Browsing by Author "Geetha, M"

Now showing 1 - 6 of 6
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    Antigen-Induced Activation of Antibody Measured by Fluorescence Enhancement of FITC Label at Fc
    (JOURNAL OF FLUORESCENCE, 2015) George, G; Geetha, M; Appukuttan, PS
    Three 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.
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    Glutaraldehyde cross-linking of lectins to marker enzymes: Protection of binding site by specific sugars
    (INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, 2000)
    The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.
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    Normal human plasma anti-beta-glucoside antibody has markedly elevated IgA content and binds fungal and yeast polysaccharides
    (IMMUNOLOGICAL INVESTIGATIONS, 2007) Geetha, M; Annamma, KI; Mathai, J; Appukuttan, PS
    Normal human plasma antibody that recognizes beta-linked glucoside moiety was purified by affinity chromatography on cellulose. The anti-beta-glucoside antibody had three times higher IgA to IgG ratio and substantially higher polymeric IgA content than total serum immunoglobulins. Cellobiose and other beta-glucosides were best inhibitors of its binding to polystyrene microwell-coated polysaccharides. In synthetic glycoproteins made by conjugating disaccharides to hemoglobin or bovine serum albumin, cellobiose, unlike lactose or maltose, was sugar-specifically recognized by the antibody. It also recognized polystyrene well-coated beta 1 -> 3 linked glycans of Saccharomyces cerevisiae, Candida albicans and of barley in decreasing order of affinity. Its sugar-binding site could thus accommodate beta-glucoside with or without substitution at C4 and C3. High IgA content along with the capacity to bind common microbial and dietary antigens pointed to the immune inflammatory potential of the antibody.
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    Plasma anti-alpha-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a)
    (GLYCOCONJUGATE JOURNAL, 2014) Geetha, M; Kalaivani, V; Sabarinath, PS; Appukuttan, PS
    Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-alpha-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the alpha-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of alpha-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O-glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O-glycan-specific lectin jacalin. O-Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to alpha-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no alpha-linked galactose.
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    Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography
    (JOURNAL OF BIOSCIENCES, 1998) Appukuttan, PS; Annamma, KI; Geetha, M; Jaison, PL
    During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.