Purification of ascitic fluid-derived murine monoclonal antibodies by anion-exchange and size-exclusion high-performance liquid chromatography.
Ascitic fluid-derived murine monoclonal antibodies (MoAbs) of immunoglobulin (Ig) M and IgG isotypes (IgG1 and IgG2a subisotypes) were previously prepared against an isolate of Actinobacillus sp (CAs8C) for the purpose of identifying and characterizing outer membrane antigens on this bacterium. An attempt was made to purify these MoAbs by anion-exchange and size exclusion high-performance liquid chromatography (HPLC). Hybridomas producing the IgG1 and IgG2a MoAbs posed unique difficulties in that they also secreted irrelevant IgG2b MoAbs that were present in the ascitic fluids. Anion-exchange chromatography (Protein-Pak DEAE-5PW column), with a simultaneous change in gradients of pH and ionic strength, was used to purify IgG and as a first step in the purification of IgM. There was good separation of IgG2b from IgG2a, but not from IgG1. Size-exclusion chromatography (Protein-Pak 300 SW column) was required to complete the purification of IgM. The presence of MoAbs in the HPLC fractions was confirmed by discontinuous gradient polyacrylamide gel electrophoresis (denatured and either reduced or non-reduced conditions) and the enzyme-linked immunosorbent assay. HPLC-purified MoAbs were free from transferrin and albumin and retained their specificity for As8C.
Hwang
H H
HH
Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University, Logan 84322-5600.
Healey
M C
MC
Johnston
A V
AV
D000188:Actinobacillus / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007202:Indicators and Reagents; D051379:Mice
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents