A zirconia-based column chromatography system optimized for the purification of IgM from hybridoma culture...

By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient concentrating and purifying of IgM from hybridoma culture supernatants. Hybridoma culture supernatants containing IgMs were diluted 3-fold with 10 mM phosphate buffer (pH 7.0) and passed through the column. During this process, zirconia particles selectively adsorbed these IgMs, and most of the contaminating proteins flowed out into the flow-through. The adsorbed IgMs were easily eluted with a small volume of 400 mM phosphate buffer (pH 8.0), and high-concentration IgM solutions were prepared. Subsequent simple processing using a Capto™ Core 400 cartridge column provided highly purified IgM. The operation is easy, and the activity of IgM is maintained because the purification process is performed using only neutral ranges of phosphate buffers. Here, we showed that anti-globoside and anti-CDw75 IgM purified by this method can be used to stain cervical cancer and Burkitt lymphoma cells that specifically express these respective tumor-associated carbohydrate antigens.