Primary and Secondary Antibody Incompatibility (ELISA (Signal Problems))

Question

Accepted Answer

No signal or minimal signal in indirect ELISA despite proper coating and blocking. Positive controls with matched antibodies work correctly. Common causes: 1) Secondary antibody is not raised against the host species of the primary antibody (e.g., anti-rabbit secondary used with mouse primary) 2) Isotype mismatch between primary and secondary antibodies 3) Secondary antibody specificity does not recognize primary antibody class or subclass 4) Fragment-specific secondary (e.g., anti-Fc) does not match primary antibody format (e.g., F(ab)2) Solutions: 1) Verify secondary antibody is raised against correct host species (e.g., anti-mouse secondary for mouse primary) 2) Confirm isotype compatibility: ensure secondary recognizes the primary antibody isotype (IgG, IgM, IgA) 3) Check that secondary antibody recognizes the correct antibody fragment (whole IgG, F(ab)2, Fc region) 4) Use species-specific and isotype-specific secondary antibodies matched to datasheet recommendations