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-peroxidase (PAP) preparations is necessary for the proper interpretation of PAP applications based on competitive binding assay. Capillary zone electrophoresis with field 300 V/cm, 40 cm capillary length (20 cm effective length), and high-performance size exclusion chromatography equipped with Superose 12 HR10/30 column revealed that a PAP preparation used for Fc gamma receptor studies contained multiple sizes of immune complexes, an excess amount of free peroxidase, and little or no free anti-peroxidase antibody. The antibody: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 well as quantitative analysis of PAP preparations.
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Department of Immunology, Sidney Kimmel Cancer Center, San Diego, CA 92121, USA.
Lei
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J
Xu
C
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Regnier
F
F
Kreisle
R
R
D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D019075:Electrophoresis, Capillary / Q000379:methods*; D004727:Endothelium / Q000166:cytology / Q000276:immunology; D006801:Humans; D007105:Immune Complex Diseases / Q000276:immunology; D009195:Peroxidase / Q000276:immunology*; D017452:Receptors, IgG / Q000032:analysis
D000936:Antigen-Antibody Complex; D017452:Receptors, IgG; D009195:Peroxidase