Immunoaffinity isolation and partial characterization of the Coccidioides immitis antigen detected by the tube precipitin and immunodiffusion-tube precipitin tests.
The antigen participating in the tube precipitin (TP) serologic test for coccidioidomycosis was isolated from mycelial-phase antigen (coccidioidin) by immunoaffinity and characterized by various analytical procedures. This was accomplished by first preparing the antigen-antibody precipitate by using antigen and human serum positive for TP (immunoglobulin M) antibody and then liberating the antigen by digestion with pronase. This protease destroyed the antibody and left the antigen intact as indicated by immunodiffusion-TP. The coccidioidal antigen was isolated from the proteolytic digest by using size exclusion chromatography. DEAE chromatography of this antigen yielded two fractions with immunodiffusion-TP reactivity which had average molecular sizes of 225 and 140 kilodaltons, respectively. The presence of carbohydrate and amino acids indicated that the antigen(s) is a glycopeptide. Compositional analysis showed that one fraction contained 3-O-methylmannose, mannose, and glucose in a ratio of 8:1.2:1, whereas the second fraction contained 3-O-methylmannose, mannose, glucose, and galactose in a ratio of 1:1:1:1. The amino acids glycine, alanine, serine, threonine, aspartic acid plus asparagine, and glutamic acid plus glutamine constituted 60 to 70% of the amino acids in both glycopeptides. Neither antigen could be detected entering the gel in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lectin affinity provided evidence of a high-mannose asparagine-linked glycopeptide in the first peak and an asparagine-linked glycopeptide with a biantennary complex-type structure in the second peak.
Zimmer
B L
BL
Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616.
Pappagianis
D
D
D000596:Amino Acids / Q000032:analysis; D000946:Antigens, Fungal / Q000032:analysis / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003045:Coccidioides / Q000032:analysis / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D011233:Precipitin Tests
D000596:Amino Acids; D000946:Antigens, Fungal