Assay / ELISA / Binding: high cv between replicates

1. Ranked causes

   1. Reagent lot / incomplete mixing before dispenseCV increases toward the later-dispensed wells in a batch; often after a lot change.
   2. Inconsistent pipetting volume between replicate wellsCV worse for manually multichannel-pipetted plates than automated ones.
   3. Edge effect — outer wells evaporate faster during incubationHigh-CV wells cluster on plate columns 1, 12 or rows A, H.

2. Recovery steps

   1. Re-pipette using reverse pipetting technique to avoid bubblesBubbles are the single largest source of well-to-well CV.
   2. Fill unused perimeter wells with buffer ("moat" plate layout)Reduces evaporation-driven edge effects on outer wells.
   3. Vortex or invert master mix immediately before every dispensePrevents settling of denser components over the plating time.

3. Controls first

   • Are replicate CVs high on the standard-curve / control wells as well as samples?If yes: Technique, edge effect, or reagent mixing — not sample biology alone.
   • Are the high-CV wells concentrated on the plate perimeter?If yes: Edge/evaporation effect — use a moat plate or edge-aware layout.

Diagnose your run

Opens Diagnostics capture with this first-party playbook behind it. Sign in if the workspace asks.