Figure 1.
Differential glycan profiles of purified IgA1.
IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was subjected to lectin microarray. IgA1-binding signals on the lectin microarray were detected with a biotinylated anti-IgA1 mAb. The relative intensity of each lectin was normalized to the maximum fluorescence intensity. mIgA-MIDD, monoclonal immunoglobulin deposition disease associated with monoclonal IgA; MPCD, monoclonal IgA plasma cell disorder; HV, healthy volunteers.
Figure 2.
Differential glycan profiles of purified IgA1 after trypsin digestion.
IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was incubated with trypsin. Each tryptic digest was labeled with Cy3-SE and subjected to the lectin microarray. The relative intensities of lectins were normalized to the maximum fluorescence intensity.
Figure 3.
Differential glycan profiles of PNGase F-treated IgA1 of mIgA-MIDD.
IgA1 purified from mIgA-MIDD serum was incubated with (left) or without (right) PNGase F. After digestion, the reaction mixture was labeled with Cy3-SE and subjected to the lectin microarray. The relative intensity of each lectin was normalized to the maximum fluorescence intensity. PNGase F, peptide N-glycosidase F.
Figure 4.
Lectin blot analysis of IgA1 purified from mIgA-MIDD serum under reducing and non-reducing conditions.
IgA1 purified from mIgA-MIDD serum was pretreated in SDS buffer with or without 2-ME, separated by SDS-PAGE under non-reducing (lane 1) or reducing (lane 2) conditions, and then subjected to western blotting with an anti-IgA1 mAb (A), ConA (B), and WFA (C). Cross-reacting bands were detected using Konica immunostaining kit (Konica, Tokyo, Japan) for anti-IgA1 mAb and ConA and Western Lightning Chemiluminescence Plus (Perkin-Elmer, Boston, MA) for WFA. 2-ME, 2-mercaptoethanol; ConA, jack bean lectin concanavalin A; WFA, Wisteria floribunda agglutinin.
Figure 5.
Sandwich lectin ELISA of sequential deglycosylated IgA1 in mIgA-MIDD.
IgA1 purified from mIgA-MIDD serum was digested with neuraminidase, and then β-galactosidase or β1,4-galactosidase. Digested and undigested samples were subjected to a sandwich lectin ELISA with HPA (A), VVA (B), PNA (C), and WFA (D) as described in the Methods. The relative intensity of each lectin was normalized to the IgA1 concentration. HPA, Helix pomatia agglutinin; VVA, Vicia villosa lectin; PNA, peanut agglutinin.
Figure 6.
Time course of PNGase F treatment of IgA1.
IgA1 purified from the sera of the mIgA-MIDD patient (A) and an MPCD patient (B) was digested partially with PNGase F for the indicated times (lanes 1–5, 7–10). Complete digestion of N-glycans was performed by incubation with PNGase F for 120 min (lanes 6, 11). Arrowheads indicate the position of PNGase F.
Figure 7.
SDS-agarose gel electrophoresis of purified IgA1.
IgA1 purified from sera of two HV, two MPCD patients, and an mIgA-MIDD patient was incubated in SDS buffer and then subjected to agarose gel electrophoresis analysis.