Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 67
Filter
1.
Anal Biochem ; 645: 114633, 2022 May 15.
Article in English | MEDLINE | ID: covidwho-1712394

ABSTRACT

The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.


Subject(s)
Aptamers, Nucleotide , COVID-19 , Aptamers, Nucleotide/genetics , COVID-19/diagnosis , Humans , SARS-CoV-2/genetics , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
Microbiol Spectr ; 10(1): e0256321, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1700249

ABSTRACT

We have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (n = 1500/month) for a 12 -month period from April 2020 until March 2021. Each sample was evaluated for SARS-CoV-2 IgG antibodies using four assays an Abbott Architect assay targeting the nucleocapsid antigen (Abbott-NP, Abbott, Chicago IL) and three in-house IgG ELISAs recognizing distinct recombinant viral antigens: full-length spike glycoprotein (Spike), spike glycoprotein receptor binding domain (RBD) and nucleocapsid (NP). We used two analytic approaches to estimate SAR-CoV-2 seroprevalence: a composite reference standard and LCA. Using LCA to estimate true seropositivity status based on the results of the four antibody tests, we estimated that seroprevalence increased from 0.8% (95% CI: 0.5-1.4%) in April 2020 to 6.3% (95% CI: 5.1-7.6%) in March 2021. Our study provides further support for the use of LCA in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable. IMPORTANCE Here, we describe an approach to estimating seroprevalence in a low prevalence setting when multiple assays are available and yet no known gold standard exists. Because serological studies identify cases through both diagnostic testing and surveillance, and otherwise silent, unrecognized infections, serological data can be used to estimate the true infection fatality ratio of a disease. However, seroprevalence studies rely on assays with imperfect sensitivity and specificity. Seroreversion (loss of antibody response) also occurs over time, and with the advent of vaccination, distinction of antibody response resulting from vaccination as opposed to antibody response due to infection has posed an additional challenge. Our approach indicates that seroprevalence on Canadian blood donors by the end of March 2021was less than 10%. Our study supports the use of latent class analysis in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.


Subject(s)
Antibodies, Viral/blood , Blood Donors/statistics & numerical data , COVID-19/blood , SARS-CoV-2/immunology , Adult , Aged , COVID-19/epidemiology , COVID-19/virology , Canada/epidemiology , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/immunology , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , SARS-CoV-2/genetics , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/immunology , Young Adult
3.
Microbiol Spectr ; 10(1): e0156421, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622004

ABSTRACT

The emergence of SARS-CoV-2 created a crucial need for serology assays to detect anti-SARS-CoV-2 antibodies, which led to many serology assays entering the market. A trans-government collaboration was created in April 2020 to independently evaluate the performance of commercial SARS-CoV-2 serology assays and help inform U.S. Food and Drug Administration (FDA) regulatory decisions. To assess assay performance, three evaluation panels with similar antibody titer distributions were assembled. Each panel consisted of 110 samples with positive (n = 30) serum samples with a wide range of anti-SARS-CoV-2 antibody titers and negative (n = 80) plasma and/or serum samples that were collected before the start of the COVID-19 pandemic. Each sample was characterized for anti-SARS-CoV-2 antibodies against the spike protein using enzyme-linked immunosorbent assays (ELISA). Samples were selected for the panel when there was agreement on seropositivity by laboratories at National Cancer Institute's Frederick National Laboratory for Cancer Research (NCI-FNLCR) and Centers for Disease Control and Prevention (CDC). The sensitivity and specificity of each assay were assessed to determine Emergency Use Authorization (EUA) suitability. As of January 8, 2021, results from 91 evaluations were made publicly available (https://open.fda.gov/apis/device/covid19serology/, and https://www.cdc.gov/coronavirus/2019-ncov/covid-data/serology-surveillance/serology-test-evaluation.html). Sensitivity ranged from 27% to 100% for IgG (n = 81), from 10% to 100% for IgM (n = 74), and from 73% to 100% for total or pan-immunoglobulins (n = 5). The combined specificity ranged from 58% to 100% (n = 91). Approximately one-third (n = 27) of the assays evaluated are now authorized by FDA for emergency use. This collaboration established a framework for assay performance evaluation that could be used for future outbreaks and could serve as a model for other technologies. IMPORTANCE The SARS-CoV-2 pandemic created a crucial need for accurate serology assays to evaluate seroprevalence and antiviral immune responses. The initial flood of serology assays entering the market with inadequate performance emphasized the need for independent evaluation of commercial SARS-CoV-2 antibody assays using performance evaluation panels to determine suitability for use under EUA. Through a government-wide collaborative network, 91 commercial SARS-CoV-2 serology assay evaluations were performed. Three evaluation panels with similar overall antibody titer distributions were assembled to evaluate performance. Nearly one-third of the assays evaluated met acceptable performance recommendations, and two assays had EUAs revoked and were removed from the U.S. market based on inadequate performance. Data for all serology assays evaluated are available at the FDA and CDC websites (https://open.fda.gov/apis/device/covid19serology/, and https://www.cdc.gov/coronavirus/2019-ncov/covid-data/serology-surveillance/serology-test-evaluation.html).


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/blood , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/virology , Diagnostic Test Approval , Humans , Laboratories , Pandemics , SARS-CoV-2/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/immunology , United States/epidemiology , United States Food and Drug Administration
4.
Molecules ; 26(22)2021 Nov 17.
Article in English | MEDLINE | ID: covidwho-1524086

ABSTRACT

COVID-19 is a highly contagious human infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the war with the virus is still underway. Since no specific drugs have been made available yet and there is an imbalance between supply and demand for vaccines, early diagnosis and isolation are essential to control the outbreak. Current nucleic acid testing methods require high sample quality and laboratory conditions, which cannot meet flexible applications. Here, we report a laser-induced graphene field-effect transistor (LIG-FET) for detecting SARS-CoV-2. The FET was manufactured by different reduction degree LIG, with an oyster reef-like porous graphene channel to enrich the binding point between the virus protein and sensing area. After immobilizing specific antibodies in the channel, the FET can detect the SARS-CoV-2 spike protein in 15 min at a concentration of 1 pg/mL in phosphate-buffered saline (PBS) and 1 ng/mL in human serum. In addition, the sensor shows great specificity to the spike protein of SARS-CoV-2. Our sensors can realize fast production for COVID-19 rapid testing, as each LIG-FET can be fabricated by a laser platform in seconds. It is the first time that LIG has realized a virus sensing FET without any sample pretreatment or labeling, which paves the way for low-cost and rapid detection of COVID-19.


Subject(s)
Biosensing Techniques/methods , COVID-19 Testing/methods , COVID-19/diagnosis , Graphite/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Transistors, Electronic/virology , COVID-19/virology , Clinical Laboratory Techniques , Humans , Lasers , Microscopy, Confocal , Microscopy, Electron, Scanning
5.
Bioelectrochemistry ; 143: 107982, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1525699

ABSTRACT

The large-scale diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for traceability and treatment during pandemic outbreaks. We developed a fast (2-3 min), easy-to-use, low-cost, and quantitative electrochemical biosensor based on carbon nanotube field-effect transistor (CNT-FET) that allows digital detection of the SARS-CoV-2 S1 in fortifited saliva samples for quick and accurate detection of SARS-CoV-2 S1 antigens. The biosensor was developed on a Si/SiO2 surface by CNT printing with the immobilization of a anti-SARS-CoV-2 S1. SARS-CoV-2 S1 antibody was immobilized on the CNT surface between the S-D channel area using a linker 1-pyrenebutanoic acid succinimidyl ester (PBASE) through non-covalent interaction. A commercial SARS-CoV-2 S1 antigen was used to characterize the electrical output of the CNT-FET biosensor. The SARS-CoV-2 S1 antigen in the 10 mM AA buffer pH 6.0 was effectively detected by the CNT-FET biosensor at concentrations from 0.1 fg/mL to 5.0 pg/mL. The limit of detection (LOD) of the developed CNT-FET biosensor was 4.12 fg/mL. The selectivity test was performed by using target SARS-CoV-2 S1 and non-target SARS-CoV-1 S1 and MERS-CoV S1 antigens in the 10 mM AA buffer pH 6.0. The biosensor showed high selectivity (no response to SARS-CoV-1 S1 or MERS-CoV S1 antigen) with SARS-CoV-2 S1 antigen detection in the 10 mM AA buffer pH 6.0. The biosensor is highly sensitive, saves time, and could be a helpful platform for rapid detection of SARS-CoV-2 S1 antigen from the patients saliva.


Subject(s)
Electrochemical Techniques/instrumentation , Nanotubes, Carbon/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Antigens, Viral/analysis , Biosensing Techniques , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
6.
Anal Bioanal Chem ; 414(3): 1313-1322, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1506326

ABSTRACT

Among the deadliest pandemics in history, coronavirus disease 2019 (COVID-19) has wreaked havoc on human lives, economies and public health systems worldwide. To temper its effects, diagnostic methods that are simple, rapid, inexpensive, accurate, selective and sensitive continue to be necessary. In our study, we developed an electrochemical biosensing platform based on gold clusters, mercaptoethanol, the spike protein of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antigen and bovine serum albumin-modified glassy carbon electrode able to detect the SARS-CoV-2 spike antibody. Moreover, during the detection of the SARS-CoV-2 spike antibody in spiked-real samples, the anodic signal of the produced biosensor at 0.85 V decreased as the amount of the SARS-CoV-2 spike antibody increased. Meanwhile, the recovery and relative standard deviation values for saliva and oropharyngeal swab samples were 97.73% and 3.35% and 102.43% and 4.63%, respectively. In 35 min, the biosensing platform could detect 0.03 fg/mL of the SARS-CoV-2 spike antibody in synthetic media and spiked-saliva or -oropharyngeal swab samples. The method thus issues a linear response to the SARS-CoV-2 spike antibody from 0.1 fg/mL to 10 pg/mL. The cross-reactivity studies with spike antigens of Middle East respiratory syndrome-coronavirus and influenza A and the antigen of pneumonia confirmed the excellent selectivity of the proposed method. The developed method was compared with the lateral flow immunoassay method in terms of sensitivity and it was found to be approximately 109 times more sensitive. Biosensing mechanism of the platform to the SARS-CoV-2 spike antibody.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Serological Testing/instrumentation , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Viral/immunology , Biosensing Techniques/instrumentation , COVID-19/immunology , Electrochemical Techniques/instrumentation , Equipment Design , Humans , Hydrogen Bonding , Models, Molecular , SARS-CoV-2/immunology , Saliva/virology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
7.
Adv Mater ; 34(3): e2104608, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1499211

ABSTRACT

Solid-state transistor sensors that can detect biomolecules in real time are highly attractive for emerging bioanalytical applications. However, combining upscalable manufacturing with the required performance remains challenging. Here, an alternative biosensor transistor concept is developed, which relies on a solution-processed In2 O3 /ZnO semiconducting heterojunction featuring a geometrically engineered tri-channel architecture for the rapid, real-time detection of important biomolecules. The sensor combines a high electron mobility channel, attributed to the electronic properties of the In2 O3 /ZnO heterointerface, in close proximity to a sensing surface featuring tethered analyte receptors. The unusual tri-channel design enables strong coupling between the buried electron channel and electrostatic perturbations occurring during receptor-analyte interactions allowing for robust, real-time detection of biomolecules down to attomolar (am) concentrations. The experimental findings are corroborated by extensive device simulations, highlighting the unique advantages of the heterojunction tri-channel design. By functionalizing the surface of the geometrically engineered channel with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody receptors, real-time detection of the SARS-CoV-2 spike S1 protein down to am concentrations is demonstrated in under 2 min in physiological relevant conditions.


Subject(s)
Biosensing Techniques/instrumentation , COVID-19/virology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Transistors, Electronic , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Immobilized , Antibodies, Viral , Bioengineering , COVID-19/blood , COVID-19/diagnosis , COVID-19 Testing/instrumentation , COVID-19 Testing/methods , Computer Simulation , Computer Systems , DNA/analysis , Equipment Design , Humans , Indium , Microtechnology , Proof of Concept Study , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Zinc Oxide
8.
Analyst ; 146(23): 7207-7217, 2021 Nov 22.
Article in English | MEDLINE | ID: covidwho-1493234

ABSTRACT

In this work, we designed and developed a method to detect S1 spike protein of SARS-CoV-2. The portable Localized Surface Plasmon Resonance instrument equipped with a two-channel system was combined with the biotin-streptavidin platform on a nanogold surface to immobilize biotinylated aptamers. The proposed assay does not utilize antibodies or enzyme-based reagents, further simplifying the detection method. Using aptamer-protein bioaffinity interactions, the aptasensor selectively and specifically detected in real-time S1 spike protein, rather than S2 spike protein, RBD spike protein, or bovine serum albumin. The dynamic range and limit of detection of the aptasensor was determined to be 1 nM-100 nM and 0.26 nM, respectively. Notably, aptasensor detected preferentially S1 protein of SARS-CoV-2 compared to SARS-CoV and detected S1 protein with >95% recovery in artificial saliva, and serum albumin, excellent repeatability and shelf-life stability. The method may provide a low-cost, rapid, and real-time detection and monitoring of viruses in the general public.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Surface Plasmon Resonance , Biotin , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/analysis
9.
Anal Chim Acta ; 1188: 339207, 2021 Dec 15.
Article in English | MEDLINE | ID: covidwho-1487555

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as 2019-nCov or COVID-19) outbreak has become a huge public health issue due to its rapid transmission making it a global pandemic. Here, we report fabricated fluorine doped tin oxide (FTO) electrodes/gold nanoparticles (AuNPs) complex coupled with in-house developed SARS-CoV-2 spike S1 antibody (SARS-CoV-2 Ab) to measure the response with Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The biophysical characterisation of FTO/AuNPs/SARS-CoV-2Ab was done via UV-Visible spectroscopy, Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). The fabricated FTO/AuNPs/SARS-CoV-2Ab immunosensor was optimised for response time, antibody concentration, temperature, and pH. Under optimum conditions, the FTO/AuNPs/Ab based immunosensor displayed high sensitivity with limit of detection (LOD) up to 0.63 fM in standard buffer and 120 fM in spiked saliva samples for detection of SARS-CoV-2 spike S1 antigen (Ag) with negligible cross reactivity Middle East Respiratory Syndrome (MERS) spike protein. The proposed FTO/AuNPs/SARS-CoV-2Ab based biosensor proved to be stable for up to 4 weeks and can be used as an alternative non-invasive diagnostic tool for the rapid, specific and sensitive detection of SARS-CoV-2 Spike Ag traces in clinical samples.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Spike Glycoprotein, Coronavirus/analysis , Electrodes , Fluorine , Gold , Humans , Immunoassay , SARS-CoV-2 , Spectroscopy, Fourier Transform Infrared , Tin Compounds
10.
Adv Sci (Weinh) ; 8(23): e2103266, 2021 12.
Article in English | MEDLINE | ID: covidwho-1479368

ABSTRACT

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.


Subject(s)
Blood Coagulation/physiology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies/chemistry , Antibodies/immunology , Antibodies/metabolism , COVID-19/diagnosis , COVID-19/virology , Endothelial Cells/chemistry , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fibrin/chemistry , Fibrin/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Interleukin-6/immunology , Liposomes/chemistry , Microfluidics/methods , Mutation , Nanoparticles/chemistry , Platelet Aggregation , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics
11.
ACS Appl Mater Interfaces ; 13(42): 49754-49761, 2021 Oct 27.
Article in English | MEDLINE | ID: covidwho-1475248

ABSTRACT

A reliable and sensitive detection approach for SARS-CoV 2 is essential for timely infection diagnosis and transmission prevention. Here, a two-dimensional (2D) metal-organic framework (MOF)-based photoelectrochemical (PEC) aptasensor with high sensitivity and stability for SARS-CoV 2 spike glycoprotein (S protein) detection was developed. The PEC aptasensor was constructed by a plasmon-enhanced photoactive material (namely, Au NPs/Yb-TCPP) with a specific DNA aptamer against S protein. The Au NPs/Yb-TCPP fabricated by in situ growth of Au NPs on the surface of 2D Yb-TCPP nanosheets showed a high electron-hole (e-h) separation efficiency due to the enhancement effect of plasmon, resulting in excellent photoelectric performance. The modified DNA aptamer on the surface of Au NPs/Yb-TCPP can bind with S protein with high selectivity, thus decreasing the photocurrent of the system due to the high steric hindrance and low conductivity of the S protein. The established PEC aptasensor demonstrated a highly sensitive detection for S protein with a linear response range of 0.5-8 µg/mL with a detection limit of 72 ng/mL. This work presented a promising way for the detection of SARS-CoV 2, which may conduce to the impetus of clinic diagnostics.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Metal-Organic Frameworks/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Base Sequence , Biosensing Techniques/instrumentation , COVID-19/diagnosis , DNA/chemistry , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Gold/radiation effects , Humans , Immobilized Nucleic Acids/chemistry , Light , Limit of Detection , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Pharynx/virology , Photochemical Processes , Porphyrins/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Ytterbium/chemistry
12.
Placenta ; 117: 47-56, 2022 01.
Article in English | MEDLINE | ID: covidwho-1474964

ABSTRACT

INTRODUCTION: Maternal anti-SARS-CoV-2 Spike antibodies can cross the placenta during pregnancy, and neonates born to infected mothers have acquired antibodies at birth. Few studies reported data on the histopathological changes of the placenta during infection and placental infection. SARS-CoV-2 infection may cause impaired development of the placenta, thus predisposing maternal and fetal unfavorable outcomes. The prospective study aims to evaluate the risk of vertical transmission of SARS-CoV-2 and placental passage of anti-Spike antibodies as well as the impact of clinical severity on placental structures. METHODS: This is a prospective cohort study on 30 pregnant women infected by SARS-CoV-2 with their neonates. The demographic features and pregnancy outcomes were collected. Gross and microscopic examinations of the placentas were done. Maternal and umbilical cord sera were obtained at the time of delivery. Nasopharyngeal swabs were collected from neonates immediately after birth. RESULTS: The concentrations of total anti-SARS-CoV-2 Spike antibodies were higher in pregnant women with moderate to severe/critical disease. The maternal total anti-SARS-CoV-2 Spike levels were correlated with those of neonatal levels. The rate of placental abnormalities is high in the mothers with severe disease, and those with positive anti-SARS-CoV-2 IgM. All neonates had negative nasopharyngeal swabs for SARS- CoV-2 infections and all placentas were negative in immunohistochemical staining for Spike protein. DISCUSSION: The maternally derived anti-SARS-CoV-2 Spike antibody can transmit to neonates born to infected mothers regardless of gestational age. Our results indicated that the disease severity is associated with ischemic placental pathology which may result in adverse pregnancy outcomes.


Subject(s)
COVID-19/complications , Placenta Diseases/virology , Pregnancy Complications, Infectious/virology , SARS-CoV-2 , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/transmission , Cohort Studies , Female , Fetal Blood/immunology , Humans , Immunity, Maternally-Acquired/immunology , Infant, Newborn , Infectious Disease Transmission, Vertical , Placenta/chemistry , Placenta/pathology , Placenta/virology , Placenta Diseases/pathology , Pregnancy , Pregnancy Complications, Infectious/pathology , Pregnancy Outcome , Premature Birth , Prospective Studies , SARS-CoV-2/isolation & purification , Severity of Illness Index , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/immunology
13.
Pathol Res Pract ; 227: 153610, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1401790

ABSTRACT

The coronavirus disease 2019(COVID-19) is recognized as systemic inflammatory response syndrome. It was demonstrated that a rapid increase of cytokines in the serum of COVID-19 patients is associated with the severity of disease. However, the mechanisms of the cytokine release are not clear. By using immunofluorescence staining we found that the number of CD11b positive immune cells including macrophages in the spleens of died COVID-19 patients, was significantly higher than that of the control patients. The incidence of apoptosis as measured by two apoptotic markers, TUNEL and cleaved caspase-3, in COVID-19 patients' spleen cells is higher than that in control patients. By double immunostaining CD11b or CD68 and SARS-CoV-2 spike protein, it was found that up to 67% of these immune cells were positive for spike protein, suggesting that viral infection might be associated with apoptosis in these cells. Besides, we also stained the autophagy-related molecules (p-Akt、p62 and BCL-2) in spleen tissues, the results showed that the number of positive cells was significantly higher in COVID-19 group. And compared with non-COVID-19 patients, autophagy may be inhibited in COVID-19 patients. Our research suggest that SARS-CoV-2 may result in a higher rate of apoptosis and a lower rate of autophagy of immune cells in the spleen of COVID-19 patients. These discoveries may increase our understanding of the pathogenesis of COVID-19.


Subject(s)
Apoptosis , Autophagy , COVID-19/pathology , SARS-CoV-2/pathogenicity , Spleen/pathology , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Autopsy , Biomarkers/analysis , CD11b Antigen/analysis , COVID-19/immunology , COVID-19/mortality , COVID-19/virology , Case-Control Studies , Caspase 3/analysis , Host-Pathogen Interactions , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Phosphorylation , Proto-Oncogene Proteins c-akt/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , SARS-CoV-2/immunology , Sequestosome-1 Protein/analysis , Spike Glycoprotein, Coronavirus/analysis , Spleen/immunology , Spleen/virology
14.
ACS Appl Mater Interfaces ; 13(36): 43696-43707, 2021 Sep 15.
Article in English | MEDLINE | ID: covidwho-1392772

ABSTRACT

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of -SH, -NH2, or -OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. KD affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.


Subject(s)
Biomarkers/analysis , COVID-19/diagnosis , Graphite/chemistry , Immunoassay/methods , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Ethylenes/chemistry , Ferritins/immunology , Ferritins/metabolism , Humans , Point-of-Care Systems , Polyamines/chemistry , Polyethylene Glycols/chemistry , Pyrenes/chemistry , Quantum Theory , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Semiconductors , Spike Glycoprotein, Coronavirus/immunology , Sulfonic Acids/chemistry , Surface Plasmon Resonance
16.
Viruses ; 12(5)2020 05 06.
Article in English | MEDLINE | ID: covidwho-1389513

ABSTRACT

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.


Subject(s)
Antibodies, Viral/immunology , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Containment of Biohazards , HEK293 Cells , Humans , Lentivirus , Peptidyl-Dipeptidase A/metabolism , Plasma/immunology
17.
STAR Protoc ; 1(3): 100214, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-1386744

ABSTRACT

This protocol describes an integrated approach for analyzing site-specific N- and O-linked glycosylation of SARS-CoV-2 spike protein by mass spectrometry. Glycoproteomics analyzes intact glycopeptides to examine site-specific microheterogeneity of glycoproteins. Glycomics provides structural characterization on any glycan assignments by glycoproteomics. This procedure can be modified and applied to a variety of N- and/or O-linked glycoproteins. Combined with bioinformatics, the glycomics-informed glycoproteomics may be useful in generating 3D molecular dynamics simulations of certain glycoproteins alone or interacting with one another. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).


Subject(s)
Glycomics/methods , Glycoproteins , Proteomics/methods , Spike Glycoprotein, Coronavirus , COVID-19/virology , Glycoproteins/analysis , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycosylation , Humans , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
18.
ACS Appl Mater Interfaces ; 13(34): 40342-40353, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1366784

ABSTRACT

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques/methods , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Coronavirus Nucleocapsid Proteins/immunology , Fluorescence , Fluorescent Dyes/chemistry , Humans , Immunoassay/methods , Limit of Detection , Magnetite Nanoparticles/chemistry , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/immunology , Quantum Dots/chemistry , Saliva/virology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
19.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1181: 122884, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1364212

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines are the most promising approach to control the COVID-19 pandemic. There are eminent needs to develop robust analytical methods to ensure quality control, as well as to evaluate the long-term efficacy and safety of vaccine. Although in vivo animal tests, such as serum-based ELISA, have been commonly used for quality control of vaccines, these methods have poor precision, are labor intensive, and require the availability of expensive, specific antibodies. Thus, there is growing interest to develop robust bioanalytical assays as alternatives for qualitative and quantitative evaluation of complex vaccine antigens. In this study, a liquid chromatography tandem mass spectrometry method was developed using optimized unique peptides for simultaneous determination of spike (S) and nucleocapsid (N) protein. Method sensitivity, linearity, repeatability, selectivity, and recovery were evaluated. The amount of S and N proteins in 9 batches of inactivated COVID-19 vaccines were quantified, and their compositions relative to total protein content were consistent. We believe this method can be applied for quality evaluation of other S and/or N protein based COVID-19 vaccine, and could be extended to other viral vector, and protein subunit-based vaccines.


Subject(s)
COVID-19 Vaccines/analysis , Chromatography, Liquid/methods , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Tandem Mass Spectrometry/methods , COVID-19/virology , Humans , Quality Control , Vaccines, Inactivated/analysis
20.
Molecules ; 26(16)2021 Aug 06.
Article in English | MEDLINE | ID: covidwho-1362397

ABSTRACT

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.


Subject(s)
Algorithms , Glycopeptides/analysis , Glycoproteins/analysis , COVID-19/metabolism , Databases, Protein , Glycopeptides/chemistry , Glycoproteins/chemistry , Glycosylation , Humans , Proteomics/methods , Proteomics/standards , SARS-CoV-2/metabolism , Software , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/chemistry , Tandem Mass Spectrometry/methods , Viral Fusion Proteins/analysis , Viral Fusion Proteins/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL