Multiplex suspension array for human anti-carbohydrate antibody profiling.
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.
University Hospital Zürich, Translational Research Group, Gynecological Research Department, Frauenklinikstrasse 10, Nord I D222, CH-8091, Zurich, Switzerland. Tatiana.Pochechueva@usz.ch
Tatiana
T
Pochechueva
Alexander
A
Chinarev
Marianne
M
Spengler
Elena
E
Korchagina
Viola
V
Heinzelmann-Schwarz
Nicolai
N
Bovin
Robert
R
Rieben
D000017:ABO Blood-Group System / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D006001:Glycoconjugates / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000097:blood*; D007075:Immunoglobulin M / Q000097:blood*; D008863:Microspheres; D011134:Polysaccharides / Q000276:immunology*; D015203:Reproducibility of Results
D000017:ABO Blood-Group System; D006001:Glycoconjugates; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011134:Polysaccharides