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.
CLE peptides are important signaling molecules that regulate the balance of proliferation and differentiation of plant cells. In this work, we conjugated gold nanospheres (15 nm) with a chemically synthesized peptide (12 amino acid residues) that the amino acid sequence corresponds to the natural peptides CLE41/44. The "golden number" for CLE41/44 was 2 Μg/mL. A stable conjugate was used to produce rabbit polyclonal antibody. By the method of dot analysis, the presence of antibodies to CLE41/44 was detected in antiserum. Using Western blot and ELISA methods, the reaction of the obtained antibody with a conjugate of CLE41/44 with BSA molecules was demonstrated. The obtained antibody is can potentially be used to the quantitative determination and identification of the localization of peptides in plant tissues.
Gold nanoparticles (GNPs) have attracted significant interest as a novel platform for various applications to nanobiotechnology and biomedicine. The conjugates of GNPs with antibiotics and antibodies were also used for selective photothermal killing of protozoa and bacteria. Also the conjugates of some antibiotics with GNPs decreased the number of bacterial growing cells. In this work was made the procedure optimization for conjugation of cefotaxime (a third-generation cephalosporin antibiotic) with GNPs (15 nm) and we examined the antimicrobial properties of this conjugate to bacteria culture of E. coli K-12. Addition of cefotaxime solution to colloidal gold does not change their color and extinction spectrum. For physiologically active concentration of cefotaxime (3 μg/mL), it was shown that the optimum pH for the conjugation was more than 9.5. A partial aggregation of the GNPs in saline medium was observed at pH 6.5-7.5. The optimum concentration of K2CO3 for conjugation cefotaxime with GNPs-15 was 5 mM. The optimum concentration of cefotaxime was at 0.36 μg/mL. We found the inhibition of the growth of E. coli K12 upon application cefotaxime-GNP conjugates.
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