Rapid separation of IgM from whole serum using spun column chromatography

Spun columns were used to separate IgM from serum samples by exploiting molecular weight and size difference between IgM and other serum antibodies. Miniature gel chromatography columns were prepared in 3 ml syringe barrels. A polyacrylamide liquid chromatography gel, Bio-Gel P-200, was chosen to exclude proteins with molecular weights greater than 200,000. IgM, 900,000 MW, was excluded while IgG, 150,000 MW, was retained in the gel column. Centrifugation of the serum-loaded columns in test tubes accomplished the separation in one step by eluting a void volume equal to the sample volume from each column into a holding tube. IgM recovery in the eluent exceeded 96% of the total serum IgM in the pre-column sample. No IgG was detected in the eluent. IgM separated from La Crosse encephalitis immune human serum retained immunological activity in a viral neutralization test. Spun column chromatography is eminently suitable for diagnostic laboratories as more than 100 sera may be fractionated in one day using inexpensive materials and a low-speed centrifuge equipped with a swinging-bucket rotor.