Eu3 labeled streptavidin

Serum ficolin-1 and ficolin-3 concentrations were determined by TRIFMA [as described by Wittenborn et al. (7 (link))] and ELISA [according to Michalski et al. (47 (link))], respectively. Ficolin-2 levels were measured by TRIFMA. Briefly, microtiter plates (Optiplate-384HB, Perkin Elmer, Waltham, MA, USA) were coated with antificolin-2 (ABS 005-16, BioPorto Diagnostics, Copenhagen, Denmark, 1 μg/ml). Plates were blocked with 0.1% BSA and then incubated with sera to be tested, pre-diluted 1:10. Biotinylated mAb (clone GN4, 1:100, Hycult Biotech, Uden, The Netherlands) and Eu³⁺-labeled streptavidin (Perkin Elmer) were used for detection. After incubation with the enhancement solution (Perkin Elmer), fluorescence values were measured using Varioskan Flash reader (ThermoFisher Scientific, Waltham, MA, USA). Serum from a healthy volunteer (ficolin-2 concentration: 3,500 ng/ml) was used as a standard.
“Low” and “high” values were arbitrarily based on 10 and 95th percentiles, respectively, determined for the control group. Consequently, concentrations <620 ng/ml (ficolin-1), <1,670 ng/ml (ficolin-2), and <12.9 μg/ml (ficolin-3) were considered “low,” while concentrations >2,900 ng/ml, >6,350 ng/ml, and >34.9 μg/ml, respectively, were considered “high.”

A FluoroNunc Maxisorp 96-well plate was coated with 1 µg/mL C11 in PBS and incubated overnight at 4 °C. Wells were blocked with 200 µL TBS containing 1 mg/ml I (Human Albumin CSL 109697) for 1 hour at RT and washed thrice with TBS/Tw (TBS containing 0.05% v/v Tween-20 (8.17072.1000, Merck)). Samples and standard (purified 9D11 were diluted in TBS/Tw containing heat agg. nhIg 100 µg/ml (Octapharma 478393, incubated 63 °C 30 min), and subsequently added in duplicates. The plate was incubated at 4 °C overnight. Wells were washed thrice in TBS/Tw and incubated with biotinylated goat-anti-mouse IgG1 (1073-08, Southern Biotech), 1 µg/ml TBS/Tw, at RT for 2 hours. Wells were washed 3 times in TBS/Tw, and Eu³⁺-labeled streptavidin (1244-360, PerkinElmer) diluted 1:1,000 in TBS/Tw containing 25 µM EDTA was subsequently added to the wells and incubated at RT for 1 hour. Finally, the wells were washed thrice with TBS/Tw, and 200 µL enhancement buffer (AMPQ99800, Amplicon) was added. The plate was shaken for 5 minutes and counts were read in a time-resolved fluorometry plate reader (Victor X5, PerkinElmer).

A FluoroNunc Maxisorp 96-well plate was coated with 1 µg/mL 9D11 in PBS and incubated overnight at 4 °C. Wells were blocked with 200 µL TBS containing 1 mg/ml I (Human Albumin CSL 109697) for 1 hour at RT and washed thrice with TBS/Tw (TBS containing 0.05% v/v Tween-20 (8.17072.1000, Merck)). Samples and standard (purified C11 were diluted in TBS/Tw containing heat agg. nhIg 100 µg/ml (Octapharma 478393), incubated 63 °C 30 min., and subsequently added in duplicates. The plate was incubated at 4 °C overnight. Wells were washed thrice in TBS/Tw and incubated with biotinylated goat-anti-mouse IgG2b (1093-08, Southern Biotech), or biotinylated goat-anti-mouse IgG2c (1077-08, Southern Biotech) or biotinylated mouse-anti-mouse IgM(b) (553519 BD), 1 µg/ml TBS/Tw, at RT for 2 hours. Wells were washed 3 times in TBS/Tw, and Eu3 + -labeled streptavidin (1244-360, PerkinElmer) diluted 1:1,000 in TBS/Tw containing 25 µM EDTA was subsequently added to the wells and incubated at RT for 1 hour. Finally, the wells were washed thrice with TBS/Tw, and 200 µL enhancement buffer (AMPQ99800, Amplicon) was added. The plate was shaken for 5 minutes and counts were read in a time-resolved fluorometry plate reader (Victor X5, PerkinElmer).