ABSTRACT
Understanding the dynamic changes in antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for evaluating the effectiveness of the vaccine and the stage for the recovery of the COVID-19 disease. A rapid and accurate method for the detection of SARS-CoV-2-specific antibodies is still urgently needed. Here, we developed a novel fluorescent lateral flow immunoassay (LFA) platform for the detection of SARS-CoV-2-specific IgM and IgG by the aggregation-induced emission carbon dots conjugated with the SARS-CoV-2 spike protein (SSP). The aggregation-induced emission carbon dots (AIE-CDs) are one of the best prospect fluorescent probe materials for exhibiting high emission efficiency in both aggregate and solid states. The AIE-CDs were synthesized and displayed dual fluorescence emission, which provides a new perspective for the design of a high sensitivity testing system. In this work, the novel LFA platform adopted the AIE carbon dots, which are used to detect SARS-CoV-2-specific IgM and IgG conveniently. Furthermore, this sensor had a low LOD of 100 pg/ml. Therefore, this newly developed strategy has potential applications in the areas of public health for the advancement of clinical research.
ABSTRACT
Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool.
Subject(s)
COVID-19 , Metal Nanoparticles , Humans , SARS-CoV-2 , COVID-19/diagnosis , Gold , Pandemics , Sensitivity and SpecificityABSTRACT
Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting. Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology. Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.
ABSTRACT
Remdesivir (RMDSVR) is a pro-drug of an ATP analog, with antiviral activity against RNA viruses. In 2016 RMDSVR is used in the treatment of Ebola and 2017 RMDSVR was tested against SARS-CoV-2. The HPLC analysis was performed on the Agilent 1100 series HPLC with Quaternary G1311 A pump, ChromosilC18 column (250 X 4.6 mm, 5μ) column, with a mixture of Acetonitrile, Methanol and 0.1% OPA in the ratio of 65:30:5 (v/v) as the mobile phase, at 0.8 mL/min flow rate and UV detection at 272 nm. Abacavir (ABVR) is considered an internal standard and the retention time was observed at 6.0 min and 9.1 min for RMDSVR and ABVR respectively. The calibration curve was obtained linearly in the concentration range of 10-70 ng/mL. the method was validated and all the validated parameters were within the acceptable limit confirms that the method is suitable for the analysis of RMDSVR in spiked human plasma. © 2021, Rasayan Journal of Chemistry, c/o Dr. Pratima Sharma. All rights reserved.
ABSTRACT
Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828â¯000 assays covering >51â¯000 proteins from 94 organisms, of which >17â¯000 proteins are present in >2400 biological pathways, and >48â¯000 mapping to >21â¯000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.
Subject(s)
Proteins , Proteomics , Amino Acid Sequence , Humans , Peptides , Reproducibility of ResultsABSTRACT
A high-performance liquid chromatography-ultraviolet detector (HPLC-UV) method has been used to quantify teicoplanin concentrations in human plasma. However, the limited analytical accuracy of previously bioanalytical methods for teicoplanin has given rise to uncertainty due to the use of an external standard. In this study, an internal standard (IS), polymyxin B, was applied to devise a precise, accurate, and feasible HPLC-UV method. The deproteinized plasma sample containing teicoplanin and an IS of acetonitrile was chromatographed on a C18 column with an acidic mobile phase consisting of NaH2PO4 buffer and acetonitrile (78:22, v/v) by isocratic elution and detection at 220 nm. The linearity was in the range 7.8-500 mg/L calculated by the ratio of the teicoplanin signal to the IS signal. This analytical method, validated by FDA guidelines with ICH Q2 (R1), was successfully applied to analyze the plasma samples of patients in the intensive care unit for treating serious resistant bacterial infectious diseases, such as those by methicillin-resistant Staphylococcus aureus and Enterococcus faecalis. The methods suggested the potential for use in routine clinical practice for therapeutic drug monitoring of teicoplanin, providing both improved accuracy and a wide range of linearity from lower than steady-state trough concentrations (10 mg/L) to much higher concentrations.