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ELISPOT moderate

White Spots in Center of Normal Spots (Donut Pattern)

Symptom
Spots develop with white or lighter centers surrounded by darker rings, creating a donut-like appearance. This is more common with enzymatic detection methods.
Common Causes
  1. 1 Coating antibody concentration insufficient for optimal capture
  2. 2 Issues during the coating step affecting antibody distribution
  3. 3 For fluorescence detection: antibody concentrations too low
Solutions
  1. 1 Increase coating antibody concentration through optimization
  2. 2 Evaluate spot quality to determine if capture antibody dilution or coating step has issues
  3. 3 For fluorescence detection: increase antibody concentrations for both capture and detection
Related Video (2)
Mabtech ★ 85
ELISpot tutorial: step-by-step full assay protocol
"Step-by-step protocol tutorial directly addresses proper technique execution including coating antibody application, which is the root cause of the donut pattern failure."
JoVE (Open Access) ★ 72
Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
"Demonstrates complete ELISPOT assay workflow with enzymatic detection methods, providing context for when donut patterns occur and how to optimize antibody coating."
Source: abcam.com ↗
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