High background in Zero (NSB, blank)

Contamination of reagents/samples

• May be contamination of reagents or samples, or cross contamination from splashing between wells. Use fresh reagents and pipette carefully.

Insufficient washing of plates

• Ensure well areas are washed adequately by filling the wells with wash buffer. Ensure all residual antibody solutions are removed before washing.

Too much antibody used leading to non-specific binding

• Check the recommended amount of antibody suggested. Try using less antibody, or diluting antibody in appropriate buffer.