Assay / ELISA / Binding: edge effects

1. Ranked causes

   1. Coating / blocking reagent lot under-fills or beads on perimeter wellsNew coating/blocking lot restores even perimeter OD; under-filled edge wells visible at coating.
   2. Coating / blocking volume short on perimeter wellsUnder-filled edge wells visible at coating; full volume removes bias.
   3. Plate washer / dispenser sprays edges differentlyManual wash of edges removes the pattern; washer service log overdue.
   4. Evaporation during incubation (uncovered or dry incubator)Edge OD drifts with longer incubations; sealing or humidity fixes it.
   5. Temperature gradient across the plate during incubationStacking plates or cold bench spots correlate with edge bias.

2. Recovery steps

   1. Seal plates and use a humidity tray / moat of buffer in unused wellsEvaporation is the dominant edge-effect driver.
   2. Verify washer manifold alignment; re-run with manual edge-aware washesSeparates instrument spray patterns from true biology.
   3. Incubate single plates on a rack (not stacked) at verified temperatureReduces thermal gradients across perimeter wells.

3. Controls first

   • Do blank / standard-curve edge wells show the same perimeter bias as samples?If yes: Evaporation, washer, or thermal gradient — not sample-specific biology.

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