Tiamulin hapten TMLO, artificial antigen, antibody and preparation method and application of tiamulin hapten TMLO
A technology of tiamulin and artificial antigen, which is applied in the biological field to achieve the effect of strong specificity, high sensitivity and wide linear range
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Embodiment 1
[0051] The preparation of embodiment 1 tiamulin hapten TMLO
[0052] 1. Experimental method
[0053] Weigh 4.05mmol of tiamulin and dissolve it in 5-10mL of anhydrous pyridine, add 5.61mmol of carboxymethyl hydroxylamine, and reflux at 60°C for 7h. After the reaction stopped, the solvent was evaporated to dryness, 50 mL of ice water was added, the pH was adjusted to 3 with 1 mol / L hydrochloric acid solution, and extracted with 100 mL of ethyl acetate three times. The organic phases were combined, washed with 50 mL of saturated brine three times, dried over anhydrous sodium sulfate, separated by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 as the developing solvent), and the product fractions were collected , after the organic reagent was removed by rotary evaporation, a dark brown solid product was obtained, which was the tiamulin hapten TMLO, and the reaction formula was as follows:
[0054]
[0055] 2. Experimental results
[0056...
Embodiment 2
[0058] The preparation of embodiment 2 tiamulin artificial antigen
[0059] The tiamulin hapten TMLO prepared in Example 1 was coupled to chicken ovalbumin OVA (albumin), hemocyanin KLH (keyhole limpet hemocyanin), bovine serum albumin BSA (Bovinealbumin), milk Ferritin LF (lactoferrin) and concanavalin ConA (concanavalin A). Specific steps are as follows:
[0060] Dissolve the tiamulin hapten TMLO in DMF, add 1.5 times (molar ratio) of NHS and EDC (molar ratio TMLH:NHS:EDC=1:1.5:1.5) to react overnight at 4°C in the solution, recorded as Liquid A: Add liquid A dropwise to the PBS buffer solution of the carrier protein, and continue to react at 4°C for 8 hours; dialyze the reaction liquid at 4°C for 3 days, change the dialysate twice a day, dialyze 6 times in total, and collect Dialyze the solution in the bag to obtain tiamulin artificial antigens (tiamulin complete antigens TMLO-OVA, TMLO-KLH, TMLO-BSA, TMHL-LF, TMLO-ConA). The structural formula of its tiamulin artificial...
Embodiment 3
[0063] Example 3 Tiamulin artificial antigen immunization and antiserum effect evaluation
[0064] 1. Experimental method
[0065] 1. Animal immunity
[0066] Healthy 6-week-old Balb / c female mice were used as experimental animals, and the complete antigens TMLO-KLH, TMLO-BSA, TMHL-LF, and TMHL-ConA were used as immunogens, and the mice were injected subcutaneously in the back of the neck and abdominal cavity. The dose for the second immunization is 0.5 mL (which contains 0.5 mg of immunogen). For the first immunization, 0.5 mL of complete Freund's adjuvant was emulsified with antigen for immunization, and 4 weeks later, 0.5 mL of incomplete Freund's adjuvant was used for booster immunization after emulsification of antigen. The blood was used for antibody quality identification. After the antibody was stabilized, the mouse with the best performance was selected for cell fusion. Three days before cell fusion, the mouse was directly injected with 0.5 mg of the immunogen intra...
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