Glycoconjugates detection

Using the same versatile modular synthetic strategy, the same group developed biotinylated bi- (438) and tetra-antennary (439) mannosylated glycoconjugates to capture and detect E. coli cells, and compared the relative capturing ability of these molecules to commercial polyclonal antibodies (Fig. 47).318 Instead of aliphatic spacers, tetraethylene glycol linkers were used to diminish nonspecific binding and to impart flexibility for a better fit in the active sites. 

D. M. Hatch, A. A. Weiss, R. R. Kale, and S. S. Iyer, Biotinylated bi- and tetra-antennary glycoconjugates for Escherichia coli detection, ChemBioChem, 9 (2008) 2433-2442. 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled... 

Figure 24.4 Lectins coated on gold particles can be used to detect specific carbohydrate sequences in cell-surface glycoconjugates.

Other molecules can be used in this type of assay approach. Hydrazide-modified (strept)avidin, lectins, biocytin, fluorescent probes and other detectable molecules can be used to detect specifically glycoconjugates in biological samples (Wilchek and Bayer, 1987). 

Gershoni, J.M., Bayer, E.A., and Wilchek, M. (1985) Blot analysis of glycoconjugates Enzyme-hydrazide— A novel reagent for the detection of aldehydes. Anal. Biochem. 146, 59-63. 

Carr SA, Annan RS, Huddleston MJ. Mapping posttranslational modifications of proteins by MS-based selective detection Application to phosphoproteomics. In Burlingame AL, ed.. Mass Spectrometry Modified Proteins and Glycoconjugates, Vol. 405, New York Academic Press, 2005, 82-115. 

Haselbeck, A. and Hosel, W. (1990) Description and application of an immunological detection system for analyzing glycoproteins on blots. Glycoconjugate J. 7, 63-74. 

Lectins, or proteins with specific binding sites for carbohydrates, can be used as targeting molecules to localize particular glycoconjugates such as glycoproteins or glycolipids on cell surfaces (Fig. 373). Labeled with gold particles, lectins are important probes for detection of cell surface components and intracellular receptors and in immunological or biochemical assay procedures (Bog-Hansen et al., 1978 Kimura et al., 1979 Nicolson, 1978 Roth, 1983 Benhamou et al., 1988 Nakajima et al., 1988). 

Anti-Lex and anti-Ley antibodies have been detected in the sera of H. pylori-infected patients, particularly those with prolonged infection, using H. pylori LPS (Appelmelk et al., 1996 Heneghan et al., 2001 Hynes et al., 2005). Importantly, the use of synthetic glycoconjugates in solution or on a solid phase does not optimally... 

Anti-a-D-mannose antibodies have been isolated from the serum of rabbits immunized with the glycoconjugate of a-D-mannose and bovine serum albumin [85], The antibodies were purified by affinity chromatography with adsorption on a mannosyl-Sepharose column and elution with a-D-mannose or methyl a-D-mannoside. Such antibodies should be especially useful for studying the detection of diseases due to the appearance of abnormal glycoproteins that contain mannose polymers. 

The Bik glycoprotein has an isoelectric point (p/) of 2.1 and is composed of a peptide and two glycoconjugate portions [30]. The predicted peptide sequence of Bik is helpful to understanding protein function. The peptide has Kunitz-binding domains I and II attached to the N-terminal peptide tail [31-33] (Fig. 2). Protein mass spectrometry by surface-enhanced laser desorption ionization (SELDI) in combination with detection by Bik antibodies has demonstrated that substantial variation exists in the Bik molecule [13,14]. 

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