of norfloxacin derivatives

[0042] A. Dissolve 1 mmol of norfloxacin in 55 ml of chloroform, add 2 mmol of DCC, appropriate amount of DMAP catalyst, 1.5 mmol of ethyl p-aminophenylacetate, stir at room temperature for 5 h, monitor the disappearance of raw materials by TLC, filter, and wash the liquid phase with water. Anhydrous NaS 2 o 4 Dry and purify by column chromatography (eluent, ethyl acetate / petroleum ether, 1 / 5).

[0043]B. Dissolve the above product in methanol, add 0.76g of NaOH, stir at room temperature at 60°C for 5h, monitor the disappearance of the raw material by TLC, remove the solvent under reduced pressure, dissolve the obtained viscous product in 1mol / L NaOH solution, adjust the pH to 3-5, Extract with ethyl acetate, dry, and purify by column chromatography (eluent, ethyl acetate / petroleum ether, 1 / 1) to obtain quinolone haptens.

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Abstract

The invention discloses an enzyme-linked immunoassay kit for detecting quinolone drugs. The enzyme-linked immunoassay kit for detecting quinolone drugs provided in the invention includes an enzyme-labelled plate coated with a quinolone drug-carrier protein conjugate, an antibody working solution, an enzyme labeled secondary antibody, quinolone series standard substances, a concentrated complex solution, a concentrated washing solution, a substrate color-developing solution, and a stopping solution. The enzyme-linked immunoassay kit for detecting quinolone drugs is a multi-residue detection kit, and can simultaneously determine the total residue of 12 quinolone drugs in aquatic product (fish, shrimp) samples. The invention also discloses a method for detecting quinolone drugs by the enzyme-linked immunoassay kit. The method comprises: first conducting sample pretreatment, then carrying out detection with the kit, and finally analyzing the detection result. The kit disclosed in the invention has the advantages of simple operation, low cost, high sensitivity, and a total operation time of only 45 minutes, can perform on-site monitoring and is suitable for screening of a large number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit for detecting quinolone drugs, which is used for detecting the content or residual amount of quinolone drugs in aquatic products (fish, shrimp, etc.). It belongs to the field of immunological detection. technical background [0002] Quinolone antibacterial drugs (4-quinolones), also known as pyroxic acids or pyridone acids, are a relatively new class of synthetic antibacterial drugs. The development of this class of drugs can be traced back to 1962. Lesher synthesized the antimalarial drug chloroquine A by-product-nalidixic acid was isolated from quinolones. After more than 40 years of development, quinolones have developed four generations of quinolones, totaling more than 50 drugs. [0003] Quinolones antibacterial drugs target bacterial deoxyribonucleic acid (DNA), and cause irreversible damage to chromosomes by selectively inhibiting bacterial DNA gyrase and topoisomerase IV, so that bacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 万宇平陶光灿吴鹏蒲小容顾蓉蓉杨昌松韩京朋王礼贵
Owner BEIJING KWINBON BIOTECH
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