The zero standard (maximum binding, no competitor) shows insufficient optical density signal, providing inadequate dynamic range for the competitive assay and poor B/B0 calculations.
Common Causes
1Antibody concentration too low for sufficient binding
2Insufficient antibody coating or capture efficiency
3Substrate incubation time too short for adequate color development
4Antibody-enzyme conjugate activity reduced due to storage conditions
Solutions
1Increase antibody concentration to enhance binding signal
2Optimize coating conditions (concentration, time, temperature) if using coated antibody
3Incubate longer with the substrate to allow fuller color development
4Verify conjugate storage conditions and use fresh aliquots if needed