A highly sensitive quantum dot (QD)-based western blot assay with extended dynamic range was developed. Bimodal size distribution QD (BQ) immunoprobes composed of small size single QD (7.3 nm) and big size QD nanobead (QB) (82.9 nm) were employed for fluorescent western blot immunoassay on a membrane. Small size QD immunoprobes contributed to wider dynamic range of assay, while big size QB immunoprobes provided higher detection sensitivity. This BQ-based western blot assay can achieve a wide dynamic range (from 7.8 to 4000 ng IgG) and is nearly as sensitive as commercial available ultrasensitive chemiluminescent methods, just using a simple gel imager with UV light (365 nm) excitation and red light filter (610 nm). The fluorescent signals of BQ western blot were stable for 10 min, while chemiluminescent signals faded after 1 min. Moreover, this BQ immunoprobe was utilized for the detection of housekeeping protein and specific target proteins in complex cell lysate samples. The limit of detection of housekeeping protein is 0.25 mu g of cell lysate, and the signal intensities were proportional to loading protein amount in a wide range from 0.61 to 80 mu g. We believe that this new strategy of bimodal size distribution nanoparticles can also be expanded for other functional nanoparticle-based biological assays to improve the sensitivity and extend the dynamic range.Graphical abstract

IgG4-related disease (IgG4-RD) is an immune-mediated disorder often showing elevated serum IgG4 concentrations, dense T and B lymphocyte infiltration, and IgG4-positive plasma cells and storiform fibrosis. We prospectively evaluated for 4 years 5 patients with histologically proven IgG4-RD of whom 3 had tubulointerstitial nephritis (TIN) and 2 had retroperitoneal fibrosis (RPF). They received an intensive B depletion therapy with rituximab. The estimated glomerular filtration rate of TIN patients after 1 year increased from 9 to 24 ml/min per 1.73 m2. IgG/IgG4 dropped from 3236/665 to 706/51 mg/dl, C3/C4 went up from 49/6 to 99/27 mg/dl, and the IgG4-RD responder index fell from 10 to 1. CD20(+) B cells decreased from 8.7 to 0.5%. A striking drop in interstitial plasma cell infiltrate as well as normalization of IgG4/IgG-positive plasma cells was observed at repeat biopsy. Both clinical and immunological improvement persisted over a 4-year follow-up. Treating these patients who were affected by aggressive IgG4-RD with renal involvement in an effort to induce a prolonged B cells depletion with IgG4 and cytokine production decrease resulted in a considerable rise in eGFR, with IgG4-RD RI normalization and a noteworthy improvement in clinical and histological features. Furthermore, the TIN subgroup was shown not to need for any maintenance therapy.