Neoglycopolymers of polyacrylamide backbone conjugated with varying densities of Gal 1/4 -3Galβ1-4Glcβ trisaccharide epitopes (α-Gal epitopes) were designed and synthesized to study the inhibition of the binding of human natural anti-Gal antibodies to either α-Gal-containing glycoproteins or α- Gal antigens on the surface of mammalian cells. An inhibition ELISA using mouse laminin and a flow cytometry assay using pig kidney cells (PK15) were established to determine the binding affinity of the synthesized polymers. In comparison to the α-Gal monomer (Galαl-3Galβ1-4GlcNHAcβ), the α-Gal polymers dramatically enhanced the inhibition of human anti-Gal antibodies (IgG, IgM, and IgA) binding to mouse laminin or mammalian cells. Increases of 7.8 x 103- and 5.0 x 104 -fold in inhibitory potential of polymer 7C to IgA and IgM (with IC50s of 7.0 and 5.6 nM respectively) were observed over the monomer in inhibition ELISA. The results also indicated that binding enhancement of α-Gal polymers is greater for anti-Gal IgA and IgM than for IgG. Such amplified binding differences among the three anti-Gal isotypes can be utilized to selectively inhibit or remove a particular isotype of anti-Gal antibodies. Moreover, it was demonstrated through the flow cytometry assay that certain α-Gal polymers are effective in inhibition of anti-Gal antibody (in human serum) binding to pig kidney (PK15) cells. Thus, such synthetic carbohydrate polymers may find practical applications in cell xenotransplantations.
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