The Glycan Array Platform as a Tool to Identify Carbohydrate Antigens

Abstract

Carbohydrate antigens are important targets for the immune system, but identification of key glycan antigens is challenging. Direct analysis of glycomes by mass spectrometry is difficult, and detection reagents, such as monoclonal antibodies and lectins, are only available for a small subset of glycans. An alternative approach involves profiling serum anti-glycan antibody populations to identify unique antibodies or changes in antibody subpopulations. Glycan microarray technology allows rapid evaluation of hundreds to thousands of antigen-antibody interactions in a single experiment. This high-throughput format is particularly useful in profiling complex anti-glycan antibodies in serum. Here we elaborate the use of this technology to explore clinically relevant carbohydrate antigens by profiling serum anti-glycan antibodies. Detailed protocols from glycan microarray fabrication to microarray binding assays and analysis of microarray data are presented.

Fig. 1. Glycan microarray binding assay.

16 arrays are printed on a single slide with hundreds of BSA-modified neoglycoproteins on one array. Prior to the assay, the slide is fitted with a 16-well module that physically separates the individual arrays. In the binding assay, the slide is first blocked to deactivate reactive functional groups on surface. After blocking, it is incubated with sample of interest, and then the captured antibodies are detected with fluorophore-labeled secondary reagents. Binding is quantitated by a fluorescent scanner. In the example shown, IgG and IgM antibodies are detected with secondary reagents labeled with different dyes

Fig. 2. Coordination of pin configuration with 384-well source plate.

Four pins are arranged in 3 × 4 configuration. Correspondingly, samples in 384-well source plate are arranged so that each sample is distributed in four wells matching with four pins. During printing, four pins are dipped into 384-well source plate, and then deliver sample onto glass slide for printing. The four-pin system allows four arrays to be printed simultaneously. A total of 16 arrays are printed on a single slide

Fig. 3

Example of different types of antibody responses observed before and after vaccination in the same patient. (a) New antibodies to antigen 1 and 2 are produced (from no binding to binding); (b) Existing antibodies to antigen 3 increase in signal (from weak binding to strong binding. IgG signals are shown in green and IgM signals are shown in red