Multiplex suspension array for human anti-carbohydrateantibody profiling

Abstract

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

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

“…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). Plasma samples from healthy donors of blood groups A, B and O (five donors each group) were pooled in equal volumes and used similarly to individual plasma samples.…”

“…The plate was washed twice with washing buffer (PBS-0.02% Tween-20 (v/v), pH 7.2) using a Bio-Plex Pro II Wash Station (Bio-Rad Laboratories Inc., Hercules, CA, USA). Various anti-glycan antibody dilutions or human serum samples, diluted to 1/40 (in accordance to (Pochechueva et al, 2011a)), or antibody diluent alone as a negative control were added to wells (in antibody diluent, 50 μl/well) and vigorously agitated for 30 s on a microplate shaker before incubation on a shaker with medium speed for 1 h at room temperature in the dark. After incubation, the plate was washed thrice with washing buffer using the Bio-Plex Wash Station.…”

“…Anti-A (A tri ), anti-B (B di ) and anti-αRha antibodies were affinity purified from pooled plasma of blood group O individuals as described previously (Obukhova et al, 2007;Pochechueva et al, 2011a). Anti-P 1 , anti-LacNAc and anti-3′-sulfo-LacNAc antibodies were affinity purified from ascites (exudative fluid from peritoneal cavity) of an ovarian cancer patient and processed by centrifugation at 3000 ×g for 15 min at 4°C.…”

“…In our previous study (Pochechueva et al, 2011a) we have compared several approaches for the immobilization, and found that the multi-step coupling procedure, i.e. the anchoring of streptavidin to the beads and the subsequent attachment of the polyacrylamide-based glycopolymer endcapped with single biotin, was the most appropriate strategy for the specific binding of serum/plasma-derived antibodies.…”

“…For the search and the identification of sensitive and ovarian cancer-specific tumor biomarkers we have previously developed "in-house" and then validated a multiplex suspension array specifically designed for the high throughput detection and profiling of anti-glycan antibodies naturally occurring in human blood serum/plasma for diagnostic purposes (Pochechueva et al, 2011a).…”