Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Abstract Background Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility. Aims In this study, we sought to optimize the methodology for producing H. capsulatum antigens, and to evaluate its applicability. Methods Antigenic preparations obtained from 12 H. capsulatum isolates were evaluated by double immunodiffusion and immunoblotting assays against homologous and heterologous sera. Results The evaluated and optimized protocol allowed a more stable production, as well as repeatable, reproducible, with shorter culture time and less costly. By double immunodiffusion and immunoblotting assays, the best pattern of reactivity was observed for antigens obtained with 33 days of culture from the isolates 200 and 406 against the M antigen and for the isolate 200 with 15 days against H antigen. The SDS-PAGE presented antigenic components of molecular masses between 17 and 119 kDa. The immunoblotting sensitivity was 95.5% and 100% with histoplasmosis sera from ill patients and sera from H. capsulatum infected but otherwise healthy patients, respectively, to the antigen derived from isolates 200 and 406. Conclusions We suggest the employment of the antigen from isolate 200, with 15 or 30 days of culture, in the double immunodiffusion and immunoblotting assays due to its good ability to discriminate both sera from patients with histoplasmosis illness and histoplasmosis infection, in addition to its high specificity against heterologous sera.