Characterization of peroxidase : anti-peroxidase immune complexes by capillary zone electrophoresis and high-performance size-exclusion chromatography

Determination of the molecular constituents of commercial peroxidase:anti-p eroxidase (PAP) preparations is necessary for the proper interpretation of PAP applications based on competitive binding assay. Capillary zone electro phoresis with field 300 V/cm, 40 cm capillary length (20 cm effective lengt h), and high-performance size exclusion chromatography equipped with Supero se12 HR10/30 column revealed that a PAP preparation used for Fc gamma recep tor studies contained multiple sizes of immune complexes, an excess amount of free peroxidase, and little or no free anti-peroxidase antibody. The ant ibody:antigen ratios of the three major immune complex components were 2:2, 1:2, and 1:1. These techniques provide useful methods of qualitative, as w ell as quantitative analysis of PAP preparations. (C) 1999 Elsevier Science B.V. All rights reserved.