scholarly journals Multiplex PCR-Based Neutralization (MPBN) Assay for Titers Determination of the Three Types of Anti-Poliovirus Neutralizing-Antibodies

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 120
Author(s):  
Hasmik Manukyan ◽  
Svetlana Petrovskaya ◽  
Konstantin Chumakov ◽  
Majid Laassri
Keyword(s):  
Multiplex Pcr ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Neutralizing Antibody ◽  
Polio Eradication ◽  
Neutralization Assay ◽  
Epidemiological Surveillance ◽  
Large Scale Analysis ◽  
Antibody Titers

Determination of poliovirus-neutralizing antibodies is an important part of clinical studies of poliovirus vaccines, epidemiological surveillance and seroprevalence studies that are crucial for global polio eradication campaigns. The conventional neutralization test is based on inhibition of cytopathic effect caused by poliovirus by serial dilutions of test serum. It is laborious, time-consuming and not suitable for large scale analysis. To overcome these limitations, a multiplex PCR-based neutralization (MPBN) assay was developed to measure the neutralizing antibody titers of anti-poliovirus sera against three serotypes of the virus in the same reaction and in shorter time. All three anti-poliovirus sera types were analyzed in a single assay. The MPBN assay was reproducible, robust and sensitive. Its lower limits of titration for the three anti-poliovirus sera types were within range of 0.76–1.64 per mL. Different anti-poliovirus sera were tested with conventional and MPBN assays; the results obtained by both methods correlated well and generated similar results. The MPBN is the first neutralization assay that specifically titrates anti-poliovirus antibodies against the three serotypes of the virus in the same reaction; it can be completed in two to three days instead of ten days for the conventional assay and can be automated for high-throughput implementation.

scholarly journals Titers of Neutralizing Antibodies against SARS-CoV-2 Are Independent of Symptoms of Non-Severe COVID-19 in Young Adults

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 284
Author(s):  
Hulda R. Jonsdottir ◽  
Michel Bielecki ◽  
Denise Siegrist ◽  
Thomas W. Buehrer ◽  
Roland Züst ◽  
...  
Keyword(s):  
Young Adults ◽  
Neutralizing Antibodies ◽  
Severe Disease ◽  
Humoral Response ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Asymptomatic Infection ◽  
Homogeneous Population ◽  
Humoral Immune ◽  
Antibody Titers

Neutralizing antibodies are an important part of the humoral immune response to SARS-CoV-2. It is currently unclear to what extent such antibodies are produced after non-severe disease or asymptomatic infection. We studied a cluster of SARS-CoV-2 infections among a homogeneous population of 332 predominantly male Swiss soldiers and determined the neutralizing antibody response with a serum neutralization assay using a recombinant SARS-CoV-2-GFP. All patients with non-severe COVID-19 showed a swift humoral response within two weeks after the onset of symptoms, which remained stable for the duration of the study. One month after the outbreak, titers in COVID-19 convalescents did not differ from the titers of asymptomatically infected individuals. Furthermore, symptoms of COVID-19 did not correlate with neutralizing antibody titers. Therefore, we conclude that asymptomatic infection can induce the same humoral immunity as non-severe COVID-19 in young adults.

scholarly journals Statistical Approach To Estimate Vaccinia-Specific Neutralizing Antibody Titers Using a High-Throughput Assay

2009 ◽  
Vol 16 (8) ◽  
pp. 1105-1112 ◽  
Author(s):  
Richard Kennedy ◽  
V. Shane Pankratz ◽  
Eric Swanson ◽  
David Watson ◽  
Hana Golding ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Large Scale ◽  
Statistical Approach ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization Assay ◽  
Antibody Titers ◽  
High Throughput Assay

ABSTRACT Because of the bioterrorism threat posed by agents such as variola virus, considerable time, resources, and effort have been devoted to biodefense preparation. One avenue of this research has been the development of rapid, sensitive, high-throughput assays to validate immune responses to poxviruses. Here we describe the adaptation of a β-galactosidase reporter-based vaccinia virus neutralization assay to large-scale use in a study that included over 1,000 subjects. We also describe the statistical methods involved in analyzing the large quantity of data generated. The assay and its associated methods should prove useful tools in monitoring immune responses to next-generation smallpox vaccines, studying poxvirus immunity, and evaluating therapeutic agents such as vaccinia virus immune globulin.

scholarly journals Commercial Serology Assays Predict Neutralization Activity Against SARS-CoV-2

2020 ◽  
Author(s):  
Raymond T Suhandynata ◽  
Melissa A Hoffman ◽  
Deli Huang ◽  
Jenny T Tran ◽  
Michael J Kelner ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Positive Serology ◽  
Neutralization Activity ◽  
Antibody Titers ◽  
Negative Controls ◽  
First Time ◽  
The Relationship ◽  
Positive Results

Background. Currently it is unknown whether a positive serology results correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods. A neutralization assay was validated in a set of PCR confirmed positive specimens and in a negative cohort. 9,530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N=164) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and the levels of neutralizing antibodies detected was correlated. Neutralizing antibody titers (ID50) were also longitudinally monitored in SARS-CoV-2 PCR confirmed patients. Results. The SARS-CoV-2 neutralization assay had a PPA of 96.6% with a SARS-CoV-2 PCR test and a NPA of 98.0% across 100 negative controls. ID50 neutralization titers positively correlated with all three clinical serology platforms. Longitudinal monitoring of hospitalized PCR confirmed COVID-19 patients demonstrates they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2% and 78.4%, respectively. Conclusions. For the first time, we demonstrate that three widely available clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in COVID-19 patients. When a two-platform screen and confirm approach was used for SARS-CoV-2 serology, nearly 80% of two-platform positive specimens had neutralization titers (ID50 >50).

scholarly journals Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Carla Frances-Gomez ◽  
Eliseo Albert ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Pseudotyped Virus ◽  
S Protein ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay ◽  
Degree Correlation ◽  
Antibody Titers

Background: Whether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. Objectives: To evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and Methods: Ninety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. Results: Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho=0.73) and moderate for the remaining assays (Rho=0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. Conclusions: the suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

scholarly journals Commercial Serology Assays Predict Neutralization Activity against SARS-CoV-2

2020 ◽  
Author(s):  
Raymond T Suhandynata ◽  
Melissa A Hoffman ◽  
Deli Huang ◽  
Jenny T Tran ◽  
Michael J Kelner ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Negative Control ◽  
Percentage Agreement ◽  
Positive Serology ◽  
Neutralization Activity ◽  
Antibody Titers ◽  
The Relationship ◽  
Positive Results

Abstract Background It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods A neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR. Results The SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively. Conclusions These 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

scholarly journals Comparative quantitative analysis of SARS-CoV-2 Spike neutralizing antibody titers following two anti COVID-19 vaccines in India

2021 ◽  
Author(s):  
Chanukya GV ◽  
Aparna Srikantam
Keyword(s):  
Quantitative Analysis ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Neutralizing Antibody ◽  
Long Term Follow Up ◽  
Antibody Titers ◽  
Age Studies ◽  
Immunogenic Response

In COVID 19 pandemic, first line of defense is effective vaccination program. Because of multiple platforms available for vaccine production we tested relative immunogenicity of two vaccines available in India, Covaxin and Covishield We performed quantitative analysis of neutralizing antibodies to SARS Cov2 spike (receptor binding domain ) protein, from sera of 53 subjects who completed vaccines schedules. There was significantly higher immunogenic response with Covishield as compared to Covaxin and are independent of age. Studies on a large scale with long term follow up are needed to further advance the knowledge in this domain.

scholarly journals A combined strategy to detect plasma samples reliably with high anti-SARS-CoV-2 neutralizing antibody titers in routine laboratories

2021 ◽  
Author(s):  
Bastian Fischer ◽  
Christoph Lichtenberg ◽  
Lisa Mueller ◽  
Joerg Timm ◽  
Johannes Fischer ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Neutralizing Antibody ◽  
Plasma Samples ◽  
Plasma Therapy ◽  
Antibody Titers ◽  
Combined Strategy ◽  
Common Cell

The determination of anti-SARS-CoV-2 neutralizing antibodies (NAbs) is of interest in many respects. High NAb titers, for example, are the most important criterion regarding the effectiveness of convalescent plasma therapy. However, common cell culture-based NAb assays are time-consuming and feasible only in special laboratories. Our data reveal the suitability of a novel ELISA-based surrogate virus neutralization test (sVNT) to easily measure the inhibition-capability of NAbs in the plasma of COVID-19 convalescents. We propose a combined strategy to detect plasma samples with high NAb titers (≥ 1:160) reliably and to, simultaneously, reduce the risk of erroneously identifying low-titer specimens. For this approach, results of the sVNT assay are compared to and combined with those acquired from the Euroimmun anti-SARS-CoV-2 IgG assay. Both assays are appropriate for high-throughput screening in standard BSL-2 laboratories. Our measurements further show a long-lasting humoral immunity of at least 11 months after symptom onset.

scholarly journals Hyperimmunized Chickens Produce Neutralizing Antibodies Against SARS-CoV-2

2021 ◽  
Author(s):  
Emily J. Aston ◽  
Daria Mochly-Rosen ◽  
Aarthi Narayanan ◽  
Sofia Egaña-Labrin ◽  
Michael G. Wallach ◽  
...  
Keyword(s):  
At Risk ◽  
Neutralizing Antibodies ◽  
Low Cost ◽  
Neutralizing Antibody ◽  
Egg Yolk ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Multiple Strategies ◽  
Effective Interventions ◽  
Antibody Titers

Abstract The novel severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV-2, is responsible for the global COVID-19 pandemic. Effective interventions are urgently needed to mitigate the effects of COVID-19 and likely require multiple strategies. Egg-extracted antibody therapies are a low-cost and scalable strategy to protect at-risk individuals from SARS-CoV-2 infection. Commercial laying hens were hyperimmunized against the SARS-CoV-2 S1 protein using three different S1 recombinant proteins and three different doses. Sera and egg yolk were collected at three and six weeks after the second immunization for enzyme-linked immunosorbent assay and plaque reduction neutralization assay to determine antigen-specific antibody titers and neutralizing antibody titers, respectively. In this study we demonstrate that hens hyperimmunized against the SARS-CoV-2 recombinant S1 and receptor binding domain (RBD) proteins produced neutralizing antibodies against SARS-CoV-2. We further demonstrate that antibody production was dependent on the dose and type of antigen administered. Our data suggest that antibodies purified from the egg yolk of hyperimmunized hens can be used as immunoprophylaxis in humans at risk of exposure to SARS-CoV-2.

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

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.

scholarly journals Durability of Response to a Third BNT162b2 Vaccine in Adults ≥60 Years

2021 ◽  
Author(s):  
Noa Eliakim Raz ◽  
Amos Stemmer ◽  
Yaara Leibovici-Weissman ◽  
Asaf Ness ◽  
Muhammad Awwad ◽  
...  
Keyword(s):  
Adverse Events ◽  
Neutralizing Antibodies ◽  
Multivariable Analysis ◽  
Vaccine Dose ◽  
Neutralizing Antibody ◽  
Clinical Frailty Scale ◽  
Younger Adults ◽  
The Third ◽  
Antibody Titers ◽  
Level 2

BACKGROUND Age and frailty are strong predictors of COVID-19 mortality. After the second BNT162b2 dose, immunity wanes faster in older (≥65 years) versus younger adults. The durability of response after the third vaccine is unclear. METHODS This prospective cohort study included healthcare workers/family members ≥60 years who received a third BNT162b2 dose. Blood samples were drawn immediately before (T0), 10-19 (T1), and 74-103 (T2) days after the third dose. Antispike IgG titers were determined using a commercial assay, seropositivity was defined as ≥50 AU/mL. Neutralizing antibody titers were determined at T2. Adverse events, COVID-19 infections, and clinical frailty scale (CFS) levels were documented. RESULTS The analysis included 97 participants (median age, 70 years [IQR, 66-74], 61% women, 58% CFS level 2). IgG titers, which increased significantly from T0 to T1 (medians, 440 AU/mL [IQR, 294-923] and 25,429 [14,203-36,114] AU/mL, respectively; P<0.001), decreased significantly by T2, but all remained seropositive (median, 8,306 AU/mL [IQR, 4595-14,701], P<0.001 vs T1). In a multivariable analysis, only time from the first vaccine was significantly associated with lower IgG levels at T2 (P=0.004). At T2, 60 patients were evaluated for neutralizing antibodies; all were seropositive (median, 1,294 antibody titer [IQR, 848-2,072]). Neutralizing antibody and antispike IgG levels were correlated (R=0.6, P<0.001). No major adverse events or COVID-19 infections were reported. CONCLUSIONS Antispike IgG and neutralizing antibodies levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults ≥60 years, although the decline in IgG is concerning. A third vaccine dose in this population should be top priority.

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