Colorimetric ELISA Protocol — Reliable Absorbance-Based Detection

Materials & Reagents

• High-binding 96-well microplate
• Coating buffer (carbonate–bicarbonate, pH 9.6)
• Capture antibody
• Blocking buffer (1–5% BSA, casein, or milk in PBS/TBS)
• Sample and standards (antigen)
• HRP-conjugated detection antibody
• TMB, OPD, or 4CN substrate solution
• Stop solution (2N H₂SO₄ or 1N HCl)
• Wash buffer (PBS-T or TBS-T, 0.05% Tween-20)
• Plate reader set to 450 nm (reference at 570 nm optional)

Step-by-Step Protocol

1. Coating: Add 100 µL/well of capture antibody diluted in coating buffer (1–10 µg/mL). Incubate overnight at 4°C or 2 hours at 37°C.
2. Wash: Wash wells 3× with PBS-T.
3. Block: Add 200 µL/well blocking buffer. Incubate 1 hour at room temperature.
4. Sample addition: Add 100 µL/well of samples and standards. Incubate 1–2 hours at RT.
5. Wash: Wash 4× with PBS-T to remove unbound analytes.
6. Detection antibody: Add HRP-conjugated detection antibody (diluted per datasheet). Incubate 1 hour at RT.
7. Wash: Wash 4–5× with PBS-T.
8. Substrate addition: Add 100 µL/well of TMB (or OPD/4CN). Incubate until sufficient color develops (typically 5–15 minutes, avoid overdevelopment).
9. Stop reaction: Add 50 µL of stop solution. The blue color turns yellow (for TMB).
10. Read plate: Measure absorbance at 450 nm using a plate reader within 30 minutes of stopping the reaction.

Optimization Tips

• Substrate selection: Use TMB for most HRP-based ELISAs; OPD for faster kinetics, 4CN for high-contrast visualization.
• Timing: Record the exact color development time for consistency.
• Temperature: Perform incubations at consistent temperature to reduce variability.
• Stop solution volume: Equalize across all wells to ensure uniform optical density.
• Plate reading: Read promptly after stopping to prevent fading or drift.

Troubleshooting

Printable Checklist

Download a printable Colorimetric ELISA checklist for tracking reagent prep, incubation, and timing consistency.