Centrifugal Precipitation Chromatography

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A new application of centrifugal precipitation chromatography for affinity separation has been demonstrated. The method uses an antibody to precipitate the analyte protein which is then dissociated by a releasing agent to elute from the column while the antibody is recovered later from the column contents. The test system uses human serum albumin as an antigen (analyte) and a rabbit antihuman IgG as an antibody. The experiment was initiated by filling the column (both channels) with 40% saturated ammonium sulfate (AS) in a phosphate buffer (pH 4.3) followed by injection of the sample mixture ( 0.2 mg of HSA + anti-HSA in one ml of distilled water) into the sample channel which was eluted with water at 0.3 ml/min. The effluent was continuously monitored by a UV detector at 280 nm and collected into test tube using a fraction collector. After an excess amount of HSA was eluted out, the mobile phase was switched with the releaser solvent of 40-50% AS solution containing 0.5M glycine at pH 10 adjusted by ammonium hydroxide to dissociate the antigen (HSA) from the antibody. The fractionated HSA is analyzed by SDS PAGE. The method was successfully applied to purification of HSA from human serum. The present method has an advantage over conventional affinity column chromatography in that the antibody is directly used for purification of the target protein without using the solid support.