Development and validation of a lectin-based assay for detection of IgG Fc glycosylation as a biomarker in lupus nephritis.

Abstract

Background: Lupus nephritis (LN) is a devastating complication of systemic lupus erythematosus (SLE). Aberrant IgG glycosylation drives podocyte injury and is a robust marker for LN. The primary method for detecting glycosylation is limited to mass spectrometry, but it is not suitable for clinical applications due to its complexity and high cost.

Methods: We developed and optimised a lectin-based enzyme-linked assay to detect IgG glycan residues. Lectins were selected based on the glycan structures detected in a SLE cohort and were differentially expressed in SLE with and without nephritis. 96-well plates were coated with protein L. Blocking solutions for each lectin were selected from 5% bovine serum albumin (BSA), deglycosylated BSA (deBSA) and carbo-free blocking solution. IgG standards range was optimised starting from 0 to 20 µg/mL. Fucose was detected by Aleuria aurantia lectin (AAL), galactose was detected by Erythrina crista-galli lectin (ECL) and sialic acid was detected by Sambucus nigra lectin (SNA). An SLE cohort was used to test the ability of these novel assays to differentiate patients with LN from non-renal SLE.

Results: The selected lectins produced reproducible standard curves with strong linearity. ELISA assays performed using IgG from a healthy donor, and a patient with LN revealed glycan differences that were consistent with mass spectrometry data and within range of the standard curves. While the AAL (fucose) binding did not differ among different SLE subgroups, ECL (galactose) binding showed significant differences in patients with active LN compared with those with active non-renal SLE and quiescent disease. SNA (sialic acid) binding also distinguished patients with active LN from those with quiescent SLE; however, it did not discriminate between renal and non-renal SLE.

Conclusions: The developed lectin-ELISA assay can detect glycosylation characteristics of IgG in a more cost-effective, clinically applicable manner compared with mass spectrometry. Our validation data suggest the potential of ECL binding assays to help diagnose and monitor patients with LN.