Background

IgA nephropathy is the most common form of primary glomerulonephritis worldwide (Lai et al., 2016). The diagnosis requires renal biopsy and there are no validated diagnostic serum or urine biomarkers for IgA nephropathy. Among recognized risk factors, high levels of galactose-deficient IgA1 (Gd-IgA1) and excessive formation of poly-IgA immune complexes in circulation are associated with kidney glomerular deposition (Magistroni et al., 2015). The observations of recurrent IgA deposition in kidney grafts in transplant recipients with IgA nephropathy strongly suggest the extrarenal origin of IgA as the source of the renal deposits (Moroni et al., 2019). This notion is further supported by evidence of circulating immune complexes in patients sharing the same immunoglobulin classes with IgA extracted from the kidney (Kanatsu et al., 1983). Local immune reactivities to the IgA deposits in the glomerulus stimulate proliferation of mesangial cells, synthesis of extracellular matrix, and infiltration of inflammatory cells (Magistroni et al., 2015).

Meanwhile, circulating IgA immune complexes are catabolized primarily by the mononuclear phagocyte system via IgA Fcα receptor I (FcαRI/CD89) (Chen et al., 2018). CD89 is a type I transmembrane glycoprotein broadly expressed on the surface of myeloid cells, including monocytes/macrophages, dendritic cells, Kupffer cells, neutrophils, and eosinophils (Maliszewski et al., 1990; Monteiro and Van De Winkel, 2003; Bakema and van Egmond, 2011). This IgA-specific receptor binds both IgA1 and IgA2 through its N-terminus Ig domain, which interacts with the Cα2/Cα3 junction of IgA (Herr et al., 2003). Previously, we and others demonstrated that CD89 exhibits higher affinities to polymeric IgA than to monomeric IgA, allowing phagocytes to selectively capture poly-IgA complexes (Reterink et al., 1997; Zhang et al., 2021).

To this end, we constructed a recombinant (r) CD89-based affinity probe for serological detection of poly-IgA immune complexes in clinical samples, namely from patients with IgA nephropathy and IgA vasculitis, formerly known as Henoch-Schӧnlein purpura (HSP) (Zhang et al., 2020). In our study, we demonstrated that rCD89 can distinctively capture poly-IgA in the plasma samples, making this rCD89 probe suitable for measuring only the disease-causing poly-IgA contents without the interference of background signals from normal IgA species. The probe was mounted on ELISA plates and the tests were performed directly with diluted plasma samples. Using this high-throughput test, we demonstrated significantly elevated levels of poly-IgA complexes in IgA nephropathy and HSP samples, as compared to either healthy controls or samples from other kidney disease types (Figure 1). The results outperformed those obtained from measurements of either total IgA or Gd-IgA1 levels regarding disease correlation. It is important to note that despite the recognition of circulating poly-IgA complexes being prone to deposition in the kidney and elsewhere, clinical tests for their levels are unavailable. Research labs have been using size-exclusion chromatography (SEC) to isolate and characterize high-molecular weight IgA complexes. However, the procedures are cumbersome and the quality of poly-IgA separation from their monomeric and dimeric counterparts by SEC is poor (Reterink et al., 1997; Novak et al., 2005, 2011). In addition, chromatography requires pre-extraction of IgA and these additional steps have the tendency to introduce experimental artifacts of IgA self-aggregation (Hui et al., 2015). In contrast, our recombinant CD89-directed ELISA is a robust assay that can be easily automated and further adapted to clinical applications. Here we describe the experimental workflow in a research lab setting.

Figure 1. Comparison of plasma poly-IgA levels among different kidney diseases. The rCD89-directed ELISA kit detects higher poly-IgA levels in plasma samples of IgAN patients, as compared to samples of healthy controls or non-IgAN kidney disease types, including membranous nephropathy (MN), lupus nephritis (LN), ANCA nephritis, Focal Segmental glomerulosclerosis (FSGS), tubulointerstitial nephritis (TIN), diabetic nephropathy (DN), and minimum change disease (MCD). (Note that the figure was adapted from Zhang et al., 2021)