A rapid procedure for purifying IgM monoclonal antibodies from murine ascites using a DEAE-disk.
A method for purifying IgM monoclonal antibodies (mAbs) from murine ascites using a DEAE-disk is described. After ammonium sulfate precipitation, ascites proteins are redissolved and loaded onto a DEAE-disk. MAb then is eluted from the disk using a stepwise NaCl gradient. IgM mAb produced by this procedure was > 95% pure as assessed by reducing SDS-PAGE analysis and was free of significant IgG contamination as determined by double radial immunodiffusion analysis. Yield of IgM mAb was approximately 2% of total ascites protein and approximately 10% of the amount of IgM contained in crude ascites. MAb retained immunoreactivity as assessed by ELISA, and the affinity index was evaluated by thiocyanate elution and remained unchanged. This two step technique for purifying IgM mAb from murine ascites is rapid, simple, and yields mAb of sufficient purity and immunoreactivity for the majority of mAb applications.
Burd
R S
RS
Department of Surgery, University of Minnesota, Minneapolis 55455.
Raymond
C S
CS
Ratz
C A
CA
Dunn
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000276:immunology*; D011232:Chemical Precipitation; D004983:Ethanolamines; D007075:Immunoglobulin M / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology
D000911:Antibodies, Monoclonal; D004983:Ethanolamines; D007075:Immunoglobulin M; C007369:2-diethylaminoethanol