Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy

Abstract

BackgroundGalactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA).MethodsRats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens.ResultsMeasurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA.ConclusionThis novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease.

Toll-Like Receptor 9 Stimulation Induces Aberrant Expression of a Proliferation-Inducing Ligand by Tonsillar Germinal Center B Cells in IgA Nephropathy

“…7,42 Serum levels of these molecules, indeed, have clinical diagnostic potential for the assessment of prognosis and disease activity for IgAN, independent of information from renal biopsy. [42][43][44][45] Reports showed abnormal glycosylation of tonsillar IgA 46,47 and aberrant cytokine profiles in tonsillar B cells, leading to the underglycosylation of IgA1 in patients with IgAN. [48][49][50][51] Because total IgA is decreased by approximately 10% on average after tonsillectomy alone in patients with IgAN 27 and because patients who showed a large decrease of serum IgA after the tonsillectomy had better clinical outcome, the palatine tonsil was hypothesized to be a major delivery source of nephritogenic IgA.…”

“…45,54,60,61 Diluted sera were added at 100 ng per well of serum IgA. The captured IgA was treated with 10 mU/ml neuraminidase (Roche Diagnostics, Indianapolis, IN) to remove terminal sialic acid residues.…”

Diagnosis and treatment of patients with IgA nephropathy in Japan

“…Yasutake et al [10] aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Levels of serum Gd-IgA1 measured by Gd-IgA1 ELISA in patients with IgA nephropathy were significantly increased compared with those in patients with other renal diseases or nonrenal diseases.…”

“…5). It appears that the novel lectin-independent method with KM55 for the measurement of serum levels of Gd-IgA1 can pave the way for a more convincing diagnosis and activity assessment of IgA nephropathy [10].…”

Biomarkers and targeted new therapies for IgA nephropathy

“…Although Gd-IgA1 is a rather sensitive and specific marker for IgAN, as reported by most studies (with a sensitivity of 56-76 % and a specificity of 89-94 %) [14][15][16], discrepancies exist likely because of the technical pitfalls for detecting Gd-IgA1 based on lectin binding assays [17]. The development of a monoclonal antibody specific to Gd-IgA1 could offer a new appropriate biomarker [18]; however, using this specific test, only one third of IgAN patients had values higher than those of other diseases. Levels of Gd-IgA1 in children were related neither to proteinuria [15] nor to treatment outcome in our IgACE trial [19].…”