A variety of factors can contribute to high background in ELISA, these include:

• Insufficient washing of wells – wash steps are crucial as it removes all unbound materials (e.g. non-specifically bound antibodies or detection regents) that can increase background noise.
• Non-specific binding of antibody – this occurs when an antibody binds to unintended protein. Non-specific binding can be reduced by using an effective blocking buffer or by diluting the antibody concentration.
• Using too much detection reagent – it is important to optimize the concentration of your detection reagent, if the concentration is too high or too low it can lead to high background.
• Using ineffective blocking buffers
• Using contaminated buffers