Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen

Abstract

Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

Cryptococcal meningitis with secondary cutaneous involvement in an immunocompetent host

“…Stains such as mucicarmine and Alcian blue are used to identify the polysaccharide capsule. A fungal colony can be grown on culture media and the varieties C. neoformans and C. gatti can be identified by a colour reaction on concanavalinglycine-thymol agar, by antibody kit for serotyping or by fingerprinting with DNA-based methods [16,17]. Cryptococcal polysaccharide antigen detection in CSF and serum is made by latex agglutination test or by ELISA.…”

Large-Scale Evaluation of the Immuno-Mycologics Lateral Flow and Enzyme-Linked Immunoassays for Detection of Cryptococcal Antigen in Serum and Cerebrospinal Fluid

“…Specimens displaying discordant results were reanalyzed with the latex-Cryptococcus antigen test (IMMY), a polyclonal LA test shown to detect serotypes A, B, C, and D (8). The manufacturer's instructions were modified slightly to accommodate repeat testing of lowvolume specimens.…”

“…Several of the assays tested, including the kit currently used in our laboratory, showed reduced sensitivity for serotype C GXM (8). The purpose of this study was to evaluate a new CrAg LFA and EIA (Immuno-Mycologics Inc.…”

“…Serotypes A and D make up the large majority of C. neoformans clinical isolates. Serotype B and C isolates are classified as C. gattii based on biochemical and molecular genetic features that differentiate them from serotype A and D isolates (7).The sensitivities of four commercially available CrAg immunoassays were recently evaluated using purified GXM isolated from serotype A, B, C, and D strains (8). Several of the assays tested, including the kit currently used in our laboratory, showed reduced sensitivity for serotype C GXM (8).…”

“…The sensitivities of four commercially available CrAg immunoassays were recently evaluated using purified GXM isolated from serotype A, B, C, and D strains (8). Several of the assays tested, including the kit currently used in our laboratory, showed reduced sensitivity for serotype C GXM (8).…”