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

Abstract

Cryptococcus neoformans glucuronoxylomannans (GXM) are capsular polysaccharides important for virulence in cryptococcosis. This study used dot enzyme assays (DEA) and enzyme-linked immunosorbent assays (ELISA) to determine the reactivity patterns of 21 murine monoclonal antibodies (MAbs) with structurally defined GXMs from five serotypes. The MAbs were categorized into eight groups on the basis of DEA and five groups on the basis of ELISA. MAbs 302, 339, and 439 were studied extensively for their binding to various native and chemically modified GXMs. Quantitative variation in the inhibitory effects of GXMs on the binding of MAbs 302, 339, and 439 were observed by competitive ELISA. O-Deacetylation of serotype A, B, and D GXM resulted in the complete loss of their inhibitory properties. Carboxyl group reduction of GXMs from serotypes A and D resulted in a significant decrease of inhibitory activity for MAb. Xylomannans and methyl glycosides exhibited no detectable inhibitory activity on MAb binding to GXM. The results indicate (i) the existence of five to eight MAb-defined distinct epitopes in C. neoformans GXM that can elicit antibody responses, (ii) MAb detection of antigenic variation within GXMs assigned to a particular serotype, (iii) good correspondence between the patterns of MAb reactivities and polyclonal rabbit factor sera, (iv) good agreement between MAb molecular structure and serotype reactivity, and (v) a dependence of the serotype reactivity profile for a given MAb on the technique used to measure binding.

Bivalency Is Required for Anticapsular Monoclonal Antibodies To Optimally Suppress Activation of the Alternative Complement Pathway by theCryptococcus neoformansCapsule

“…These results support the argument that cross-linking of the capsular matrix is a critical component of antibody-mediated suppression of the alternative pathway. The mildly anomalous behavior of Fab fragments of MAb 471 may be related to the fact that the serotype specificity of MAb 471 is slightly different from the specificities of MAbs 3C2 and 439 (1). Although it is a less likely explanation, our data do not exclude the alternative possibility that regulation of C3 deposition is a function of a large domain on the polysaccharide located near, but not at, the binding site of the suppressive MAb.…”

“…Three anti-GXM MAbs were used in this study, i.e., MAbs 439, 3C2, and 471. The characteristics of these antibodies have been previously described (1,2,10,35). The MAbs were isolated from mouse ascites fluid and purified as described elsewhere (31).…”

Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by theCryptococcus neoformansCapsule

“…These antibodies fall into three groups on the basis of molecular structure and reactivity with polysaccharides of the four major cryptococcal serotypes. The properties of the antibodies have been described previously (2,5,15,50) and are summarized in Table 1. The molecular groupings shown in Table 1 are based on antibody variable region usage, which determines antibody molecular structure (5).…”

Allergen1 regulates polysaccharide structure in Cryptococcus neoformans

“…Given the conformational differences in the exo‐PS, we studied the binding ability of a panel of anti‐GXM monoclonal antibodies (mAb) that recognize distinct epitopes on the polysaccharide capsule. These mAbs yield defined staining patterns that correlate with specific serotype‐specific SRGs (Belay et al ., ). Previous work documented that binding of mAbs can be greatly influenced by the degree of PS branching and conformation (Cordero et al ., ).…”