beta 1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay.
Lymphocytic beta 1,4-galactosyltransferase (beta 1,4-GalTase, EC 2.4.1.38) activity was measured in B cells using a neoglyco-protein, N-acetylglucosamine-phenylisothiocyanate-bovine serum albumin (GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linked immunosorbent assay (ELISA)-based method. This assay proved to be much simpler to use than the lengthy and expensive radiochemical assays commonly used, and has the additional advantage that it specifically detects the enzyme mediating transfer via the Gal beta 1,4GlcNAc linkage. A F(ab')2 antibody against GalTase was able to specifically inhibit the reaction. Greater sensitivity for beta 1,4-GalTase activity was obtained using GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin. Low levels of beta-galactosidase activity were detectable in lymphocyte cell lysates at acidic pH, although such activity was not detectable at the neutral pH used in the beta 1,4-GalTase activity assay. Using this assay with the GlcNAc-pITC-BSA acceptor, similar beta 1,4-GalTase activities were observed in CD19+ B cells from patients with rheumatoid arthritis (RA) to those seen in normal control individuals.
Keusch
J
J
Department of Immunology, University College London Medical School, UK.
Lydyard
P M
PM
Isenberg
D A
DA
Delves
P J
PJ
D000328:Adult; D000368:Aged; D018941:Antigens, CD19 / Q000032:analysis; D001172:Arthritis, Rheumatoid / Q000201:enzymology; D001402:B-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D002236:Carbohydrate Conformation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D017879:Isothiocyanates; D008297:Male; D008875:Middle Aged; D009237:N-Acetyllactosamine Synthase / Q000097:blood*; D010047:Ovalbumin / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D013379:Substrate Specificity; D013861:Thiocyanates / Q000378:metabolism
D018941:Antigens, CD19; D017879:Isothiocyanates; D012709:Serum Albumin; D013861:Thiocyanates; C025616:glucosylated serum albumin; C005441:phenylisothiocyanate; D010047:Ovalbumin; D009237:N-Acetyllactosamine Synthase