Before you start
- Plan the plate: standards in duplicate (7-point 2-fold series + blank), samples in duplicate, and spike or known controls.
- Dilute samples so they fall in the middle of the standard curve.
Reagents and materials
Totals are for 96 wells (+10% extra). Per-well amounts are in the middle column.
| Reagent | Per well | Total |
|---|---|---|
| Capture antibody in coating buffer (1–4 µg/mL) | 100 µL | 100 µL |
| Blocking buffer (1% BSA in PBS) | 250 µL | 250 µL |
| Standard or sample | 100 µL | 100 µL |
| Biotinylated detection antibody (0.25–2 µg/mL) | 100 µL | 100 µL |
| Streptavidin-HRP (working dilution) | 100 µL | 100 µL |
| TMB substrate | 100 µL | 100 µL |
| Stop solution (1 M H₂SO₄) | 50 µL | 50 µL |
| Wash buffer (PBS + 0.05% Tween-20)about 5–6 washes | 2 mL | 2 mL |
Buffers and recipes
Carbonate–bicarbonate coating buffer, pH 9.6 per 1 L
| Na₂CO₃ | 1.59 g |
| NaHCO₃ | 2.93 g |
| Ultrapure water | to 1 L |
Check pH 9.6. PBS is an alternative for many antibody pairs.
Wash buffer (PBST) per 1 L
| PBS | 1 L |
| Tween-20 (0.05%) | 0.5 mL |
Equipment
- High-binding 96-well ELISA plate
- Plate sealer
- Plate washer or multichannel pipette
- Plate reader (450 nm with 570 nm reference)
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Coat
Add 100 µL capture antibody per well, seal and incubate overnight at 4 °C.
Wash
Wash 3× with PBST; blot on paper towels.
Block
Add 200–300 µL blocking buffer and incubate 1–2 h at room temperature.
Add standards and samples
Wash 3×, add 100 µL standards and samples and incubate 2 h at room temperature (or overnight at 4 °C).
Detection antibody
Wash 3–4×, add 100 µL detection antibody and incubate 1 h at room temperature.
Streptavidin-HRP
Wash 3–4×, add 100 µL streptavidin-HRP and incubate 20–30 min at room temperature in the dark.
Substrate
Wash 5× (soak 30 s each), add 100 µL TMB and watch the colour develop (5–30 min, dark).
Stop and read
Add 50 µL stop solution (blue turns yellow) and read at 450 nm with 570 nm correction within 30 min.
Analyse
Subtract the blank, fit a four-parameter logistic curve to the standards and interpolate samples; multiply by the dilution factor.
Critical points
- Thorough washing is the main factor for low background.
- Do not let wells dry out between steps.
- Report only values within the standard range.
Troubleshooting
| Problem | What to check or change |
|---|---|
| High background | More/longer washes, fresh blocking buffer, lower HRP concentration, shorter TMB time. |
| No signal | Check antibody pair orientation, reagent order and that HRP was not inhibited (avoid sodium azide). |
| Poor duplicates | Calibrate pipettes, mix samples, avoid bubbles and edge effects. |
Safety
Sulfuric acid is corrosive. TMB is an irritant.
Further reading
- Crowther JR. The ELISA Guidebook (Methods in Molecular Biology).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.