ELISA kit for detecting quinolones in animal derived food

Abstract

The invention provides an agent box for detecting fluorine drugs of animal foodstuff, which uses enzyme immune method to detect the preprocessed animal organization, shrimp, fish and blood product. The enzyme immune agent box comprises: enzyme mark plate which coats fluorine drugs antigen, enzyme mark antibody, ciproloxacin standard solution, base material color developing solution, fluorine drugs antibody, compression cleaning liquid, ending solution and compression twin solution. The invention also discloses a method for applying the detecting method, which comprises: first doing sample front process, then using the agent box to detect, at last analyzing the detected result.

Description

technical field

[0001] The invention relates to an ELISA kit and a detection method for detecting fluoroquinones in animal-derived foods including animal tissues (muscle, liver), shrimp, fish and blood products. Background technique

[0002] Fluoroquinone antibacterial drugs (also known as quinolone antibacterial drugs, QNS), which have a broad antibacterial spectrum and strong antibacterial effect, have become one of the most important antibiotics in the livestock and aquaculture industry. However, due to the drug resistance and potential carcinogenicity caused by the residues of fluoroquinones, the European Union, Japan and my country have all established the maximum residues in food. Fluoroquinone residue detection has become a mandatory inspection item for eels exported to Japan from my country.

[0003] The residual markers of fluoroquinones in tissues were enrofloxacin and ciprofloxacin, with the highest residual concentration in liver tissue and kidney tissue, follow...

Examples

Embodiment 1

[0083] Example 1 Component preparation of the enzyme-linked immunoimmunoassay kit for detecting the residual amount of fluoroquinones

[0084] 1. Antigen Synthesis

[0085] a. Synthesis of hapten: take fluoroquinone drug ciprofloxacin as intermediate and adopt succinic anhydride method to synthesize fluoroquinone drug hapten. The preparation process is as follows:

[0086] (1) In 70% 200mL ethanol, add the ciprofloxacin hapten with O-(carboxymethyl) hydroxyl group and ketone group, so that its concentration is 10mmol / L and 4mmol / L respectively;

[0087] (2) Reflux heating for 90min;

[0088] (3) rotary evaporation, reduce the volume, then add water to 50mL, dichloromethane extraction;

[0089] (4) Wash the dichloromethane extract with water, wash with NaSO 4 Dries to white powder.

[0090] b. Immunogen synthesis: the fluoroquinone drug hapten and thyroid protein were coupled by the mixed anhydride method to obtain the immunogen, and the preparation process was as follows: ...

Embodiment 2

[0118] Embodiment 2 detects the formation of the enzyme-linked immunosorbent assay kit of fluoroquinones

[0119] An enzyme-linked immunosorbent assay kit for detecting fluoroquinones was set up to include the following components:

[0120] (1) Enzyme plates coated with fluoroquinone antigens;

[0121] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0122] (3) 6 bottles of ciprofloxacin standard solution, the concentrations were 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L and 81 μg / L;

[0123] (4) Chromogenic solution A liquid is hydrogen peroxide, and chromogenic liquid B liquid is o-phenylenediamine;

[0124] (5) Fluoroquinone antibody working solution with a protein concentration of 0.5 μg / L;

[0125] (6) The concentrated washing solution is a phosphate buffer containing 0.8% Tween 20;

[0126] (7) The stop solution is a sulfuric acid solution of 1mol / L;

[0127] (8) The concentrated complex solution is 0.01M phosphate buffer containing 1% gelatin....

Embodiment 3

[0128] Embodiment 3 detects the formation of the ELISA kit of fluoroquinones

[0129] An enzyme-linked immunosorbent assay kit for detecting fluoroquinones was set up to include the following components:

[0130] (1) Enzyme plates coated with fluoroquinone antigens;

[0131] (2) Goat anti-mouse anti-antibody labeled with alkaline phosphatase;

[0132] (3) 6 bottles of ciprofloxacin standard solution, the concentrations were 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L and 81 μg / L;

[0133] (4) The substrate solution is p-nitrophosphate buffer;

[0134] (5) Fluoroquinone antibody working solution with a protein concentration of 5.0 μg / L;

[0135] (6) The concentrated washing solution is a phosphate buffer containing 1.2% Tween 20;

[0136] (7) The stop solution is a sodium hydroxide solution of 2mol / L;

[0137] (8) The concentrated complex solution is 0.01M phosphate buffer containing 1% gelatin.

technical field

[0001] The invention relates to an ELISA kit and a detection method for detecting fluoroquinones in animal-derived foods including animal tissues (muscle, liver), shrimp, fish and blood products. Background technique

[0002] Fluoroquinone antibacterial drugs (also known as quinolone antibacterial drugs, QNS), which have a broad antibacterial spectrum and strong antibacterial effect, have become one of the most important antibiotics in the livestock and aquaculture industry. However, due to the drug resistance and potential carcinogenicity caused by the residues of fluoroquinones, the European Union, Japan and my country have all established the maximum residues in food. Fluoroquinone residue detection has become a mandatory inspection item for eels exported to Japan from my country.

[0003] The residual markers of fluoroquinones in tissues were enrofloxacin and ciprofloxacin, with the highest residual concentration in liver tissue and kidney tissue, follow...

Examples

Embodiment 1

[0083] Example 1 Component preparation of the enzyme-linked immunoimmunoassay kit for detecting the residual amount of fluoroquinones

[0084] 1. Antigen Synthesis

[0085] a. Synthesis of hapten: take fluoroquinone drug ciprofloxacin as intermediate and adopt succinic anhydride method to synthesize fluoroquinone drug hapten. The preparation process is as follows:

[0086] (1) In 70% 200mL ethanol, add the ciprofloxacin hapten with O-(carboxymethyl) hydroxyl group and ketone group, so that its concentration is 10mmol / L and 4mmol / L respectively;

[0087] (2) Reflux heating for 90min;

[0088] (3) rotary evaporation, reduce the volume, then add water to 50mL, dichloromethane extraction;

[0089] (4) Wash the dichloromethane extract with water, wash with NaSO 4 Dries to white powder.

[0090] b. Immunogen synthesis: the fluoroquinone drug hapten and thyroid protein were coupled by the mixed anhydride method to obtain the immunogen, and the preparation process was as follows: ...

Embodiment 2

[0118] Embodiment 2 detects the formation of the enzyme-linked immunosorbent assay kit of fluoroquinones

[0119] An enzyme-linked immunosorbent assay kit for detecting fluoroquinones was set up to include the following components:

[0120] (1) Enzyme plates coated with fluoroquinone antigens;

[0121] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0122] (3) 6 bottles of ciprofloxacin standard solution, the concentrations were 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L and 81 μg / L;

[0123] (4) Chromogenic solution A liquid is hydrogen peroxide, and chromogenic liquid B liquid is o-phenylenediamine;

[0124] (5) Fluoroquinone antibody working solution with a protein concentration of 0.5 μg / L;

[0125] (6) The concentrated washing solution is a phosphate buffer containing 0.8% Tween 20;

[0126] (7) The stop solution is a sulfuric acid solution of 1mol / L;

[0127] (8) The concentrated complex solution is 0.01M phosphate buffer containing 1% gelatin....

Embodiment 3

[0128] Embodiment 3 detects the formation of the ELISA kit of fluoroquinones

[0129] An enzyme-linked immunosorbent assay kit for detecting fluoroquinones was set up to include the following components:

[0130] (1) Enzyme plates coated with fluoroquinone antigens;

[0131] (2) Goat anti-mouse anti-antibody labeled with alkaline phosphatase;

[0132] (3) 6 bottles of ciprofloxacin standard solution, the concentrations were 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L and 81 μg / L;

[0133] (4) The substrate solution is p-nitrophosphate buffer;

[0134] (5) Fluoroquinone antibody working solution with a protein concentration of 5.0 μg / L;

[0135] (6) The concentrated washing solution is a phosphate buffer containing 1.2% Tween 20;

[0136] (7) The stop solution is a sodium hydroxide solution of 2mol / L;

[0137] (8) The concentrated complex solution is 0.01M phosphate buffer containing 1% gelatin.

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