PMC:7108609 / 31640-33104 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7108609","sourcedb":"PMC","sourceid":"7108609","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7108609","text":"Human plasma preparation\nPlasma samples from two blood group O healthy donors with anti-A titers 1/256 by classical hemagglutination were used. The presence of anti-A natural antibodies was confirmed by ELISA on synthetic A type 2 tetrasaccharide conjugated to polyacrylamide (Lectinity, Moscow, Russia). The polyacrylamide conjugate at 10 μg/mL in a carbonate–bicarbonate buffer, 100 mM, pH 9.6, was coated onto Maxisorp ELISA plates (NUNC, Roskilde, Denmark) by overnight incubation at 37°C. After three washes with PBS containing 0.05% Tween 20 (TPBS), wells were incubated with 5% defatted milk in PBS for 1 h at 37°C. PBS 2-fold serially diluted plasma samples were then incubated for 1 h at 37°C. After washing with TPBS, peroxidase-labeled anti-human IgG (H + L) (Uptima, Montluçon, France) diluted at 1/10,000 were incubated for 1 h at 37°C. After final washes with TPBS, reactivities were detected using TMB (5-tetramethylbenzidine) as a substrate (BD Bioscience, San Jose, CA) and read at A450 nm. To adsorb the anti-A natural antibodies, 1600 μL of plasma diluted 1/4 in PBS were incubated onto 120 mg of silica beads conjugated with either synthetic A type 2 tetrasaccharide or a methyl group (kind gift from the late Pr. R.U. Lemieux, Edmonton, Canada). The latter being used as mock adsorbed controls. After a 1-h incubation under gentle agitation and centrifugation at 13,000 × g for 10 min, the supernatant was collected and kept at 4°C until used.","divisions":[{"label":"title","span":{"begin":0,"end":24}}],"tracks":[]}