Retention of Activity by Antibodies Immobilized on Gold Nanoparticles of Different Sizes: Fluorometric Method of Determination and Comparative Evaluation.

Anatoly V Zherdev,Nadezhda A Byzova,Dmitriy V Sotnikov,Boris B Dzantiev

doi:10.3390/nano11113117

Anatoly V Zherdev, Nadezhda A Byzova + Show 2 more

Open Access

https://doi.org/10.3390/nano11113117

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Abstract

Antibody–nanoparticle conjugates are widely used analytical reagents. An informative parameter reflecting the conjugates’ properties is the number of antibodies per nanoparticle that retain their antigen-binding ability. Estimation of this parameter is characterized by a lack of simple, reproducible methods. The proposed method is based on the registration of fluorescence of tryptophan residues contained in proteins and combines sequential measurements of first the immobilized antibody number and then the bound protein antigen number. Requirements for the measurement procedure have been determined to ensure reliable and accurate results. Using the developed technique, preparations of spherical gold nanoparticles obtained by the most common method of citrate reduction of gold salts (the Turkevich–Frens method) and varying in average diameter from 15 to 55 nm have been characterized. It was shown that the number of antibodies (immunoglobulins G) bound by one nanoparticle ranged from 30 to 194 during adsorptive unoriented monolayer immobilization. C-reactive protein was considered as the model antigen. The percentage of antibody valences that retained their antigen-binding properties in the conjugate increased from 17 to 34% with an increase in the diameter of gold nanoparticles. The proposed method and the results of the study provide tools to assess the capabilities of the preparations of gold nanoparticles and their conjugates as well as the expediency of seeking the best techniques for various practical purposes.

Highlights

Antibody–marker complexes are effective reagents for bioanalytical purposes
We propose an integral technique in which both the binding of antibodies to nanoparticles and the subsequent interaction of a protein antigen with immobilized antibodies are characterized by the same approach: registration of tryptophan fluorescence
The method for determining the composition of gold nanoparticles (GNPs) conjugates with antibodies and their antigen-binding activity is based on measuring tryptophan fluorescence twice: in solutions obtained after the conjugates’ centrifugation and in the same solutions after adding a known amount of protein to them

Summary

Introduction

Antibody–marker complexes are effective reagents for bioanalytical purposes. They combine high-affinity and selective antibody interaction with the target analyte and highly sensitive detection of the formed complex, provided by the marker’s properties [1]. Their use makes it possible to implement various detection methods and register immune complexes at extremely low concentrations [2,3,4]. The problems of antibody inactivation during immobilization with the subsequent deterioration of immunosensoric systems’ analytical characteristics have been noted in many studies and have prompted various approaches for indirect oriented antibody immobilization [5,6,7,8,9]. Such actions involve the use of additional reagents and methodological complications. Many variants of direct immobilization on nanoparticles continue to be widely used, and assessments of the resulting preparations’

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