Antigen and antibody for multi-residue detection of quinolones and sulfonamides, preparation method and application thereof
A quinolone and sulfonamide technology, applied in the field of food safety, can solve the problems of inability to meet the detection of multiple veterinary drugs at the same time, waste of resources, etc., and achieve the effects of easy popularization, good accuracy and sensitivity, and broad application prospects.
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Embodiment 1
[0041] Example 1 Preparation of sulfonamide haptens
[0042] Weigh 3.5g of acetaminobenzenesulfonyl chloride and place it in a round-bottom single-necked flask, dissolve it with 30mL of anhydrous pyridine, then add 4.0g of 4-aminobutyric acid, reflux for 5 hours, evaporate the solvent to dryness, add water, and extract with ethyl acetate 2-3 times, combine the organic phases, evaporate the organic phases to dryness, add 30mL 10% NaOH solution and stir overnight, adjust the pH of the solution to slightly acidic with 6N HCl the next day, a large amount of solids precipitate out, filter and wash the solids. After drying, a yellowish solid is obtained, which is the sulfonamide hapten. The mass spectrum of the sulfonamide hapten is as follows figure 2 shown. Prove that it has the molecular structure shown in formula (II):
[0043]
Embodiment 2
[0044] Embodiment 2 Preparation method of quinolones and sulfonamides combined antigen
[0045] S1. Weigh 33mg of ciprofloxacin and dissolve it in 1mL DMF, stir and add 36mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 25mg of N-N-hydroxysuccinate Imide (NHS), stirred and reacted at room temperature for 8 hours. After the reaction, 0.25 mL of the reaction solution was added to 50 mg of pH7.4 BSA phosphate buffer solution, stirred at room temperature for 6 hours, and the reaction solution was put into a dialysis bag. Dialyze with pH7.4 phosphate buffer solution for 3 days, change the water 2 to 3 times a day, and obtain the complete antigen of quinolones;
[0046] S2. Weigh 26 mg of the sulfonamide hapten prepared in Example 1 and dissolve it in 1 mL of DMF, stir and add 36 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) , 25mg N, N-hydroxysuccinimide (NHS), stirred and reacted at room temperature for 8h to obtain a reaction sol...
Embodiment 3
[0048] Example 3 Preparation and identification of antibodies
[0049] Mix the immunogen with an equal dose of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations), emulsify completely with a mixer, and immunize healthy white rabbits at multiple points on the back, Afterwards, the immunization was boosted once every two weeks, and the serum titer and inhibition rate were measured by indirect ELISA. After the titer was stable, the immunization was boosted again. Blood was collected from the heart 10 days after the last immunization, and the serum was retained by centrifugation. The serum was purified by octanoic acid-ammonium sulfate salting-out method to obtain highly specific antibodies.
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