Thermo Fisher Microplate Reader Software Operation

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

Detection System Lacks Sensitivity for Target Concentration

Low or no signal despite proper assay setup. High concentration samples may show weak signal while low concentration samples are undetectable.

💡 4 · ✓ 4

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

High Coefficient of Variation at Low Standard Concentrations

Replicate wells at the bottom of the standard curve (low analyte, high OD) show high CVs >15-20%, while mid-curve and high-concentration points demonstrate acceptable reproducibility.

💡 4 · ✓ 4

moderate

Wrong Instrument Settings for Detection Wavelength

No signal or very low signal readings from plate reader despite visible color development or expected fluorescence. Signal does not correlate with visual observations.

💡 4 · ✓ 4

moderate

Excessive Substrate Concentration or Incubation Time

Rapid color development with all wells turning dark quickly. Signal may exceed linear range of reader (OD >2.0). Background wells show significant color development.

💡 4 · ✓ 5

moderate

Biological Sample Not in Detectable Range

Standard curve appears normal, but sample wells show no signal or signal below the lowest standard. This occurs when the analyte concentration in samples is below the assay detection limit.

💡 4 · ✓ 4

moderate

High Variability Between Experimental Runs

Standard curves or sample values differ significantly between experiments run on different days, despite using the same reagents and protocol, showing poor reproducibility.

💡 5 · ✓ 5

moderate

Edge and Drift Effects on ELISA Plate

Wells at the plate edges show systematically higher or lower absorbance than center wells, or a gradient pattern appears across the plate, affecting data quality especially for samples in edge positions.

💡 5 · ✓ 5

moderate

High Coefficient of Variation at Lower Standard Curve

Replicate measurements at the bottom of the standard curve (highest OD values, lowest analyte concentrations) show excessive variability with high coefficient of variation (CV) between replicates.

💡 4 · ✓ 4

moderate

Standard Curve Fitting Model Not Appropriate

The recommended curve-fitting model (typically Four-Parameter Logistic) does not adequately fit the standard data points, resulting in poor correlation even with properly prepared standards.

💡 4 · ✓ 5

moderate

B/B0 Ratio at Standard Curve Endpoint Out of Range

The B/B0 ratio (bound/maximum bound) at the lowest or highest standard concentration is either >95% or <5%, indicating the standard curve dynamic range is not properly positioned.

💡 3 · ✓ 3

moderate

High CVs at Bottom of Standard Curve

Coefficient of variation (CV) among replicates is excessively high at the lowest standard concentrations (highest OD in competitive format), reducing precision at the sensitive end of the curve.

💡 4 · ✓ 4

moderate

High CV from Poorly Maintained Plate Reader

High coefficient of variation (>15%) across the plate with no obvious pattern. Signal intensity may drift over time or show unexpected variability between identical samples.

💡 5 · ✓ 5

minor

Standard Curve OD Values Differ From Datasheet

The optical density (OD) measurement values for the standard curve vary considerably from the examples shown on the kit datasheet or protocol booklet, causing user concern about assay validity.

💡 4 · ✓ 4

minor

Sample Values Above Assay Range (Hook Effect Excluded)

Sample absorbance readings exceed the top standard, reading off the curve, while the standard curve itself appears normal with proper shape and dynamic range.

💡 2 · ✓ 3

minor

Plate Contamination or Optical Interference

Random high background in specific wells or patterns. Fingerprints, dust, or residue visible on plate bottom. Erratic readings not following expected pattern.

💡 4 · ✓ 5