Production of polyclonal antibody against ergosterol hemisuccinate using Freund's and Titermax adjuvants

Abstract

Ergosterol is the principal sterol in fungi and an important component of cell membranes. This sterol has been used previously to measure fungal growth in foods. In an attempt to develop antibodies for an immunoassay for ergosterol, we first derivatized the compound to its hemisuccinate (Erg-HS) and conjugated it to bovine serum albumin (Erg-HS-BSA). Six white female New Zealand rabbits were immunized by subcutaneous injections of Erg-HS-BSA conjugate followed by four booster injections. Freund's and Hunter's TiterMax adjuvants were used for stimulating strong and prolonged responses. An indirect enzyme-inked immunosorbent assay (ELISA) was used to assess the titers. Antibody titers to Erg-HS rose much earlier and were markedly higher when Freund's adjuvant was used than when TiterMax was used. Although titers as high as 6,400 were detectable by indirect ELISA, binding of these antibodies to an Erg-HS-BSA solid phase could not be inhibited by free ergosterol in a competitive indirect ELISA. The antibody was also evaluated for applicability in an enzyme-linked immunocytochemical assay for a variety of fungi but failed to detect the presence of ergosterol in the membranes of these organisms. Thus, although antibodies could be generated that showed high specificity for the Erg-HS-BSA conjugate, they could not detect free ergosterol.