El312 bio kinetics microplate reader

Urine spots were collected on the day of renal biopsy and stored at -80°C until measurement. Urinary CFH concentrations were measured by ELISA described previously, with minor modifications [15 (link)]. Briefly, plates (Nalge-Nunc, Rochester, NY) were coated with goat anti-human factor H (Calbiochem, Darmstadt, Germany) at a concentration of 10μg/mL in 0.05 M bicarbonate buffer (pH = 9.6). Plates coated with bicarbonate buffer alone were also prepared simultaneously as antigen-free controls. The reaction volume was 100 μL. Urine samples from patients with IgAN and healthy controls were added to wells in duplicate. After incubation and washing, 1:1000 diluted mouse anti-human CFH (US Biological, Swampscott, MA, USA) was added. Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) was added as a second antibody following three washes. The absorbance was measured at 405 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Commercially available highly purified human factor H was used to establish the standard curve.
The urinary CFH measurements were corrected by urinary creatinine concentration, and the ratios of urinary CFH over urinary creatinine (uCFH/uCr) were used for analysis.

The level of SA-IgG in plasma was determined using ELISA according the slightly modified procedure (ab100547; Abcam, UK). Briefly, 100 μL of diluted plasma was added into each well and incubated for 2.5-hs at room temperature. After washing, 100 μL diluted biotinylated-SNA (1:50, VECTOR, USA) was added for 1 h. The horseradish peroxidase (HRP)-streptavidin solution was added for 45 minutes, followed by incubation with TMB solution and the stop solution. The optical densities (ODs) were determined at 450 nm with an EL312 Bio-Kinetics microplate reader (Bio-TekInstruments, Winooski, VT).