Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 8)

2. Remove any unbound Ag by flooding the wells of the assay
plates with PBS. Use a plastic wash bottle or an automatic
plate washer. When using a wash bottle, fill each well, soak
for 15 seconds, then vigorously shake off solution in a sink
(flick-washing). Repeat 2 times.
3. To prevent nonspecific binding, fill all the wells with 300 µl
blocking solution(1% BSA-PBS). Let stand at room temperature
for 30 minutes, then flick-wash the plate 3x with PBS-tween.
4. Add hybridoma culture fluid (undiluted) or ascites (diluted)
samples, 100 µl/well, using the suggested format outlined in
Figure 1. Six wells of columns 2-11 are used (1 sample per
column). Column 1 is reserved for substrate blank and column
12 for positive control (
i.e., mouse serum). Cover and incubate
the plate for 1 hour at room temperature.
5. Empty the plates of hybridoma supernatant or diluted ascites
fluid. Flick-wash the plate 3x with PBS-Tween.
6. Add appropriate rabbit anti-mouse panel reagents using the
format in Figure 1. All of rows A-H are filled with respective
panel reagent, 100 µl/well. Incubate the covered microplate for
1 hour at room temperature.
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