摘要
目的了解不同辛酸浓度下盐析法、不同离心力对抗河豚毒素(TTX)单克隆抗体的纯化效果以及纯化后酶标抗体性能的影响,优化出纯化抗TTX单克隆抗体的方法,为免疫学方法检测TTX提供依据。方法在传统辛酸-硫酸铵蛋白纯化方法的基础上进行改进,考察不同的辛酸浓度、不同的离心力对抗TTX单克隆抗体纯化效果的影响,并将纯化后抗体与辣根过氧化物酶(HRP)连接,用间接竞争酶联免疫吸附试验法(ELISA)检测酶标抗体的生物学活性及各项性能指标并比对。结果不同方法纯化获得的抗体及其酶结合物的各项指标参数、活性不同,当腹水稀释液与辛酸体积比为1 000∶11、纯化过程中离心力10 000 r/min时纯化获得的抗体虽然产量有所下降但纯度较高,酶标抗体的生物活性、各项指标均最优。结论经过对抗TTX单克隆抗体纯度、抗体中蛋白含量、酶标抗体各项性能指标的对比,优化出用于抗TTX抗体纯化用腹水稀释液与辛酸比值1 000∶11(V/V)、离心力10 000 r/min的试验条件。
Objective To understand the effects of different caprylic acid-ammonium sulfate concentration and centrifugal force on purification of HRP-labeled monoclonal antibody against tetrodotoxin （ TTX ） and to optimize the purification protocol for the antibody in order to provide the technical basis for TTX detection by immunoassay. Methods Under different centrifugal force and concentration of caprylic acid-ammonium sulfate, the antibody was purified and linked with the horseradish peroxidase enzyme （HRP） , and the biochemical properties and parameters of HRP-labeled antibody were determined with indirect enzyme-linked immuno sorbent assay. Results The purity of antibody and HRP-labeled antibody obtained by different methods were different as well as other parameters and bioactivities. The best experiment conditions for high productivity and bioactivity of the monoclonal antibody were ascites diluent： lipoic acid （1 000 ： 11, V/V） and 10 000 r/min centrifugation. Conclusion The purification protocol was optimized for the dilution and centrifugal conditions based on the productivity, purity and bioactivity.
出处
《中国食品卫生杂志》
北大核心
2013年第3期194-197,共4页
Chinese Journal of Food Hygiene
基金
国家高技术研究发展计划(863计划:2012AA101603)
