Abstract
Increased levels of hypo-galactosylated immunoglobulin (Ig)A1 (HG-IgA1) in IgA nephropathy (IgAN) have been detected using a Helix aspersa agglutinin lectin enzyme-linked immunosorbent assay (ELISA). In this study, we developed monoclonal antibodies to evaluate the HG-IgA1 in IgA nephropathy, aiming to gain a more consistent and reproducible assay. As an analogue to the HG-IgA1 hinge region, a 19 mer synthetic peptide with five GalNAc (sHGP) residues at positions 4, 7, 9, 11 and 15 [VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT (GalNAc)PSPS-NH2] was synthesized. Two monoclonal antibodies against sHGP (35A12 and 44H8) that reacted with human IgA were developed. Also, their reactivities to serum IgA from IgAN patients (n = 49), patients with other forms of kidney diseases (OKD, n = 48), and healthy controls (HC, n = 41) were evaluated using ELISA assays. The binding levels of the two monoclonal antibodies against serum IgA were significantly higher (all comparisons, p < 0.0001, Steel–Dwass non-parametric test) in IgAN patients compared to HC and OKD patients. In each individual, there was a close correlation of IgA binding levels between 35A12 and 44H8 (R 2 = 0.737). These results indicate that the monoclonal antibodies recognize similar epitopes in HG IgA1, which is found predominantly in IgAN patients. The developed antibodies are proposed as a clinically useful tool for IgAN screening.
Highlights
Immunoglobulin (Ig)A nephropathy (IgAN) diagnosis is currently confirmed using an invasive method of renal biopsy, as the presence of predominant mesangial IgA deposits is the gold standard for diagnosis
It has been suggested that the presence of truncated O-glycans with an exposed GalNAc residue is more common on the IgA1 of IgA nephropathy (IgAN) patients
O-glycans in the hinge region of IgA1 were evaluated with an enzyme-linked immunosorbent assay (ELISA) using the Helix aspersa agglutinin (HAA) lectin, which binds to GalNAc residues [13, 14]
Summary
Immunoglobulin (Ig)A nephropathy (IgAN) diagnosis is currently confirmed using an invasive method of renal biopsy, as the presence of predominant mesangial IgA deposits is the gold standard for diagnosis. Several candidates of serum and urinary markers for identifying IgA nephropathy have been proposed, such as anti-IgA antibody [1, 2] and anti-IgA1 hinge peptide antibody [3, 4]. They exhibit insufficient specificity for clinical application to identify IgAN. It has been suggested that the presence of truncated O-glycans with an exposed GalNAc residue is more common on the IgA1 of IgAN patients. O-glycans in the hinge region of IgA1 were evaluated with an enzyme-linked immunosorbent assay (ELISA) using the Helix aspersa agglutinin (HAA) lectin, which binds to GalNAc residues [13, 14]. The results indicated that there is increased binding of HAA to serum IgA1 from IgAN patients. We attempted to develop monoclonal antibodies that recognize hypo-galactosylated IgA1, which is found predominantly in IgAN patients
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