鬼臼毒素人工抗原的合成与鉴定(英文)

Synthesis and identification of antigenic conjugates of podophyllotoxin

目的 合成和鉴定鬼臼毒素人工抗原,以获取具有特异性和高度亲和性的鬼臼毒素多克隆抗体。方法通过酯化反应合成半抗原鬼臼毒素半琥珀酰酯;利用活泼酯法和混合酸酐法偶联成免疫和包被所需的人工抗原;按常规免疫法,从免疫的兔血清中获取高效的抗鬼臼毒素抗体。结果 质谱、核磁共振和红外分析表明两种不同方法均成功地合成了半抗原鬼臼毒素半琥珀酰酯。人工抗原的偶联比因载体蛋白和合成方法的不同而有所不同,Hapten- BSA1为88. 6,Hapten BSA2为40. 3,Hapten OVA1为17 8和Hapten- OVA2为54 2。利用兔M4440产生的抗血清建立的ELISA方法对鬼臼毒素的最低检测浓度为0 .12μg·mL-1,半数抑制浓度为2. 21μg·mL-1。结论 成功地合成了鬼臼毒素人工抗原,并获得了抗鬼臼毒素抗体,为鬼臼毒素免疫分析方法的进一步研究提供了条件。

Aim To synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody. Methods The hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits. Results The results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA ~1, 40.3 for Hapten-BSA ~2, 17.8 for Hapten-OVA ~1, and 54.2 for Hapten-OVA ~2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC ~50 value of 2.21 μg·mL^-1 with a detection limit of 0.12 μg·mL^-1. Conclusion Antigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.