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Quinolone drug chemiluminescence enzyme-linked immunodetection kit

A technology of chemiluminescent enzymes and quinolones, which is applied in the field of immunological detection, can solve problems such as inability to achieve, and achieve the effect of increased sensitivity and high sensitivity

Active Publication Date: 2013-04-03
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 the method of instrumental analysis, and the 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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  • Quinolone drug chemiluminescence enzyme-linked immunodetection kit
  • Quinolone drug chemiluminescence enzyme-linked immunodetection kit
  • Quinolone drug chemiluminescence enzyme-linked immunodetection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of derivatives, immunogens, coating agents and monoclonal antibodies

[0047] (1) Synthesis of norfloxacin derivatives

[0048] A. Dissolve 1 mmol of norfloxacin in 15 mL of chloroform, add 2 mmol of DCC, catalytic amount of DMAP, 1.5 mmol of tert-butyl allanine, stir at room temperature for 5 h, monitor the disappearance of raw materials by TLC, filter, wash the liquid phase with water, Dry with water NaS2O4, and purify by column chromatography (eluent, ethyl acetate / petroleum ether, 1 / 5).

[0049] B. Dissolve the above product in glacial acetic acid, stir at room temperature for 2 hours, monitor the disappearance of raw materials 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, and dry. Purify by column chromatography (eluent, ethyl acetate / petroleum ether, 1 / 1) to obtain quinolone haptens.

[0050] (2) Synthesis of immunogen

[...

Embodiment 2

[0060] Embodiment 2: the establishment of ELISA detection method

[0061] (1) Optimization of antibody and coated antigen concentration (square matrix)

[0062] Serially dilute each coated antigen longitudinally at 80.0 μg / mL, 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL Coat the ELISA plate with 100 μL / well, place it in a 37°C incubator for 2 hours, and then pat it dry; seal it with 150 μL / well blocking solution, place it in a 37°C incubator for 2 hours, wash the plate once, and pat it dry; add 50 μL / well A series of diluted antibodies (1:1000 to 1:512000), then add 50 μL / well of 1:2000 horseradish peroxidase-goat anti-mouse IgG antibody, incubate at room temperature (20-25°C) for 15 minutes, wash the plate 5 pat dry for the last time; add 100 μL / well of chemiluminescence solution, and measure the luminescence intensity value. Specificity determination was carried out with the coating antigen concentration and antibody dilution having ob...

Embodiment 3

[0072] Example 3: Chemiluminescence enzyme-linked immunosorbent assay kit for detecting quinolones

[0073] (1) The composition of the chemiluminescent ELISA kit for detecting quinolones

[0074] A, the solid phase carrier (elisa plate) that is coated with coating former (QNS-OVA);

[0075] B, Standard solutions of quinolones: 0ng / mL, 0.1ng / mL, 0.3ng / mL, 0.9ng / mL, 2.7ng / mL and 8.1ng / mL.

[0076] C. Enzyme-labeled goat anti-mouse antibody solution: Enzyme-labeled goat anti-mouse antibody is horseradish peroxidase-goat anti-mouse IgG stock solution, filled in, and prepared with washing solution to a working concentration of 1:2000 when used.

[0077] D, quinolone drug antibody solution: the monoclonal antibody prepared by immunizing animals with artificial immune antigen (QNS-BSA), and the obtained quinolone drug antibody was diluted with washing solution to a working concentration of 1:32000.

[0078] E, luminescent solution: A solution is tris(hydroxymethyl)aminomethane solu...

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Abstract

The present invention discloses a quinolone drug chemiluminescence enzyme-linked immunodetection kit, which comprises a kit body, an enzyme label plate placed inside the kit body, and reagents placed inside the kit body, and is characterized in that every hole of the enzyme label plate is coated with coating antigen, the coating antigen is a norfloxacin derivative and carrier protein conjugate, and the reagents comprise quinolone drug monoclonal antibody, horseradish peroxidase-labeled goat anti-mouse antibody, a series of quinolone drug standard solutions, a concentrated phosphate buffer, a concentrated washing solution and a chemiluminescence solution. The quinolone drug chemiluminescence enzyme-linked immunodetection kit has characteristics of high sensitivity, simple and rapid detection, high accuracy, and more drug detection types, provides a substantially reduced operation time compared to the conventional colorimetric ELISA method, and can be used for detection of residues of the 12 quinolone drugs in animal tissues (pork, chicken, pork liver and chicken liver), aquatic products (fish and shrimp) and honey.

Description

technical field [0001] The invention relates to a chemiluminescence immunoassay kit for detecting quinolones, which is used for detecting the content of quinolones in animal tissues (pork, chicken, pig liver, chicken liver), aquatic products (fish, shrimp, etc.), and honey or residues. It belongs to the field of immunological detection. Background technique [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 b...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/76
Inventor 万宇平罗贵昆杨秀贤韩锐扶胜冯月君文雪静孙倩倩
Owner BEIJING KWINBON BIOTECH
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