# Reactivity of Antibodies Immobilized on Gold Nanoparticles: Fluorescence Quenching Study

## TL;DR

This study evaluates how well antibodies attached to gold nanoparticles retain their reactivity using a fluorescence quenching method.

## Contribution

A new method using fluorescence quenching is introduced to assess antibody reactivity on gold nanoparticles.

## Key findings

- Antibody reactivity retention ranged from 11% to 58% depending on conjugation conditions.
- Bovine serum albumin outperforms mercapto polyethylene glycol in preserving antibody activity.
- Lower antibody densities showed up to a twofold difference in activity retention between blocking agents.

## Abstract

Antibodies labeled with gold nanoparticles are widely used in analytical systems. Consequently, the choice of methods for producing such conjugates requires a precise determination of the retained reactivity of the antibodies. Existing methods give highly variable results, necessitating new, simple, and accurate approaches. This study demonstrates how a fluorescein (FL) and anti-FL antibody pair can be used to comparatively evaluate conjugation conditions. The method is based on the quenching of fluorescein emission after binding to antibodies, enabling real-time monitoring of interactions in the reaction medium. Using this approach, we compared a series of conjugates of gold nanoparticles (average diameter 24 nm) and anti-FL antibodies that were obtained with antibody concentrations of 5, 10, and 20 μg/mL during the conjugation. The proportion of antibodies that retained their reactivity varied from 11% to 58%. We also estimated the blocking of the conjugates’ surfaces by two widely used reagents, bovine serum albumin and mercapto polyethylene glycol. It was found that the former provides better retention of antigen-binding activity of immobilized antibodies. The difference between these two kinds of preparations is most pronounced—reaching up to a twofold change—at lower antibody densities.