Engineering antibody conjugation and valency for optimized nanoparticle-based immunoassays.

Area of Science:

• Nanoparticle-based immunoassays
• Bioconjugation chemistry
• Assay development and optimization

Background:

• Nanoparticles (NPs) enhance immunoassay performance.
• Current NP immunoassays lack precise conjugation control and understanding of antibody valency effects.

Purpose of the Study:

• Develop a site-specific antibody conjugation strategy for NPs.
• Investigate the impact of antibody valency on immunoassay performance.
• Optimize an immunoassay for detecting quinolones in milk.

Main Methods:

• Integrated cysteine-engineered single-chain variable fragment (scFv), click chemistry, and biotin-streptavidin interactions for site-specific NP conjugation.
• Quantitatively studied antibody valency effects on homogeneous chemiluminescence immunoassays (HCLIA).
• Developed and validated an HCLIA for 17 quinolone detection.

Main Results:

• Novel strategy yielded superior conjugate uniformity, antibody functionality, sensitivity, and reproducibility compared to traditional methods.
• Optimal sensitivity was achieved with medium antibody valency (10-50% coupling).
• High antibody valency (>50% coupling) enhanced robustness but reduced sensitivity.
• Developed HCLIA detected 17 quinolones with limits of detection from 0.010 to 0.131 μg/L.

Conclusions:

• Presents a controllable antibody conjugation strategy and advances understanding of antibody valency in NP immunoassays.
• Provides guidelines for rational design of future NP-based immunoassays.
• The HCLIA method offers a practical tool for detecting quinolone residues in milk.