Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof
A technology of enzyme-linked immunosorbent reagents and sulfonamides, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex instrument equipment pretreatment process, unsatisfactory specificity of sulfonamides, etc.
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Embodiment 1
[0078] Embodiment 1 Preparation of kit components
[0079] 1. Antigen Synthesis
[0080] a, sulfonamide drug hapten synthesis (synthetic route such as figure 2 )
[0081] 1) Put 10g of para-acetaminobenzenesulfonyl chloride into a 100ml round bottom flask, add 25ml of pyridine to dissolve, add 55ml of pyridine solution containing 0.52g of p-aminobenzoic acid dropwise under vigorous stirring, heat up the oil bath, and react at 44°C 1h. After the reaction stopped, cool to room temperature, spin evaporate, remove pyridine, dissolve with 0.01mol / L sodium hydroxide, add ethyl acetate for extraction, separate the organic phase, wash with water to remove sodium hydroxide, and evaporate to dryness to obtain a yellow solid substance of 0.72 g;
[0082]2) Dissolve 6g of the yellow product in 30ml of 0.1mol / L NaOH aqueous solution, raise the temperature in the oil bath, and reflux for 3 hours. After the reaction, cool to room temperature, adjust the pH value to weak acidity with 1mol...
Embodiment 2
[0115] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting sulfonamides
[0116] The enzyme-linked immunosorbent assay kit for detecting sulfonamides was set up to include the following components:
[0117] (1) Enzyme plates coated with sulfa drug-conjugated antigens;
[0118] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;
[0119] (3) Monoclonal antibody working solution of sulfa drugs;
[0120] (4) 6 bottles of sulfamethoxazole standard solution, the concentrations are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L;
[0121] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid tetramethylbenzidine;
[0122] (6) The stop solution is 2mol / L hydrochloric acid;
[0123] (7) The concentrated washing solution is a carbonate buffer solution with a pH value of 7.2, containing 0.7-1.0% Tween-2...
Embodiment 3
[0125] The detection of sulfa drugs in the sample of embodiment 3
[0126] 1. Sample pretreatment
[0127] (1), chicken, pork, fish, shrimp sample pretreatment method
[0128] Weigh 2.0±0.05g of the homogeneous substance into a 50ml polystyrene centrifuge tube, add 8ml of ethyl acetate, shake immediately with a shaker for 5min, and centrifuge at 3000g or more for 5min; take 4ml of the supernatant and put it in a clean glass test tube of 10ml. Blow dry in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane, vortex for 30s with a vortexer to dissolve the dry residue, then add 1ml of complex solution working solution, vortex for 30s with a vortexer, then transfer to a 2ml centrifuge tube, Above 3000g, centrifuge at room temperature (20-25°C) for 5min; remove the upper n-hexane phase, and take 50μl of the lower aqueous phase for analysis.
[0129] (2) Honey sample pretreatment method
[0130] Weigh 1.0±0.05g honey sample into a 50ml polystyrene centrifuge tube; add...
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