Schuler G, Reinacher M
Institut für Veterinärpathologie der Justus-Liebig-Universität, Giessen, Germany.
J Chromatogr. 1991 Nov 29;587(1):61-70. doi: 10.1016/0021-9673(91)85198-o.
Protein A affinity chromatography is a standard method of purifying murine monoclonal antibodies (mabs), primarily because it can be performed easily and achieves high-purity levels. Because of its high concentration capacity, it lends itself particularly well to the isolation of mabs from the supernatants of hybridoma cultures. Unfortunately, murine immunoglobulin (Ig) G1 antibodies, a subclass which occurs frequently in the IgG mabs of mice, binds very poorly to protein A, leading to problems in this isolation procedure. For this reason an attempt was made to increase the effectiveness of protein A affinity chromatography in purifying mabs of this IgG subclass by optimizing the binding conditions. The influence of ionic strength, pH and temperature on the binding capacity of a protein A column was studied. The results show the significance of temperature in the binding of the murine IgG1 mab tested to protein A. Further investigations were carried out to optimize the elution conditions and to study the contamination of mab preparations obtained with non-specific bovine protein A reactive Igs originating from culture medium supplement (10% foetal calf serum). An optimized, automatic single-step procedure to obtain highly purified murine IgG1 mabs from hybridoma culture supernatants was developed.
蛋白A亲和层析是纯化鼠单克隆抗体（mab）的标准方法，主要是因为它操作简便且能达到高纯度水平。由于其高浓缩能力，特别适合从杂交瘤培养上清液中分离单克隆抗体。不幸的是，鼠免疫球蛋白（Ig）G1抗体是小鼠IgG单克隆抗体中常见的一个亚类，它与蛋白A的结合能力很差，导致在这个分离过程中出现问题。因此，人们试图通过优化结合条件来提高蛋白A亲和层析在纯化该IgG亚类单克隆抗体方面的有效性。研究了离子强度、pH值和温度对蛋白A柱结合能力的影响。结果表明温度对所测试的鼠IgG1单克隆抗体与蛋白A的结合具有重要意义。进一步开展研究以优化洗脱条件，并研究从培养基补充物（10%胎牛血清）中获得的非特异性牛蛋白A反应性Ig对所得单克隆抗体制剂的污染情况。开发了一种优化的自动单步程序，用于从杂交瘤培养上清液中获得高度纯化的鼠IgG1单克隆抗体。
