Immunochemical studies of Aspergillus fumigatus mycelial antigens by polyacrylamide gel electrophoresis and Western blotting techniques

Abstract

Differences were detectable among strains of the opportunist fungal pathogen Aspergillus fumgatus when watersoluble (WS) preparations were analysed by combined SDS-PAGE and Western blotting procedures. A wide range of molecules of apparent molecular masses from approximately 20 to > lo0 kDa showed specific binding to antibodies raised in rabbits to A. fumgatus wall and cytoplasmic components. The ability to bind antibody was markedly reduced by treatment of these antigens with sodium periodate or with specific proteases or glucanases. Pretreatment of blotted antigens with either concanavalin A (ConA) or wheat germ agglutinin (WGA) did not, however, inhibit subsequent antibody binding. The antigens of subfractions prepared from a single strain of A. fumigatus WS material were also susceptible to periodate oxidation and enzymic hydrolysis. Slight crossreactivity was apparent when crude preparations of cellular or culture filtrate antigens, used in this laboratory to detect antibodies to C d i h albkarts, Coccidioides immitis and Cryptococcus neoformans, were probed with hyperimmune rabbit antisera to A. fumigatus. Efforts were made to characterize the WS preparations of A. fumgatus, used as diagnostic antigens in many laboratories. The electrophoretically separated antigenic moieties were shown to be predominantly glycoproteins. Binding of cytoplasmic antigens to antibodies raised to wall material showed the presence of many common components in both wall and cytosol. Antiserum to wall components revealed most differentiation among A. fumigatus strains.

Cross-Reactivity between <i>Aspergillus fumigatus</i> and <i>Penicillium</i>

“…Most of the A. fumigatus antigens arc glycoproteins [26] and several of them contain galactofuranosyl side chains [ 13]. It seems obvious to assume that anti-Penicillium reacts predominantly with these carbohydrate antigens, because they occur for example in Aspergillus, Penicillium and Tri chophyton [13].…”

Use of Recombinant Mitogillin for Improved Serodiagnosis ofAspergillus fumigatus-Associated Diseases

“…Most immunoassays for detection of circulating antibodies are based on crude extracts of A. fumigatus. These extracts contain complex and undefined mixtures of polysaccharide and protein components and are derived from various sources such as conidial, mycelial, cytoplasmic, metabolic, or cell wall fractions of the fungus (13)(14)(15)28). Consequently, there is little knowledge about the role of the humoral immune response, the cross-reactivities of different epitopes in these crude antigenic preparations, the diagnostic value of class-specific antibody detection, and antibody kinetics in human aspergillosis (7).…”

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“…Various staining methods such as immunoperoxidase staining [6] and Gomori's methenamine silver (GMS) stain [2] are also used for detecting pathogens. The use of other diagnostic methods such as auto¯uorescence [7], PCR [8], thin-layer chromatography and high-performance liquid chromatography for toxin detection [9], SDS-PAGE and Western blotting procedures [10] has been reported. In practice, the detection of pathogenicity of Aspergillus spp.…”