Home › Failure Case Library › Excessive Signal - Entire Plate Turned Blue
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)
Thermo Fisher Scientific ★ 85
How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial
"Step-by-step ELISA protocol tutorial directly addresses proper washing procedures and substrate development to prevent non-specific signal"
Bilibili (China-Accessible Mirrors) ★ 82
DuoSet ELISA — Sandwich ELISA Hands-on Protocol (Bio-Techne)
"Hands-on sandwich ELISA demonstration shows critical washing steps between antibody incubations and substrate development, directly relevant to failure cause"
Thermo Fisher Scientific ★ 78
How to coat your own plate and run an Invitrogen ELISA kit
"ELISA plate coating and kit protocol video covers full workflow including washing steps essential to prevent background signal saturation"
Source: rndsystems.com ↗
← Back to all cases