📋 Protocol
Full flow at a glance, add products directly1
Coating (Capture Antibody)
过夜 / 2 hDilute capture antibody in coating buffer, add to plate, incubate 4°C overnight or 37°C 2 h.
💡 Tip Wash 3× with wash buffer after coating to remove unbound antibody.
⚠️ Caution Optimize antibody concentration; too high wastes reagent, too low is unsaturated.
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2
Blocking
1 hAdd BSA/non-fat milk blocking buffer to cover unbound sites and reduce non-specific binding.
💡 Tip Using blocking buffer from the same species reduces cross-reactivity.
⚠️ Caution Insufficient blocking causes high background.
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3
Add Sample (Antigen/Standards)
1–2 hAdd standards (for the standard curve) and samples, incubate at room temperature.
💡 Tip Make serial dilutions of standards; run samples in duplicates.
⚠️ Caution Add samples quickly and consistently to reduce well-to-well error.
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4
Add Detection Antibody
1–2 hAdd biotin-labeled detection antibody to bind the captured antigen.
💡 Tip Detection and capture antibodies must recognize different epitopes (key to sandwich ELISA).
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5
Add Enzyme Conjugate (HRP-Streptavidin)
30 minAdd HRP-streptavidin, which binds biotin with high affinity.
💡 Tip Wash thoroughly after this step; unbound conjugate causes high background.
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6
Develop & Read
30 minAdd TMB substrate, stop with stop solution, read OD at 450 nm, quantify via standard curve.
💡 Tip Fix development time (e.g. 15–20 min) and stop on time for comparability.
⚠️ Caution Protect TMB from light; stop solution (acid) is corrosive.
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⚠️ 4 Common Beginner Mistakes (Pitfall Guide)
- ① Insufficient washing: high background, false positives — wash 3× each step.
- ② Edge effect: uneven temperature at edge wells skews readings — use plate sealer.
- ③ Poor standard curve: bad dilution, R² < 0.99 — redo.
- ④ Sample out of linear range: OD beyond curve — dilute and re-measure.
❓ FAQ (Troubleshooting)
+High background?
Check: (1) washing thoroughness, (2) blocking sufficiency, (3) antibody concentration.
+Low standard-curve R²?
Check standard dilution accuracy and expiry; re-dilute serially with replicates.
+Weak or no signal?
Check: (1) wrong antibody pair, (2) missed component, (3) sample lacks analyte, (4) substrate expired.