Isotype Control Does Not Match Test Antibody Background (Flow Cytometry (Controls))

Question

Accepted Answer

Isotype control fails to accurately represent the non-specific binding background of the test antibody. Background levels measured by isotype control differ significantly from actual test antibody background. Common causes: 1) Background depends on multiple factors making it difficult to find matching isotype control 2) Isotype control antibody has different binding characteristics than test antibody despite matching isotype 3) Isotype controls by themselves do not account for fluorescence spillover from other channels 4) Test antibody and isotype control have different protein concentrations or conjugation ratios Solutions: 1) Match isotype control as closely as possible to test antibody in terms of isotype, fluorochrome, and concentration 2) Use isotype controls in combination with compensation to account for both non-specific binding and spillover 3) Consider biological controls as an alternative when isotype matching is problematic 4) Run multiple control types (isotype, FMO, biological) during assay development to compare background measurements 5) Validate that isotype control accurately represents test antibody background before relying on it for gating decisions