Production of Monoclonal Antibodies for Detection of a Secreted Aspartyl Proteinase fromCandidaspp. in Biologic Specimens

Abstract

Secreted acid proteinases (SAP) constitute an important group of virulence factors in Candida albicans. In the present work, an acid proteinase from C. albicans was sequentially purified from the supernatant of a yeast culture by precipitation with ammonium sulfate, ion exchange chromatography, and molecular exclusion chromatography, yielding a specific enzymatic activity of 204.1 IU/mg on bovine serum albumin (BSA). The molecular mass of the purified proteinase was estimated at 43 kd after exclusion chromatography and at 41 kd by nondenaturating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteinase was able to degrade BSA at pH 2.5, but was not active on collagen, and it was significantly inhibited by pepstatin A. The immunization of BALB/c mice with the purified proteinase and later fusion of their spleen cells with myeloma cells resulted in 19 monoclonal antibody secreting hybridomas (MAbs) capable of detecting SAP in enzyme-linked immunosorbent assay (ELISA) assays. All MAbs obtained are isotype IgG1 kappa (kappa) immunoglobulins and develop a 41 kd protein band by Western blot (WB) in samples of SAP obtained from C. albicans (12-A) and C. dubliniensis (strain 778) crude extracts. The anti-SAP MAbs were used in capture ELISA and two combinations of these antibodies proved suitable for SAP detection, that is, MAP1 (1B1B3) or MAP2 (2D2C10) as coat antibodies, and biotinylated MAP3 (2A6E8) as detect antibody. Capture ELISA using these sets of MAbs detected over 32 ng/mL protein in purified SAP samples as well as in crude C. albicans and C. dubliniensis extracts. The results herein obtained allow for the prediction of how this set of antibodies can be useful for SAP detection in biologic specimens.

Novel nanoscale bacteriophage-based single-domain antibodies for the therapy of systemic infection caused by Candida albicans

“…However, another two bands were detected in total cell lysates, which were also observed when reacted with anti-rSap2 pAb. These indicated that the scFv-phages might only target Sap2, and the two lower molecular weight bands might be the autodegradation components of Sap2 as reported before31. Moreover, the cross reaction of anti-rSap2 scFv-phages against cell wall lysates was also excluded as no band was observed (Fig.…”

Coriandrum sativum L. (Coriander) Essential Oil: Antifungal Activity and Mode of Action on Candida spp., and Molecular Targets Affected in Human Whole-Genome Expression

“…By using circular end of a 8-mm-diameter properly sterilized glass rod, yeasts colonies were picked up from Agar Sabouraud Dextrose plates and printed onto Petri plates containing BSA/YNB agar (0.2% bovine-serum albumin, 1.45 g Yeast Nitrogen Base without ammonium sulfate and amino acids, 20 g glucose, 20 g agar per liter) at pH 4.0. Plates were incubated at 37°C for 72 hrs [26]. The presence of proteases was detected by the production of a translucent zone around the yeast colony.…”

Kinin release from human kininogen by 10 aspartic proteases produced by pathogenic yeast Candida albicans

“…By applying multiple infection models and different methodologies, the available data are both diverse and controversial. Moreover, most studies primarily focus on the expression of Sap-encoding mRNA [ 50 , 51 ] and only occasionally quantify protein products using western blot analysis [ 52 ], but these studies do not reveal anything about the in situ proteolytic activities of individual Saps. However, the hypothesis that soluble Sap3 alone, or membrane-bound Sap9 with the assistance of several soluble Saps, controls kinins at the sites of candidal infections seems to be relatively insensitive to the discrepancies in the literature because it is difficult to find any studies that definitely exclude the expression of both SAP3 and SAP9 from any infection model.…”