title: Conjugation of A and B Blood Group Structures to Silica Microparticles for the Detection of Antigen-Specific B Cells.
abstract: Silica microparticles were functionalized with A and B blood group carbohydrate antigens (A type I, A type II, B type I, and B type II) to enable the detection and monitoring of ABO antigen-specific B cells. Microparticles were prepared via the Stöber synthesis, labeled with an Alexafluor fluorescent dye, and characterized via TEM and fluorescence microscopy. The silica microparticles were functionalized with (3-aminopropyl)trimethoxysilane (APTMS), followed by the use of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based linker. The terminal Fmoc moiety of the PEG-based linker was then deprotected, yielding free amino groups, to which the A and B antigens were coupled. The carbohydrate antigens were synthesized with a p-nitrophenol ester to enable conjugation to the functionalized silica microparticles via an amide bond. The number of free amine groups available for coupling for a given mass of PEG-functionalized silica microparticles was quantified via reaction with Fmoc-glycine. The antigen-functionalized microparticles were then evaluated for their specificity in binding to A and B antigen-reactive B-cells via flow cytometry, and for blocking of naturally occurring antibodies in human serum. Selective binding of the functionalized microparticles to blood group-reactive B cells was observed by flow cytometry and fluorescence microscopy. The modular approach outlined here is applicable to the preparation of silica microparticles containing any carbohydrate antigen and alternative fluorophores or labels. This approach therefore comprises a novel, general platform for screening B cell populations for binding to carbohydrate antigens, including, in this case, the human A and B blood group antigens.
authors.affiliation: National Institute for Nanotechnology (NINT), National Research Council , 11421 Saskatchewan Drive, Edmonton, Alberta, Canada T6G 2M9.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Slaney
authors.forename: I Esmé
authors.initials: IE
authors.lastname: Dijke
authors.forename: Mylvaganam
authors.initials: M
authors.lastname: Jeyakanthan
authors.forename: Caishun
authors.initials: C
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Zou
authors.forename: Patrice
authors.initials: P
authors.lastname: Plaza-Alexander
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Meloncelli
authors.affiliation: National Institute for Nanotechnology (NINT), National Research Council , 11421 Saskatchewan Drive, Edmonton, Alberta, Canada T6G 2M9.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Bau
authors.affiliation: Pathology and Laboratory Medicine, Faculty of Medicine, Vancouver General Hospital, JP Pavilion North, University of British Columbia , 855 West 12th Avenue, Vancouver, British Columbia, Canada V5Z 1M9.
authors.forename: Lenka L
authors.initials: LL
authors.lastname: Allan
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
authors.forename: Lori J
authors.initials: LJ
authors.lastname: West
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cairo
authors.affiliation: National Institute for Nanotechnology (NINT), National Research Council , 11421 Saskatchewan Drive, Edmonton, Alberta, Canada T6G 2M9.
authors.forename: Jillian M
authors.initials: JM
authors.lastname: Buriak
mesh_terms: D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D046529:Microscopy, Electron, Transmission; D008863:Microspheres; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D000941:Antigens; D012822:Silicon Dioxide