scholarly journals Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

2020 ◽  
Author(s):  
Delyan Hristov ◽  
Hom Rijal ◽  
Jose Gomez-Marquez ◽  
Kimberly Hamad
Keyword(s):  
Test Line ◽  
Human Saliva ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Signal Quality ◽  
Binding Pattern ◽  
Rt Pcr ◽  
Global Pandemic ◽  
Antigen Assay ◽  
Assay Design

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location dependent, time consuming and relatively expensive. Thus, there is a need for a more flexible method which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here we present the first steps towards a paper-based test which can differentiate between different between the Spike protein of various coronaviruses, SARS-CoV-1, SARS-CoV-2 and CoV-HKU1 with negligible cross reactivity for HCoV-OC43 and HCoV-229E in a single assay which takes less than 30 minutes. Furthermore, our test can distinguish between fractions of the same Spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration and antigen interference is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality. <br>

scholarly journals Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

2020 ◽  
Author(s):  
Delyan Hristov ◽  
Hom Rijal ◽  
Jose Gomez-Marquez ◽  
Kimberly Hamad
Keyword(s):  
Test Line ◽  
Human Saliva ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Signal Quality ◽  
Binding Pattern ◽  
Rt Pcr ◽  
Global Pandemic ◽  
Antigen Assay ◽  
Assay Design

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location dependent, time consuming and relatively expensive. Thus, there is a need for a more flexible method which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here we present the first steps towards a paper-based test which can differentiate between different between the Spike protein of various coronaviruses, SARS-CoV-1, SARS-CoV-2 and CoV-HKU1 with negligible cross reactivity for HCoV-OC43 and HCoV-229E in a single assay which takes less than 30 minutes. Furthermore, our test can distinguish between fractions of the same Spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration and antigen interference is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality. <br>

scholarly journals Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies

2021 ◽  
Author(s):  
Sophie Edouard ◽  
Rita Jaafar ◽  
Nicolas Orain ◽  
Philippe Parola ◽  
Philippe Colson ◽  
...  
Keyword(s):  
Negative Control ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Rt Pcr ◽  
Substantial Agreement ◽  
Western Immunoblotting ◽  
Elisa Kit ◽  
Line Test ◽  
Serologic Assay ◽  
Control Samples

AbstractELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The JessTM Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2.

scholarly journals Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257351
Author(s):  
Carolina de la Guardia ◽  
Giselle Rangel ◽  
Alcibiades Villarreal ◽  
Amador Goodridge ◽  
Patricia L. Fernández ◽  
...  
Keyword(s):  
Area Under The Curve ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Close Relative ◽  
Rt Pcr ◽  
Characteristic Analysis ◽  
Serum Samples ◽  
Health Authorities ◽  
Significant Difference

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase–quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

scholarly journals High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

2020 ◽  
Author(s):  
Tiancheng Liu ◽  
Jessica Hsiung ◽  
Su Zhao ◽  
Jessica Kost ◽  
Deepika Sreedhar ◽  
...  
Keyword(s):  
Near Infrared ◽  
Human Saliva ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Igg Antibody ◽  
Antibody Maturation ◽  
Global Pandemic ◽  
Rapid Spread

AbstractThe outbreak and rapid spread of SARS-CoV-2 virus has led to a dire global pandemic with millions of people infected and ~ 400,000 deaths thus far. Highly accurate detection of antibodies for COVID-19 is an indispensable part of the effort to combat the pandemic1,2. Here we developed two-plex antibody detection against SARS-CoV-2 spike proteins3 (the S1 subunit and receptor binding domain RBD) in human serum and saliva on a near-infrared nano-plasmonic gold (pGOLD) platform4–8. By testing nearly 600 serum samples, pGOLD COVID-19 assay achieved ~ 99.78 % specificity for detecting both IgG and IgM with 100 % sensitivity in sera collected > 14 days post disease symptom onset, with zero cross-reactivity to other diseases. Two-plex correlation analysis revealed higher binding of serum IgM to RBD than to S1. IgG antibody avidity toward multiple antigens were measured, shedding light on antibody maturation in COVID-19 patients and affording a powerful tool for differentiating recent from remote infections and identifying re-infection by SARS-CoV-2. Just as important, due to high analytical sensitivity, the pGOLD COVID-19 assay detected minute amounts of antibodies in human saliva, offering the first non-invasive detection of SARS-CoV-2 antibodies.

scholarly journals Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies

2020 ◽  
Author(s):  
S. Edouard ◽  
R. Jaafar ◽  
N. Orain ◽  
P. Parola ◽  
P. Colson ◽  
...  
Keyword(s):  
Negative Control ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Rt Pcr ◽  
Substantial Agreement ◽  
Western Immunoblotting ◽  
Elisa Kit ◽  
Line Test ◽  
Serologic Assay ◽  
Control Samples

ABSTRACTELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The Jess™ Simple Western system, an automated capillary-based assay was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as antigen, and IgT detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposition of people to HCoVs including SARS-CoV-2.

scholarly journals Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity

2021 ◽  
Author(s):  
Vipul Kumar ◽  
Jasdeep Singh ◽  
Seyed E. Hasnain ◽  
Durai Sundar
Keyword(s):  
Binding Energy ◽  
Immune Escape ◽  
Spike Protein ◽  
Wild Type ◽  
Rt Pcr ◽  
Dynamic Simulations ◽  
Human Antibodies ◽  
Global Pandemic ◽  
Hydrogen Interaction ◽  
The Stability

AbstractThe Severe Acute syndrome corona Virus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has caused alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests, which also might lead to inefficacy of the available drugs. It has also increased the chances of reinfection and immune escape. We have performed Molecular Dynamic simulations of three different Spike-ACE2 complexes, namely Wildtype (WT), B.1.1.7 variant (N501Y Spike mutant) and B.1.617 variant (L452R, E484Q Spike mutant) and compared their dynamics, binding energy and molecular interactions. Our result shows that mutation has caused the increase in the binding energy between the Spike and hACE2. In the case of B.1.617 variant, the mutations at L452R and E484Q increased the stability and intra-chain interactions in the Spike protein, which may change the interaction ability of human antibodies to this Spike variant. Further, we found that the B.1.1.7 variant had increased hydrogen interaction with LYS353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.

scholarly journals Possible Link between Higher Transmissibility of Alpha, Kappa and Delta Variants of SARS-CoV-2 and Increased Structural Stability of Its Spike Protein and hACE2 Affinity

2021 ◽  
Vol 22 (17) ◽  
pp. 9131 ◽  
Author(s):  
Vipul Kumar ◽  
Jasdeep Singh ◽  
Seyed E. Hasnain ◽  
Durai Sundar
Keyword(s):  
Binding Energy ◽  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Spike Protein ◽  
Rt Pcr ◽  
Dynamic Simulations ◽  
Global Pandemic ◽  
Hydrogen Interaction ◽  
Alpha Variant ◽  
The Stability

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.

scholarly journals Performance of the LIAISON® SARS-CoV-2 Antigen Assay vs. SARS-CoV-2-RT-PCR

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 658
Author(s):  
Melanie Fiedler ◽  
Caroline Holtkamp ◽  
Ulf Dittmer ◽  
Olympia E. Anastasiou
Keyword(s):  
Reference Method ◽  
Respiratory Viruses ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Loads ◽  
Antigen Assay ◽  
Polymerase Chain

We aimed to evaluate the LIAISON® SARS-CoV-2 antigen assay (DiaSorin), comparing its performance to real-time polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2 RNA. 182 (110 PCR-positive and 72 PCR-negative) nasopharyngeal swab samples were taken for the detection of SARS-CoV-2. RT-PCR and antigen assay were performed using the same material. The sensitivity and specificity of the antigen assay were calculated for different cut-offs, with RT-PCR serving as the reference method. Stored clinical samples that were positive for other respiratory viruses were tested to evaluate cross-reactivity. One third (33/110, 30%) were falsely classified as negative, while no false positives were found using the 200 TCID50/mL cut-off for the SARS-CoV-2 antigen as proposed by the manufacturer. This corresponded to a sensitivity of 70% (60–78%) and a specificity of 100% (94–100%). Lowering the cut-off for positivity of the antigen assay to 22.79 or 57.68 TCID50/mL increased the sensitivity of the method, reaching a sensitivity of 92% (85–96%) vs. 79% (70–86%) and a specificity of 81% (69–89%) vs. 99% (91–100%), respectively. The antigen assay reliably detected samples with high SARS-CoV-2 viral loads (≥106 copies SARS-CoV-2/mL), while it cannot differentiate between negative and low positive samples. Cross-reactivity toward other respiratory viruses was not detected.

scholarly journals Thrombotic Thrombocytopenia after COVID-19 Vaccination: In Search of the Underlying Mechanism

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 559
Author(s):  
Piotr Rzymski ◽  
Bartłomiej Perek ◽  
Robert Flisiak
Keyword(s):  
Direct Interaction ◽  
Underlying Mechanism ◽  
The United States ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Vaccine Hesitancy ◽  
Future Research ◽  
Platelet Factor ◽  
Precautionary Measures ◽  
Adenoviral Proteins

The rollout of COVID-19 vaccines brings hope for successful pandemic mitigation and getting the transmission of SARS-CoV-2 under control. The vaccines authorized in Europe displayed a good safety profile in the clinical trials. However, during their post-authorization use, unusual thrombotic events associated with thrombocytopenia have rarely been reported for vector vaccines. This led to the temporary suspension of the AZD1222 vaccine (Oxford/AstraZeneca) in various European countries and the Ad26.COV2 vaccine (Janssen/Johnson&Johnson) in the United States, with regulatory bodies launching investigations into potential causal associations. The thromboembolic reactions were also rarely reported after mRNA vaccines. The exact cause of these adverse effects remains to be elucidated. The present paper outlines the hypotheses on the mechanisms behind the very rare thrombotic thrombocytopenia reported after the COVID-19 vaccination, along with currently existing evidence and future research prospects. The following are discussed: (i) the role of antibodies against platelet factor 4 (PF4), (ii) the direct interaction between adenoviral vector and platelets, (iii) the cross-reactivity of antibodies against SARS-CoV-2 spike protein with PF4, (iv) cross-reactivity of anti-adenovirus antibodies and PF4, (v) interaction between spike protein and platelets, (vi) the platelet expression of spike protein and subsequent immune response, and (vii) the platelet expression of other adenoviral proteins and subsequent reactions. It is also plausible that thrombotic thrombocytopenia after the COVID-19 vaccine is multifactorial. The elucidation of the causes of these adverse events is pivotal in taking precautionary measures and managing vaccine hesitancy. It needs to be stressed, however, that the reported cases are currently sporadic and that the benefits of COVID-19 vaccines vastly outweigh their potential risks.

scholarly journals CT findings of 795 COVID-19 positive cases: a multicenter study in Egypt

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Youssriah Yahia Sabri ◽  
Mohamed Mohsen Tolba Fawzi ◽  
Eman Zaki Nossair ◽  
Safaa Mohamed El-Mandooh ◽  
Amira Aly Hegazy ◽  
...  
Keyword(s):  
Virus Disease ◽  
Chest Ct ◽  
Imaging Features ◽  
Rt Pcr ◽  
Relevant Research ◽  
Non Invasive ◽  
Ct Findings ◽  
Global Pandemic ◽  
Polymerase Chain ◽  
Ct Studies

Abstract Background Corona Virus Disease 2019 (COVID-19) outbreak was officially announced as a global pandemic by the WHO on March 11th 2020. Thorough understanding of CT imaging features of COVID-19 is essential for effective patient management; rationalizing the need for relevant research. The aim of this study was to analyze the chest CT findings of patients with real-time polymerase chain reaction (RT-PCR) proved COVID-19 admitted to four Egyptian hospitals. The recently published RSNA expert consensus statement on reporting COVID-19 chest CT findings was taken into consideration. Results Normal CT “negative for COVID-19” was reported in 26.1% of our RT-PCR proved COVID-19 cases. In descending order of prevalence, imaging findings of the positive CT studies (73.9%) included GGO (69%), consolidation (49.7%), crazy paving (15.4%), and peri-lobular fibrosis (40.6%). These showed a dominantly bilateral (68.2%), peripheral (72.4%), and patchy (64.7%) distribution. Remarkably, thymic hyperplasia was identified in 14.3% of studies. According to the RSNA consensus, CT findings were classified as typical in 68.9%, indeterminate in 3.6%, and atypical in 1.4% of the evaluated CT studies. Conclusion Although COVID-19 cannot be entirely excluded by chest CT, it can be distinguished in more than two-thirds of cases; making CT a widely available, non-invasive, and rapid diagnostic tool.

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