Sulfonamide drug hapten and artificial antigen and preparation method and application thereof
A technology of sulfonamide drugs and artificial antigens, which is applied in the field of immunoassays, can solve the problems of few types of SAs, uneven cross-reaction rates, and poor sensitivity
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Embodiment 1
[0071] Preparation and identification of embodiment 1 sulfa drug hapten
[0072] 1. Mix 100 g of ethyl 4-bromobutyrate (compound 1) and 150 g of triphenylphosphine, and reflux in 1 L of toluene for 24 hours. The precipitate was filtered, recovered, and washed with petroleum ether, and the operation was repeated 3 times. Drying in vacuo gave the product (compound 2) as a white solid.
[0073] 2. Dissolve 200g of 2,4-pentanedione (compound 3) and 200g of guanidine hydrochloride (compound 4) in 2L of water, and gradually add 550g of potassium carbonate. The reaction mixture was stirred at 40 °C for 24 h. The precipitate was filtered, washed with water, and repeated 3 times. Drying under vacuum afforded an off-white solid (Compound 5).
[0074] 3. Add 150 g of compound 5 and 200 g of N-bromosuccinimide into 1 L of acetonitrile, and heat at 80° C. for 6 h. The precipitate was filtered, washed with cold acetonitrile, and dried under vacuum to give a white solid (compound 6).
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Embodiment 2
[0081] Example 2 Preparation of sulfa drugs immune antigen sulfamethazine valeric acid-hemocyanin
[0082] 1. Add 0.1mmol sulfamethazine valeric acid, 40mg N,N'-dicyclohexylcarbodiimide (DCC), 25mg N-hydroxysuccinimide (NHS) into 1mL DMF, stir overnight at room temperature to esterify (activate) the carboxyl group of the hapten.
[0083] 2. Take out the activated product and centrifuge at 4000rpm for 10min.
[0084] 3. Weigh 100mg of KLH and dissolve it in 20mL of pre-cooled PBS.
[0085]4. Take the centrifuged supernatant of the activated product, slowly add it dropwise to the KLH solution, and stir overnight at 4°C.
[0086] 5. Put the above reaction product in a dialysis bag, and dialyze overnight in 5L of dialysate.
[0087] 6. Take out the product, aliquot it and store it in a freezer at -70°C, that is, the immunogen sulfamethazine valeric acid-KLH. Example 3 Preparation of coated original sulfamethazine valeric acid-bovine serum albumin for detection of sulfa drugs
Embodiment 3
[0087] 6. Take out the product, aliquot it and store it in a freezer at -70°C, that is, the immunogen sulfamethazine valeric acid-KLH. Example 3 Preparation of coated original sulfamethazine valeric acid-bovine serum albumin for detection of sulfa drugs
[0088] 1. Take 0.5mmol sulfamethazine valeric acid, 200mg N,N'-dicyclohexylcarbodiimide (DCC), 100mg N-hydroxysuccinimide (NHS) and add it to 1mL DMF, stir overnight at room temperature to esterify (activate) the carboxyl group of the hapten.
[0089] 2. Take out the activated product and centrifuge at 4000rpm for 10min.
[0090] 3. Weigh 500mg of BSA and dissolve it in 20mL of pre-cooled PBS.
[0091] 4. Take the centrifuged supernatant from the activated product, slowly add it dropwise to the BSA solution, and stir overnight at 4°C.
[0092] 5. Put the above reaction product in a dialysis bag, dialyze for three days, and change the dialysate every 8 hours.
[0093] 6. Take out the product, subpackage it and store it in a...
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