Very Rapid Microanalysis of IgG in Ascites Fluids by HPLC Using a Novel Anion-Exchange Column

Abstract

An HPLC system has been optimized for the rapid resolution and quantitation of IgG in mouse ascites fluids, making use of newly developed HPLC columns (MA7P). Chromatography using MA7P columns is characterized by remarkably narrow band widths and very short retention times. The HPLC system is capable of analyzing the IgG content of a typical ascites fluid with a cycle-to-cycle time of under 5 minutes, regardless of IgG subtype. Separation times under 1 minute are possible. Recoveries of 100 μg injections of IgG and other proteins are quantitative. Using a UV monitor, the system was optimized to generate a linear plot of peak area vs. amount of IgG injected from 100 ng to 3 μg per injection. These properties make the MA7P column very useful for both analytical and micropreparative applications. Ascites fluids from eleven different IgG₁-producing hybridomas were analyzed. The IgG concentrations in those eleven ascites varied considerably. Retention times of the IgG varied slightly but significantly from hybridoma to hybridoma. Some monoclonals produce heterogeneous IgG. This heterogeneity can be detected by chromatography using MA7P columns.