261 Characterization of Glycosylation Patterns of Single IgA Molecules Using Single-Molecule Fluorescence Microscopy

Abstract

OBJECTIVES/GOALS: IgA1 nephropathy, which can lead to kidney failure, is caused by complexes formed between aberrant galactose-deficient IgA1 and antibodies directed against it. Our goals are to characterize shifting glycosylation patterns at the level of single IgA1 molecules and to apply this to patient samples for early detection and understanding of the disease. METHODS/STUDY POPULATION: To characterize glycosylation patterns on single IgA1 molecules, labelled IgA1 in low concentration was physisorbed to borosilicate glass in a fluidic cell and labelled Jacalin was flowed in to bind with Glycans on IgA1. The samples were observed with a Nikon TiE epi-fluorescence microscope. FRET images were created by exciting the Jacalin dye with a blue laser and recording the red emission of the IgA1 dye with an EMCCD camera. FRET emission intensities of individual IgA1 molecules over time were analyzed to determine how frequent and how long Jacalin binds to each of them. The rate of binding is roughly inversely proportional to the amount of abnormal glycans on a given IgA1 molecule. After the method is perfected, we intend to compare the glycosylation patterns of healthy and diseased patient samples. RESULTS/ANTICIPATED RESULTS: Addition of the competitive binder Galactose to the solution led to an increase in the off times of Jacalin and IgA1 and a decrease in the on times in a concentration-dependent manner, yielding an increase in the dissociation constant. Dissociation constants of individual molecules within a single experiment vary by 3 orders of magnitude, which cannot be attributed to stochastic fluctuations but rather reflects differences in the adsorption geometry. Nevertheless, the unaffected dissociation constant can be identified. We expect that when this method is applied to samples from healthy and IgA1 Nephropathy patients, specific IgA1 molecules from patients will have higher dissociation constants for Jacalin compared to those from healthy patients. DISCUSSION/SIGNIFICANCE: The binding rates of Jacalin to single IgA1 vary by 3 orders of magnitude. The observed heterogeneity shows the Jacalin probe can differentiate between different IgA1 populations. An understanding of which IgA1 molecules in patient samples are problematic and what their distribution of Glycans is can lead to discovering biomarkers and treatments.