Purification of immunoprecipitated pemphigus foliaceus antigen fragment and its use in radioimmunoassay.
A major difficulty in biochemical studies of the pemphigus foliaceus (PF) antigen is the lack of a method for its quantitative determination. Immunofluorescence blocking and immunoprecipitation methods are semiquantitative and time consuming. Radioimmunoassay (RIA) methods are quantitative but they require pure and stable antigen preparations that have not been available for PF. The present investigation shows the further purification of a previously described preparation of PF antigen fragment obtained from trypsinization media of mouse skin (Con A Frn) and demonstrates its usefulness in a RIA method for quantitation of the antigen. The major contaminants of the 45-kD tryptic fragment of the PF antigen (tf-PF) in immunoprecipitates of the Con A Frn with PF sera were identified as H and L chains of murine IgG and mannose-binding lectins. The IgG contaminants could be removed by avoidance of blood contamination during preparation of the Con A Frn and/or pre-absorption of the Con A Frn with protein A Sepharose. The lectins could be removed by affinity chromatography of the Con A Frn on asialofetuin column and washing the immunoprecipitates with 0.2 M alpha-methyl-mannoside. Using the purified, labeled Con A Frn in RIA, we showed that standard curves could be established and the amounts of PF antigen could be determined in different extracts without the need for electrophoresis, autoradiography, or scanning. This RIA method is rapid and can be easily used to analyze many samples, e.g., chromatographic fractions and extracts made from different tissues.
D000818:Animals; D000941:Antigens / Q000302:isolation & purification*; D001323:Autoantibodies / Q000032:analysis; D000939:Epitopes / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010392:Pemphigus / Q000276:immunology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011233:Precipitin Tests; D011863:Radioimmunoassay / Q000379:methods
D000941:Antigens; D001323:Autoantibodies; D000939:Epitopes; D010446:Peptide Fragments