Primary-secondary antibody incompatibility preventing detection

Symptom

No signal detected despite proper primary antibody binding. Secondary antibody fails to recognize or bind to the primary antibody, breaking the detection chain.

1. 1 Secondary antibody not raised against primary antibody host species
2. 2 Isotype mismatch between primary and secondary antibodies
3. 3 Secondary antibody conjugate incompatible with detection system
4. 4 Wrong secondary antibody selected for primary antibody class (IgG, IgM, etc.)

1. 1 Ensure secondary antibody is raised against primary antibody species (e.g. anti-rabbit for rabbit primary)
2. 2 Verify isotype compatibility between primary and secondary antibodies
3. 3 Confirm secondary antibody conjugate matches detection method (HRP for DAB, fluorophore for IF)
4. 4 Check that secondary antibody recognizes primary antibody class (IgG, IgM, etc.)
5. 5 Run positive control with validated antibody pair to confirm detection system

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