Troubleshooting ELISA results
Elisa Wells
If your analysis workflow is solid but the numbers still do not behave, it is usually because the plate performance is telling you something real. The key is to diagnose patterns first, then choose the smallest change that will fix the issue.
Below are common failure modes and what to check next.
Low OD values or weak signal
What it looks like
- Standards and samples are all close to background
- Positive control is low or absent
- Little separation between standard points
Most common causes
- A critical reagent was missed or added in the wrong order (detection antibody, conjugate, substrate, stop)
- Substrate or conjugate activity is compromised (age, light exposure, contamination)
- Incubation time or temperature was too short or too cold for your protocol
- Capture or detection reagents are too dilute for the target abundance
- Excessively harsh washing or aspiration disturbs bound complexes (more likely in some in-house coatings)
What to check next
- Confirm the reagent addition sequence against the plate map and worksheet
- Verify that the positive control rises well above background before interpreting samples
- Check substrate and conjugate handling: storage conditions, light protection, and expiration
- If the assay is in-house, confirm coating conditions and that the plate type matches the protocol
- Consider a longer incubation or a higher concentration of capture or detection reagents, one change at a time
High background or elevated signal in negatives
What it looks like
- Blank or zero standard is higher than expected
- Negative controls read well above background
- Standards compress because the baseline is lifted
Most common causes
- Incomplete wash removal (residual conjugate or sample remains in wells)
- Detection antibody or conjugate concentration is too high
- Blocking is insufficient for the plate chemistry or sample matrix
- Incubation is too long or too warm, increasing non specific interactions
- Substrate incubation is uneven across the plate, producing drift
What to check next
- Inspect washing technique: soak time, aspiration depth, and consistency across rows
- Confirm that wash buffer is correct and freshly prepared if made in-house
- Titrate detection antibody or conjugate down rather than changing multiple steps
- Strengthen blocking or switch blockers based on the assay and sample matrix
- Reduce incubation time or tighten temperature control before changing reagents
Standard curve problems or unreliable curve fitting
What it looks like
- Standards are not monotonic (a point goes up then down)
- Replicates diverge at specific points
- The curve fits but back calculated standards are inconsistent
- Samples fall in regions where the curve is flat or unstable
Most common causes
- Standard dilutions were prepared inaccurately or not mixed thoroughly
- Standards span the wrong range for the samples (too narrow or shifted)
- Edge effects or plate trends distort some standards
- The wrong curve model is used for the assay format (especially competitive assays)
What to check next
- Re-make the standards with careful mixing and fresh tips at each step
- Use more points and ensure they bracket the expected sample range
- Place standards in a layout that reduces positional bias (avoid only outer wells if edge effects are suspected)
- Confirm the curve model matches the assay behavior (many sandwich assays fit 4PL or 5PL, competitive assays can invert)
- Evaluate fit using residuals and back calculated standards, not only R squared
High replicate variation or high intra-assay CV
What it looks like
- Duplicate or triplicate wells disagree
- CV spikes for certain samples but not others
- Variation clusters on one side of the plate
Most common causes
- Pipetting inconsistency, especially during standards, conjugate, or substrate additions
- Inadequate mixing of samples or standards (layering, precipitation, viscous matrices)
- Timing drift when using single channel pipetting across many wells
- Bubbles, splashing, or partial well drying during washing
- Edge effects from evaporation or temperature gradients
What to check next
- Use a multi-channel pipette where possible and keep addition timing consistent
- Mix standards and samples immediately before loading, especially in serum or plasma matrices
- Visually inspect wells for bubbles before reading and burst them carefully if present
- Use plate seals and stable incubation conditions to reduce evaporation
- If edge effects persist, reserve edge wells for buffer or controls rather than critical samples
Out of range samples
What it looks like
- Sample ODs are above the top standard or at the upper plateau
- Sample ODs are indistinguishable from background
Most common causes
- Dilution factor does not match the expected concentration
- Matrix effects reduce apparent signal at low concentrations
- The assay sensitivity is not sufficient for the sample type
What to do
- For high samples, dilute further and rerun until the OD falls in the quantifiable range
- For low samples, consider concentrating the sample, increasing sample volume if allowed, or using a more sensitive assay
- If you suspect matrix interference, compare a sample dilution series or use matrix matched standards when possible
Remember
When troubleshooting, change one variable at a time and keep your controls constant. The fastest way to lose a day is to adjust wash conditions, antibody concentration, incubation time, and substrate timing all at once and then not know what helped.
