In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens

Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were f ractionated in a fast protein liquid chromatography (FPLC) system, using Q- Sepharose anion-exchange resin, in order to characterize antigenic fraction s that could elicit cell reactivity and antibody recognition in human parac occidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solut ion (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system wa s able to resolve 7 fractions, enumerated from 0 to VI, according to the el ution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showe d that fractions 0 to V were constituted by multiple protein bands with mol ecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contai ned antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to inve stigate the capacity of these fractions to induce cell proliferation. It wa s demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononu clear cells, while fraction 0 induced the lowest proliferative response amo ng patients with PCM, in either acute, acute treated, or chronic forms. (C) Elsevier, Paris.