Sandwich ELISA
The target is sandwiched between a plate-bound capture antibody and a detection antibody, then quantified by an enzyme colour reaction. The standard curve is S-shaped, so diluting samples into its middle is the key step.
Key points
- Dilute samples into the middle of the curve — run 2–3 dilutions if unsure
- Washing controls background and replicate error
- Fit the curve with 4PL, not a straight line
Materials
- Capture/detection antibody pair (or kit)
- Standard protein
- ELISA 96-well plate
- Wash buffer (PBS + 0.05% Tween-20)
- Blocking buffer (e.g. 1% BSA/PBS)
- Streptavidin-HRP
- TMB substrate, stop solution (acid)
- Plate reader at 450 nm (570 nm correction if available)
Steps
- 💡 Plan the standard series with the "Serial dilution" tab of the reagent calculator.
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Sample dilution
What it isHow much to dilute. The steep middle of the S-curve is precise; the flat ends are not.
Less dilutionToo little: above the top standard and saturated — concentration unknown.
More dilutionToo much: near the detection limit, reads as zero or varies wildly.
Typical choiceThe middle of the curve. If unknown, run both ×2 and ×10.
Washing
What it isRemoves unbound antibody and enzyme.
Lower · lessToo little: high background and scattered replicates.
Higher · moreToo harsh or wells dry out: bound antigen/antibody is lost and edge wells deviate.
Typical choice3–5 washes per step; add the next reagent before wells dry.
Incubation time · temperature
What it isTime for antigen–antibody binding.
Lower · lessShort or cold: weaker signal and lower sensitivity.
Higher · moreLong or warm: higher background and saturated top standards.
Typical choiceExactly as the kit states, identical for all plates.
TMB development time
What it isTime the enzyme turns substrate blue; stopped with acid.
Lower · lessShort: low standards cannot be told apart.
Higher · moreLong: top standards saturate (OD > 3) and the upper curve flattens.
Typical choiceStop when the top standard is clearly blue, around OD 2–2.5.
Curve model
What it isThe equation that converts absorbance to concentration.
Typical choice4PL (or 5PL).
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| High background everywhere | Insufficient washing or blocking, too much HRP, cross-contamination | Wash more, change tips, check HRP dilution |
| Flat standard curve | Degraded standard, reagent order mistake, old TMB | Fresh standard, check order, confirm TMB is colourless before use |
| All samples above the top standard | Not diluted enough | Dilute further and repeat |
| Replicate CV above 15% | Pipetting error, bubbles, uneven washing | Multichannel pipette, remove bubbles, check the washer |
⚠ Safety The stop solution (acid) is corrosive — wear eye protection and gloves.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
Standard curve calculator (ELISA 4PL) → Serial dilution calculator → Group comparison statistics →