Lectin Immobilization Research Articles

Flow-injection assay of the pathogenic bacteria using lectin-based quartz crystal microbalance biosensor

A novel flow-injection assay of the pathogenic enterobacteria using novel lectin-based quartz crystal microbalance (QCM) biosensor has been proposed. The biosensing part of the analytical device contained the lectins – Concanavalin A, lectins from Ulex europeus, Maackia amurensis, Lens culinaris, wheat germ agglutinin – immobilized on the gold surface of quartz crystal electrode which served as a transducer. The immobilization of lectins was carried out using amine coupling on the surface of the crystal modified with 11-mercaproundecanoic acid. The biosensor makes it possible to identify the presence of different bacterial using the lectins immobilized on the surface of QCM crystal which bind specifically to the certain oligosaccharides present on the cell wall of the bacteria injected. The working conditions of the biosensor – pH of buffer solutions, concentration of the immobilized lectins, dilution of the bacterial cells, regeneration solution and flow rate – were optimized. The use of solution of glycine (pH 2.5) makes it possible to remove the formed complex from the crystal surface to make it reusable and ready for the next experiment. The proposed biosensor is able to detect 10 3 cells. The flow-injection assay of the bacterial cells takes about 30 min.

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

A Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au–cystamine–glutaraldehyde–lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 ± 0.2) × 10 4 M − 1 for Jacalin–Fetuin and (4.5 ± 0.1) × 10 2 M − 1 for ConA–maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin–carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides.

Lectin-immobilization strategies for affinity purification and separation of glycoconjugates

Given the high specificity of plant lectins, it is not surprising that their immobilized forms are more and more frequently used for affinity-based analysis of glycoproteins. Common lectin-immobilization techniques range from reversible, non-covalent attachments to covalent immobilization onto a vast palette of substrates (e.g., agarose, silica, and polymeric materials). This article gives an overview of recently reported strategies in lectin immobilization to different stationary phases in chromatography and electrophoresis in order to support lectin-affinity-interaction-based bioseparation methodologies.

Glycoprofiling with micro-arrays of glycoconjugates and lectins.

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.

Preparation of reversibly glucose-responsive hydrogels by covalent immobilization of lectin in polymer networks having pendant glucose

Glucose-responsive hydrogels were prepared by copolymerization of a monomer having a pendant glucose with modified lectin (concanavalin A (ConA)) having vinyl groups. Swelling behavior of ConA-copolymerized glucosyloxyethyl methacrylate (GEMA) hydrogels was discussed from the viewpoint of their synthetic condition and structures. The swelling ratio of the ConAcopolymerized GEMA hydrogels was strongly dependent on the glucose concentration in a buffer solution. As free glucose resulted in the dissociation of the complex between ConA and pendant glucose in the networks and the cross-linking density in the hydrogel decreased, the hydrogels swelled gradually in the presence of free glucose. Leak of ConA from the ConA-entrapment hydrogel and ConA-copolymerized hydrogel was examined in order to discuss the reversible changes of their glucose-responsive behavior. During swelling in the presence of free glucose, ConA leaked out of the ConA-entrapment GEMA hydrogel but did not out of the ConA-copolymerized GEMA hydrogel. As a result, the ConA-copolymerized GEMA hydrogel showed the reversible swelling changes in response to a stepwise change in the glucose concentration. This study revealed that covalent immobilization of ConA in the GEMA networks is very important for development of reversibly glucose-responsive hydrogels.

Polyacrylamide Gel Affinity Electrophoresis for Separation of Enzyme Isoforms.

Affinity electrophoresis of differently glycosylated isoforms of enzymes using lectin as affinity ligand has been reported on support media such as cellulose acetate membrane (CAM) or agarose gel. We report a method for affinity electrophoresis in polyacrylamide gel (PAG) using wheat germ agglutinin (WGA). WGA is added to acrylamide-Bis mixture and incubated for 10 minutes at room temperature. This causes WGA to react covalently with acrylamide and Bis. Polymerization is initiated with addition of N,N,N,N-tetramethylethylene diamine (TEMED) and ammonium persulphate to give polyacrylamide gel with immobilized lectin. This gel has been found to effectively separate differently glycosylated isoforms of alkaline phosphatase (ALP). Concanavalin - A, similarly immobilized, did not give effective separation of ALP isoforms. The immobilization of lectin on polyacrylamide as support media requires less amount of lectin in comparison to CAM and agarose. Additional advantage of affinity electrophoresis on PAG is separation of biomolecules according to size.

Synthetic glycosyl polymers in isolation, characterization and immobilization of lectins

A review is given on preparative procedures and practical applications in the field of lectins of two types of synthetic polymers: (1) Glycosyl polyacrylamide derivatives - insoluble (cross-linked) and water-soluble (linear) - that are obtained by copolymerization of alkenyl glycosides and acrylamide, and (2) glycosyloxyethyl dimethacrylate copolymers prepared by glycosylation of the commercially available macroporous glycol methacrylate copolymer, Separon. Both types of copolymers are efficient affinity carriers for isolation of lectins. Separon derivatives can be used after partial periodate oxidation of the glycosyl groups for covalent coupling of lectins. Affinity carriers thus obtained can be employed in separation and purification of glycoproteins and other glycosylated macromolecules. Soluble glycosyl polyacrylamide derivatives are useful in precipitation of lectins and, in general, as synthetic model substances for studying interactions of lectins with sugar ligands.

Polyacrylhydrazido-agarose as a support for immobilization of enzymes, lectins, antibodies, and small ligands

Enzymes, antibodies, lectins, and small ligands were coupled to glutaraldehyde-activated polyacrylhydrazido-agarose. High yields of binding and activity were obtained. The bound enzymes exhibited increased stability to heat and 6 M urea when compared with the respective soluble enzyme as well as enzymes coupled to cyanogen bromide-activated agarose. Antibodies and lectins were also found to maintain specificity for the appropriate antigens and glycoprotein receptors, and were used effectively for affinity chromatography purifications. Polyacrylhydrazido-agarose provides a new carrier that combines advantages of both agarose and acrylamide in one resin.