Excessive Substrate Concentration or Incubation Time (ELISA (High Background))

Question

Accepted Answer

Rapid color development with all wells turning dark quickly. Signal may exceed linear range of reader (OD >2.0). Background wells show significant color development. Common causes: 1) Substrate concentration too high for enzyme activity present 2) Substrate incubation time too long allowing excessive product formation 3) Substrate incubation temperature too high accelerating reaction 4) Delay in reading plate after stop solution addition causing signal drift Solutions: 1) Dilute substrate working solution to reduce reaction rate 2) Decrease substrate incubation time (typically 15-30 minutes) 3) Perform kinetic reading at 600-620nm, add stop solution when OD reaches ~1.0 4) Read plate immediately within 5-10 minutes after adding stop solution 5) Incubate substrate at room temperature, avoid elevated temperatures