Combinatorial Glycoarray

Abstract

Glycolipid–protein interactions are increasingly recognised as critical to numerous and diverse biological processes, including immune recognition, cell–cell signalling, pathogen adherence, and virulence factor binding. Previously, such carbohydrate–lectin interactions have been assessed in vitro largely by assaying protein binding against purified preparations of single glycolipids. Recent observations show that certain disease-associated autoantibodies and other lectins bind only to complexes formed by two different gangliosides. However, investigating such 1:1 glycolipid complexes can prove technically arduous. To address this problem, we have developed a semi-automated system for assaying lectin binding to large numbers of glycolipid complexes simultaneously. This employs an automated thin-layer chromatography sampler. Single glycolipids and their heterodimeric complexes are prepared in microvials. The autosampler is then used to print reproducible arrays of glycolipid complexes onto polyvinylidene difluoride membranes affixed to glass slides. A printing density of 300 antigen spots per slide is achievable. Following overnight drying, these arrays can then be probed with the lectin(s) of interest. Detection of binding is by way of a horseradish peroxidase-linked secondary antibody driving a chemiluminescent reaction rendered on radiographic film. Image analysis software can then be used to measure signal intensity for quantification.

Key words

• Glycolipid
• Ganglioside
• Ganglioside complexes
• Microarray
• Glycoarray
• Lectin
• Immunoassay
• Bacterial toxins