Non-specific Background Signal Too High in Competitive ELISA (ELISA (Competitive))

Question

Accepted Answer

High background optical density is observed across wells, reducing signal-to-noise ratio and making it difficult to distinguish specific binding from non-specific interactions. Common causes: 1) Detector conjugate binding non-specifically to the plate surface 2) Excessive concentration of detector conjugate antibody 3) Inadequate blocking of unoccupied binding sites on plate 4) Insufficient washing cycles allowing unbound conjugate to remain Solutions: 1) Run control wells omitting primary antibody and sample to test non-specific binding of detector conjugate 2) Decrease the concentration of the detector conjugate antibody 3) Optimize blocking buffer formulation or increase blocking time/temperature 4) Increase the number and/or duration of wash cycles