Experimental principle 62.5mgTS, 34.5mg N-hydroxysuccinimide ester and 45.3mg 1,3-dicyclohexylcarbodiimide were reacted in 1.5ml of dry N, N-dimethylamide for 18h, 4o C, filtered through The removal of the resulting precipitate of dicycloethylurea resulted in a filtrate. The filtrate was added to 2ml of PBS containing 65mg LPH, and the pH was adjusted to 7.6 with NaOH. The mixture was stirred at 4o C for 24h. Check whether the sulfa drug has polymerized by TLC. Because of the low basic free sulfamic acid cluster (for example, sulfathiazole pka is 2.36), especially the amino cluster of lysine (pka 10.28). It may be the main reason why each amino acid forms an amide. After 24h, the reaction mixture was transferred to dialysate, dialyzed against 1L of 8M urea for 12h, the dialysate was transferred to 4L, 50mM ammonia bicarbonate, and then to 4L, 25mM ammonia bicarbonate, each dialysate At least 8h. The contents of the dialysis bag are lyophilized. TS-bsa uses the same method, bsa instead of LPH. Experimental reagent Anhydrous sodium sulfate; 150ml methanol; 100ml hydrochloric acid; 2.1g 2-amino-4-thiazole acetic acid; 2.65g N-Acetylsulfanilyl chloride (ASC); 50ml pyridine; 100ml dichloromethane layer; 5g silica gel; 50g silica column (50 * 2.4); 200ml 1: 4 methanol-chloroform elution 50ml 2MNAOH solution; 100ml 6N hydrochloric acid. Experimental Materials 50 ml round bottom flask 250 ml three-necked flask Experimental procedure Synthesis of 2-amino-4-thiazole-methyl acetate: 1. In an ice bath, add 100ml of methanol dried over anhydrous sodium sulfate to a 250ml three-necked flask. 2. Add hydrochloric acid gas to the flask containing methanol, stirring constantly, generating a large number of bubbles. When the mass of the solution increases by about 60g, stop adding hydrochloric acid. 3. Slowly add 2.1g of 2-amino-4-thiazoleacetic acid to the flask, remove the ice bath, and stir for 10 minutes. 4. The solution in the flask was refluxed for 6 hours, at this time a slightly yellow solution. 5. Transfer the solution to a rapid evaporator, add 25ml of dry methanol, and remove the solvent. 6. The solid recrystallized from ethyl acetate and methanol is a synthetic solid (melting point 169-171o C), which is methyl 2-amino-thiazole acetate, light yellow. Synthesis of N4-acetyl-N1- {4-[(methoxyhydroxy) methyl] -2-thiazole} -sulfonamide: 1. Add 2.65g of N-Acetylsulfanilyl chloride (ASC) to a 50ml round-bottom flask and add 5ml of dry pyridine with stirring. 2. Slowly add 1.50g of ethyl 2-amino-4-thiazole ethyl acetate to the pyridine solution, maintaining the temperature at no more than 40 ° C. The solution was brown, refluxed for 1.5h, and cooled to room temperature. 3. With slow stirring, add 30ml of warm water and extract with dichloromethane (3 * 25). 4. The methylene chloride layer was dried with sodium sulfate and filtered, while most of the solvent was removed with a fast rotary evaporator. Add about 5g of silica gel and remove the remaining solvent, leaving some light yellow powder. These powders passed through a 50g silica column (50 * 2.4), which was eluted with 150ml 1: 4 methanol-chloroform. Remove these solvents to obtain N4-acetyl-N1- {4-[(A Oxyhydroxy) methyl] -2-thiazole} -sulfonamide (1.6 g, 50% yield). Synthesis of N- [4- Carboxymethy1) -2-thiazolyl] sulfanilamide (TS): 25ml, 2MNAOH solution was added to purified 0.95g N4-acetyl-N1- {4-[(methoxyhydroxy) methyl] -2-thiazole} -sulfonamide, refluxed for 2h, after cooling to room temperature, using 6N hydrochloric acid Adjust the PH to 4.0. The resulting cloudy solution was tested for the presence of epitope density and free aromatic amino groups using ethyl acetate conjugates. 1. Antibody preparation 1) Four 10-week-old female rabbits, one rabbit was injected with 0.8ml of sterile PBS and FCA (1: 1) oily mixture; two rabbits were injected with TS-conjugated LPH mixture; the other rabbit was used ts-bsa is used as an immunogen. The mice were injected with 0.5 mg of the mixed solution (2 * 0.2 ml) of the scapula and 0.8 ml of the oily mixture of gluteal muscles (2 * 0.4 ml). Rabbits received the same vehicle injection, except that FIA replaced FCA, with an interval of 2 to 4 weeks after the first injection. 2) After 4 weeks, draw blood from the rabbit's ear-derived large vein, about 15 ml per mouse. At 6 weeks, collect blood through the heart about 70 ml per rabbit. The collected blood is left at room temperature for 1.5 hours, and the serum is centrifuged at 1,000 rpm At 5 minutes, clear yellow serum and red blood cell deposits appeared. The serum was collected in a 1.5ml sealed container and stored at -20 ° C. 2. Isolation of IgG from rabbit serum 0.6 ml of Protein A packing was packed into a 10 ml disposable polypropylene column, which was rinsed with 10 ml of PBS containing 0.02% sodium methionate. The filtered 0.5ml of serum was slowly added from the upper end of the protein A column, staying for about 15 minutes. It takes 15ml of PBS containing azide to elute the serum proteins except IgG for about 3 minutes. 1ml of serum protein was collected. Next, elute IgG with approximately 5 ml of acetic acid. The eluate was transferred to a dialysis bag (4L, 25mM ammonium bicarbonate for 24h, changing the fluid every 4h). The contents of this dialysis bag were freeze-dried and stored at -20o C. Approximately 2 mg of IgG can be recovered from 0.5 ml of rabbit serum. 3. Production of immunoglobulin Fab fragments 1) 0.5ml papain for fixation is added to a glass detection tube. Digestion buffer (2.76g sodium dihydrogen phosphate, 3.80g edetate powder tetrandrine salt and 3.51g cysteine ​​hydroxide, 1M sodium hydroxide adjusted to pH 7.0) was added to the detection tube In the process, papain can be separated by buffer enzyme, and the enzyme can be eluted again with digestion buffer. The fixed papaya enzyme was suspended in 0.5 ml of digestive juice. The lyophilized IgG (4 mg) was dissolved in 2 ml of fresh digestive juice and added to the detection tube pretreated with papain. The enzyme-IgG mixture was incubated in a 37 ° C shaking water bath for 5h. After 5 hours, 1.5 ml of 10 mM trisaminomethane hydrochloric acid buffer ph7.5 was added to the IgG enzyme digestion solution, and the Fab fragments were separated. 2) Fab fragments, non-hydrolyzed IgG and Fc fragments are loaded on top of the protein A packing, elute the column with 10ml PBS, the speed is 1 mL / 3 min, the first 1 mL is discarded, the next 3 mL is collected and transferred Into the dialysis bag. Use 4L, 25mM ammonium bicarbonate for 24h, and change the fluid every 4h. The material in this dialysis bag was lyophilized and 1.1 mg Fab was recovered from 0.5 mL of rabbit serum (confirmation of whether the Fab was purified by gel isoelectric focusing electrophoresis). 4. Indirect ELISA, 96-well microtitration 1) Add 200 µL of 0.01% thimerosal PBS containing 20 µg / mL (TS-OV, NS-OV, CS-OV), coat at 4 ° C overnight, and put a wet bag on each plate. The next day, the coating solution was discarded, and 200 µL of 1% BSA-containing thimerosal in PBS was added to each well, and then placed in the same bag at room temperature for 1 hour. Discard the coating solution, wash the plate 3 times, and add 100 µl of thiomersal in PBS containing 0-25 µg / mL sulfa drug. 2) The plate diluted with serum to 0.05% BSA is stored in a plastic bag at room temperature for 2h, and washed three times with thimerosal PBS. Goat anti-rabbit antibody diluted 3000 times was added, and thimerosal PBS was added to 200μL / well per well. The blank group was also stored in a plastic bag at room temperature for 2h and washed three times with thimerosal PBS. 3) Enzyme acting substrate, 0.4 mg / mL (o-phe-nylenediamine) and ammonium peroxide (1.0 mg / mL), 0.1M citrate, PH4.75 are added to each well, 200µL / well, in After 30 minutes of reaction, the absorbance was measured with a microplate reader at room temperature, and the absorbance was measured at 450 nm. 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November 11, 2020