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ELISA Development severe

Poor Assay-to-Assay Reproducibility

Symptom
Inconsistent results between different assay runs with same samples
Common Causes
  1. 1 Insufficient washing
  2. 2 Automatic plate washer ports clogged
  3. 3 Variations in incubation temperature
  4. 4 Variations in protocol between runs
  5. 5 Reused plate sealers with residual HRP
  6. 6 Improper calculation of standard curve dilutions
  7. 7 Contaminated buffers
Solutions
  1. 1 Follow washing procedure from ELISA Development Guide page 4
  2. 2 If using automatic plate washer, check all ports are clean and free of obstructions
  3. 3 Adhere to recommended incubation temperature
  4. 4 Avoid incubating plates in areas where environmental conditions vary
  5. 5 Adhere to same protocol from run to run
  6. 6 Use fresh plate sealer for each step
  7. 7 Check calculations and make new standard curve
  8. 8 Use internal controls
  9. 9 Make fresh buffers
Related Video (3)
Thermo Fisher Scientific ★ 82
How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial
"Step-by-step ELISA protocol tutorial directly addresses proper technique execution, including washing procedures critical to reproducibility"
Bilibili (China-Accessible Mirrors) ★ 79
DuoSet ELISA — Sandwich ELISA Hands-on Protocol (Bio-Techne)
"Hands-on sandwich ELISA demonstration explicitly covers all protocol steps including plate coating, washing, and substrate development where insufficient washing causes assay-to-assay variability"
Thermo Fisher Scientific ★ 71
How to coat your own plate and run an Invitrogen ELISA kit
"Plate coating and ELISA kit execution video provides practical guidance on the foundational steps where washing protocol deficiencies originate"
Source: rndsystems.com ↗
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