Chemiluminescent ELISA Protocol — High-Sensitivity Signal Detection

Chemiluminescent ELISA Protocol — High-Sensitivity Signal Detection

Overview: Chemiluminescent ELISA offers ultra-sensitive detection using enzyme-catalyzed light emission. HRP or alkaline phosphatase reacts with luminol-based substrates to produce light that is measured by a luminometer, providing superior dynamic range and lower detection limits than colorimetric assays.

Materials & Reagents

  • High-binding 96-well plate (opaque white recommended)
  • Capture antibody diluted in carbonate–bicarbonate coating buffer (pH 9.6)
  • Blocking buffer (1–5% BSA or casein in PBS/TBS)
  • Sample and standards
  • HRP-conjugated detection antibody
  • Chemiluminescent substrate (e.g., luminol + enhancer or ECL reagent)
  • Wash buffer (PBS-T or TBS-T, 0.05% Tween-20)
  • Luminometer or plate reader with luminescence detection mode

Step-by-Step Protocol

  1. Coating: Add 100 µL/well of capture antibody (1–10 µg/mL) in coating buffer. Incubate overnight at 4°C or 2 hours at 37°C.
  2. Wash: Wash plate 3× with PBS-T.
  3. Block: Add 200 µL/well of blocking buffer. Incubate 1 hour at room temperature.
  4. Sample addition: Add 100 µL/well of standards and samples. Incubate 1–2 hours at RT or overnight at 4°C for higher sensitivity.
  5. Wash: Wash 4–5× with PBS-T to remove unbound analytes.
  6. Detection antibody: Add 100 µL/well of HRP-conjugated detection antibody. Incubate 1 hour at RT.
  7. Wash: Wash 4–6× with PBS-T to minimize background.
  8. Substrate addition: Add 100 µL/well of chemiluminescent substrate. Incubate in the dark for 1–5 minutes (follow manufacturer’s guidance).
  9. Read plate: Measure luminescence immediately using a plate luminometer. Use integration times of 0.5–2 seconds per well.
  10. Data analysis: Generate a standard curve (log-log or four-parameter fit) and interpolate sample concentrations.

Optimization Tips

  • Plate type: Use white opaque plates for maximum light reflection and sensitivity.
  • Timing: Read immediately after substrate addition — chemiluminescence decays rapidly.
  • Substrate handling: Prepare luminol substrate fresh and protect from light.
  • Background control: Include blank wells with buffer only to assess baseline luminescence.
  • Temperature: Keep assay consistent (RT or 37°C) as temperature affects enzyme kinetics and light output.

Troubleshooting

Issue Possible Cause Solution
Low signal intensity Expired substrate or low HRP activity Use fresh substrate; check conjugate stability and dilution
High background Incomplete washing or substrate exposure to light Increase wash cycles; protect plate from ambient light
Rapid signal decay Long delay before reading Read immediately after substrate addition

Printable Checklist

Download a ready-to-use Chemiluminescent ELISA checklist for your workflow planning and reagent tracking.

Download Chemiluminescent ELISA Checklist (PDF)
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