Abstract
Administration of MoAbs to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) can alter the course of infection in mouse models. However, the effectiveness of these antibodies appears to depend on isotype and specificity. Comparison of isotype protection efficacy requires families of MoAbs with identical fine specificity and different constant region domain. The generation of such families by hybridoma technology is not always possible because the immune response produces MoAbs of limited classes or subclasses. In these instances isotype switch variants can be isolated in vitro. Unfortunately, standard methods of recovering spontaneous switch variants are often unsuccessful, mainly because of the low frequency of switching. In this study we demonstrate that acridine orange stimulation of an IgG3 anti-C. neoformans-producing hybridoma can be used to recover the entire set of isotype switch variants: IgGl, IgG2b, IgG2a, IgE and IgA. All isotype switch variants bind to GXM; fine specificity mapping, using an 11 amino acid peptide polysaccharide mimetope, revealed conservation of binding site specificity. Furthermore, all isotype switch variants reacted with an anti-idiotopic MoAb. The functional activity of this set of MoAbs was demonstrated by their ability to enhance phagocytosis and antifungal efficacy of human macrophage-like THP-1 cells, with IgG3 being the most effective and IgE being the least effective.