sepharose and Glomerulonephritis--IGA

IgA nephropathy (IgAN) is the most common primary glomerulonephritis, with various pathological phenotypes. Our previous study suggested that aberrant glycosylation of serum IgA1 was associated with different pathological phenotypes of IgAN, and substantial evidence indicated that deglycosylated IgA1 had an increased tendency to form macromolecules. The aim of the current study was to investigate the composition of IgA1-containing macromolecules in different pathological phenotypes of IgAN. Sera from 10 patients with mild mesangial proliferative IgAN (mIgAN), 10 with focal proliferative sclerosing IgAN (psIgAN) and 10 healthy blood donors were collected. The sera were applied and IgA1 binding proteins (IgA1-BP) were eluted from the columns immobilized with desialylated IgA1 (DesIgA1/Sepharose) or desialylated/degalactosylated IgA1 (DesDeGalIgA1/Sepharose), respectively. The amounts of IgA1 and IgG and the glycoform of IgA1 in the IgA1-BP were detected by enzyme-linked immunosorbent assays (ELISAs) and were compared between patients with different pathological phenotypes and normal controls. The amount of IgA1 in IgA1-BP eluted from both columns was significantly higher in patients with both pathological phenotypes of IgAN than in normal controls. In IgA1-BP eluted from DesDeGalIgA1/Sepharose, the desialylation of IgA1 was much more pronounced in patients with both pathological phenotypes of IgAN than in normal controls, while the degalactosylation of IgA1 was much more pronounced only in patients with psIgAN than in normal controls. Furthermore, the amount of IgG in IgA1-BP eluted from DesDeGalIgA1/Sepharose was significantly higher in patients with psIgAN than in normal controls. In patients with psIgAN, the amount of IgG eluted from DesDeGalIgA1/Sepharose was much greater than from DesIgA1/Sepharose. In conclusion, self-aggregated deglycosylated IgA1 with or without IgG were associated with the development of IgAN.

Topics: Enzyme-Linked Immunosorbent Assay; Glomerulonephritis, IGA; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Glycosylation; Humans; Immunoglobulin A; Immunoglobulin G; Lymphokines; Neuraminidase; Phenotype; Polysaccharides; Sepharose

The immunoglobulin A (IgA)-fibronectin aggregates, detected by enzyme-linked immunosorbent assay using either antifibronectin or collagen I as binding protein, were previously found to be raised in the circulation of patients with IgA nephropathy (IgAN). It has been suggested that IgA-fibronectin aggregates are involved in the pathogenesis and that the plasma IgA-fibronectin level may even be of diagnostic value in IgAN. Nevertheless, a recent report has questioned the specificity of these assays as plasma IgA may interact with immobilized IgG and these assays detect not only IgA-fibronectin, but also total plasma IgA. These doubts render the interpretation of raised IgA-fibronectin aggregates in IgAN impossible. We isolated total IgA, in plasma by jacalin-agarose. Monomeric and polymeric IgA1 were distinctly separated by fast protein liquid chromatography. When the fast protein liquid chromatography fractions were analyzed for IgA-fibronectin using the antifibronectin capture assay, increased optical density values were predominantly observed in polymeric IgA but not in monomeric IgA. Similar findings were found when the fast protein liquid chromatography fractions were studied using a novel gelatin-anti-IgA assay that avoided nonspecific interaction between plasma IgA and immobilized IgG used as the capture antibody in antifibronectin capture assay. Using our gelatin-anti-IgA assay, we failed to demonstrate a diagnostic increase in IgA-fibronectin aggregates in polymeric IgA from patients with IgAN compared with controls. Our finding of circulating IgA-fibronectin aggregates in patients with IgAN comparable to those of healthy controls did not support the notion that these aggregates may have a pathogenetic role or diagnostic value in IgAN.

Topics: Antibodies; Biomarkers; Carrier Proteins; Chromatography, Liquid; Collagen; Enzyme-Linked Immunosorbent Assay; Female; Fibronectins; Gelatin; Glomerulonephritis, IGA; Humans; Immunoglobulin A; Immunoglobulin G; Lectins; Male; Plant Lectins; Sensitivity and Specificity; Sepharose

IgA antibodies from patients with primary IgA nephropathy bind to collagens I, II, and IV. Here we show that this binding is mediated by the collagen-binding site of fibronectin, which occurs in the circulation in complex with IgA. No antibodies binding directly to collagen were identified. The complexes were isolated by affinity chromatography on gelatin-Sepharose and heparin-Sepharose, both with affinity for fibronectin, followed by adsorption to anti-human IgA immobilized on agarose gel. The presence of fibronectin and IgA antibodies in the isolated complexes is shown by enzyme-linked immunosorbent assay, gel electrophoresis, and electrophoretic transfer followed by immunostaining. The presence of an IgA-fibronectin complex in serum and the binding of this complex to collagen demonstrate the necessity of removing fibronectin from serum prior to identifying anti-collagen antibodies.

Topics: Adsorption; Antibody Specificity; Antigen-Antibody Complex; Autoantibodies; Binding Sites; Chromatography, Affinity; Collagen; Fibronectins; Gelatin; Glomerulonephritis, IGA; Heparin; Humans; Immunoglobulin A; Sepharose