Rabbit anti human fibrinogen antibody

Patient plasma fibrinogen was analyzed by SDS-PAGE under reduced conditions (10% polyacrylamide gel) and immunoblots using a rabbit-anti-human fibrinogen antibody (DAKO, Carpinteria, CA, USA) or rabbit anti-human Bβ-chain antibody (Chemicon International, Temecula, CA, USA) [17 (link)]. Reacting species were visualized with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Medical and Biological Laboratories Ltd., Nagoya, Japan) and enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were then exposed on Hyperfilm-ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK).

ECM proteins in coating buffer (50 μl) were added to wells of a high‐binding 96‐well plate (Immulon 2HB) and incubated for 16 hr at 4°C. Wells were washed once in TBSC and non‐specific binding sites were blocked with 3% BSA in TBSC for 1 hr at 37°C. Wells were washed once in TBSC and recombinant protein (0–5 μg) diluted in TBSC was applied to the wells and incubated for 1 hr at 37°C. Unbound protein was removed and wells washed once in TBS. Primary antibody diluted in TBST was added to the wells and incubated for 1 hr at 37°C. Wells were washed twice in TBST before adding HRP‐linked secondary antibody diluted in TBST containing 3% BSA and incubating for 1 hr at 37°C. Wells were washed once in TBST, twice in TBS, and detection reagent (0.102 M Na₂HPO₄, 0.049 M citric acid, 0.012% H₂O₂, 3.7 mM o‐phenylenediamine) was added to wells. Plates were incubated in the dark for 10 min at room temperature, 0.56 M H₂SO₄ was added to stop the reactions and A₄₉₀ measured. x6His‐tagged proteins were detected using anti‐tetraHis antibody (Qiagen) at 1:1000 dilution and HRP‐conjugated anti‐mouse antibody (Dako) at 1:2000 dilution. Fibrinogen was detected using rabbit anti‐human fibrinogen antibody (Dako) at 1:1000 dilution and HRP‐conjugated swine anti‐rabbit antibody (Dako) at 1:2000 dilution.