Preparation of human sera containing one single IgG subclass using affinity chromatography.
Monoclonal antibodies reactive against one or three human IgG subclasses were immobilised on agarose beads, put into columns and used in combination with coupled polyclonal anti-IgA and anti-IgM reactive gels to obtain serum preparations devoid of IgA, IgM and all but one IgG subclass. This was possible after one single run over the appropriate combination of columns; only for IgG4 preparations was a second purification step sometimes required to reach purity. Negative affinity chromatography was used throughout, thus the resulting preparations had not been exposed to high ionic strength conditions, non-physiological pH buffers or chaotropic agents in concentrations ordinarily used to elute bound material from affinity columns. The yield was approximately 50% and 20-30% if two runs were required. Regeneration of the columns permitted repetitive use, so far up to 30 times without substantial loss of activity. The protocol offers an easy, comparatively fast and reproducible method to obtain human serum preparations containing only IgG1,2,3, or 4.
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007070:Immunoglobulin A / Q000302:isolation & purification; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D007074:Immunoglobulin G / Q000145:classification / Q000302:isolation & purification*; D007075:Immunoglobulin M / Q000302:isolation & purification; D009994:Osmolar Concentration
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007075:Immunoglobulin M