Biotinylated goat anti mouse igg1

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).

Ig-secreting cells were quantified in Ig-specific assays as described previously.^{24 (link)} In brief, purified goat anti-mouse total Ig (10 μg/ml: SouthernBiotech) was used to coat 96-well plates overnight at 4°C. After blockade with 1% gelatin in PBS, serial dilutions of the purified cell populations were cultured overnight at 37 °C in triplicates. Cells were incubated with either a biotinylated goat anti-mouse IgM or with a biotinylated goat anti-mouse IgG1 (Southern Biotechnology Associates), and then revealed with streptavidin-conjugated alkaline phosphatase (1 h, 37 °C: SouthernBiotech) followed by 5-bromo-4-chloro-3-indolyl-phosphate in 4% low-melt agarose (Sigma-Aldrich, St. Louis, MO, USA).