IgA nephropathy: characterization of IgG antibodies specific for galactose-deficient IgA1.
The circulating immune complexes in IgA nephropathy (IgAN) are composed of galactose (Gal)-deficient IgA1 bound to IgG or IgA1 antibodies specific for hinge-region O-linked glycans of Gal-deficient IgA1. To analyze properties of the anti-glycan antibodies, we determined the binding of serum IgG and IgG secreted by Epstein-Barr virus (EBV)- immortalized B cells from patients with biopsy-proven IgAN (n = 12) and healthy controls (n = 5) to a panel of antigens coated on ELISA plates. These antigens were: (1) enzymatically desialylated and degalactosylated IgA1 myeloma protein (dd-IgA1), (2) Fab fragment of Gal-deficient IgA1 containing part of the hinge region with O-glycans (Fab-IgA1), (3) synthetic hinge-region peptide linked to bovine albumin (HR-BSA), and (4) synthetic hingeregion glycopeptide with three GalNAc residues linked to BSA (HR-GalNAc-BSA). IgG-secreting EBV-immortalized cell lines were subcloned by limiting dilution. The concentration of total IgG and distribution of IgG subclasses were measured by ELISA. The levels of IgG in sera and supernatants directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (p < 0.01). IgG from IgAN patients exhibited strong reactivity with HR-GalNAc-BSA, but not with HR-BSA. The IgG-secreting cell lines produced antibodies specific to dd-IgA1; the antigen-specific IgG was most frequently of the IgG2 subclass. In summary, sera and supernatants from IgG-secreting cell lines from patients with IgAN were characterized by high levels of IgG antibodies with specificity to the Gal-deficient O-linked glycans of IgA1. The immortalized cell lines will provide a stable and convenient source of IgG for molecular studies of antibodies specific to the aberrant O-glycans in IgA1.
Suzuki
Hitoshi
H
University of Alabama at Birmingham, Department of Microbiology, Birmingham, AL 35294-2170, USA. shitoshi@uab.edu
Moldoveanu
Zina
Z
Hall
Stacy
S
Brown
Rhubell
R
Julian
Bruce A
BA
Wyatt
Robert J
RJ
Tomana
Milan
M
Tomino
Yasuhiko
Y
Novak
Jan
J
Mestecky
Jiri
J
D000918:Antibody Specificity; D002461:Cell Line, Transformed; D005690:Galactose / Q000276:immunology*; D005922:Glomerulonephritis, IGA / Q000276:immunology*; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*
D007070:Immunoglobulin A; D007074:Immunoglobulin G; D005690:Galactose