Sandwich ELISA Protocol — Capture & Detect for Sensitive Quantitation

Sandwich ELISA Protocol — Capture & Detect for Sensitive Quantitation

Overview: Sandwich ELISA uses a capture antibody immobilized on the plate and a separate detection antibody to quantify antigen with high specificity and sensitivity. Ideal for complex matrices and low-abundance analytes.

Materials & Reagents

  • High-binding 96-well microplate (e.g., Nunc MaxiSorp)
  • Capture antibody (unlabeled), optimized coating concentration
  • Detection antibody (unlabeled) — ideally from a different epitope; may be biotinylated or enzyme-conjugated
  • Streptavidin-HRP (if detection antibody is biotinylated)
  • Recombinant antigen or purified standard for standard curve
  • Coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) or PBS, as validated
  • Blocking buffer (1–5% BSA, casein, or specialized blockers)
  • Wash buffer (PBS-T or TBS-T, 0.05% Tween-20)
  • Substrate (TMB recommended for HRP); stop solution (1N HCl or 2N H₂SO₄)
  • Microplate reader (450 nm for TMB stop)

Step-by-Step Protocol (Typical)

  1. Coat with capture antibody: Dilute capture antibody in coating buffer to optimized concentration (typical 0.5–5 µg/mL). 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 to prevent non-specific binding.
  4. Wash: Wash 3× with wash buffer.
  5. Add standards and samples: Prepare a standard curve (serial dilutions of known antigen) and add 100 µL/well of standards, controls, and samples (usually in duplicate or triplicate). Incubate 1–2 hours at RT or overnight at 4°C depending on sensitivity needs.
  6. Wash: Wash 4–5× with wash buffer to remove unbound antigen.
  7. Detection antibody: Add 100 µL/well of detection antibody diluted in blocking buffer. If using a biotinylated detection antibody, incubate 1 hour at RT (or as optimized).
  8. Wash: Wash 4–5× with wash buffer.
  9. Streptavidin-HRP (if applicable): Add 100 µL/well streptavidin-HRP diluted according to manufacturer instructions (usually 1:2000–1:10000). Incubate 20–30 minutes at RT.
  10. Wash: Wash 5× with wash buffer (increase washes if background persists).
  11. Substrate: Add 100 µL/well TMB. Incubate 5–20 minutes protected from light, monitoring color development.
  12. Stop & read: Add 50 µL/well stop solution. Read OD at 450 nm within 30 minutes.

Optimization Tips

  • Antibody pair selection: Choose capture and detection antibodies that bind distinct, non-overlapping epitopes. If possible, validate pairs with a sandwich-pair matrix.
  • Checkerboard titration: Perform checkerboard titration of capture vs detection antibody concentrations to identify the optimal combination with high signal-to-noise.
  • Standard curve range: Design the standard curve to bracket expected sample concentrations and include a blank and multiple replicates for QC.
  • Blocking: Test different blockers (BSA, casein, fish gelatin, commercial blockers) — matrix-specific effects can alter background.
  • Incubation conditions: Longer, cooler incubations (overnight at 4°C) can improve sensitivity for low-abundance analytes.
  • Minimize matrix effects: Include matrix-matched standards or use sample diluent with serum/plasma blockers to reduce interference.

Troubleshooting — Common Problems & Fixes

Problem Possible Cause Recommended Fix
High background Insufficient blocking; cross-reactivity of detection antibody; excess streptavidin-HRP Increase blocking strength/time; reduce detection or streptavidin-HRP concentration; add extra washes; include detergent in blocker.
Low or no signal Capture antibody not binding; antigen degraded; incorrect epitope pair Confirm capture coating by capture-check assay (e.g., add labeled antigen); test antigen integrity; try alternative antibody pairs.
Poor standard curve fit Inaccurate standard prep; adsorption losses; pipetting errors Prepare fresh standards; use low-binding tubes; increase replicates; check pipettes and dilution technique.
High variability Uneven washing; edge effects; inconsistent incubation times Use consistent timing across wells; use plate shaker; avoid using outer wells for critical samples; ensure even plate sealing.

Printable Checklist

Use this bench checklist to keep track of reagent lot numbers, dilutions, and incubation times.

Download Sandwich ELISA Checklist (PDF)
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