Streptavidin eu

Activation of the AP was followed by measuring the deposition of C3 fragments onto microtiter well surfaces. A 96-well plate was coated with 20 µg/mL zymosan in 50 mM sodium carbonate pH 9.6 and incubated overnight at 4°C. To block remaining protein binding sites, the plate was subsequently incubated in TBS/Tween (10 mM TRIS pH 7.4, 145 mM NaCl, 0.05% Tween 20) with 1 mg/mL human serum albumin for 1 h at room temperature. The plate was washed three times in TBS/Tween and added 100 µL normal human serum diluted to 11% (v/v) in AP deposition buffer (10 mM HEPES pH 7.5, 140 mM NaCl, 10 mM EGTA, 5 mM MgCl₂) either in the presence or absence of nanobody in concentrations from 4.9-1200 nM. The plate was incubated for 1 h at 37°C in a humid chamber and subsequently washed three times in TBS/Tween. Next, 100 µL 0.75 µg/mL biotinylated anti hC3d (DAKO) in TBS/Tween was added to the wells followed by a 16 h incubation period at room temperature. The wells were washed three times in TBS/Tween, then incubated with 1 µg/mL streptavidin-Eu (PerkinElmer) for 1 h at room temperature. Subsequently, the plate was washed three times in TBS/Tween, followed by incubation with enhancement buffer (Ampliqon) for 2 min. The signal of the europium in the wells was subsequently read by time-resolved fluorometry on a VICTOR 5 plate reader (PerkinElmer) and was given as counts per second.