新型氨基甲酸乙酯人工抗原的制备与表征

目的合成新型氨基甲酸乙酯(ethylcarbamate,EC)人工抗原并对其效果进行表征分析。方法用4-(二苯基羟甲基)苯甲酸对EC衍生化合成半抗原,用核磁氢谱(proton nuclear magnetic resonance, NMR)、核磁碳谱和质谱法(massspectrometry,MS)对其结构进行表征;用活化酯法将EC半抗原与牛血清蛋白(bovine serum albumin, BSA)偶联得到EC人工抗原。用紫外光谱和荧光光谱法对人工抗原进行表征并计算偶联比,用考马斯亮蓝法测定蛋白含量,间接竞争酶联免疫法鉴定其免疫活性。结果 EC半抗原结构与理论结构一致,半抗原与BSA成功偶联,偶联比为13:1,冻干粉的蛋白含量为0.695 mg/mg,用该新型人工抗原免疫小鼠,得到的抗血清效价为1:10000,且特异性较好。结论该新型氨基甲酸乙酯半抗原具有较好的免疫活性。

Synthesis and identification of a novel ethyl carbamate artificial antigen

Objective To synthesis and characterize a new ethyl carbamate (EC) artificial antigen. Methods EC was derivatized with 4- (diphenylhydroxymethyl) benzoic acid to synthesize hapten, and its structure was characterized by proton nuclear magnetic resonance (NMR), NMR carbon spectrum and mass spectrometry (MS). EC was coupled with bovine serum albumin (BSA) by activated ester method to obtain EC artificial antigen. The artificial antigen was characterized by ultraviolet and fluorescence spectroscopy and the coupling ratio was calculated. The protein content was determined by the Coomassie Brilliant Blue method, and its immunological activity was identified by indirect competitive ELISA. Results The structure of the EC hapten was consistent with the theoretical structure. The hapten was successfully coupled with BSA, and the coupling ratio was 13:1, and the protein content of the lyophilized powder was 0.695 mg/mg. The mice were immunized with the new artificial antigen, and the antiserum was obtained. The titer was 1:10000 and the specificity was good. Conclusion The novel urethane hapten has good immune activity.