Quantitative detection of neutralizing antibodies against polioviruses and non-polio enteroviruses (NPEV) using an automated microneutralization assay: A seroepidemiologic survey

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.

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

10.21203/rs.3.rs-858163/v1 ◽

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.

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Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus

Journal of Clinical Virology ◽

10.1016/j.jcv.2009.09.020 ◽

2009 ◽

Vol 46 (4) ◽

pp. 314-317 ◽

Cited By ~ 7

Author(s):

Ann R. Falsey ◽

Maria A. Formica ◽

Edward E. Walsh

Keyword(s):

Neutralizing Antibodies ◽

Human Metapneumovirus ◽

Microneutralization Assay

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Immunogenicity and safety of a tetravalent dengue vaccine in dengue-naïve adolescents in Mexico City

Revista Panamericana de Salud Pública ◽

10.26633/rpsp.2021.67 ◽

2021 ◽

Vol 45 ◽

pp. 1

Author(s):

Shibadas Biswal ◽

Jorge Fernando Mendez Galvan ◽

Mercedes Macias Parra ◽

Juan-Francisco Galan-Herrera ◽

Monica Belisa Carrascal Rodriguez ◽

...

Keyword(s):

Adverse Events ◽

Injection Site ◽

Mexico City ◽

Neutralizing Antibodies ◽

Geometric Mean ◽

Dengue Vaccine ◽

Microneutralization Assay ◽

To Receive

Objective. To describe the immunogenicity and safety of a tetravalent dengue vaccine (TAK-003) in healthy adolescents living in Mexico City, an area considered non-endemic for dengue (NCT03341637). Methods. Participants aged 12–17 years were randomized 3:1 to receive two doses (Month 0 and Month 3) of TAK-003 or placebo. Immunogenicity was assessed by microneutralization assay of dengue neutralizing antibodies at baseline, Months 4 and 9. Solicited and unsolicited adverse events (AEs) were recorded after each vaccination. Serious (SAEs) and medically-attended AEs (MAAEs) were recorded throughout the study. Results. 400 adolescents were enrolled, 391 (97.8%) completed the study. Thirty-six (9%) were baseline seropositive to ≥1 serotypes (reciprocal titer ≥10). Geometric mean titers (GMTs) in baseline seronegative TAK-003 recipients were 328, 1743, 120, and 143 at Month 4, and 135, 741, 46, and 38 at Month 9 against DENV-1, -2, -3, and -4, respectively. Placebo GMTs remained <10. Tetravalent seropositivity rates in vaccine recipients were 99.6% and 85.8% at Months 4 and 9, respectively. One MAAE in each group was considered treatment-related (TAK-003: injection-site erythema, and placebo: pharyngitis). Conclusion. TAK-003 was immunogenic against all four serotypes and was well tolerated in dengue-naïve adolescents living in Mexico City.

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Chemiluminescence Immunoassay Based Serological Immunoassays for Detection of SARS-CoV-2 Neutralizing Antibodies in COVID-19 Convalescent Patients and Vaccinated Population

Viruses ◽

10.3390/v13081508 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1508

Author(s):

Qiangling Yin ◽

Yecheng Zhang ◽

Lijun Lian ◽

Yuanyuan Qu ◽

Wei Wu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Detection Methods ◽

Quantitative Detection ◽

Antibody Concentration ◽

Inhibition Assay ◽

Healthy Donors ◽

Kappa Value ◽

Human Sera ◽

Highly Correlated

The development of rapid serological detection methods re urgently needed for determination of neutralizing antibodies in sera. In this study, four rapid methods (ACE2-RBD inhibition assay, S1-IgG detection, RBD-IgG detection, and N-IgG detection) were established and evaluated based on chemiluminescence technology. For the first time, a broadly neutralizing antibody with high affinity was used as a standard for the quantitative detection of SARS-CoV-2 specific neutralizing antibodies in human sera. Sera from COVID-19 convalescent patients (N = 119), vaccinated donors (N = 86), and healthy donors (N = 299) confirmed by microneutralization test (MNT) were used to evaluate the above methods. The result showed that the ACE2-RBD inhibition assay calculated with either ACE2-RBD binding inhibition percentage rate or ACE2-RBD inhibiting antibody concentration were strongly correlated with MNT (r ≥ 0.78, p < 0.0001) and also highly consistent with MNT (Kappa Value ≥ 0.94, p < 0.01). There was also a strong correlation between the two evaluation indices (r ≥ 0.99, p < 0.0001). Meanwhile, S1-IgG and RBD-IgG quantitative detection were also significantly correlated with MNT (r ≥ 0.73, p < 0.0001), and both methods were highly correlated with each other (r ≥ 0.95, p < 0.0001). However, the concentration of N-IgG antibodies showed a lower correlation with the MNT results (r < 0.49, p < 0.0001). The diagnostic assays presented here could be used for the evaluation of SARS-CoV-2 vaccine immunization effect and serological diagnosis of COVID-19 patients, and could also have guiding significance for establishing other rapid serological methods to surrogate neutralization tests for SARS-CoV-2.

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Impact of the Second Epidemic Wave of SARS-CoV-2: Increased Exposure of Young People

Frontiers in Public Health ◽

10.3389/fpubh.2021.715192 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lisandru Capai ◽

Shirley Masse ◽

Toscane Fourié ◽

Dorine Decarreaux ◽

Jean Canarelli ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Herd Immunity ◽

Wave Period ◽

Quantitative Detection ◽

Igg Antibodies ◽

Cross Sectional ◽

Clinical Laboratories ◽

Serological Surveillance ◽

Second Wave ◽

Neutralization Antibody Titer

We aimed to use serological surveillance based on serial cross-sectional sampling of residual sera obtained from clinical laboratories to compare the differences in age and sex profiles of infected persons in the first and second waves of SARS-CoV-2 in Corsica, France. Residual sera were obtained, including samples from individuals of all ages collected for routine screening or clinical management by clinical laboratories. All the sera collected were tested for the presence of anti-SARS-CoV-2 IgG using a kit for semi-quantitative detection of IgG antibodies against the S1 domain of the viral spike protein (ELISA-S). Samples that were borderline and positive in ELISA-S were tested with an in-house virus neutralization test. During the second-wave period, we collected between 6 November, 2020 and 12 February, 2021, 4,505 sera from patients aged 0–101 years (60.4% women). The overall weighted seroprevalence of residual sera collected during the second-wave period [8.04% (7.87–9.61)] was significantly higher than the overall weighted seroprevalence estimated at the end of the first wave between 16 April and 15 June, 2020 [5.46% (4.37–7.00)] (p-value = 0.00025). Ninety-eight (30.1%) of the 326 samples tested in the VNT assay had a positive neutralization antibody titer. Estimated seroprevalence increased significantly for men [odds ratio (OR) OR = 1.80 (1.30–2.54); p-value = 0.00026] and for people under 30 years of age [OR = 2.17 (1.46–3.28); p-value = 0.000032]. This increase was observed in young adults aged 20–29 years among whom antibody frequencies were around four-fold higher than those observed at the end of the first wave. In conclusion, our seroprevalence estimates, including the proportion of the participants who had produced neutralizing antibodies, indicate that in February, 2021 the population of Corsica was still far from being protected against SARS-Cov-2 by “herd immunity.”

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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.

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