Assay / ELISA / Binding: saturated curve

1. Ranked causes

   1. Sample concentration too high — off the top of the standard curveMultiple sample dilutions all read at the assay's OD ceiling.
   2. Detector conjugate lot concentration too high, driving max signal earlyCurve compresses toward saturation at all but the lowest standards.
   3. Substrate development run to completion (all-or-none color)Standards also plateau at the same OD.

2. Recovery steps

   1. Serially dilute samples 1:5 to 1:50 and re-readBrings concentrated samples back onto the linear/sigmoidal portion of the curve.
   2. Read the plate earlier, before substrate fully developsPrevents standards themselves from plateauing at OD ceiling.
   3. Titrate detection conjugate down 2-foldWidens the assay's dynamic range before saturation.

3. Controls first

   • Do the standard-curve / positive-control wells also plateau at the top OD?If yes: Assay-level saturation (substrate/detector), not just sample concentration.
   • Do the standard-curve / positive-control wells also plateau at the top OD?If no: Sample-specific — dilute and re-read.

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