Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunotechnology

Background:

• Direct time-resolved fluorescence measurement in immunoassays offers advantages over solution-based signal generation.
• Epi-fluorometric detection in microtiter wells is limited by small signal collection areas, impacting assay sensitivity with large coating volumes.

Purpose of the Study:

• To develop an improved immunoassay method using concentrated binding surfaces.
• To enhance assay sensitivity and detection limits by condensing labeled antibodies onto specific spots.

Main Methods:

• Generated streptavidin-coated spots (native SAv and high-capacity GA-SAv) in microtiter well indentations (2.5-4.5 mm diameter).
• Utilized adsorption of liquid droplets containing streptavidin to create concentrated binding sites.
• Performed a sandwich immunoassay for thyroid-stimulating hormone (TSH) to evaluate the spot assay performance.

Main Results:

• Streptavidin-coated spots condensed labeled antibodies for optimal excitation.
• GA-SAv spots achieved higher Eu-biotin binding densities (0.47 pmol/mm²) compared to native SAv (0.080 pmol/mm²).
• The spot assay demonstrated a fivefold to sixfold increase in signal-to-background ratio and improved detection limit (DL < 0.01 mU/L) for TSH.

Conclusions:

• Streptavidin-coated spots effectively enhance immunoassay sensitivity and detection limits.
• This spot-based approach offers a significant improvement over conventional microtiter well assays.
• The method holds promise for more sensitive detection of biomarkers in various immunoassay applications.