Complete Absence of Standard Curve Signal with Normal Zero (ELISA (Competitive))

Question

Accepted Answer

All standard concentrations show no detectable signal (very low OD), while the zero standard (B0, maximum binding) produces normal expected signal, indicating complete competition failure. Common causes: 1) Pipetting error causing omission of standards from wells during plate setup 2) Serial dilution error resulting in incorrect standard concentrations 3) Standard concentration vastly exceeds assay dynamic range causing 100% competition at all points 4) Standard stock solution degraded or incorrectly prepared Solutions: 1) Verify standard curve pipetting protocol with careful visual confirmation of additions to each well 2) Recalculate and re-prepare serial dilutions using fresh standard stock solution 3) Decrease starting standard concentration by 10-100 fold to bring into assay dynamic range 4) Confirm standard stock concentration and activity using alternative analytical method (mass spec, HPLC)