ABSTRACT
Background: The VITROS IgG Quant assay∗ is for the quantitative detection of SARS-CoV-2 IgG antibodies with calibration traceable to the first World Health Organization International Standard for Anti- SARS-CoV-2 antibody. Results are reported in both qualitative (reactive/ non-reactive) and quantitative values (Binding Antibody Units [BAU]/ml). PRNT is considered the gold standard method for determining neutralizing antibody (nAb) titers. Aims: This study was designed to assess the correlation of the VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Quantitative Assay (VITROS IgG Quant) to a plaque reduction neutralization test developed at Colorado State University (CSU PRNT). Methods: VITROS IgG Quant: The VITROS IgG Quant assay is a fully automated, high-throughput method run on the VITROS family of immunoassay analyzers. First, antibodies to SARS-CoV-2 present in the sample bind with the S1 subunit of the Spike protein coated on wells. After washing, horseradish peroxidase (HRP)- labelled murine monoclonal anti-human IgG antibodies are added. Following a final wash, bound HRP conjugates are detected using the VITROS signal reagent. The amount of conjugate directly correlates to the amount of SARS-CoV-2 IgG antibody present and is reported in BAU/ml. CSU PRNT: Samples were heat-inactivated for 30 min at 56°C, and serial two-fold dilutions were prepared in a 96-well plate. Viral stock (strain hCoV-19/USA/WA1/2020) containing ∼200 pfu per 0.1 ml was added to each well containing serum dilutions. Following incubation at 37°C in 5% CO2, 6-well plates containing recently confluent Vero cells were inoculated with the virus-serum mixtures. After a second incubation at 37°C, 2 ml of overlay was added to each well. After 24 h incubation at 37°C, a second overlay containing neutral red was dispensed into each well and the number of plaques was counted 48-72 h after initial inoculation. The highest dilution of serum that inhibited plaque formation by 50% (PRNT50) was determined based upon the titre of the samples and the number of plaques present at each dilution. Samples with PRNT50 titers less than or equal to 1:20 are considered negative for nAbs. One hundred forty-nine samples were blind-tested with both the VITROS IgG Quant assay and the CSU PRNT, 74 known positive for SARS-CoV-2 antibody and 75 negatives. The correlation of the VITROS IgG Quant values to CSU PRNT50 was determined. Results: The VITROS IgG Quant results ranged from <2 to 2009 BAU/ml. The CSU PRNT50 results ranged from <1:20 to >1:2560. Pearson correlation coefficient was calculated to be 0.867, demonstrating a good correlation between the VITROS IgG Quant results and the CSU PRNT50 titers. Summary/Conclusions: The VITROS Anti-SARS-CoV-2 IgG Quant assay demonstrates a strong correlation to PRNT50 for the measurement of SARS-CoV-2 nAb titers.
ABSTRACT
Background: The SARS-CoV-2 pandemic has sickened over 245 million people, and has killed more than 5 million worldwide. Recent data proves that vaccinations are highly effective in preventing Covid-19 disease, however antigenic drift and other functional mutations in the virus genome reduce the efficacy of vaccines, indicating that the development of antiviral treatments remain a crucial priority. We report potent antiviral activity against SARS-CoV-2 for a promising, novel class of nitrogen-based heterocyclic compounds. Methods: 232 compounds based on the same class of nitrogen-based hetereocyclic molecules were synthesized to final purity of greater than 99%. This library was screened for antiviral phenotypes in a cytopathic effect (CPE) assay using VeroE6 cells and the SARS-CoV-2 WA1 isolate. Based on the results of the WA1 CPE screen, 47 lead candidates were structurally analyzed, and this information was utilized to design 56 additional compounds. A second antiviral CPE-based screen was performed using these 103 candidates in VeroE6 cells with the SARS-CoV-2 delta variant. Antiviral assays studying SARS-CoV-1 (Urbani) and MERS-CoV were performed in Vero 76 cells utilizing a Neutral Red cytopathic effect assay. Results: Within the same class of structurally related small molecules, we tested an initial set of 232 compounds using a CPE-based assay with VeroE6 cells and the USA/WA1 SARS-CoV-2 isolate. Of the compounds tested, 124 demonstrated potency 10 to 540-times higher than a Remdesivir control tested in parallel. Importantly, we observed no detectable toxicity for the vast majority of these compounds when tested up to a concentration of 30 μ M. The lead candidate in this screen displayed an IC50 of 0.02 μ M and a selectivity index of >1,500. Based on structural analysis of an initial 47 lead candidates, we synthesized 56 new molecules, and tested all 103 in a CPE-based assay using the delta variant, also observing efficacy against this variant of concern. Examples of this same class of compounds also display antiviral activity against SARS-CoV-1 (Urbani) and MERS-CoV in cell-based assays. Conclusion: We have identified a novel class of antiviral compounds with potent activity against SARS-CoV-2. High potency against both the early WA1 isolate and the more recent delta variant, as well as efficacy against SARS-CoV-1 and MERS-CoV, suggest that this class of antiviral compounds has pan-Coronavirus antiviral activity.
ABSTRACT
Synthesis of bio-based environmental remedial and antimicrobial products is an urgent need of the 21st century in the COVID-19 pandemic world. Keeping this in mind, cellulose-supported Ag bionanocomposites (AGC NCs) were synthesized by using cellulose as a reducing and stabilizing agent. AGC NCs showed potential antimicrobial activity against Candida albicans, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Bacillus subtilis with a MIC of 15, 15, 35, 15, and 30 mu g ml(-1) respectively. AGC NCs efficiently degraded harmful dyes, Orange G, Phenol red, Brilliant blue FCF, Giemsa stain, Neutral red, and 2-nitro aniline in the presence of sunlight with a rate constant of 0.229 x 10(-2) min(-1), 1.147 x 10(-2) L mol(-1) min(-1), 0.447 x 10(-2) L mol(-1) min(-1), 4.144 x 10(-2) mol L-1 min(-1), 0.317 x 10(-2) L mol(-1) min(-1), and 0.785 x 10(-2) L mol(-1) min(-1) in 60 min respectively. AGC NCs also showed efficient antioxidant activity in DPPH assay with an IC50 value of 52.67 mu g ml(-1). Formation of Ag NPs was confirmed by observing the UV-Visible absorption peak at 418 nm. The FCC structure of AGC NCs was confirmed by the X-ray diffraction (XRD) pattern with well-defined peaks at angles 38.24 degrees, 44.40 degrees, 64.64 degrees, and 77.28 degrees corresponding to the planes 111, 200, 220, and 311, with a d-spacing of 2.35, 2.04, 1.44, and 1.23 (JCPDS no. 00-001-1164). The presence of cellulose in AGC NCs was determined by Fourier transform infrared spectroscopy (FTIR) with bands at 3421.90 cm(-1) and 2899.3 cm(-1) due to O-H stretching and the methylene (-CH2-) group respectively and at 1076-1023 cm(-1) and 903 cm(-1) due to -C-O-C- pyranose ring skeletal vibration and beta-glycosidic linkages. The morphology, shape and size (13.21 nm), and elemental composition of the nanocomposites were determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive spectroscopy (EDS) respectively. The thermal properties (exothermic peaks appear at 335 degrees C and 440 degrees C due to the thermal degradation of Ag NPs and cellulose respectively), surface area (13.892 m(2) g(-1)), stability (-18.43 +/- 0.850 mV), and hydrodynamic diameter (399.10 +/- 30.49 nm) and polydispersity index (PDI) value (0.565 +/- 0.193) of the composites were determined by thermogravimetric analysis (TGA) and differential thermal analysis (DTA), Brunauer-Emmett-Teller (BET) method, Zeta potential studies, and dynamic light scattering (DLS) respectively.