🧪 Direct ELISA Protocol (Step by Step)

1️⃣ Plate Coating

🟦 Add "antigen solution" (1–10 µg/mL) to each well

⏱ Incubate overnight at 4°C or 1–2 hr at 37°C

💡 Use high-binding ELISA plates

2️⃣ Washing

💧 Wash plate 3–5x with PBS/TBST

🧼 Add 0.05% Tween-20 to reduce background

3️⃣ Blocking

🛡 Add blocking buffer (1–5% BSA or milk)

⏱ Incubate 1 hr at RT

💡 Prevents non-specific binding

4️⃣ Primary Antibody Binding

🧬 Add enzyme-conjugated primary antibody (HRP/AP)

⏱ Incubate 1–2 hr RT or overnight at 4°C

💡 Optimize antibody concentration for best signal

5️⃣ Washing

💦 Wash plate 3–5x with PBS/TBST

6️⃣ Substrate Addition

🔬 Add enzyme substrate (TMB for HRP)

⏱ Incubate until color develops (5–30 min)

💡 Watch carefully to avoid overdevelopment

7️⃣ Stop Reaction

🛑 Add stop solution (1M H₂SO₄ for TMB)

🎨 Color turns yellow with TMB/HRP

8️⃣ Read Plate

📊 Measure absorbance at 450 nm using plate reader

💡 Include blanks + standards for accuracy

9️⃣ Data Analysis

📈 Plot standard curve

🧮 Calculate antigen concentration in samples

✅ Quick Flow Recap:

🟦 Coat → 💧 Wash → 🛡 Block → 🧬 Bind → 💦 Wash → 🔬 Substrate → 🛑 Stop → 📊 Read → 📈 Analyze