High levels of serum glycans monovalent IgG immune complexes detected by dissociative ELISA in experimental visceral leishmaniasis

Abstract

Summary Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme‐linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000–3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6–13 sugar residues using MALDI‐TOF analysis. Glycan‐BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.

Serum antibodies blocked by glycan antigens in canine visceral leishmaniasis serology are mostly IgA immune complexes

“…The greater reactivity associated with the use of PSE to detect IgG antibodies is probably related to a greater quantitative exposure to L. (L.) infantum antigenic epitopes, considering the variety of protein and glycan antigens. GBC has antigenic glycans of low molecular weight of <5 kDa (de Carvalho et al, 2019). These characteristics are compatible with glycoinositol phospholipid (GILP) molecules that play an important role in the modulation of the VL immune response (Cabezas et al, 2015).…”

“…IgG antibodies often form ICs, but other antibodies, such as IgM, IgA and IgE, also have this ability (Aibara and Ohyama, 2020). The binding of specific antibodies to protein or glycan antigens forms different ICs in VL and represents a limiting factor for conventional serological methods (de Carvalho et al, 2013(de Carvalho et al, , 2019. Acid dissociation in serum samples is an alternative for indirect detection of protein or glycan ICs in VL and is performed by ELISA, as previously reported (de Carvalho et al, 2013;Aparecida de Carvalho et al, 2019).…”

“…In parallel with the use of PSE, we used a glycan antigen conjugated to bovine serum albumin (BSA) or GBC. Briefly, GBC was produced by a low molecular weight fraction of PSE, which we characterized as a glycan antigen that was chemically linked to a support protein, as described in a previously published work (de Carvalho et al, 2019).…”

“…We then added 25 μL of 0.5 M TRIS/HCl (pH 8.0) to the wells and reacted for 1 h at 37°C. For control wells, instead of acid and buffer, we added 50 μL of phosphate buffered saline with 0.3% skimmed milk and 0.05% Tween 20 (de Carvalho et al, 2019). Anti-dog peroxidase conjugates were used at specific dilutions: IgG (1/20 000), IgA (1/10 000) and IgM (1/80 000).…”

“…Acid dissociation in serum samples is an alternative for indirect detection of protein or glycan ICs in VL and is performed by ELISA, as previously reported (de Carvalho et al, 2013;Aparecida de Carvalho et al, 2019). Glycan haptens from L. (L.) infantum promastigotes conjugated to a carrier protein allow their application to solid support in ELISA and the detection of specific anti-Leishmania antibodies and ICs in experimental hamster infection (de Carvalho et al, 2019).…”