Browsing by Author "George, G"
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Item Antigen-Induced Activation of Antibody Measured by Fluorescence Enhancement of FITC Label at Fc(JOURNAL OF FLUORESCENCE, 2015) George, G; Geetha, M; Appukuttan, PSThree anti-carbohydrate antibodies of defined specificity isolated from plasma were used to demonstrate that macromolecular antigen binding caused considerable enhancement of fluorescence of FITC-labeled antibody. Mono and disaccharide antigens which could compete with the large antigens in antibody binding could not however produce any increase in fluorescence. Fluorescence enhancement in a given antibody sample increased with the size of the occupying macromolecular antigen. Conversely in antibody samples of same ligand specificity isolated from plasma of different individuals, fluorescence enhancement produced by the same antigen correlated with specific activity of the antibody sample. Removal of Fc part of antibody, confirmed by electrophoresis and Fc-specific antibody binding, caused abolition of most of the antigen-driven fluorescence increase. Since antigen binding sites of antibodies were protected during FITC labeling, the above results suggest that conformational shift in Fc produced by occupation of binding sites by large antigens resulted in the enhancement of fluorescence of FITC tags on Fc. Data provides a tool for detection and measurement of specific ligands using fluorolabeled whole antibodies.Item Plasma anti-alpha-galactoside antibody mediates lipoprotein(a) binding to macrophages(GLYCOCONJUGATE JOURNAL, 2016) Sheela, B; George, G; Mandagini, G; Appukuttan, PSLipoprotein (a) [Lp(a)] is the dominant lipid in atherosclerotic plaques though it is much less numerous than LDL or HDL in circulation. Molecular mechanism of selective uptake of Lp(a) into macrophages is unclear. Lp(a) was reported to form circulating immune complexes with the IgG-dominated plasma anti-alpha-galactoside antibody (anti-Gal) using the serine- and threonine-rich peptide sequences ( STPS) on its apo(a) subunit as surrogate ligand but left the other binding site of antibody free. We examined if these monovalent immune complexes could bind to smaller STPS-containing molecules on macrophage surface. Using placental membrane O-glycosylated proteins (PMOP) isolated by lectin affinity chromatography as model it was shown that human cell surface glycoproteins were small enough to occupy both binding sites of anti-Gal since they increased the fluorescence of FITC label at Fc part of anti-Gal and inhibited binding of anti-Gal and Griffonia simplicifolia lectin of similar specificity to immobilized ligands. Pre-incubation with anti-Gal facilitated Lp(a) attachment to macrophages unless anti-Gal-specific sugar was present. Anti-Gal-mediated attachment of apo(a) to macrophages increased with the number of apo(a) subunits. Further, anti-Gal-mediated binding of the same sample of apo(a) increased with the specific activity of anti-Gal sample. Finally binding of anti-Gal and anti-Gal-apo(a) complex to PMOP and macrophages respectively was mostly inhibited by LDL suggesting STPS as major anti-Gal epitopes on the cell surface. Results indicated that circulating Lp(a)-anti-Gal immune complexes anchor on macrophages using STPS-bearing cell surface glycoproteins as ligands and offer a pathway for Lp(a) sequestration into macrophages.