Tishchenko G A, Bleha M, Skvor J, Bostík T
Institute of Macromolecular Chemistry of the Academy of Sciences of the Czech Republic, Prague.
J Chromatogr B Biomed Sci Appl. 1998 Feb 27;706(1):157-66. doi: 10.1016/s0378-4347(97)00453-2.
A three-stage process, consisting of an ammonium sulfate precipitation step, dialysis desalination with microporous anion-exchange Neosepta membranes and anion-exchange chromatography on DEAE-cellulose DE-52 was used for the isolation of mouse monoclonal antibodies specific against different antigens. The ascites fluids contained monoclonal antibodies against human IgG, against horseradish peroxidase and against the heavy chain of human IgM. The effect of the salt concentration gradient in the elution buffer was examined with the aim of optimizing chromatographic conditions. The quality of separation of protein zones was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The immunoreactivity of purified monoclonal antibodies was determined by enzyme-linked immunosorbent assay using a solid-phase adsorbed antigens against which each monoclonal antibody type was directed.
采用三步法从腹水液中分离针对不同抗原的小鼠单克隆抗体，该方法包括硫酸铵沉淀步骤、使用微孔阴离子交换Neosepta膜进行透析脱盐以及在DEAE - 纤维素DE - 52上进行阴离子交换色谱。腹水液中含有抗人IgG、抗辣根过氧化物酶和抗人IgM重链的单克隆抗体。为优化色谱条件，研究了洗脱缓冲液中盐浓度梯度的影响。在非还原条件下，使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定蛋白质区带的分离质量。通过酶联免疫吸附测定法，使用每种单克隆抗体所针对的固相吸附抗原，测定纯化单克隆抗体的免疫反应性。
