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Production of Paracoccidioides brasiliensis exoantigens for immunodiffusion tests
Z De Camargo1, C Unterkircher, S P Campoy
1Disciplina de Micologia, Escola Paulista de Medicina, São Paulo, Brazil.
Abstract:
Growth curves of the yeast form of Paracoccidioides brasiliensis B-339 based on total and viable cell counts were determined. Crude culture filtrate antigens were obtained after 7, 10, 15, 20, 25, and 30 days of incubation. Different patterns of proteins were obtained by affinity chromatography on Sepharose 4B-immunoglobulin G complex made with immunoglobulin G from patients with paracoccidioidomycosis, with subsequent analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Three major proteins were excreted during the time course of a 30-day culture: a doublet at 20 to 21 kilodaltons (kDa) and molecules of 43 and 52 kDa. The 43-kDa antigen was present throughout the growth period, and its level reached a peak on days 15 to 20 and then decreased considerably toward day 30. The antigenic preparations collected on days 7, 10, 15, and 20 gave better reactions in immunodiffusion tests than those collected on days 25 and 30. The 7-day exoantigen gave a sensitivity of 97.1% and specificity of 100% on immunodiffusion. The main line of precipitation had a very high intensity, showing a total identity with that of a previously purified glycoprotein of 43 kDa. A 7-day crude exoantigen displayed a high level of sensitivity and specificity, being reproducible from batch to batch and retaining its activity for years when kept lyophilized. A protocol is recommended for the production of a stable diagnostic antigen to be used in immunodiffusion tests for paracoccidioidomycosis.
Insights
A 7-day crude exoantigen from Paracoccidioides brasiliensis demonstrated high sensitivity and specificity for diagnosing paracoccidioidomycosis. This stable, reproducible antigen is recommended for immunodiffusion tests, offering a reliable diagnostic tool.
Area of Science:
- Mycology
- Immunology
- Biochemistry
Background:
- Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis.
- Accurate serological diagnostic tools are crucial for effective disease management.
- Characterizing fungal antigens is essential for developing sensitive and specific diagnostic assays.
Purpose of the Study:
- To determine the growth curves of Paracoccidioides brasiliensis yeast.
- To identify and characterize major excreted antigens during culture.
- To evaluate the diagnostic potential of crude exoantigens in immunodiffusion tests.
Main Methods:
- Growth curves determined by total and viable cell counts.
- Crude culture filtrate antigens collected at various incubation days (7-30).
- Protein analysis using affinity chromatography, SDS-PAGE, and scanning densitometry.
Main Results:
- Three major proteins identified: a 20-21 kDa doublet, 43 kDa, and 52 kDa.
- The 43 kDa antigen was consistently present, peaking at 15-20 days.
- Antigens from days 7-20 showed superior immunodiffusion reactions; 7-day antigen yielded 97.1% sensitivity and 100% specificity.
Conclusions:
- A 7-day crude exoantigen is a highly sensitive and specific diagnostic marker for paracoccidioidomycosis.
- This antigen is reproducible, stable when lyophilized, and suitable for immunodiffusion tests.
- A protocol for producing this stable diagnostic antigen is recommended.