OR32. Differential detection of glycosylation of HLA donor specific antibodies (DSA) using single antigen microbeads

Aim Differential glycosylation of Immunoglobulin G (IgG) is known to affect the functions of IgG by modulating the interactions with its binding proteins, such as C1q and Fc receptors. Our research aim was to develop an assay to analyze glycosylation of donor specific antibodies (DSA) using conventional single antigen microbead technology, and subsequently to analyze glycosylation profiles of kidney transplant patients with DSA and biopsy data for graft function assessment. Methods Conventional Class I and II LABScreen® single antigen beads were pretreated to eliminate glycans on the Class I and II antigens. Patient serum was incubated with the pretreated beads according to the standard assay. Glycans of the DSA bound to the beads were detected with biotinylated lectins known to specifically detect sialic acid, galactose, or fucose residues, along with neutravidin-PE (phycoerithrin). Data acquisition was performed using a LABScan 3D™. Results Pretreated Class I and II single antigen beads maintained DSA binding capabilities comparable to the untreated conventional beads when PAN IgG-PE was used as the detection agent. Using pretreated beads, glycans on the bound DSA were specifically detectable with lectin/neutravidin based reporters with slight modification of conventional LABScreen® single antigen protocol (Figure: Detection of Class I DSA with lectin specific to galactose vs. PAN IgG). Conclusions We have demonstrated a simple and robust assay for detection of glycans on DSA using modifications of conventional LABScreen® single antigen technology. We used lectins as specific glycan probes and neutravidin-PE as a reporter. Further research is in progress to assess the clinical validity of the assay and to assess the predictive value of glycosylation profiles of Class I and II DSA on the outcome of kidney transplant recipients. Download high-res image (109KB) Download full-size image