D val leu lys p nitroanilide dihydrochloride

Plasmin formation was evaluated according to protocols described by Vieira et al. (2009 (link)), with slight modification. Leptospires were grown in EMJH medium without rabbit serum to avoid PLG contamination, recovered by centrifugation (10,000 × g, 15 min), washed twice, and resuspended in PBS. Bacterial suspensions were standardized to OD_420nm of 0.5 in 500 μL of PBS. Next, 2 μg of PLG were added followed by incubation for 2 h. Cells were pelleted by centrifugation, washed once, and resuspended in 250 μL of PBS, to which 50 ng of urokinase-type PLG activator (uPA) and 250 μL of plasmin (PLA) substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride (Sigma) were added to a final concentration of 0.4 mM for amidolytic activity determination. For experimental controls, either PLG, uPA, or leptospires were omitted from the reactions. After a 16-h reaction, bacteria were pelleted and the supernatant was collected for absorbance readings at 405 nm in microdilution plates, in quintuplicate.

Human glu-plasminogen was obtained from Haematologic Technologies (Essex Junction, VT, USA). Plasminogen was activated to plasmin using urokinase plasminogen activator (uPA) from Merck Millipore, Darmstadt, Germany. Both the chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) and fibrinogen were purchased from Sigma-Aldrich (Steinheim, Germany). Goat anti-fibrinogen antiserum was purchased from Acris Antibodies (Herford, Germany), mouse anti-His antiserum was obtained from GE Healthcare (Munich, Germany). Horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).

Human glu-plasminogen was obtained from Haematologic Technologies (Essex Junction, VT, USA). Plasminogen was activated to plasmin using urokinase plasminogen activator (uPA) from Merck Millipore, Darmstadt, Germany. Both the chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) and fibrinogen were purchased from Sigma-Aldrich (Steinheim, Germany). Purified C3b was obtained from Complement Technology, Tyler, TX, USA. A. baumannii Tuf was detected using a polyclonal rabbit antiserum raised against Streptococcus pneumoniae Tuf [29 (link)]. C3 and fibrinogen polyclonal antisera were purchased from Acris Antibodies (Herford, Germany). The monoclonal hexahistidine antibody was obtained from GE Healthcare (Munich, Germany). Horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany) and Alexa Fluor 488-conjugated anti-rabbit immunoglobulins from Life Technologies (Darmstadt, Germany).