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

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.

Download Full-text

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

PLoS ONE ◽

10.1371/journal.pone.0259551 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0259551

Author(s):

Syed Hani Abidi ◽

Kehkashan 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

Background Individuals recovering from COVID-19 are known 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 although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for 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 a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target 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, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

Download Full-text

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

Diagnostics ◽

10.3390/diagnostics11060994 ◽

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.

Download Full-text

Scalable, Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

10.1101/2021.03.05.434152 ◽

2021 ◽

Cited By ~ 1

Author(s):

Richard S. Bennett ◽

Elena N. Postnikova ◽

Janie Liang ◽

Robin Gross ◽

Steven Mazur ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Imaging System ◽

Neutralization Assay ◽

Qualitative Assessment ◽

Clinical Samples ◽

Therapeutic Approaches ◽

Serum Samples ◽

Specific Test ◽

Multiple Virus ◽

Infected Cells

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was expanding, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Download Full-text

First Isolation of Cytopathogenic Bovine Torovirus in Cell Culture from a Calf with Diarrhea

Clinical and Vaccine Immunology ◽

10.1128/cvi.00475-06 ◽

2007 ◽

Vol 14 (8) ◽

pp. 998-1004 ◽

Cited By ~ 24

Author(s):

Masaki Kuwabara ◽

Kazumasa Wada ◽

Yukiko Maeda ◽

Ayako Miyazaki ◽

Hiroshi Tsunemitsu

Keyword(s):

Neutralizing Antibodies ◽

Rectal Adenocarcinoma ◽

Rt Pcr ◽

Adenocarcinoma Cell Line ◽

Serum Samples ◽

Reverse Transcription Pcr ◽

Pcr Products ◽

Infected Cells ◽

Pcr Targeting ◽

S Genes

ABSTRACT A cytopathogenic virus (designated the Aichi/2004 strain) was isolated in a human rectal adenocarcinoma cell line (HRT-18) from the ileum contents of a calf with diarrhea. Oval and elongated particles, approximately 100 to 170 nm in diameter, with club-shaped projections were seen in the infected culture supernatant, and torovirus-like (tubular and torus nucleocapsid) structures were seen in the infected cells by electron microscopy. An antiserum against bovine torovirus (BToV) reacted with the infected cells by immunofluorescence and neutralized the isolate. However, antisera against bovine coronavirus (BCV) failed to react with the infected cells by immunofluorescence or did not neutralize the isolate. Further, the isolate was positive for BToV by reverse transcription-PCR (RT-PCR) targeting fragments of the nucleocapsid (N), membrane (M), and spike (S) genes. Comparison of the nucleotide sequences of the PCR products with those of the published N, M, and S genes (476 to 497, 672, and 687 to 690 nucleotides, respectively) of toroviruses showed high sequence identities (up to 99.4%, 98.7%, and 94.9% for the N, M, and S genes, respectively) between the isolate and BToVs. In contrast, the isolate was negative for BCV by RT-PCR. In a serological survey of serum samples from 355 calves at 33 farms, 92% of calves were positive for neutralizing antibodies to the isolate. These results indicate that the isolate in this study was BToV and that BToV infection might be common in cattle in Japan. To our knowledge, this is the first isolation of BToV in tissue culture.

Download Full-text

Evaluation of SARS‐CoV‐2 neutralizing antibodies using a CPE‐based colorimetric live virus micro‐neutralization assay in human serum samples

Journal of Medical Virology ◽

10.1002/jmv.25986 ◽

2020 ◽

Vol 92 (10) ◽

pp. 2096-2104 ◽

Cited By ~ 17

Author(s):

Alessandro Manenti ◽

Marta Maggetti ◽

Elisa Casa ◽

Donata Martinuzzi ◽

Alessandro Torelli ◽

...

Keyword(s):

Human Serum ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Serum Samples ◽

Live Virus ◽

Human Serum Samples

Download Full-text

A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

10.1101/2021.04.08.21255150 ◽

2021 ◽

Author(s):

Craig Fenwick ◽

Priscilla Turelli ◽

Celine Pellaton ◽

Alex Farina ◽

Jeremy Campos ◽

...

Keyword(s):

High Throughput ◽

Neutralizing Antibodies ◽

Large Scale ◽

Competitive Inhibition ◽

Natural Infection ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Antibody Responses ◽

Serum Samples ◽

Live Virus

The detection of SARS-CoV-2-specific antibodies in the serum of an individual indicates prior infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral Spike are far more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, poorly flexible and potentially biohazardous. Here we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 Spike protein binding to the angiotensin converting enzyme 2 (ACE2) viral receptor. This high-throughput method matches the performance of the gold standard live virus infectious assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific IgG titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 Spike variants of concern (VOC), which is otherwise unpredictable even in individuals displaying robust neutralizing antibody responses. Profiling serum samples from 59 hospitalized COVID-19 patients, we found that although most had high activity against the 2019-nCoV Spike and to a lesser extent the B.1.1.7 variant, only 58% could efficiently neutralize a Spike derivative containing mutations present in the B.1.351 variant. In conclusion, we have developed an assay that has proven its clinical relevance in the large-scale evaluation of effective neutralizing antibody responses to VOC after natural infection and that can be applied to the characterization of vaccine-induced antibody responses and of the potency of human monoclonal antibodies.

Download Full-text

Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

Viruses ◽

10.3390/v13050893 ◽

2021 ◽

Vol 13 (5) ◽

pp. 893

Author(s):

Richard S. Bennett ◽

Elena N. Postnikova ◽

Janie Liang ◽

Robin Gross ◽

Steven Mazur ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Imaging System ◽

Neutralization Assay ◽

Clinical Samples ◽

Therapeutic Approaches ◽

Serum Samples ◽

Specific Test ◽

High Content Imaging ◽

Multiple Virus ◽

Infected Cells

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Download Full-text

Infectious Middle East Respiratory Syndrome Coronavirus Excretion and Serotype Variability Based on Live Virus Isolates from Patients in Saudi Arabia

Journal of Clinical Microbiology ◽

10.1128/jcm.01368-15 ◽

2015 ◽

Vol 53 (9) ◽

pp. 2951-2955 ◽

Cited By ~ 36

Author(s):

Doreen Muth ◽

Victor M. Corman ◽

Benjamin Meyer ◽

Abdullah Assiri ◽

Malak Al-Masri ◽

...

Keyword(s):

Middle East ◽

Infectious Virus ◽

Virus Isolation ◽

Vero Cells ◽

Prototype Strain ◽

Middle East Respiratory Syndrome ◽

Serum Samples ◽

Live Virus ◽

Virus Isolates ◽

Rna Concentration

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 1,082 people, including 439 fatalities. So far, no empirical virus isolation study has been done to elucidate infectious virus secretion or serotype variability. Here, we used 51 respiratory samples from 32 patients with confirmed MERS-CoV infection for virus isolation in Vero B4 and Caco-2 cells. We found Caco-2 cells to significantly enhance isolation success over routinely used Vero cells. Isolation success correlated with viral RNA concentration and time after diagnosis as well as with the amount of IgA antibodies secreted in respiratory samples used for isolation. Results from plaque reduction neutralization assays using a representative range of serum samples and virus isolates suggested that all circulating human MERS-CoV strains represent one single serotype. The choice of prototype strain is not likely to influence the success of candidate MERS-CoV vaccines. However, vaccine formulations should be evaluated for their potential to induce IgA.

Download Full-text

Development and utilization of a surrogate SARS-CoV-2 viral neutralization assay to assess mRNA vaccine responses.

10.1101/2021.08.05.21261616 ◽

2021 ◽

Author(s):

Adam V Wisnewski ◽

Jian Liu ◽

Carolina V Lucas ◽

Jon Klein ◽

Akiko Iwasaki ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Assay ◽

Competitive Elisa ◽

Prior History ◽

Viral Neutralization ◽

Neutralizing Activity ◽

Vaccine Responses ◽

Protein Receptor ◽

History Of ◽

High Level

Background: Tests for SARS-CoV-2 immunity are needed to help assess responses to vaccination, which can be heterogeneous and may wane over time. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring serum neutralizing antibodies but requires high level biosafety, live viral cultures and days to complete. We hypothesized that competitive enzyme linked immunoassays (ELISAs) based on SARS-CoV-2 spike protein receptor binding domain (RBD) attachment to its host receptor, the angiotensin converting enzyme 2 receptor (ACE2r), would correlate with PRNT, given the central role of RBD-ACE2r interactions in infection and published studies to date, and enable evaluation of vaccine responses. Methods and Findings: Configuration and development of a competitive ELISA with plate-bound RBD and soluble biotinylated ACE2r was accomplished using pairs of pre/post vaccine serum. When the competitive ELISA was used to evaluate N=32 samples from COVID-19 patients previously tested by PRNT, excellent correlation in IC50 results were observed (rs= .83, p < 0.0001). When the competitive ELISA was used to evaluate N=41 vaccinated individuals and an additional N=14 unvaccinated recovered COVID-19 patients, significant differences in RBD-ACE2r inhibitory activity were associated with prior history of COVID-19 and type of vaccine received. In longitudinal analyses pre and up to 200 days post vaccine, surrogate neutralizing activity increased markedly after primary and booster vaccine doses, but fell substantially, up to <12% maximal levels within 6 months. Conclusions: A competitive ELISA based on inhibition of RBD-ACE2r attachment correlates well with PRNT, quantifies significantly higher activity among vaccine recipients with prior COVID (vs. those without), and highlights marked declines in surrogate neutralizing activity over a 6 month period post vaccination. The findings raise concern about the duration of vaccine responses and potential need for booster shots.

Download Full-text

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants

PLoS Medicine ◽

10.1371/journal.pmed.1003656 ◽

2021 ◽

Vol 18 (7) ◽

pp. e1003656

Author(s):

Fiona Tea ◽

Alberto Ospina Stella ◽

Anupriya Aggarwal ◽

David Ross Darley ◽

Deepti Pilli ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Rt Pcr ◽

Breakthrough Infection ◽

Nucleocapsid Proteins ◽

Viral Neutralization ◽

Polymerase Chain ◽

Serial Samples ◽

High Responders ◽

Neutralization Response ◽

Selection Of

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

Download Full-text