Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
1.
Microbiol Spectr ; 9(3): e0113121, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1522926

ABSTRACT

Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organization (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralization test (MNT). We also compared the MNT results of two laboratories. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13 to 150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA, 100% specificity and sensitivity were obtained for a shorter time window (13 to 36 and 13 to 28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralizing antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ = 0.77 to 0.84, P < 2.2 × 10-16) with NAb titers, and the two laboratories' NAb titers displayed a very strong correlation (ρ = 0.95, P < 2.2 × 10-16). Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against the WHO international standard did not, however, improve the comparability of FMIA and EIA results. IMPORTANCE SARS-CoV-2 serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers. Neutralization tests are still considered the gold standard for SARS-CoV-2 serological assays, but our assay can identify samples with neutralizing antibodies with 100% sensitivity and 96% specificity without the need for laborious and slow biosafety level 3 (BSL-3) facility-requiring analyses.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , Fluorescent Antibody Technique/methods , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Nucleocapsid Proteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Humans , Nucleoproteins , Phosphoproteins/immunology , SARS-CoV-2 , Sensitivity and Specificity
2.
Mol Biotechnol ; 64(4): 339-354, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1469770

ABSTRACT

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.


Subject(s)
COVID-19 Testing/methods , Biosensing Techniques/methods , COVID-19 Testing/instrumentation , CRISPR-Cas Systems , Electrochemical Techniques , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique/methods , Humans , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Neutralization Tests , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Surface Plasmon Resonance
3.
STAR Protoc ; 2(3): 100499, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1275771

ABSTRACT

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).


Subject(s)
COVID-19/prevention & control , Fluorescent Antibody Technique/standards , Germinal Center/drug effects , Immunization, Secondary/methods , SARS-CoV-2/immunology , Spleen/drug effects , Animals , COVID-19/diagnostic imaging , COVID-19/immunology , COVID-19/virology , Fluorescent Antibody Technique/methods , Germinal Center/immunology , Germinal Center/pathology , Germinal Center/virology , Immunogenicity, Vaccine , Male , Mice , Spleen/immunology , Spleen/pathology , Spleen/virology
4.
Viruses ; 13(5)2021 04 24.
Article in English | MEDLINE | ID: covidwho-1201926

ABSTRACT

As of April 2021, the COVID-19 pandemic has swept through 213 countries and infected more than 132 million individuals globally, posing an unprecedented threat to human health. There are currently no specific antiviral treatments for COVID-19 and vaccination programmes, whilst promising, remain in their infancy. A key to restricting the pandemic is the ability to minimize human-human transmission and to predict the infection status of the population in the face of emerging SARS-CoV-2 variants. Success in this area is dependent on the rapid detection of COVID-19 positive individuals with current/previous SARS-CoV-2 infection status. In this regard, the ability to detect antibodies directed against the SARS-CoV-Spike protein in patient sera represents a powerful biomarker for confirmation of infection. Here, we report the design of a proof-of-concept cell-based fluorescent serology assay (termed C19-S-I-IFA) to detect SARS-CoV-2 infection. The assay is based on the capture of IgG antibodies in the serum of COVID-19-positive patients using cells exogenously expressing SARS-CoV-2-Spike and their subsequent fluorescent detection. We validate the assay in 30 blood samples collected in Oxford, UK, in 2020 during the height of the pandemic. Importantly, the assay can be modified to express emerging Spike-variants to permit assessments of the cross-reactivity of patient sera to emerging SARS-CoV-2 strains.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Fluorescent Antibody Technique/methods , SARS-CoV-2/isolation & purification , A549 Cells , Antibodies, Viral/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19/immunology , Cross Reactions , Humans , Immunoglobulin G/blood , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics
5.
J Clin Virol ; 139: 104818, 2021 06.
Article in English | MEDLINE | ID: covidwho-1164017

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) endgame may benefit from simple, accurate antibody testing to characterize seroprevalence and immunization coverage. OBJECTIVES: To evaluate the performance of the lateral flow QIAreach anti-SARS-CoV-2 Total rapid nanoparticle fluorescence immunoassay compared to reference isotype-specific IgG, IgM, and IgA SARS-CoV-2 ELISA using S1 or receptor binding domain (RBD) as antigens. STUDY DESIGN: A diagnostic comparison study was carried out using 154 well-characterized heparin plasma samples. Agreement between assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient. RESULTS: Overall agreement between the QIAreach anti-SARS-CoV-2 Total and any anti-spike domain (S1 or RBD) antibody isotype was 96.0 % (95 % CI 89.8-98.8), the positive percent agreement was 97.6 % (95 % CI 91.0-99.9), the negative percent agreement was 88.2 % (95 % CI 64.4-98.0). The kappa coefficient was 0.86 (95 % CI 0.72 to 0.99). CONCLUSION: The QIAreach anti-SARS-CoV-2 Total rapid antibody test provides comparable performance to high-complexity, laboratory-based ELISA.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Fluorescent Antibody Technique/methods , SARS-CoV-2/immunology , Adult , Aged , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Nanoparticles
6.
J Med Virol ; 93(7): 4219-4241, 2021 07.
Article in English | MEDLINE | ID: covidwho-1151934

ABSTRACT

The potential zoonotic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false-negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine-tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high-throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS-CoV-2 pandemic.


Subject(s)
COVID-19/diagnosis , High-Throughput Screening Assays/methods , Severe Acute Respiratory Syndrome/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Genome, Viral/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS Virus/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Viral Plaque Assay/methods
7.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1046770

ABSTRACT

Coronaviruses (CoV) are a family of viral pathogens that infect both birds and mammals, including humans. Seven human coronaviruses (HCoV) have been recognized so far. HCoV-229E, -OC43, -NL63, and -HKU1 account for one-third of common colds with mild symptoms. The other three members are severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. These viruses are responsible for SARS, MERS, and CoV disease 2019 (COVID-19), respectively. A variety of diagnostic techniques, including chest X-rays, computer tomography (CT) scans, analysis of viral nucleic acids, proteins, or whole virions, and host antibody detection using serological assays have been developed for the detection of these viruses. In this review, we discuss conventional serological tests, such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence assay (IFA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA), as well as biosensor-based assays that have been developed for diagnosing HCoV-associated diseases since 2003, with an in-depth focus on COVID-19.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Middle East Respiratory Syndrome Coronavirus/immunology , SARS-CoV-2/immunology , Severe Acute Respiratory Syndrome/diagnosis , Antibodies, Viral/immunology , Biosensing Techniques/methods , Blotting, Western/methods , COVID-19/virology , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Humans , Luminescent Measurements/methods , SARS Virus/immunology , Severe Acute Respiratory Syndrome/virology
8.
Cytometry B Clin Cytom ; 100(1): 33-41, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1006421

ABSTRACT

Over a remarkably short period of time, a great deal of knowledge about severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection has been acquired, through the focused and cooperative effort of the international scientific community. Much has become known about how the immune response is coordinated to fight infection, and how it becomes dysregulated in severe disease. In this review, we take an in-depth look at the many immune features associated with the host response to SARS-CoV2, as well as those that appear to mark severe disease.


Subject(s)
COVID-19/diagnostic imaging , COVID-19/immunology , Flow Cytometry/methods , Fluorescent Antibody Technique/methods , SARS-CoV-2/immunology , Biomarkers/analysis , COVID-19/pathology , COVID-19/therapy , Chemokines/analysis , Chemokines/metabolism , Cytokines/analysis , Cytokines/metabolism , Fluorescent Antibody Technique/trends , Host-Pathogen Interactions/immunology , Humans , Immunity/physiology , Metabolomics/methods , Metabolomics/trends , Risk Assessment , Severity of Illness Index
9.
J Med Virol ; 93(5): 2838-2847, 2021 05.
Article in English | MEDLINE | ID: covidwho-996231

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) epidemic has made a huge impact on health, economies, and societies all over the world. Although reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid detection has been primarily used in the diagnosis of COVID-19, it is time-consuming with limited application scenarios and must be operated by qualified personnel. Antibody test, particularly point-of-care antibody testing, is a suitable complement to nucleic acid test as it provides rapid, portable, and cost-effective detection of infections. In this study, a Rapid Antibody Test Kit was developed based on fluorescence immunochromatography for the sensitive, accurate, and automated detection of immunoglobulin M (IgM) and IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human serum, plasma, and whole blood samples within 10 min. The sensitivity, specificity, precision, and stability of the test kit were of good performance. No cross-activity and no interference was observed. In the multiple-center parallel study, 223 samples from hospitalized patients were used to evaluate the clinical specificity of the test. Both SARS-CoV-2 IgM and IgG achieved a clinical specificity of 98.21%. The clinical sensitivities of SARS-CoV-2 IgM and IgG were 79.54% and 87.45%, respectively, among 733 reverse transcription-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 samples. For the combined IgM and IgG assays, the sensitivity and specificity were 89.22% and 96.86%, respectively. Our results demonstrate that the combined use of IgM and IgG could serve as a more suitable alternative detection method for patients with COVID-19, and the developed kit is of great public health significance for the prevention and control of the COVID-19 pandemic.


Subject(s)
Antibodies, Viral/blood , COVID-19 Testing/methods , COVID-19/diagnosis , Fluorescent Antibody Technique/methods , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Reagent Kits, Diagnostic , Adolescent , Adult , Aged , Aged, 80 and over , Animals , COVID-19/immunology , Child , Child, Preschool , Female , Fluorescence , Humans , Male , Mice , Middle Aged , Point-of-Care Testing , Recombinant Proteins , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Young Adult
10.
PLoS One ; 15(10): e0240395, 2020.
Article in English | MEDLINE | ID: covidwho-890178

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging threat worldwide. This study aims to assess the serologic profiles and time kinetics of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with COVID-19 using two immunoassays. METHODS: A total of 97 samples serially collected from 17 patients with COVID-19 and 137 negative control samples were analyzed for IgM and IgG against SARS-CoV-2 using the AFIAS COVID-19 Ab (Boditech Med Inc., Chuncheon, Republic of Korea) and the EDI™ Novel Coronavirus COVID-19 ELISA Kit (Epitope Diagnostics, Inc., San Diego, CA). RESULTS: With both assays, IgM and IgG rapidly increased after 7 days post symptom onset (PSO). IgM antibody levels reached a peak at 15-35 d PSO and gradually decreased. IgG levels gradually increased and remained at similar levels after 22-35 d. The diagnostic sensitivities of IgM/IgG for ≤14d PSO were 21.4%/35.7~57.1% and increased to 41.2~52.9%/88.2~94.1% at >14 d PSO with specificities of 98.5%/94.2% for AFIAS COVID-19 Ab and 100.0%/96.4% for EDI™ Novel Coronavirus COVID-19 ELISA Kit. Among 137 negative controls, 12 samples (8.8%) showed positive or indeterminate results. CONCLUSIONS: The antibody kinetics against SARS-CoV-2 are similar to common findings of acute viral infectious diseases. Antibody testing is useful for ruling out SARS-CoV-2 infection after 14 d PSO, detecting past infection, and epidemiologic surveys.


Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Serologic Tests/methods , Adult , Aged , Aged, 80 and over , COVID-19 , Coronavirus Infections/virology , Enzyme-Linked Immunosorbent Assay/methods , Female , Fluorescent Antibody Technique/methods , Follow-Up Studies , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Kinetics , Male , Middle Aged , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , Young Adult
11.
Euro Surveill ; 25(28)2020 07.
Article in English | MEDLINE | ID: covidwho-874407

ABSTRACT

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Subject(s)
Antibodies, Monoclonal/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , Blotting, Western , COS Cells , COVID-19 , Chlorocebus aethiops , Conserved Sequence , Coronavirus Infections/genetics , Coronavirus Infections/virology , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Genome, Viral , Mice , Pandemics , Peptidyl-Dipeptidase A/immunology , Plasmids , Pneumonia, Viral/genetics , Recombinant Proteins/immunology , SARS Virus/genetics , SARS-CoV-2 , Sequence Alignment , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/genetics , Transfection , Vero Cells , Virus Integration
12.
Placenta ; 100: 69-74, 2020 10.
Article in English | MEDLINE | ID: covidwho-728807

ABSTRACT

INTRODUCTION: While the COVID-19 pandemic continues to have a significant global health impact, rates of maternal to infant vertical transmission remain low (<5%). Parenchymal changes of placentas from COVID-19 infected mothers have been reported by several groups, but the localization and relative abundance of SARS-CoV-2 viral proteins and cellular entry machinery has not been fully characterized within larger placental tissue cohorts. METHODS: An extended placental tissue cohort including samples from 15 COVID-19 positive maternal-fetal dyads (with n = 5 cases with evidence of fetal transmission) in comparison with 10 contemporary COVID-19 negative controls. Using comparative immunofluorescence, we examined the localization and relative tissue abundance of SARS-CoV2 spike glycoprotein (CoV2 SP) along with the co-localization of two SARS-CoV2 viral entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). RESULTS/CONCLUSIONS: CoV2 SP was present within the villous placenta in COVID-19 positive pregnancies with and without evidence of fetal transmission. We further identified the predominance of ACE2 expression in comparison with TMPRSS2. Importantly, both CoV2 SP and ACE2 expression consistently localized primarily within the outer syncytiotrophoblast layer placental villi, a key physiologic interface between mother and fetus. Overall this study provides an important basis for the ongoing evaluation of SARS-CoV-2 physiology in pregnancy and highlights the importance of the placenta as a key source of primary human tissue for ongoing diagnostic and therapeutic research efforts to reduce the global burden of COVID-19.


Subject(s)
Betacoronavirus/chemistry , Chorionic Villi/chemistry , Coronavirus Infections , Pandemics , Peptidyl-Dipeptidase A/analysis , Pneumonia, Viral , Serine Endopeptidases/analysis , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2 , COVID-19 , Female , Fetus , Fluorescent Antibody Technique/methods , Humans , Infectious Disease Transmission, Vertical , Placenta/chemistry , Pregnancy , Pregnancy Complications, Infectious/virology , Receptors, Virus/analysis , SARS-CoV-2 , Trophoblasts/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL