Insufficient Signal from Zero Standard (B0) (ELISA (Competitive))

Question

Accepted Answer

The zero standard (no competing antigen, maximum antibody binding) produces OD values that are too low, resulting in compressed standard curve with poor sensitivity. Common causes: 1) Antibody concentration too low to produce adequate coating or detection signal 2) Insufficient substrate incubation time preventing full color development 3) Antibody activity lost due to improper storage or freeze-thaw cycles 4) Substrate reagent expired or improperly stored reducing enzymatic activity Solutions: 1) Increase coating antibody concentration by 1.5-2 fold or increase detector conjugate concentration incrementally 2) Extend substrate incubation time from 15 minutes to 30 minutes monitoring color development 3) Use freshly thawed aliquots of antibody stored at -20°C or -80°C avoiding repeated freeze-thaw 4) Prepare fresh substrate solution immediately before use and verify substrate reagent expiration dates