96 well high binding microloan elisa plates

Concentrations of human MPO in neutrophil supernatants were measured by commercial ELISA kit (R&D Systems, Minneapolis, MN). Serial dilutions prepared from MPO standard provided in the kit (stock: 125ng/ml) were used to quantify MPO concentrations of unknown samples. HNE release was assessed by ELISA. Supernatant samples diluted with coating buffer (25mM carbonate, 25mM bicarbonate, pH 9.6) were incubated overnight at 4°C in 96-well high binding microloan ELISA plates (Greiner bio-one, Germany). After blocking with 1% BSA for 1hr, anti-HNE rabbit polyclonal antibody (1:500 in PBS, Calbiochem, 481001, EMD Millipore, MA) was added for 2hrs at RT. After repeated washes, samples were incubated with horse radish peroxidase-linked donkey anti-rabbit antibody (1:2000 in PBS, NA934V, GE Healthcare, UK) for 1hr. Blue coloration developed in the presence of TMB (Thermo Scientific, Rockford, IL) peroxidase substrate solution. Reaction was stopped by adding 1N hydrochloric acid (Sigma, St. Louis, MO) and absorption was read at 450nm wavelength with Eon microplate photometer (BioTek, Winooski, VT). Purified HNE standard (Cell Sciences, Canton, MA; stock: 1mg/ml) was used to determine HNE concentrations in unknown samples.