The separation of immunoglobulin M from human serum by fast protein liquid chromatography

A fast protein liquid chromatography (FPLC) system was evaluated as a method for rapid separation of serum immunoglobulin M (IgM) from immunoglobulin G (IgG) and immunoglobulin A (IgA). The system incorporates the use of a strong anion exchanger. Evaluation was carried out in 3 ways. The effect of increasing the serum percentage in the 500 microliters volumes loaded on to the column was tested. Samples containing up to 60% serum resulted in only small concentrations of contaminating IgG and IgA in the IgM fraction. Reproducibility was tested by fractionating the same serum sample several times; the coefficient of variation (CV) of the IgM concentration in the IgM fraction was 6%. A number of sera which varied considerably in immunoglobulin concentration were fractionated without any significant adverse effects on the immunoglobulin ratios in the IgM fraction. One serum sample containing a high concentration of IgG and IgA was included. In contrast to gel filtration chromatography, FPLC can separate IgM from IgG and IgA within 6 min. On loading 500 microliters samples containing from 20 to 60% serum, less than 0.01 g/l IgG was detected in the IgM fractions when tested by the radial immunodiffusion method.