Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide

Abstract

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin Gl isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.

The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies.

“…(27) . For the ak, calculation, we assumed a molecular mass of 800,000 daltons for the polysaccharide (41) . For 4H3, an IgG3 mAb, we observed considerable variability in the shape of the curves, and suspect that this variability reflects cooperative binding effects that can occur with this subclass (26) .…”

Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay

“…In recent years, monoclonal antibodies (MAbs) specific for GXM have been generated in various laboratories. The available MAbs have been studied by enzyme-linked immunosorbent assay (ELISA) (8,11,17,24,25,46,48), immunodiffusion (24,25,46), and immunofluorescence (23-25, 41, 48). Several MAbs are now used in diagnostic tests for capsular polysaccharide (28,33,47).…”

“…Preliminary studies of serotype specificities of the anti-C. neoformans MAbs against GXMs have been done (8,9,11,24,25,46,48). However, a comprehensive comparison of the specificities of the various MAbs under standardized conditions has not been reported.…”

Redistribution of a glucuronoxylomannan epitope towards the capsule surface coincides with Titanisation in the human fungal pathogenCryptococcus neoformans

“…Identical gene segment usage is not entirely surprising given the restricted set of antibody gene segments utilised by antibodies specific to capsular polysaccharides (29), however the two mAbs were produced in response to GXM derived from different serotypes of Cryptococcus . Whereas mAb 302 was generated following the immunisation of a mouse with serotype D GXM (ATCC 24064) (43), we generated Crp127 through the immunisation of a mouse with a cocktail containing both serotype A (H99) and serotype B (R265) GXM. Whichever serotype of GXM activated the B cell from which Crp127 derives, the sequence similarities between mAbs Crp127 and 302 demonstrate that nearly identical antibodies can be elicited during infection by at least two different serotypes of Cryptococcus .…”