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

Tong He1, Gelin Liu1, Yanwen Zhao1

  • 1State Key Laboratory of Veterinary Public Health and Safety, Beijing Key Laboratory of Detection Technology for Animal-Derived Food, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.

Analytica Chimica Acta
|February 5, 2026
PubMed
Summary

A new nanoparticle conjugation strategy precisely controls antibody loading for improved immunoassays. Antibody valency optimization enhances sensitivity and robustness, enabling sensitive detection of quinolones in milk.

Keywords:
Homogeneous chemiluminescence immunoassaysNanoparticle-based immunoassaysSite-specific couplingValency

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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.