Open AccessGeneration of biologically active anti‐ Cryptococcus neoformans IgG, IgE and IgA isotype switch variant antibodies by acridine orange mutagenesis
Author(s) -
SPIRA G.,
PAIZI M.,
MAZAR S.,
NUSSBAUM G.,
MUKHERJEE S.,
CASADEVALL A.
Publication year - 1996
Publication title -
clinical & experimental immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.329
H-Index - 135
eISSN - 1365-2249
pISSN - 0009-9104
DOI - 10.1046/j.1365-2249.1996.d01-786.x
Subject(s) - isotype , cryptococcus neoformans , immunoglobulin e , immunology , acridine orange , antibody , biology , virology , microbiology and biotechnology , monoclonal antibody , genetics , staining
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: IgG1, 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 anti‐fungal efficacy of human macrophage‐like THP‐1 cells, with IgG3 being the most effective and IgE being the least effective.