Identification of the active precipitin components in a purified preparation of the A antigen of Blastomyces dermatitidis

Abstract

A purified A-antigen preparation of Blastomyces dermatitidis was determined to be composed of five major glycoprotein bands, visible with Coomassie blue and periodic acid-Schiff staining of polyacrylamide gels. At least 20 additional protein bands were detected by using a silver stain, which was 100 times more sensitive than the Coomassie method. Two components of this mixture were determined to be associated with the A-antigenic activity of B. dermatitidis. Of several antigen preparations examined in Ouchterlony precipitation tests, those reactive with a reference anti-A antiserum contained the slowest moving of the Coomassie blue bands. The antigen preparations without precipitin reactivity lacked this protein band. Two protein bands were shown to disappear from an antigen preparation after incubation with an affinity gel linked to the reference anti-A serum. One of the bands was the slowest Coomassie blue band, and the other was a fast-migrating protein detectable only with the silver stain. Characterization of the components responsible for the A-antigenic activity has important applications in the production and standardization of serological reagents for the diagnosis of blastomycosis.

Immunological and chemical characterization of glycoproteins in IEF fractions of Blastomyces dermatitidis yeast lysate antigen

“…[ 161, but Hurst and Kaufman [ 131 showed that these epitopes in an A antigen fraction of B. dematitidis were resistant even to rather strong periodate oxidation. Because of the disparity between these two closely related organisms, this study was designed to look for the presence of cross-reactive carbohydrate moieties in the mycoses 40, 83-90 (1997) Young and Larsh [28] showed the presence of five glycoprotein bands in their B. dermatitidis cytoplasmic antigen, which may correspond to the five glycoproteins described here. Unfortunately, these investigators did not determine the molecular masses of the glycoproteins, nor were the bands individually screened for cross-reactivity, so no accurate comparisons could be made with regard to similarities among these components.…”

Production and characterization of a hybridoma-derived antibody to Blastomyces dermatitidis

“…Although the A and ASWS preparations have improved the diagnosis of blastomycosis (4,8), they still contain numerous separate proteins (5,16; K. D. Young and H. W. Larsh, Mycopathologia, in press). It is possible that these polyvalent preparations may lack or obscure important protective or diagnostic components (2).…”

“…prepared by mechanical disruption of B. dermatitidis yeast cells in a Braun homogenizer as previously described (16) Okla.). This 009BY preparation was derived from Merthiolate-killed yeast cells of the Centers for Disease Control isolate A 373 and contained 1.0 mg of protein per ml.…”

“…Additional procedures. Polyacrylamide gel electrophoresis, Ouchterlony immunodiffusion, and affinity chromatography have been described previously (14,16). Rabbit anti-mouse IgG, anti-mouse IgM, and pure mouse IgG and IgM from myeloma lines MOPC-21 and MOPC-104C were obtained from Bionetics Laboratory Products.…”

“…The solid arrow designates the protein which corresponds to the band eluted from an OU32/B6 antibody-linked affinity column. The open arrow marks the position of the slowest-moving specific component of purified A antigen(16). DF, Dye front; C, protein bands which also stain for carbohydrate by the periodic acid-Schiff procedure.…”