A simple agglutination system for rapid antigen detection from large sample volumes with enhanced sensitivity

Area of Science:

• Biotechnology
• Assay Development
• Point-of-Care Diagnostics

Background:

• Lateral flow assays (LFAs) are widely used for rapid diagnostics but suffer from limited sensitivity due to small sample volumes.
• Increasing sample volume in LFAs can improve sensitivity by concentrating target analytes.
• Adapting LFAs for larger volumes requires methods to efficiently capture and concentrate analytes.

Purpose of the Study:

• To develop a simple agglutination system to enable LFAs to process larger sample volumes.
• To enhance the sensitivity of LFAs for improved diagnostic performance.
• To create a proof-of-concept assay for SARS-CoV-2 detection in saliva.

Main Methods:

• A biotinylated antibody (Ab) and streptavidin (SA) agglutination system was designed to form large aggregates.
• Dynamic light scattering (DLS) was used to characterize aggregate size.
• Monte Carlo modeling and experiments determined critical factors for agglutination (high valency, equivalent concentrations).
• A 3D-printed device integrated the agglutination system with conventional immunoassays for antigen capture and filtration.

Main Results:

• The agglutination system formed aggregates exceeding 0.5 μm (up to 1.5 μm with poly-SA).
• High valency and equivalent component concentrations were crucial for efficient agglutination.
• The developed agglutination assay demonstrated a 10-fold sensitivity enhancement for SARS-CoV-2 nucleocapsid antigen detection.
• The assay achieved a limit of detection (LoD) of 10 pg/mL (250 fM) and 250 genome copies/μL in saliva.

Conclusions:

• The proposed agglutination system effectively captures antigens from large sample volumes.
• This method significantly improves LFA sensitivity by enabling processing of larger volumes.
• The agglutination assay offers a versatile platform for enhancing existing LFAs for various diagnostic applications.
• The technology holds promise for improved point-of-care and at-home testing, particularly for infectious diseases.