scholarly journals Validation of high-throughput, semiquantitative solid phase SARS coronavirus-2 serology assays in serum and dried blood spot matrices

Bioanalysis ◽  
2021 ◽  
Author(s):  
Leo Maritz ◽  
Nicholas J Woudberg ◽  
Amber C Bennett ◽  
Andreia Soares ◽  
Florian Lapierre ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Solid Phase ◽  
Dried Blood Spot ◽  
Sars Coronavirus ◽  
Diagnostic Specificity ◽  
Global Pandemic ◽  
Specificity And Sensitivity ◽  
Infection Monitoring ◽  
Blood Spot

Aim: Serological assays for the detection of anti-SARS coronavirus-2 (SARS-CoV-2) antibodies are essential to the response to the global pandemic. A ligand binding-based serological assay was validated for the semiquantitative detection of IgG, IgM, IgA and neutralizing antibodies (nAb) against SARS-CoV-2 in serum. Results: The assay demonstrated high levels of diagnostic specificity and sensitivity (85–99% for all analytes). Serum IgG, IgM, IgA and nAb correlated positively (R2 = 0.937, R2 = 0.839, R2 = 0.939 and R2 = 0.501, p < 0.001, respectively) with those measured in dried blood spot samples collected using the hemaPEN® microsampling device (Trajan Scientific and Medical, Victoria, Australia). In vitro SARS-CoV-2 pseudotype neutralization correlated positively with the solid phase nAb signals in convalescent donors (R2 = 0.458, p < 0.05). Conclusion: The assay is applicable in efficacy studies, infection monitoring and postmarketing surveillance following vaccine rollout.

scholarly journals A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amelia E. Sancilio ◽  
Richard T. D’Aquila ◽  
Elizabeth M. McNally ◽  
Matthew P. Velez ◽  
Michael G. Ison ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Venous Blood ◽  
Host Cells ◽  
Dried Blood Spot ◽  
Blood Collection ◽  
Widespread Application ◽  
Live Virus ◽  
Dilution Series ◽  
Wide Range ◽  
Blood Spot

AbstractThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

scholarly journals Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

2020 ◽  
Author(s):  
Jun-Gyu Park ◽  
Fatai S. Oladduni ◽  
Kevin Chiem ◽  
Chengjin Ye ◽  
Michael Pipenbrink ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Health Impact ◽  
In Vitro Assays ◽  
Global Pandemic ◽  
Novel Coronavirus ◽  
Human Infections ◽  
Identification And Characterization

AbstractTowards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

scholarly journals Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

2020 ◽  
Author(s):  
Hua-Long Xiong ◽  
Yang-Tao Wu ◽  
Jia-Li Cao ◽  
Ren Yang ◽  
Jian Ma ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Cell Number ◽  
New Drugs ◽  
Human Society ◽  
Angiotensin Converting Enzyme 2 ◽  
Global Pandemic ◽  
Vesicular Stomatitis

AbstractThe global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Comparison and validation of Dried Blood Spot (DBS) and Solid Phase Extraction (SPE) techniques

2010 ◽  
Vol 196 ◽  
pp. S294-S295 ◽  
Author(s):  
F. Pastori ◽  
D. Salerno ◽  
G. Oberto ◽  
S. Villa ◽  
R. Ricci ◽  
...  
Keyword(s):  
Solid Phase Extraction ◽  
Solid Phase ◽  
Dried Blood Spot ◽  
Phase Extraction ◽  
Blood Spot

scholarly journals A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

2021 ◽  
Author(s):  
Amelia Sancilio ◽  
Richard D’Aquila ◽  
Elizabeth M. McNally ◽  
Matt E Velez ◽  
Michael G. Ison ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Venous Blood ◽  
Host Cells ◽  
Dried Blood Spot ◽  
Blood Collection ◽  
Widespread Application ◽  
Live Virus ◽  
Dilution Series ◽  
Wide Range ◽  
Blood Spot

ABSTRACTBackgroundThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample.MethodsSamples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV).ResultsDilution series produced the expected pattern of dose-response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples.ConclusionsLarge-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

scholarly journals A Peptide Mimic of a Protective Epitope of Respiratory Syncytial Virus Selected from a Combinatorial Library Induces Virus-Neutralizing Antibodies and Reduces Viral Load In Vivo

1998 ◽  
Vol 72 (3) ◽  
pp. 2040-2046 ◽  
Author(s):  
Daniel Chargelegue ◽  
Obeid E. Obeid ◽  
Shiou-Chih Hsu ◽  
Michael D. Shaw ◽  
Andrew N. Denbury ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Neutralizing Antibodies ◽  
Solid Phase ◽  
Effective Vaccine ◽  
Peptide Mimic ◽  
Rsv Infection ◽  
Multiple Antigen Peptides ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 × 109 and 4.93 × 109 M−1 for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.

scholarly journals Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders

2020 ◽  
Vol 6 (2) ◽  
pp. 26 ◽  
Author(s):  
Stuart J. Moat ◽  
Roanna S. George ◽  
Rachel S. Carling
Keyword(s):  
Metabolic Disorders ◽  
Ultra Performance Liquid Chromatography ◽  
Dried Blood Spot ◽  
Specificity And Sensitivity ◽  
Low Concentrations ◽  
Inherited Metabolic Disorders ◽  
The 1960S ◽  
Analytical Technology ◽  
Test Result ◽  
Blood Spot

Monitoring of patients with inherited metabolic disorders (IMDs) using dried blood spot (DBS) specimens has been routinely used since the inception of newborn screening (NBS) for phenylketonuria in the 1960s. The introduction of flow injection analysis tandem mass spectrometry (FIA–MS/MS) in the 1990s facilitated the expansion of NBS for IMDs. This has led to increased identification of patients who require biochemical monitoring. Monitoring of IMD patients using DBS specimens is widely favoured due to the convenience of collecting blood from a finger prick onto filter paper devices in the patient’s home, which can then be mailed directly to the laboratory. Ideally, analytical methodologies with a short analysis time and high sample throughput are required to enable results to be communicated to patients in a timely manner, allowing prompt therapy adjustment. The development of ultra-performance liquid chromatography (UPLC–MS/MS), means that metabolic laboratories now have the capability to routinely analyse DBS specimens with superior specificity and sensitivity. This advancement in analytical technology has led to the development of numerous assays to detect analytes at low concentrations (pmol/L) in DBS specimens that can be used to monitor IMD patients. In this review, we discuss the pre-analytical, analytical and post-analytical variables that may affect the final test result obtained using DBS specimens used for monitoring of patients with an IMD.

scholarly journals A robust multiplexed assay to quantify C1-inhibitor, C1q, and C4 proteins for in vitro diagnosis of hereditary angioedema from dried blood spot

2021 ◽  
Vol 195 ◽  
pp. 113844
Author(s):  
Yongquan Lai ◽  
Guodong Zhang ◽  
Neil Inhaber ◽  
Jonathan A. Bernstein ◽  
Michael Cwik ◽  
...  
Keyword(s):  
Hereditary Angioedema ◽  
Dried Blood Spot ◽  
C1 Inhibitor ◽  
Blood Spot ◽  
In Vitro Diagnosis

scholarly journals Immunoglobulin fragment F(ab’)2 against RBD potently neutralizes SARS-CoV-2 in vitro

2020 ◽  
Author(s):  
Xiaoyan Pan ◽  
Pengfei Zhou ◽  
Tiejiong Fan ◽  
Yan Wu ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
High Titer ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Ovary Cells ◽  
Global Pandemic ◽  
Potent Inhibitory Effect

AbstractCOVID-19 caused by the emerging human coronavirus, SARS-CoV-2, has become a global pandemic, leading a serious threat to human health. So far, there is none vaccines or specific antiviral drugs approved for that. Therapeutic antibodies for SARS-CoV-2, was obtained from hyper immune equine plasma in this study. Herein, SARS-CoV-2 RBD with gram level were obtained through Chinese hamster ovary cells high-density fermentation. The binding of RBD to SARS-CoV-2 receptor, human ACE2, was verified and the efficacy of RBD in vivo was tested on mice and then on horses. As a result, RBD triggered high-titer neutralizing antibodies in vivo, and immunoglobulin fragment F(ab’)2 was prepared from horse antisera through removing Fc. Neutralization test demonstrated that RBD-specific F(ab’)2 inhibited SARS-CoV-2 with EC50 at 0.07 μg/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlights as RBD-specific F(ab’)2 as therapeutic candidate for SARS-CoV-2.

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