Biotinylated goat anti mouse igg2c

Leishmania major-specific IgG1 and IgG2c levels were determined by ELISA in the sera of mice harvested at the termination of the experiment, as previously described (26 (link)), Biotinylated goat anti-mouse IgG2c (Southern Biotech) and biotinylated rat anti-mouse IgG1 (BD Pharmingen) were used. Plates were read at the optical density of 490 nm. Titration curve were performed for all samples.

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).

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).