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ELISA Development severe

Excessive Signal - Entire Plate Turned Blue

Symptom
Whole plate turned uniformly blue indicating non-specific signal saturation
Common Causes
  1. 1 Insufficient washing or skipped washing step leaving unbound peroxidase
  2. 2 Substrate solution mixed too early and turned blue prematurely
  3. 3 Excessive streptavidin-HRP concentration
  4. 4 Reused plate sealers or reagent reservoirs with residual HRP
  5. 5 Buffers contaminated with metals or HRP
Solutions
  1. 1 Follow proper washing procedure from ELISA Development Guide page 4
  2. 2 Mix and use substrate solution immediately before use
  3. 3 Check streptavidin-HRP dilution and titrate if necessary
  4. 4 Use fresh plate sealer and reagent reservoir for each step
  5. 5 Make fresh buffers
Related Video (3)
Bilibili (China-Accessible Mirrors) ★ 88
DuoSet ELISA — Sandwich ELISA Hands-on Protocol (Bio-Techne)
"Hands-on sandwich ELISA protocol covering washing and substrate development steps, directly showing where excess signal from insufficient washing would occur."
Thermo Fisher Scientific ★ 78
How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial
"Step-by-step Invitrogen ELISA kit tutorial demonstrating proper washing and incubation technique to avoid non-specific peroxidase signal."
Thermo Fisher Scientific ★ 62
How to coat your own plate and run an Invitrogen ELISA kit
"Shows plate coating and running an Invitrogen ELISA kit, providing context for the assay steps where the washing failure arises."
Source: rndsystems.com ↗
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