Materials and Methods

Patients. This study enrolled 52 subjects (42 males, 10 females) with biopsy-proven IgAN who underwent kidney transplantation 0.5-13 (mean 3.2) years before and 40 transplanted patients with non-IgA nephropathy. Among the IgAN patients, 32 had no histologic and/or clinical sign of recurrence (nonrecurrent group), 9 developed IgA mesangial deposits 12-111 (mean 41) months after transplantation in the grafted kidney (recurrent group) and 6 had urinary abnormalities suggesting a recurrence but had not undergone biopsy and therefore were not included in the calculation when presence or absence of recurrence was considered. The remaining 5 showed other de novo glomerulonephritides, including membranous (n= 2), mesangiocapillary (n= 2) and focal sclerosis (n= 1). IgC Detection. The detection of conglutinin binding IgAIC (K-IgAIC)[28] and macromolecular IgA measured in 2.5% polyethylene glycol (PEG-IgAIC)[29] are described in detail elsewhere.

Mixed IgA/IgGIC Complexes. A solid-phase jacalin assay was used to detect mixed IgA/IgGIC. Jacalin 50g/ml in 0.1 M bicarbonate buffer (BB), pH 9.6, prepared from dried jackfruit seeds (Madagascar) by saline extraction (44) was used for coating microplate wells. Sera diluted 1/50 were incubated in the coated plates, which were then washed and incubated with 1: 3,000 diluted alkaline phosphatase (AP)-labeled rabbit anti-human IgG (Dako, Denmark). After two additional washes, NPP 1.5 mg/ml in 0.1 M BB, pH 9.6, was added to each well and absorbance at 405 was read. IgA-Fibronectin Aggregates. The BioCarb Diagnostics (Lund, Sweden) modification of the ELISA described by Cederholm et al.[17] was used. Micro plate wells were coated with the IX, CB7 fragment of type I collagen. The bound IgA-fibronectin aggregates were detected by alkaline phosphatase-conjugated swine anti-human IgA (Orion Diagnostica, Helsinki, Finland). After a 60-min incubation, the absorbance was determined at 405 nm. IgA Binding to Mesangial Matrix Components. Microplate wells were coated with human fibronectin, laminin or type IV collagen (Sigma) 10 g/ml in 0.1 M BB, pH 9.6, and the test was performed as previously described [23]. Total IgA. Serum IgA levels were measured by nephelometry (Beckman Array Protein System; Brea, Calif., USA).