Purification of a murine monoclonal antibody of the IgM class

Abstract

• A simple method is described for the purification of murine monoclonal antibodies of the IgM class. Ascites fluid was subjected to ammonium sulfate precipitation. The precipitate was redissolved and dialyzed and subsequently subjected to gel filtration chromatography on Ultrogel AcA22 and finally ion exchange chromatography on DEAE-Sepharose. The purity of the antibody was assessed by reducing SDS gel electrophoresis, and estimated to be greater than 85% pure after gel filtration, and greater than 95% pure after the ion exchange chromatography. The Immuno-activity of the antibody was assessed throughout the purification scheme by the ability of the antibody to bind to immunogen immobilized to nitrocellulose. • Approximately 50% antibody activity was recovered from this purification scheme after gel filtration, but only 10% of the initial activity could be recovered after ion exchange chromatography. This result with ion exchange chromatography underscores the lability of IgM antibodies to immobilization. Therefore, if the recovery of immunoreactive IgM antibody is a goal, purification schemes of IgM antibodies should avoid ion exchange or affinity chromatography.