Home Failure Case Library High CVs at Bottom of Standard Curve
ELISA (Competitive) moderate

High CVs at Bottom of Standard Curve

Symptom
Coefficient of variation (CV) among replicates is excessively high at the lowest standard concentrations (highest OD in competitive format), reducing precision at the sensitive end of the curve.
Common Causes
  1. 1 Higher OD values inherently demonstrate greater variance in competitive format
  2. 2 Pipetting errors more pronounced at small volume differences
  3. 3 Standard concentrations approaching detection limit of the assay
  4. 4 Samples falling outside optimal measurement range of curve
Solutions
  1. 1 Accept that higher ODs (lower concentrations) naturally show more variance in replicates
  2. 2 Increase the concentration range of the standard curve to shift measurement window
  3. 3 Dilute samples less so they fall in the middle of the standard curve
  4. 4 Use technical triplicates or quadruplicates at low concentrations to improve precision
Related Video (2)
Bilibili (China-Accessible Mirrors) ★ 72
Thermo Fisher Microplate Reader Software Operation
"Microplate reader software operation directly addresses data acquisition and OD measurement precision issues that underlie high CV problems at high OD values in competitive ELISA"
Bilibili (China-Accessible Mirrors) ★ 71
R&D Systems Quantikine ELISA Operation Guide
"R&D Systems Quantikine ELISA protocol includes troubleshooting guidance and complete workflow that contextualizes standard curve construction and precision challenges in competitive formats"
Source: abcam.com ↗
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