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

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.

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes

“…To clarify points for improvement, let us consider this process in more detail. The concentration of IgG as the main class of immunoglobulins in human blood is typically about 6–20 mg/mL [ 36 ], while the sorption capacity of gold nanoparticles with a diameter of 30 nm (as the optimal LFIA label) does not exceed 5 μg/mL per unit of the OD [ 37 ]. Therefore, even if the OD of the conjugate is 10, it binds less than 1% of the IgG fraction from the sample.…”

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes

“…Let's consider this process in more detail to clarify points for its improvement. The concentration of IgG as the main class of immunoglobulins in human blood is normally about 6-20 mg/mL [34] while the sorption capacity of gold nanoparticles with a diameter of 30 nm (as the optimal LFIA label) does not exceed 5 µg/mL per unit of the OD [35]. Therefore, even if the OD of the conjugate is 10, it binds less than 1% of the IgG fraction from the sample.…”

Peroxidase mimicking activity ofSaccharum officinarumL. capped gold nanoparticles usingo-dianisidine as a substrate

“…Several conventional chemical methods such as the Turkevich Frens method, 10 Brust Schiffrin method, 11 Martin method, 12 Laser ablation, etc. 13 involving reducing and stabilizing agents have been reported for the synthesis of Au NPs.…”

“…7,8 Au NPs have unique optoelectronic properties, high surface to volume ratio, good biocompatibility, and stability to denaturation. 9 Several conventional chemical methods such as the Turkevich Frens method, 10 Brust Schiffrin method, 11 Martin method, 12 Laser ablation, etc. 13 involving reducing and stabilizing agents have been reported for the synthesis of Au NPs.…”