A Time-Resolved Immunofluorometric Method for the Measurement of Sialyl Lewis x-Synthesizing α1,3-Fucosyltransferase Activity

Enforced fucosylation of neonatal CD34+ cells generates selectin ligands that enhance the initial interactions with microvessels but not homing to bone marrow

“…For detection of FucT activity in sorted CD34 ϩ cells, we modified the method described by Räbinä et al, 37 which allows one to quantify FucT activity lysates derived from relatively few cells. Briefly, 6 ϫ 10 5 cells were resuspended in 100 L of 50 M morpholinepropanesulfonic acid (MOPS)-NaOH pH 7.4, 150 mM NaCl buffer, and lysed by sonication on ice by two 6-second bursts on a microprobe sonicator.…”

“…37 This assay measures the ability of a given cell extract to add the high-energy fucose donors (GDP-fucose) to 3Јsialyl-N-acetyllactosamine acceptors. The reaction generates the sLex antigen which can then be recognized by the HECA-452 antibody and detected by ELISA.…”

A Time-Resolved Immunofluorometric Assay of Sialyl Lewis x-Degrading α2,3-Sialidase Activity

“…Anti-Lex antibody PM-81 was from Medarex (Annandale, NJ) and anti-sLex antibody 2H5 was from Pharmingen (San Diego, CA). Rat anti-mouse IgM antibody (Sanbio, Uden, The Netherlands) was labeled with europium as described previously (28). Arthrobacter ureafaciens sialidase was from Boehringer Mannheim (Mannheim, Germany).…”

Application of Time-Resolved Fluorometry of Lanthanide in Glycobiology

“…The amount of Eu (measured with the DELFIA technique) will be the basis of quantification. Instead of lectins, carbohydrate-specific antibodies can be used also (21).…”