Non-specific Background Signal Too High in Competitive ELISA

Symptom

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.

1. 1 Detector conjugate binding non-specifically to the plate surface
2. 2 Excessive concentration of detector conjugate antibody
3. 3 Inadequate blocking of unoccupied binding sites on plate
4. 4 Insufficient washing cycles allowing unbound conjugate to remain

1. 1 Run control wells omitting primary antibody and sample to test non-specific binding of detector conjugate
2. 2 Decrease the concentration of the detector conjugate antibody
3. 3 Optimize blocking buffer formulation or increase blocking time/temperature
4. 4 Increase the number and/or duration of wash cycles

Related Video (3)

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