Silver nanoparticles enhanced enzyme-linked immunosorbent assay (ELISA) detection of cancer testis antigens (CTAs).

OBJECTIVE: The Enzyme-Linked Immunosorbent Assay (ELISA) has been a cornerstone technique in laboratory medicine for over 55 years, relying on the specific binding of antibodies to antigens. ELISA's widespread use stems from its ability to detect low concentrations, its specificity, reproducibility, and potential for high-throughput screening. However, its sensitivity has limitations, prompting the exploration of innovative methods to improve the limit of detection (LOD). Nanoparticles provide a promising platform for enhancing ELISA sensitivity. Due to their high surface-to-volume ratio, they offer increased binding sites for capture elements and reporting tags, leading to amplified analytical signals. Recent studies have demonstrated improved sensitivity in ELISA through nanoparticle application, yielding faster detection times and enhanced sensitivities. This study investigates the potential of 50 nm citrate-capped silver nanoparticles to enhance ELISA's performance in quantifying cancer testis antigens (CTAs). PATIENTS AND METHODS: In our study, we used the Human NY-ESO-1 ELISA kit (for research purposes) to determine the concentration of CTAs in randomly selected samples from healthy (n=89) and oncological (n=80) subjects, aged 18-75. We employed 50 nm citrate-capped silver nanoparticles (AGCB50-1M, BioPure Silver Nanoparticles - bare citrate, nano-Composix, San Diego, CA, USA). ELISA reactions followed the manufacturer's instructions, and data processing aligned with the same guidelines. Absorbance (OD) measurements occurred at 450 nm, influencing nanoparticle selection. Each ELISA well contained 5 ml of nanoparticles' stock solution with specified concentrations. CTAs concentrations were derived from the standard curve through CurveExpert Basic software. Statistical analysis was performed using SPSS v. 27 software, with p-values indicating significance if <0.03. The study adhered to Helsinki Declaration principles and received ethical approval. Participants provided informed written consent. RESULTS: The increased concentration values of CTAs for healthy individuals and cancer patients were determined in the case of the application of silver nanoparticles. CONCLUSIONS: The usage of nanoparticles can enhance the sensitivity of the ELISA method and positively influence its specific detection limit.

The detection of IgG class antibodies against SARS-CoV-2 nucleocapsid protein by application of nanoparticles.

OBJECTIVE: Enzyme-linked immunosorbent assay (ELISA) is a widely used biochemical analytical method for the detection of a biomarker, through a specific antigen-antibody reaction. A common with ELISA is the amount of concrete biomarkers falling below the detection limit. Thus, the approach that will contribute to enhanced sensitivity of enzyme-linked immunosorbent assay is of great importance for medical practice. To address this issue, we used nanoparticles to improve the detection limit of traditional ELISA. MATERIALS AND METHODS: 80 samples were used, for which the presence of IgG antibodies against SARS-CoV-2 nucleocapsid protein were already determined qualitatively.  We tested the samples using an in vitro ELISA kit [SARS-CoV-2 IgG ELISA, COVG0949 (NovaTec, Leinfelden-Echterdingen, Germany)]. Additionally, we tested the same sample with the same ELISA kit but with the addition of 50 nm diameter citrate-capped silver nanoparticles. The reaction was performed, and data were calculated according to manufacturer guidelines. To measure ELISA results absorbance (optical density - OD) at 450 nm was read. RESULTS: Greater absorbance values have been revealed in case of silver nanoparticles application (66 cases, 82.5%, p<0.05). ELISA with application of nanoparticles classified 19 equivocal cases as positive and 3 equivocal ones as negative, 1 negative case as equivocal. CONCLUSIONS: Our findings suggest that nanoparticles can be used to improve the sensitivity of ELISA method and increase the detection limit. Thus, it is logical and desirable to enhance the sensitivity of ELISA method by application of nanoparticles; the approach is low cost and with a positive impact on accuracy.

PREVALENCE OF PAH MUTATIONS IN GEORGIAN PKU PATIENTS COMPARED TO MOST FREQUENT PAH MUTATIONS IN EUROPEAN POPULATIONS.

The aim of the study was to compare Georgian PAH mutation spectrum to the most frequent European mutations. Population study publications were reviewed and 20 most frequent European PAH mutations were determined. Mutations were detected in 40 Georgian PKU patients using Sanger sequencing. PAH mutations were detected on all 80 alleles, clinical diagnose of PKU was confirmed in all 40 patients. Detected mutations in Georgian population was: P281L in 37.5%, IVS10-11G>A in 17.5%, R261X in 10%, L48S in 8.75%, E280K in 5%, R270K in 3.75%, E390G in 3.75% and mutations R252W, IVS12+1G>A, R243Q, R261Q, 1089delG, Y387H, EX5del, IVS7-5T>C, IVS12+1G>A, G171R, IVS2+5G>C each in 1.25%. Study revealed that the most common Georgian PAH mutations spectrum differs from the European one. 9 out of 18 detected mutations coincide with the European panel. At the same time more than half (55%) of the mutations found in Georgians were not identified as the most common mutations in Europe. These findings may indicate the necessity for the development of diagnostic panels specific to the Georgian population, including both 9 frequent European PAH mutations and 9 mutations more common for the Georgian population, which will significantly improve the quality of PKU diagnostics in Georgia. The results have been obtained are of an intermediate nature, which propose to continue and complete this research by studying the entire Georgian PKU population.