Immunoaffinity centrifugal precipitation chromatography.
Purification of proteins based on immunoaffinity has been performed using a solid support coated with antibody against the target proteins. The method requires immobilizing the antibody onto the solid support using protein A or G, and has a risk of adsorptive loss of target proteins onto the solid support. Centrifugal precipitation chromatography has been successfully used to purify enzymes, such as ketosteroid isomerase and hyaluronidase without the use of solid support. The purpose of this study is to demonstrate that immunoaffinity centrifugal precipitation chromatography is capable of isolating an antigen by exploiting antigen-antibody binding. The separation was initiated by filling both channels with 40% saturated ammonium sulfate (AS) of pH 4-4.5 followed by loading 20 microl of human plasma (National Institutes of Health blood bank) mixed with 2 mg of rabbit anti-HSA (human serum protein) antibody (Sigma). Then, the sample channel was eluted with water at 0.03 ml/min and AS channel with 40% AS solution of pH 4-4.5 at 1 ml/min until all non-binding components were eluted. Then, the releasing reagent (50% AS solution containing 0.5 M glycine and 10% ammonium hydroxide at pH 10) was introduced through the AS channel to release the target protein (HSA). The retained antibody was recovered by eluting the sample channel with water at 1 ml/min. A hollow fiber membrane device at the outlet (MicroKros, Spectrum, New Brunswick, NJ, USA) was provided on-line dialysis of the eluent before fractions were collected, so that the fractions could be analyzed by SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) without further dialysis. The current method does not require immobilizing the antibody onto a matrix, which is used by the conventional immunoaffinity chromatography. This method ensures full recovery of the antigen and antibody, and it may be applied to purification of other proteins.
Qi
Lin
L
Office of New Drug Quality Assessment, OPS/CDER/FDA, 10903 New Hampshire Avenue, Silver Spring, MD 20993-0002, USA.
Ito
Yoichiro
Y
D000645:Ammonium Sulfate / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D012709:Serum Albumin / Q000737:chemistry
D000906:Antibodies; D012709:Serum Albumin; D000645:Ammonium Sulfate