Analytical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies

Protein A based rapid affinity chromatography for quantitation of various immunoglobulins of class G (IgG) is described. Three-minute analysis using either citrate or phosphate buffers and detection with 220- or 280-nm ultraviolet absorption was found to be optimum for quantitation of IgG from 0.25 to 250 micrograms of IgG on-column with a percent relative standard deviation (% RSD) of 2-3% RSD. The method has a detection limit estimated to be 100 ng of IgG on-column. It has been used to analyze a variety of IgG-containing samples from such diverse sources as hybridoma selection, media cultivation, and purification studies. Gradient elution studies and the relationship of IgG elution to IgG isoelectric point (pI) are also described.