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ELISA kit for detecting sulfanilamides residue in animal derived food

A technology for detecting sulfonamide drugs and animals, which is applied in the field of immunological detection, can solve the problems of carcinogenicity, cumbersome handling and measurement operations, and restrictions on popularization and use, and achieve the effect of reducing the loss of enzyme activity

Active Publication Date: 2007-10-24
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the serious side effects of sulfa drugs residues, the long-term existence of sulfa drugs in the human body will cause many bacteria to develop resistance to sulfa drugs, and they are potentially carcinogenic. Developed countries such as Europe and the United States have banned or strictly prohibited their use
The No. 235 document of the Ministry of Agriculture of my country stipulates that the residue limit is 100 μg / kg. Due to the cumbersome sample pretreatment and high cost of instrument analysis methods such as high performance liquid chromatography and gas chromatography, the popularization and use are limited.

Method used

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  • ELISA kit for detecting sulfanilamides residue in animal derived food
  • ELISA kit for detecting sulfanilamides residue in animal derived food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] The preparation of embodiment 1 kit components

[0086] 1. Synthesis of Coatogen

[0087] (1) Dissolve 2 g of the sulfonamide drug hapten in 30 ml of 50% N, N'-dimethylformamide solution.

[0088] (2) Dissolve 0.5 ml of isobutyl chloroformate in 5 ml of anhydrous dioxane and add it to the hapten solution, and react with stirring at room temperature for 4 hours.

[0089] (3) 32 g of carrier protein hemocyanin was dissolved in 70 ml of carbonate buffer solution of pH 9.6.

[0090] (4) Add the carrier protein solution dropwise to the hapten and stir overnight at 4°C.

[0091] (5) Dialyze the reacted artificial antigen against 0.2M phosphate buffer for 7 days, and change the solution 3 to 4 times a day. Finally the antigen is concentrated or lyophilized.

[0092] (6) Purification and preservation.

[0093] 2. Preparation of mouse monoclonal antibody against sulfa drugs

[0094] Animal immunization procedure: Balb / c mice were used as immunized animals, the sulfonamide ...

Embodiment 2

[0108] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting sulfonamides

[0109] The enzyme-linked immunosorbent assay kit for detecting sulfonamides was set up to include the following components:

[0110] (1) Enzyme plates coated with sulfonamide antigens;

[0111] (2) Antibodies to sulfa drugs with a protein concentration of 0.5 μg / L;

[0112] (3) 6 bottles of sulfamethazine standard solution, the concentrations are respectively 0ppb, 1ppb, 3ppb, 9ppb, 27ppb and 81ppb;

[0113] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0114] (5) The substrate chromogenic solution A liquid is hydrogen peroxide, and the substrate chromogenic liquid B liquid is o-phenylenediamine;

[0115] (6) The concentrated washing solution is a phosphate buffer containing 0.8% Tween 20;

[0116] (7) The stop solution is a sulfuric acid solution of 2mol / L;

[0117] (8) The concentrated extract is 0.01-0.05 mol / L phosphate buffer solution.

Embodiment 3

[0118] Example 3 The formation of the enzyme-linked immunosorbent assay kit for detecting sulfa drugs

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

[0120] (1) A microtiter plate coated with goat anti-mouse anti-antibody;

[0121] (2) Antibodies to sulfa drugs with a protein concentration of 5.0 μg / L;

[0122] (3) 6 bottles of sulfamethazine standard solution, the concentrations are respectively 0ppb, 1ppb, 3ppb, 9ppb, 27ppb and 81ppb;

[0123] (4) extracting alkaline phosphatase-labeled sulfa drugs with bacteria;

[0124] (5) The substrate chromogenic solution is p-nitrophosphate buffer;

[0125] (6) The concentrated washing solution is a phosphate buffer containing 1.2% Tween 20;

[0126] (7) The stop solution is a sodium hydroxide buffer solution of 2mol / L;

[0127] (8) The concentrated extract is 0.01-0.05 mol / L phosphate buffer solution.

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Abstract

The invention provides an enzyme immune agent box for detecting sulpha drugs of animal organization, which uses enzyme immune method to detect the preprocessed animal organization, honey, urine, milk. The enzyme immune agent box comprises: enzyme mark plate which coats sulpha drugs antigen or antibody, sulpha drugs mouse monoclonal antibody working solution, enzyme mark antibody or sulpha drugs antigen solution, sulpha drugs standard solution, base material color developing solution, compression cleaning liquid, ending solution and compression extracting 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 the field of immunological detection, in particular to an enzyme-linked immunoimmunoassay kit for detecting sulfa drugs in animal-derived foods and a detection method thereof. Background technique [0002] Sulfonamides are widely used antibiotics, which once played an important role in the control and treatment of livestock and poultry diseases. However, due to the serious side effects of sulfa drugs residues, the long-term existence of sulfa drugs in the human body will cause many bacteria to develop resistance to sulfa drugs, and they are potentially carcinogenic. Developed countries such as Europe and the United States have banned or strictly prohibited their use. The No. 235 document of the Ministry of Agriculture of my country stipulates that the residue limit is 100 μg / kg. Due to the cumbersome pretreatment and high cost of sample pretreatment and determination by instrumental analysis methods such as high performance liq...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577G01N33/535
Inventor 沈建忠何方洋冯才伟万宇平吴小平冯才茂汪善良李军赵正苗张照亮史为民张素霞丁双阳孙倩罗晓琴
Owner BEIJING WANGER BIOTECH
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