Streptavidin peroxidase

Neutrophil elastase (NE) concentration was analyzed in culture supernatants collected after 2 h of cell culture as described in Souwer et al. [3 (link)]. Briefly, flat-bottom EIA/RIA 96-well plates were coated overnight with polyclonal rabbit IgG directed against NE (1.5 ng/mL, Sanquin) at 4 °C. Plates were washed with PT thrice before adding culture supernatants, usually diluted 250 times in PTG (PT-0.2% v/w gelatin), and incubated for 1 h. Next, plates were washed with PT and incubated for 1 h with biotinylated rabbit anti-human elastase diluted in PTG (1 ng/mL, Sanquin blood Supply). Subsequently, after a washing step, plates were incubated for 30 min with streptavidin-peroxidase diluted 1:1000 in PTG (Amersham Life Science, Buckinghamshire, UK). Finally, the ELISA was completed using TMB as a substrate and measured as described above.

PdC1-INH (Cetor^®) was obtained from the Sanquin Blood Supply Foundation, The Netherlands. Monoclonal antibody (mAb) RII and polyclonal antibodies against C1-INH were produced previously [36 (link),37 (link)]. C1s for kinetic assays and ELISA was purchased from Calbiochem (La Jolla, CA, USA). C1s and pAb against C1-INH were biotinylated using EZ-Link^® Sulfo-NHS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) or Pierce (Rockford, ILL) following the manufacturer protocol. Human Factor XIIa and kallikrein were purchased from Kordia (Leiden, The Netherlands). Agarose-bound RCA₁₂₀ and biotinylated-RCA₁₂₀ were from Vector Labs, USA. Streptavidin-peroxidase was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Streptavidin-polymerized peroxidase (poly-HRP) was obtained from Sanquin (Business Unit Reagents, Amsterdam, The Netherlands). Chromogenic substrates S2251, S-2302 and S2366 were from DiaPharma (Beckett Ridge, Ohio, USA); C1s substrate H-D-Val-Ser-Arg.p-NA.2HCl was custom synthesized by Biomatik (Wilmington, DE, USA).