Application of time-resolved fluorescence for imaging-based multisample and multianalyte detection in single...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Enzyme-linked immunosorbent assays (ELISAs) are standard for specific protein detection.
• Multianalyte protein arrays allow simultaneous detection of multiple targets but suffer from antibody cross-reactivities.
• Existing methods often require larger sample volumes and can compromise assay specificity.

Purpose of the Study:

• To develop a novel immunoassay principle for highly specific multianalyte detection.
• To reduce the required sample volume for complex immunoassays.
• To eliminate antibody cross-reactivities inherent in traditional protein arrays.

Main Methods:

• A new assay principle utilizing biotinylated time-resolved fluorophore EuLH (as a PEG11-dye conjugate) with ExtrAvidin® for high signal-to-background ratios.
• Detection of the model protein epidermal growth factor (EGF) using a sandwich immunoassay format.
• Spatial resolution of assay signals for imaging-based analysis in microtiter wells.

Main Results:

• The novel assay principle successfully detected the model protein EGF with parameters comparable to commercial assays.
• Elimination of antibody cross-reactivities was achieved by analyzing analytes in separate wells.
• Demonstrated reduction of sample volume to 1 μL per analyte, enabling up to 100 analytes from 100 μL.
• Spatial resolution allowed for imaging-based analysis of individual spots within a single 96-microtiter well.

Conclusions:

• The presented assay principle offers a robust solution for specific multianalyte detection with significantly reduced sample volumes.
• This method overcomes the limitations of traditional protein arrays by preventing antibody cross-reactivities.
• The system enables sensitive, spatially resolved analysis, advancing laboratory diagnostics and high-throughput screening.