Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Abstract

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galalpha1-4GalNAcbeta1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and weakly by Galalpha1-4Gal. However, the type 1 antibodies are strongly inhibited by Galalpha1-4Galbeta1-3Gal-R and weakly by Galalpha1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galalpha1-4GalNAcbeta1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several alpha-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-alphaGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates

“…The purified glycosphingolipids were solubilized in chloroform/methanol (2:1, v / v ), applied to HPTLC plates (Kieselgel 60, Merck, Darmstadt, Germany) and analyzed by high-performance thin-layer chromatography (HPTLC), using chloroform/methanol/water (55:45:9, v / v / v ) for development. The dried plates were immersed in 0.05% polyisobutylmethacrylate (Sigma-Aldrich, St. Louis, MO, USA) in hexane for 1 min, dried, rinsed with 1X TBS (0.05 M Tris, 150 mM NaCl, pH 7.4) and blocked in 5% human serum albumin (HSA) in TBS for 1 h. For antibody assays, plates were overlaid with (1) mouse anti-P1 or human anti-P1 antibody or mouse anti-NOR (clone nor118, [ 40 ]) antibody (diluted 1:100 with 1% BSA/TBS); (2) biotinylated goat anti-mouse IgG antibody or goat anti-human IgG antibody, diluted 1:1000 with 1% BSA/TBS, used in the case of anti-P1 and anti-NOR antibodies (for Stx1B assay, this step was preceded by incubation with anti-6xHis, diluted 1:1000); (3) ExtrAvidin-alkaline phosphatase conjugate diluted 1:5000 with 1% BSA/TBS/0.05% Tween20 for 1 h; and (4) the substrate NBT/BCIP solution (Sigma-Aldrich, St. Louis, MO, USA). Each HPTLC experiment was repeated at least three times.…”

Alpha-Gal Bound Aptamer and Vancomycin Synergistically Reduce Staphylococcus aureus Infection In Vivo

“…α-gal is a carbohydrate structure not produced by humans, Old World monkeys, or apes, but it is commonly found across many other mammals and bacteria [ 18 ]. Diet and the gut microbiota are thought to be the primary source of α-gal exposure, and given the consistent contact with both, there is likely continuous antigenic stimulation in the host [ 19 , 20 , 21 , 22 , 23 , 24 ]. This consistent α-gal exposure in humans leads to high levels of anti-α-gal Abs, comprising up to 10% of total circulating Abs and up to 1% of total IgG in humans [ 19 , 20 , 25 ].…”