Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies

Abstract

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P1, a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P1 antibody binding profiles displayed much lower concordance. Whilst anti-P1 antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p = 0.004), we got only similar results using SA (p = 0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.

Printed glycan array: antibodies as probed in undiluted serum and effects of dilution

“…We have previously found [3] that the concentration of anti-glycan IgM in blood is several fold higher than that of IgG, and that IgM affinities are much lower. Thus it is reasonable to expect competition between IgM and IgG for glycans in PGAs [4].…”

“…We believe that optimal stoichiometry between immobilized glycan and antibody in solution [4] is important.…”

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array

“…The set of glycans included P 1 (Galα1-4Galβ1-4GlcNacβ), a trisaccharide which we have previously identified using PGA as significantly associated with ovarian cancer (Jacob et al, 2012). We found that the SGA, when compared to the PGA, exhibited a similar or, in some cases, even higher sensitivity and specificity in the detection of plasma anti-glycan antibodies (Pochechueva et al, 2011b;Jacob et al, 2012), which is one benefit of SGA. The other benefits of SGA are the opportunity to assess multiple analytes in a single sample, the wide dynamic range, the feasibility of the assay reconfiguration, and the minute consumption of glycans and glycan-binding proteins, making SGA an attractive tool for biomedical and diagnostic applications.…”

“…Specimens were processed and stored as described previously (Pochechueva et al, 2011b;Jacob et al, 2012). Human plasma samples were used in all the experiments in the dilution of 1/40, as described previously (Pochechueva et al, 2011a).…”

“…Each bead's region was embedded with a precise ratio of red and infrared fluorescent dyes allowing its identification using a Bio-Plex 200 suspension array system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Coupling of biotinylated glycopolymers was accomplished similarly to the procedure developed for non-magnetic Bio-Plex carboxylated beads (Pochechueva et al, 2011b). Briefly, the stock vial of microspheres (1.25 × 10 7 microspheres/ml) was vigorously vortexed for 30 s and sonicated for 15 s in a water bath prior to its use.…”