单克隆抗体免疫亲和层析法纯化人的凝血因子Ⅸ

Immunoaffinity Chromatographic Purification of Human Coagulation Factor IX (FIX) by Using Conformation-specific Monoclonal Anti-body

本文报告一种对连接钙(或镁)离子的因子Ⅸ(FⅨ)之构型具有特异性的单克隆抗体(MAb),并将其偶联Affi-Gel 15凝胶,组成亲和层析柱.将钙(或镁)离子加入含有FⅨ的原料液内,混合液流进层析柱时,唯独FⅨ被吸附,而且易被不含钙(或镁)离子的缓冲液洗脱.如洗脱液内含1 mM EDTA,则FⅨ洗脱峰十分陡峭.1 ml凝胶能吸附0.1mg的FⅨ,其吸附容量是理论容量的8%.FⅨ制品比活性为140～180活性单位/mg蛋白.在还原和非还原的SDS-PAGE电泳图谱上呈现一条谱带.此方法快速,且仅需一步即可从人血浆内提取纯化10000倍的FⅨ.

An immunoaffinity method has been developed that allows for the rapid purification of human FIX. A specific monoclonal antibody for the calcium(or magnesium)-stabilized conformation of FIX was coupled to Affi-Gel 15. The immobilized anti-body was capable of binding FIX from several different sources in the presence of either Ca^(++) or Mg^(++). FIX was eluted by the buffer in the absence of Ca^(++) and Mg^(++). If there are 1mM EDTA in the elution buffer, a sharp peak of FIX will be obtained. The capacity of the column for FIX was determined to be 0.1 mg of protein per ml of resin which represents about 8% of the theoretical capacity of the column based on 2 moles of FIX binding per mole of antibody. The specific activity of the FIX product ranged between 140 and 180 units/mg and behaved as a single band on reduced and nonreduced SDS-PAGE. This immunopurification resulted in a 10,000-fold one step purification of the fully functional zymogen and has potential application for the large scale purification of FIX for specific replacement therapy in hemophilia B.