Agraz A, Duarte C A, Costa L, Pérez L, Páez R, Pujol V, Fontirrochi G
Biopharmaceutics Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
J Chromatogr A. 1994 Jun 24;672(1-2):25-33. doi: 10.1016/0021-9673(94)80591-1.
A murine monoclonal antibody developed for the purification of recombinant hepatitis B surface antigen was immobilized on a chromatographic support and used to adsorb and purify the recombinant antigen from yeast. The adsorption-elution behaviour was first investigated using monoclonal antibody-coated enzyme-linked immunosorbent assay plates and performing adsorption, washing and elution procedures with different elution agents. It was found that 3 M KSCN and 8 M urea at neutral pH disrupted antigen-antibody interactions in both systems. The procedure for washing the immunoaffinity column was optimized, using different salts and detergents. The best results were obtained by applying the starting material in 1 M NaCl and washing with the same buffer. The use of 0.1% sodium deoxycholate in the washing buffer reduced about 20-fold lipopolysaccharide contamination in the eluates as compared with washing without detergent. The relationship between bed height and the adsorption capacity of the column was studied, and it was found that the dynamic capacity decreased twice on reducing its length/diameter ratio tenfold. The recovery of antigen was not affected by increasing the flow-rate up to 25 cm/h but decreased at higher values. Using the optimum conditions, the affinity column was able to purify the recombinant hepatitis B surface antigen to more than 90% purity and a 65% antigen recovery was obtained.
一种用于纯化重组乙肝表面抗原的鼠单克隆抗体被固定在色谱支持物上，并用于从酵母中吸附和纯化重组抗原。首先使用包被有单克隆抗体的酶联免疫吸附测定板，并使用不同的洗脱剂进行吸附、洗涤和洗脱程序，研究其吸附 - 洗脱行为。发现在中性pH条件下，3 M KSCN和8 M尿素会破坏两个系统中的抗原 - 抗体相互作用。使用不同的盐和去污剂对免疫亲和柱的洗涤程序进行了优化。通过在1 M NaCl中加入起始材料并用相同缓冲液洗涤可获得最佳结果。与不使用去污剂洗涤相比，在洗涤缓冲液中使用0.1%脱氧胆酸钠可使洗脱液中的脂多糖污染降低约20倍。研究了柱床高度与柱吸附容量之间的关系，发现将其长度/直径比降低10倍时，动态容量降低了两倍。流速增加到25 cm/h之前，抗原回收率不受影响，但流速更高时回收率降低。在最佳条件下，亲和柱能够将重组乙肝表面抗原纯化至纯度超过90%，抗原回收率为65%。
