ELISA stop solution (2 N H₂SO₄) recipe
Stops the TMB reaction and turns blue to yellow for reading at 450 nm: 2 N (1 M) sulfuric acid, added in the same volume as substrate.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Concentrated sulfuric acid (95–98%, ≈18 M) | 5.43 mL | 1 M (2 N) |
| Water (distilled) | to final volume |
Steps
- Put about 80% of the final volume of water in a glass vessel standing in ice water.
- In a fume hood, add the concentrated acid very slowly while stirring (very exothermic — acid into water).
- When cool, bring to volume and store in a glass bottle at room temperature.
Good to know
- Read 450 nm (reference 540 or 570 nm) within 30 min of stopping.
- 1 N HCl also works as a stop solution.
⚠ Safety Concentrated sulfuric acid is highly corrosive and can boil violently with water — wear goggles, gloves and a lab coat, and never add water to acid.
FAQ
Can I read before stopping?
You can read the blue at 650 nm, but it keeps developing; stopping fixes the signal and boosts it about 2–3×.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.