Complete Absence of Standard Curve Signal with Normal Zero

Symptom

All standard concentrations show no detectable signal (very low OD), while the zero standard (B0, maximum binding) produces normal expected signal, indicating complete competition failure.

1. 1 Pipetting error causing omission of standards from wells during plate setup
2. 2 Serial dilution error resulting in incorrect standard concentrations
3. 3 Standard concentration vastly exceeds assay dynamic range causing 100% competition at all points
4. 4 Standard stock solution degraded or incorrectly prepared

1. 1 Verify standard curve pipetting protocol with careful visual confirmation of additions to each well
2. 2 Recalculate and re-prepare serial dilutions using fresh standard stock solution
3. 3 Decrease starting standard concentration by 10-100 fold to bring into assay dynamic range
4. 4 Confirm standard stock concentration and activity using alternative analytical method (mass spec, HPLC)

Related Video (3)

Thermo Fisher Scientific ★ 80

How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial

"Full step-by-step ELISA protocol tutorial directly shows correct reagent and standard pipetting procedures that, if omitted, cause the described failure."

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R&D Systems Quantikine ELISA Operation Guide

"Official hands-on Quantikine ELISA demonstration covers standard preparation and plate setup, illustrating how standards should be added and what normal signal should look like."

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"Concise benchwork demonstration makes pipetting steps and well placement clearly visible, useful for comparing against a no-standard-signal setup error."