Chen F M, Naeve G S, Epstein A L
Department of Pathology, School of Medicine, University of Southern California, Los Angeles 90033.
J Chromatogr. 1988 Jul 1;444:153-64. doi: 10.1016/s0021-9673(01)94018-x.
Nine immunoglobulin M (IgM) monoclonal antibodies (MAbs) produced in ascites fluids or in cell culture supernatants, have been purified on a fast protein liquid chromatography (FPLC) system using anion-exchange, size-exclusion, or mixed-mode chromatography matrices. The use of a mixed-mode ABx column provided an IgM that had a purity of greater than 99% after a single purification step. Anion-exchange chromatography using a Mono Q column, provided a partial purification of the IgM which could subsequently be purified to a product of ca. 90% purity (determined from sodium dodecyl sulfate polyacrylamide gel electrophoresis) by size-exclusion chromatography on a Superose-6 column. Alternatively, the ascites containing the IgM was ammonium sulfate precipitated and chromatographed on the Superose-6 column under normal- as well as high-ionic strength conditions, which also yielded a product of ca. 90% purity. The purification of IgM from concentrated cell culture supernatants was evaluated using the Superose-6 or the ABx column. IgM purified from this source was greater than 99% pure when chromatographed on the mixed-mode column and ca. 60% pure on the size-exclusion column. MAbs from each of the procedures retained their immunoreactivity, as shown by indirect immunofluorescence staining of fixed cell preparations. A comparison of these methods revealed that mixed mode chromatography was simple, efficient, and yielded a product of high purity. The optimization of these methods facilitates the large-scale purification of mouse IgM MAbs and provides practical procedures for generating IgMs for use as diagnostic and therapeutic reagents.
在腹水或细胞培养上清液中产生的9种免疫球蛋白M（IgM）单克隆抗体（MAb），已使用阴离子交换、尺寸排阻或混合模式色谱基质在快速蛋白质液相色谱（FPLC）系统上进行了纯化。使用混合模式ABx柱，单步纯化后可得到纯度大于99%的IgM。使用Mono Q柱进行阴离子交换色谱，可对IgM进行部分纯化，随后通过在Superose-6柱上进行尺寸排阻色谱，可将其纯化至约90%纯度的产物（由十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定）。或者，将含有IgM的腹水进行硫酸铵沉淀，并在正常和高离子强度条件下在Superose-6柱上进行色谱分离，也可得到约90%纯度的产物。使用Superose-6柱或ABx柱评估了从浓缩细胞培养上清液中纯化IgM的方法。从该来源纯化的IgM在混合模式柱上进行色谱分离时纯度大于99%，在尺寸排阻柱上纯度约为60%。如固定细胞制剂的间接免疫荧光染色所示，各方法得到的MAb均保留了其免疫反应性。这些方法的比较表明，混合模式色谱简单、高效，且产物纯度高。这些方法的优化有助于大规模纯化小鼠IgM MAb，并为制备用作诊断和治疗试剂的IgM提供了实用方法。
