Preparation of combined extract of cell wall and cytosol antigens of Candida albicans for immunoblot analysis

Abstract

Immunoblot analysis is not in wide use for diagnosis of invasive candidiasis, mostly because the procedure is not standardized and hence not reliable. This work describes a standardized method for C. albicans antigen extract preparation and immunochemical detection. The major improvement of the method is the preparation of combined antigen extract - consisting of the cell wall and cytosol antigens. The fungal cells and lysis buffer were mixed at a 1:3 ratio and disintegrated by ultrasound for six cycles of one minute each. After centrifugation, cytosol antigens were obtained in the supernatant and cell wall antigens were in the precipitate. Precipitate was dissolved in lysis buffer with 2% sodium dodecyl sulfate (SDS) and boiled at 100°C for 2 min. After centrifugation, the supernatant was combined with the previous one, so the extract of the combined antigens was obtained in the mixture. With those combined antigen extracts and with sera of three different groups of patients, immunoblot analysis showed sensitivity of 90.2%, specificity of 84.4%, and accuracy of 88.0%.