Combined use of Paracoccidioides brasiliensis recombinant rPb27 and rPb40 antigens in an enzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosis.

Abstract

Paracoccidioidomycosis (PCM) is one of themost important endemicmycoses in LatinAmerica; it's usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified,well-characterized antigens appears preferable andmay yield good results. In the present study combinations of the previously described 27-kDa recombinant antigen(rPb27) and a recombinant 40-kDa-molecular-mass antigen(rPb40) fromthis fungus, that was identified by Goes et al. (2005) through the AST strategy as a homolog of Neurospora crassa calcineurin B, were used in an indirect enzyme linked immunosorbent assay (ELISA) for diagnosis and follow-up of patients with PCM. The complete coding cDNA of rPb40 and rPb27 were cloned into a pET-21a and a pET-DEST 42 plasmid, respectively, expressed in E. coli with a his-tag and purified by affinity chromatography. Among 109 PCM serum samples analyzed, a homogeneous IgG response to these proteinswas observed. 62 serumsamples frompatientswith other diseases, 18 frompatientswith othermycosis and 23 fromhealthy individualswere also studied. Detection of anti-rPb27 and anti-rPb40 antibodies in sera of patientswith PCMby ELISA using a combination of the two purified proteins showed a sensitivity of 96% with a specificity of 100% in relation to control normal human sera andto sera frompatientswith other systemicmycosis and 93.5%to sera from patients with diverse infections. The use of this two proteins combination provided an excellent immunodiagnosis assaywith great values of sensitivity and specificity, even in relation to sera frompatients with othermycosis,making possible the standadization of a new methodology to diagnose this important mycosis, with a good confiability and reprodutibility.