Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay

Abstract

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI1) for the detection of GPI1-specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

“…Based on the promising results obtained with the BBMA using human sera and the strong performance of TgSAG1 bio [37,39], we strived for a transfer of this assay to other animal species, including chickens, to establish an improved method for large scale, e cient serological monitoring. Although there is a number of BBMAs for veterinary purposes, these tests mainly focus on viral infections [41][42][43][44] and cannot be easily compared with our assay, which, to the best of our knowledge represents the rst BBMA focusing on parasitic pathogens in chickens.…”

“…16.67 μg/10 6 MagPlex® beads, region 34), chicken serum albumin (CSA, Sigma-Aldrich, Darmstadt, Germany; 12 µg/10 6 MagPlex® beads, region 54) as a negative control, or chicken IgY (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 6.67 µg/10 6 MagPlex® beads, region 52) as a positive control followed the instructions of the xMAP® Cookbook [38,39]. Prior to coupling, bead stocks were vortexed for 30 s and sonicated for 30 s in a water-bath.…”

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

“…The coupling of recombinant Sav (Anaspec; 25 µg/1,5 x 10 6 MagPlex ® beads, region 33) and performing the BBMA proceeded according to the instructions of the xMAP ® Cookbook [70] and have been described in detail previously [69]. We did not observe notable differences in binding of biotinylated antigens and concomitant maximal signal intensities with standard sera and different batches of custom-prepared Sav beads (data not shown).…”

“…The description and evaluation of human sera used in this study as either seropositive or -negative using either the VIDAS TOXO IgG enzyme-linked uorescent immunoassay (ELIFA; bioMérieux) or the anti-Toxoplasma gondii-IgG ELISA (Euroimmun, Lübeck, Germany) were published previously [14,69].…”

“…Depending on the preparation, between 10-100 ng SAG1 bio -His 6 (or SAG2A bio -His 6 ) were added to 1,500 Sav-coated beads (per well) and bound human antibodies were detected with a 1:333 dilution of goat IgG anti-human IgG (Fc)-RPE. Human serum albumin (Sigma-Aldrich) or unconjugated goat IgG anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories) coupled to different bead regions were included as negative or positive controls, respectively [69].…”