scholarly journals Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hideki Tani ◽  
Miyuki Kimura ◽  
Long Tan ◽  
Yoshihiro Yoshida ◽  
Tatsuhiko Ozawa ◽  
...  
Keyword(s):  
Virus Infection ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
High Specificity ◽  
Hospitalized Patients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Live Virus ◽  
Specificity And Sensitivity

Abstract Background SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. Methods To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). Results The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. Conclusions In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

scholarly journals Evaluation of SARS-CoV-2 Neutralizing Antibodies Using a Vesicular Stomatitis Virus Possessing SARS-CoV-2 Spike Protein

2020 ◽  
Author(s):  
Hideki Tani ◽  
Long Tan ◽  
Miyuki Kimura ◽  
Yoshihiro Yoshida ◽  
Hiroshi Yamada ◽  
...  
Keyword(s):  
Virus Infection ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
High Specificity ◽  
Immunofluorescence Assay ◽  
Hospitalized Patients ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Live Virus ◽  
Specificity And Sensitivity

Abstract Background:SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. Methods:To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). Results:The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. Conclusions:In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

scholarly journals Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein

2020 ◽  
Author(s):  
Hideki Tani ◽  
Long Tan ◽  
Miyuki Kimura ◽  
Yoshihiro Yoshida ◽  
Hiroshi Yamada ◽  
...  
Keyword(s):  
Virus Infection ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
High Specificity ◽  
Immunofluorescence Assay ◽  
Hospitalized Patients ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Live Virus ◽  
Specificity And Sensitivity

AbstractSARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

scholarly journals SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikail Dogan ◽  
Lina Kozhaya ◽  
Lindsey Placek ◽  
Courtney Gunter ◽  
Mesut Yigit ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
High Specificity ◽  
Spike Protein ◽  
Humoral Immune ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Specific Igg ◽  
Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

scholarly journals Evaluation of a Pseudovirus Neutralization Assay for SARS-CoV-2 and Correlation with Live Virus-Based Micro Neutralization Assay

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 994
Author(s):  
Ahmed Majdi K. Tolah ◽  
Sayed S. Sohrab ◽  
Khaled Majdi K. Tolah ◽  
Ahmed M. Hassan ◽  
Sherif A. El-Kafrawy ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Epidemiological Studies ◽  
Neutralization Assay ◽  
Serum Samples ◽  
Live Virus ◽  
Spike Gene ◽  
Microneutralization Assay ◽  
Viral Particles ◽  
Reliable Performance ◽  
Prior Infection

The unusual cases of pneumonia outbreak were reported from Wuhan city in late December 2019. Serological testing provides a powerful tool for the identification of prior infection and for epidemiological studies. Pseudotype virus neutralization assays are widely used for many viruses and applications in the fields of serology. The accuracy of pseudotype neutralizing assay allows for its use in low biosafety lab and provides a safe and effective alternative to the use of wild-type viruses. In this study, we evaluated the performance of this assay compared to the standard microneutralization assay as a reference. The lentiviral pseudotype particles were generated harboring the Spike gene of SARS-CoV-2. The generated pseudotype particles assay was used to evaluate the activity of neutralizing antibodies in 300 human serum samples from a COVID-19 sero-epidemiological study. Testing of these samples resulted in 55 positive samples and 245 negative samples by pseudotype viral particles assay while microneutralization assay resulted in 64 positive and 236 negative by MN assay. Compared to the MN, the pseudotyped viral particles assay showed a sensitivity of 85.94% and a specificity of 100%. Based on the data generated from this study, the pseudotype-based neutralization assay showed a reliable performance for the detection of neutralizing antibodies against SARS-CoV-2 and can be used safely and efficiently as a diagnostic tool in a biosafety level 2 laboratory.

scholarly journals Serum MicroRNA Signature as a Diagnostic and Therapeutic Marker in Patients with Psoriatic Arthritis

2020 ◽  
Vol 47 (12) ◽  
pp. 1760-1767
Author(s):  
Sarah M. Wade ◽  
Trudy McGarry ◽  
Siobhan C. Wade ◽  
Ursula Fearon ◽  
Douglas J. Veale
Keyword(s):  
Psoriatic Arthritis ◽  
Characteristic Curve ◽  
High Specificity ◽  
Serum Samples ◽  
Rna Molecules ◽  
Serum Mirna ◽  
Serum Microrna ◽  
Specificity And Sensitivity ◽  
European League Against Rheumatism ◽  
Noninvasive Markers

ObjectiveMicroRNA (miRNA) are small endogenous regulatory RNA molecules that have emerged as potential therapeutic targets and biomarkers in autoimmunity. Here, we investigated serum miRNA levels in patients with psoriatic arthritis (PsA) and further assessed a serum miRNA signature in therapeutic responder versus nonresponder PsA patients.MethodsSerum samples were collected from healthy controls (HC; n = 20) and PsA patients (n = 31), and clinical demographics were obtained. To examine circulatory miRNA in serum from HC and PsA patients, a focused immunology miRNA panel was analyzed utilizing a miRNA Fireplex assay (FirePlex Bioworks Inc.). MiRNA expression was further assessed in responders versus nonresponders according to the European League Against Rheumatism response criteria.ResultsSix miRNA (miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, miR-26a-5p, and miR-21-5p) were significantly higher in PsA compared to HC (all P < 0.05), with high specificity and sensitivity determined by receiver-operating characteristic curve analysis. Analysis of responder versus nonresponders demonstrated higher baseline levels of miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, and miR-26a-5p were associated with therapeutic response.ConclusionThis study identified a 6-serum microRNA signature that could be attractive candidates as noninvasive markers for PsA and may help to elucidate the disease pathogenesis.

Multicolor enzyme-linked immunoassay method for visual readout of carbendazim

2021 ◽  
Author(s):  
Haoran Liu ◽  
Yiwen Wang ◽  
Ruijie Fu ◽  
Jing Zhou ◽  
Yanlin Liu ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Enzyme-linked immunosorbent assay (ELISA) with high specificity and sensitivity is one of the most popular techniques for detecting carbendazim (CBD), a commonly used benzimidazole fungicide in agriculture. However, the traditional...

scholarly journals A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring

2019 ◽  
Author(s):  
Lihua Wang ◽  
Shijiang Mi ◽  
Rachel Madera ◽  
Llilianne Ganges ◽  
Manuel V. Borca ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Confidence Interval ◽  
Excellent Agreement ◽  
Neutralizing Antibodies ◽  
Vaccination Campaign ◽  
Classical Swine Fever ◽  
Enzyme Linked Immunosorbent Assay ◽  
Live Virus ◽  
Post Vaccination ◽  
Neutralizing Monoclonal Antibody

Abstract Background: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n=445) and C-strain VNT positive pig sera (n=70), the 6B211 based cELSIA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be detected in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n=139) in parallel, the cELISA showed excellent agreement (Kappa=0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r 2 =0.903, p<0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). Conclusions: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

scholarly journals A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

2021 ◽  
Author(s):  
Syed Hani Abidi ◽  
Kehkeshan Imtiaz ◽  
Akbar Kanji ◽  
Shama Qaiser ◽  
Erum Khan ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Neutralization Assay ◽  
Dilution Method ◽  
Rt Pcr ◽  
Serum Samples ◽  
Live Virus ◽  
Viral Neutralization ◽  
Microneutralization Assay ◽  
Infected Cells

Abstract Background Individuals recovering from COVID-19 are shown to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow the determination of virus neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike receptor binding domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.

scholarly journals Immunoglobulin A Antibody Responses in Dengue Patients: a Useful Marker for Serodiagnosis of Dengue Virus Infection

2005 ◽  
Vol 12 (10) ◽  
pp. 1235-1237 ◽  
Author(s):  
M. Nawa ◽  
T. Takasaki ◽  
M. Ito ◽  
S. Inoue ◽  
K. Morita ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Immunoglobulin A ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Infections ◽  
Serum Samples ◽  
Dengue Virus Infection ◽  
Iga Antibody ◽  
Antibody Capture

ABSTRACT We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

2003 ◽  
Vol 10 (3) ◽  
pp. 439-442 ◽  
Author(s):  
F. Roodbari ◽  
M. H. Roustai ◽  
A. Mostafaie ◽  
H. Soleimanjdahi ◽  
R. Sarrami Foroshani ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Neutralizing Antibodies ◽  
Measles Virus ◽  
Clinical Symptoms ◽  
Maculopapular Rash ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Vaccination Programs ◽  
Elisa System

ABSTRACT Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).

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