Isotype Control Does Not Match Test Antibody Background

Symptom

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.

1. 1 Background depends on multiple factors making it difficult to find matching isotype control
2. 2 Isotype control antibody has different binding characteristics than test antibody despite matching isotype
3. 3 Isotype controls by themselves do not account for fluorescence spillover from other channels
4. 4 Test antibody and isotype control have different protein concentrations or conjugation ratios

1. 1 Match isotype control as closely as possible to test antibody in terms of isotype, fluorochrome, and concentration
2. 2 Use isotype controls in combination with compensation to account for both non-specific binding and spillover
3. 3 Consider biological controls as an alternative when isotype matching is problematic
4. 4 Run multiple control types (isotype, FMO, biological) during assay development to compare background measurements
5. 5 Validate that isotype control accurately represents test antibody background before relying on it for gating decisions

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