B/B0 Ratio Outside Acceptable Range at Curve Endpoints (ELISA (Competitive))

Question

Accepted Answer

The B/B0 ratio (bound/maximum bound) for the terminal point of the standard curve is either >95% or <5%, indicating insufficient competitive displacement range. Common causes: 1) Standard concentration range not optimized for the competitive binding dynamics 2) Highest standard concentration insufficient to achieve >95% competition 3) Lowest standard concentration causing excessive competition beyond useful detection limit Solutions: 1) Increase standard concentration by one dilution factor (e.g., 2× or 3×) to shift curve upward if B/B0 >95% 2) Decrease standard concentration by one dilution factor to shift curve downward if B/B0 <5% 3) Re-establish standard curve with 7-8 points spanning 2-3 orders of magnitude around expected sample concentrations