ELISA Experimental Protocol Complete Series (5 Episodes)

critical

Poor or Non-Linear Standard Curve

Standard curve shows irregular shape, poor linearity, or inconsistent serial dilution response. R-squared value below 0.95 or curve does not follow expected sigmoidal or linear pattern across dynamic range.

💡 5 · ✓ 5

critical

Complete Absence of Standard Curve Signal with Normal Zero

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.

💡 4 · ✓ 4

severe

Excessive Non-Specific Background Signal in Competitive ELISA

High background optical density readings are observed across wells, obscuring the difference between standards and samples. Signal persists even in wells without primary antibody or sample.

💡 4 · ✓ 4

severe

High CV from Inconsistent Pipetting Technique

High intra-assay coefficient of variation (>15%) between technical replicates. Standard curve shows poor reproducibility between duplicate points.

💡 5 · ✓ 5

severe

Serial Dilution Calculation or Execution Error

Standard curve shows irregular pattern or non-monotonic response, with individual points deviating significantly from expected sigmoidal or linear trend.

💡 5 · ✓ 6

severe

B/B0 Ratio at Curve Endpoint Outside 5-95% Range

The B/B0 ratio (bound/maximum bound signal) for the highest or lowest standard concentration exceeds 95% or falls below 5%, indicating the standard curve dynamic range does not adequately cover the analyte concentration range.

💡 4 · ✓ 4

moderate

Standard Curve Plateau at High Concentration End

The top of the standard curve (high analyte concentration, low OD) shows a plateau with minimal signal change across multiple high-concentration standards, reducing dynamic range.

💡 4 · ✓ 4

moderate

Bacterial Contamination in Wash or Incubation Buffers

Sporadic signal loss or high background that worsens over time during multi-day experiments. Cloudy or turbid buffer appearance. Inconsistent results when using same buffer batch.

💡 4 · ✓ 5