Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose.
A three-stage process, consisting of an ammonium sulfate precipitation step, dialysis desalination with microporous anion-exchange Neosepta membranes and anion-exchange chromatography on DEAE-cellulose DE-52 was used for the isolation of mouse monoclonal antibodies specific against different antigens. The ascites fluids contained monoclonal antibodies against human IgG, against horseradish peroxidase and against the heavy chain of human IgM. The effect of the salt concentration gradient in the elution buffer was examined with the aim of optimizing chromatographic conditions. The quality of separation of protein zones was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The immunoreactivity of purified monoclonal antibodies was determined by enzyme-linked immunosorbent assay using a solid-phase adsorbed antigens against which each monoclonal antibody type was directed.
Tishchenko
G A
GA
Institute of Macromolecular Chemistry of the Academy of Sciences of the Czech Republic, Prague.
Bleha
M
M
Skvor
J
J
Bostík
T
T
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000276:immunology; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D012965:Sodium Chloride; D006735:Horseradish Peroxidase