Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 12)

Problem Probable Cause Recommended Solution
C. Monoclonal 1. Impure sample. 1. Use fresh source of cul-
exhibits multiple ture supernatant. Purify
sub-isotype ( i.e. media or ascites fluid. 5-7
assay shows one 2. Sample too con- 2. Dilute supernanants and
clone to be both centrated ascites fluid, repeat test.
an IgG1 and 3. Sample is polyclonal. 3. Re-clone hybridoma cells
IgM). by limited dilution, 8 and
repeat assay.
Notes to Troubleshooting Guide
1. Activity Test for Horseradish Peroxidase Substrate Solution.
Combine 1.0 ml of substrate solution with 10 ml of antibody
conjugate. If no color develops in 5 minutes, substrate solution
is at fault.
2. Activity Test for Antibody Conjugate Solution.
Combine 1.0 ml of horseradish peroxidase substrate solution
(tested above) and 1.0 ml of diluted antibody conjugate
solution. If no color develops in 5 minutes, the conjugate is
suspect. Repeat procedure using fresh conjugate antibody
dilution.
3. Activity Test for Monoclonal Antibody Solution.
Use RID, Ouchterlony precipitation, or ELISA assay to
determine reactivity. Repeat procedure with more
concentrated monoclonal antibody solution.
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