Fluorescence ELISA Protocol

Fluorescence ELISA Protocol

A high-sensitivity approach for quantifying low-abundance analytes using fluorescent signal detection

Overview

Fluorescence ELISA (FELISA) enhances traditional ELISA sensitivity by replacing colorimetric detection with fluorescence-based readouts. This allows quantification of low-concentration analytes and supports multiplexing applications where multiple targets are detected simultaneously.

Principle

In a fluorescence ELISA, antigen-antibody binding occurs on a solid phase similar to a conventional ELISA. The key difference lies in the detection system—fluorescently labeled secondary antibodies or enzyme-substrate reactions generate measurable fluorescence, providing higher dynamic range and sensitivity.

Materials Required

  • High-binding 96-well microplate
  • Capture antibody (coating antibody)
  • Blocking buffer (e.g., 1% BSA or casein)
  • Sample or standard antigen
  • Fluorescent-labeled secondary antibody or enzyme conjugate (e.g., HRP + Amplex Red substrate)
  • Fluorescence microplate reader (with appropriate excitation/emission filters)
  • Wash buffer (PBS + 0.05% Tween-20)

Protocol Steps

  1. Coating: Add 100 µL of capture antibody (1–10 µg/mL) to each well and incubate overnight at 4°C.
  2. Blocking: Add 200 µL blocking buffer to each well; incubate 1 hour at room temperature.
  3. Sample Addition: Add 100 µL of sample or standards; incubate 1–2 hours at room temperature.
  4. Detection Antibody: Add 100 µL fluorescent-labeled detection antibody and incubate 1 hour.
  5. Wash: Wash 3–5 times with wash buffer.
  6. Readout: Measure fluorescence at the appropriate excitation/emission wavelength pair.

Tips for Optimization

  • Optimize antibody concentrations to prevent quenching or background fluorescence.
  • Use black, flat-bottom plates to reduce cross-talk between wells.
  • Minimize exposure to light during incubation and storage to prevent fluorophore bleaching.

Troubleshooting

  • Low signal: Verify fluorophore activity; use fresh substrates or higher gain settings.
  • High background: Improve blocking efficiency or reduce antibody concentration.
  • Inconsistent wells: Ensure uniform plate coating and washing.

Applications

Fluorescence ELISA is widely used in cytokine quantification, neurodegenerative biomarker detection (e.g., GFAP, UCH-L1, S100B), and multiplex assays in diagnostics and research.

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