PURIFICATION OF 2 MURINE MONOCLONAL-ANTIBODIES OF THE IGM CLASS BY HYDROXYLAPATITE CHROMATOGRAPHY AND GEL-FILTRATION

A two-step method using hydroxylapatite chromatography and gel filtrat ion is described for the purification of two murine monoclonal antibod ies of the IgM class. Ascites fluid from each hybridoma was diluted in sodium phosphate buffer (0.01 M, pH 6.8), loaded onto a hydroxylapati te column and eluted with a stepwise sodium phosphate gradient. The im munoreactive protein peaks were concentrated and subjected to gel filt ration using either Sephadex G-200 or Sephacryl S-200HR. The biologica l activity of the end-products was confirmed by complement lysis assay and by indirect immunofluorescence and flow cytometry. The purity of the end-products as assessed by SDS polyacrylamide gel electrophoresis and densitometry was at least 90%. The methods described produced imm unoreactive material with a high level of purity. The procedure for ea ch antibody was reproducible and provides a reliable method for purifi cation of monoclonal antibodies of the IgM class.