Competitive ELISA Protocol — Quantitative Detection via Antigen Competition

Competitive ELISA Protocol — Quantitative Detection via Antigen Competition

Overview: The Competitive ELISA is designed for quantifying small molecules or antigens with a single epitope. It relies on the competition between sample antigen and labeled antigen for limited antibody binding sites, producing an inverse relationship between signal and analyte concentration.

Materials & Reagents

  • High-binding 96-well microplate (e.g., Nunc MaxiSorp)
  • Purified antigen (for coating and standard curve)
  • Primary antibody specific to the antigen
  • HRP-conjugated antigen or HRP-conjugated secondary antibody
  • Blocking buffer (1–5% BSA, casein, or fish gelatin)
  • Wash buffer (PBS-T or TBS-T, 0.05% Tween-20)
  • TMB substrate and stop solution (1N HCl or 2N H₂SO₄)
  • Plate reader (450 nm)

Step-by-Step Protocol

  1. Coating: Add 100 µL/well of antigen diluted in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) at an optimized concentration (1–10 µg/mL). Incubate overnight at 4°C.
  2. Wash: Wash plate 3× with PBS-T to remove unbound antigen.
  3. Block: Add 200 µL/well of blocking buffer and incubate 1 hour at RT to prevent non-specific binding.
  4. Prepare competition mix: In a separate plate or tubes, mix sample antigen (unknown) with a fixed amount of antibody (and labeled antigen if applicable). Incubate 1 hour at RT for competition to occur.
  5. Add competition mixture: Transfer 100 µL/well of the antigen–antibody mix to the coated and blocked plate. Incubate 1–2 hours at RT.
  6. Wash: Wash 3–5× with wash buffer.
  7. Add HRP-conjugate: If the primary antibody is unlabeled, add HRP-conjugated secondary antibody (1:2000–1:10000 dilution). Incubate 1 hour at RT.
  8. Wash: Wash 4–5× with wash buffer.
  9. Develop color: Add 100 µL/well of TMB substrate. Incubate 5–20 minutes protected from light.
  10. Stop & read: Add 50 µL/well stop solution. Read at 450 nm within 30 minutes.

Data Analysis

Plot the absorbance values against the logarithm of standard antigen concentration. The signal decreases with increasing antigen concentration in a competitive ELISA. Fit data with a 4-parameter logistic (4PL) curve to determine unknown sample concentrations.

Optimization Tips

  • Antibody–antigen ratio: Optimize the ratio of antibody to labeled antigen for dynamic range and sensitivity.
  • Coating concentration: Adjust antigen coating level to maximize signal-to-noise without saturation.
  • Competition time: Longer incubation allows full equilibrium and improved precision.
  • Blocking buffer choice: Use blockers that reduce cross-reactivity without affecting specific binding.
  • Signal inversion: Remember, higher antigen = lower OD — check curve orientation during analysis.

Troubleshooting

Issue Possible Cause Fix
Low signal overall Too low coating antigen concentration; insufficient antibody Increase coating concentration or antibody amount in mix.
No inverse correlation in standard curve Competition step incomplete or incorrect reagent order Increase incubation time for equilibrium; verify antigen and antibody preincubation.
High background Incomplete washing or poor blocking Increase wash cycles; optimize blocking reagent.

Printable Checklist

Download a printable version of this competitive ELISA workflow to use at your bench.

Download Competitive ELISA Checklist (PDF)
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