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

Abstract

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.

Keywords: Leishmania (Leishmania) infantum chagasi; glycan; hapten; hypergammaglobulinemia; immune complexes.

Figure 1

Anti‐promastigote soluble extract (PSE) IgG detection by conventional ELISA (cELISA) and dissociative ELISA (dELISA) in hamster serum samples at 1 : 1000 (a) and 1 : 2000 (b) dilutions. Open dots cELISA, solid dots dELISA. Labels show days after infection and negative controls (C = cELISA and D = dELISA). The bars represent the standard deviation and the asterisks indicate the level of statistical significance between the results using Bonferroni analysis of variance post‐tests.

Figure 2

Chromatography purification and MALDI‐TOF analysis of 1000–3000 MW antigens from promastigote soluble extract (PSE). (a) PSE analytical G‐25 chromatogram showing A_280nm (protein) and Schiff reaction (carbohydrate) of individual fractions. (b) PSE preparative G‐25 chromatogram. A = high molecular weight protein and glycoproteins; Pool P2 = pooled fractions with salts and < 3000 MW antigens. (c) Pool P2 (B chromatogram) preparative Bio‐gel P2 chromatogram. B = pooled fraction desalted and containing 1000–3000 MW antigens. (d) MALDI‐TOF analysis of pooled fractions (C chromatogram) with molecular weight and estimation of sugar residue numbers between parentheses.

Figure 3

Glycan–bovine serum albumin complex (GBC) ELISA standardization. (a) Detection of anti‐leishmania IgG in rabbit sample using various concentrations of GBC (open points), promastigote soluble extract (PSE) (closed points) and bovine serum albumin (BSA) (open points) in solid phase. Δ values (asterisks) represent the difference between the reactivity of rabbit serum to GBC and BSA. (b) Reactivity in GBC and PSE ELISA of hamster (square) or rabbit (circle) serum dilutions for anti‐leishmania IgG detection, analysed by sigmoidal dose models. GBC (dashed line) and PSE (continuous line).

Figure 4

IgG anti‐glycan–bovine serum albumin complex (GBC) detection by conventional ELISA (cELISA) and dissociative ELISA (dELISA) in hamster serum samples at 1 : 1000 (a) and 1 : 2000 (b) dilutions. Open dots cELISA, solid dots dELISA. Labels show days after infection or negative controls (C = cELISA and D = dELISA) The bars represent the standard deviation and the asterisks the level of statistical significance between the results using Bonferroni analysis of variance post‐tests.