Assay / ELISA / Binding: hook effect

1. Ranked causes

   1. Analyte concentration above the assay’s hook threshold1:10–1:100 dilution raises apparent concentration into the standard range.
   2. Antibody pair affinity/concentration set too low for high-load samplesRicher detection antibody reduces hooking on the same samples.
   3. Insufficient wash leaving unbound analyte that blocks detectionExtra washes plus dilution both move results upward.
   4. Standard curve too narrow to flag the hook visuallyExtending standards higher reveals non-monotonic sample behaviour.

2. Recovery steps

   1. Re-assay samples at 1:10 and 1:100; report the dilution that falls on-curveDilution test is the definitive hook check.
   2. Increase detection antibody concentration one step if dilutions still hookSome kits need richer detection for high-load matrices.
   3. Extend the standard curve upward for future runs of this sample typeMakes hooking visible before results are reported.

3. Controls first

   • Do standard-curve / positive-control wells look normal while neat high samples read paradoxically low?If yes: Hook/prozone behaviour — dilute samples and re-report; standards are not the failure.
   • Do diluted samples read higher (on-curve) than the neat sample?If yes: Confirmed hook — report the on-curve dilution.

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