A kind of tiamulin hapten tmlh, artificial antigen, antibody and its preparation method and application
A tiamulin and artificial antigen technology, applied in hydrazone preparation, chemical instruments and methods, animal/human protein, etc., to achieve high sensitivity, good affinity, and improve immunogenicity
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Embodiment 1
[0048] The preparation of embodiment 1 tiamulin hapten TMLH
[0049] 1. Experimental method
[0050] Weigh 4.05mmol of 8α-hydroxymiaolin and dissolve it in 5-10mL of methanol, add 4.05mmol of hydrazine hydrate under stirring, after stirring at room temperature for 2 hours, add 4.05mmol of succinic anhydride, stir at room temperature overnight, evaporate to remove organic The residual solid after the solvent was separated by 200-300 mesh silica gel column chromatography (the developing solvent is petroleum ether: ethyl acetate = 1:1), and the product was collected by spotting the plate, and the organic reagent was removed by rotary evaporation to obtain the product , the tiamulin hapten TMLH. The reaction formula is as follows:
[0051]
[0052] 2. Experimental results
[0053] The mass spectrum and NMR data of tiamulin hapten TMLH are as follows: ESI-MS(m / z):435.30[M+H] + ; 1 H NMR (500MHz, DMSO-d 6 )δ5.79–5.69(m,1H),5.11(dd,J=16.9,2.4Hz,1H),5.04(dd,J=9.9,2.4Hz,1H),3....
Embodiment 2
[0055] The preparation of embodiment 2 tiamulin artificial antigen
[0056] The tiamulin hapten TMLH 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:
[0057] Dissolve the tiamulin hapten TMLH in DMF, add 1.5 times (molar ratio) NHS and EDC (molar ratio TMLH:NHS:EDC=1:1.5:1.5) to react overnight at 4°C in the solution, denoted as A solution; add solution A dropwise into the PBS buffer solution of the carrier protein, and continue to react at 4°C for 8 hours; dialyze the reaction solution at 4°C for 3 days, change the dialysate twice a day, dialyze 6 times in total, and collect the dialyzed The solution in the bag is the tiamulin artificial antigen (tiamulin complete antigen TMLH-OVA, TMLH-KLH, TMLH-BSA, TMLH-LF, TMLH-ConA). The structural formula of its tiamulin artifi...
Embodiment 3
[0060] Example 3 Tiamulin artificial antigen immunization and antiserum effect evaluation
[0061] 1. Experimental method
[0062] 1. Animal immunity
[0063] Healthy 6-week-old Balb / c female mice were used as experimental animals, and the complete antigens TMLH-KLH, TMLH-BSA, TMLH-LF, and TMLH-ConA were used as immunogens, and subcutaneously injected in the back of the neck and abdominal cavity of the mice. 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 intrape...
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