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The enzyme immunoassay (EIA) is one of the most sensitive methods for the detection of molecules (antigens) based on the use of specific antibodies and linked enzymes. In our opinion, the use of gold nanoparticles (AuNP) functionalized simultaneously by two types of protein molecules (antibody and enzyme) is a very promising direction in the development of EIA. The preparation of such bifunctional particles is simpler than chemical conjugations of two types of proteins, and the use is more efficient in comparison with standard antibody–AuNP conjugates. This work aimed to obtain stable AuNPs functionalized horseradish peroxidase (HRP) and antibodies against a bacterial O-antigen. At the first stage, the “golden number” for AuNPs and HRP solutions was determined by the salt method. Graphically, according to the dependence of the optical density of the mixture solution at 620 nm on the protein concentration, it was calculated that the "golden number" is 31.3 μg/mL. A mixture of nanoparticles with 10 μg/mL of HRP (concentration below the "golden number") was prepared, to which an equal volume of antibody solution was added. The resulting solution of nanoparticles was stable to salt aggregation, interacted with a specific antigen, and possessed enzymatic activity. The use of this conjugate in the dot assay demonstrated an 8-fold increase in the antigen detection sensitivity with the addition of a substrate solution for HRP as compared to staining with only functionalized AuNPs without enzyme reaction.
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