🧪 Direct ELISA Protocol (Step by Step)

Materials & Reagents

• High-binding 96-well microplate (e.g., Nunc MaxiSorp, Greiner)
• Antigen (purified protein) at coating concentration
• Coating buffer (e.g., 0.05 M carbonate-bicarbonate, pH 9.6)
• Blocking buffer (1–5% BSA or 5% nonfat dry milk in PBS or TBS)
• Primary antibody — enzyme-conjugated (e.g., HRP-conjugated)
• Wash buffer (PBS-T or TBS-T, 0.05% Tween-20)
• Substrate (TMB for HRP) and stop solution (1N HCl or 2N H2SO4)
• Microplate reader (450 nm for TMB stop)

Step-by-Step Protocol

1. Plate coating: Dilute antigen to the optimized coating concentration (typical range 0.1–5 µg/mL) in coating buffer. Add 100 µL/well and incubate 1–24 hours at 4°C or 1–2 hours at 37°C.
2. Wash: Remove coating solution and wash wells 3× with 200 µL wash buffer.
3. Block: Add 200 µL/well blocking buffer and incubate 1 hour at room temperature (RT) with gentle shaking.
4. Wash: Wash 3× with wash buffer.
5. Primary antibody: Add 100 µL/well of enzyme-conjugated primary antibody diluted in blocking buffer. Incubate 1 hour at RT or overnight at 4°C.
6. Wash: Wash 5× with wash buffer to minimize background.
7. Substrate: Add 100 µL/well TMB. Incubate 5–20 minutes protected from light, monitoring color development.
8. Stop: Add 50 µL/well stop solution. Read OD at 450 nm within 30 minutes.

Optimization Tips

• Titrate antigen (e.g., serial 2-fold) to find minimal coating that gives robust signal.
• Titrate enzyme-conjugated antibody to reduce background and cost.
• Test blocking buffers (BSA, casein, milk) — some antigens bind better with specific blockers.
• Increase wash stringency (more washes or longer soak) if background is high.

Troubleshooting — Quick Checks

Checklist & Resources

Download a printable checklist for the Direct ELISA protocol (materials, timings, and QC checks) to use at the bench.