Single Molecule Kinetic Fingerprinting of Glycans on IgA1 Antibodies.

Immunoglobulin A (IgA) nephropathy is the most common form of primary glomerulonephritis and is triggered by damage to glomeruli from deposition of complexes formed between glycosylated IgA1 antibodies that are "galactose-deficient" and antibodies directed to these aberrant proteins. Currently, galactose deficiencies are detected with ensemble measurements, e.g., via mass spectrometry or liquid chromatography, which only measure average glycan-IgA1 ratios, but cannot resolve heterogeneity of O-glycosylation between different IgA1 populations. To resolve these differences at the single molecule level, we developed an assay to detect the glycosylation state of individual IgA1 using single molecule fluorescence microscopy. By using fluorescence resonance energy transfer (FRET), high concentrations of fluorescently labeled probes with low binding rates can be employed to observe the binding of protein probes to surface adhered target molecules and obtain their kinetic fingerprints. We measured the binding and unbinding rates of jacalin (a lectin binding to O-linked glycans) to individual IgA1 molecules on a glass surface. Adding galactose decreased binding, which demonstrated that the jacalin probe binds specifically to O-linked glycans on the hinge region of IgA1. This result is a first step toward using kinetic fingerprinting to sequence glycans on IgA1.