ELISA kit for detecting sulfanilamides residue in animal derived food
A sulfonamide drug, a technology for detecting animals, applied in the field of immunological detection, can solve the problems of carcinogenicity, cumbersome processing and measurement operations, and restrictions on promotion and use, and achieve the effect of reducing the loss of enzyme activity
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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] Embodiment 2 detects the formation of the ELISA kit of sulfa drugs
[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] Embodiment 3 detects the formation of the ELISA kit of 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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