On-line post-capillary affinity detection of immunoglobulin G subclasses and monoclonal antibody variants for...

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-alpha 1-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.