scholarly journals A Simple and High-Throughput ELISA-Based Neutralization Assay for the Determination of Anti-Flavivirus Neutralizing Antibodies

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 297
Author(s):  
Jean Claude Balingit ◽  
Minh Huong Phu Ly ◽  
Mami Matsuda ◽  
Ryosuke Suzuki ◽  
Futoshi Hasebe ◽  
...  
Keyword(s):  
High Throughput ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Vaccine Trials ◽  
Antibody Levels ◽  
Flavivirus Infection

Mosquito-borne flavivirus infections, including dengue virus and Zika virus, are major public health threats globally. While the plaque reduction neutralization test (PRNT) is considered the gold standard for determining neutralizing antibody levels to flaviviruses, the assay is time-consuming and laborious. This study, therefore, aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test (EMNT) for the detection of neutralizing antibodies to mosquito-borne flaviviruses. The inhibition of viral growth due to neutralizing antibodies was determined colorimetrically by using EMNT. Given the significance of Fcγ-receptors (FcγR) in antibody-mediated neutralization and antibody-dependent enhancement (ADE) of flavivirus infection, non-FcγR and FcγR-expressing cell lines were used in the EMNT to allow the detection of the sum of neutralizing and immune-enhancing antibody activity as the neutralizing titer. Using anti-flavivirus monoclonal antibodies and clinical samples, the utility of EMNT was evaluated by comparing the end-point titers of the EMNT and the PRNT. The correlation between EMNT and PRNT titers was strong, indicating that EMNT was robust and reproducible. The new EMNT assay combines the biological functional assessment of virus neutralization activity and the technical advantages of ELISA and, is simple, reliable, practical, and could be automated for high-throughput implementation in flavivirus surveillance studies and vaccine trials.

scholarly journals Neutralizing Antibody Responses in COVID-19 Convalescent Sera

2020 ◽  
Vol 223 (1) ◽  
pp. 47-55 ◽  
Author(s):  
William T Lee ◽  
Roxanne C Girardin ◽  
Alan P Dupuis ◽  
Karen E Kulas ◽  
Anne F Payne ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
High Titer ◽  
Experimental Treatment ◽  
Neutralizing Antibody ◽  
Maximal Activity ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
United States Food ◽  
Antibody Titers ◽  
Antibody Levels

Abstract Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration’s (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31–35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.

scholarly journals Can individuals with low antibody responses to vaccines against other viruses acquire adequate SARS-CoV-2 antibody after vaccination with the BNT162b2 mRNA vaccine?

2021 ◽  
Author(s):  
Wataru Ogura ◽  
Kouki Ohtsuka ◽  
Sachiko Matsuura ◽  
Takahiro Okuyama ◽  
Satsuki Matsushima ◽  
...  
Keyword(s):  
Cohort Study ◽  
Neutralizing Antibodies ◽  
Healthcare Workers ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Antibody Activity ◽  
Low Responders ◽  
Before And After ◽  
Positive Threshold ◽  
Antibody Levels

Objective In Japan, healthcare workers (HCWs) are vaccinated against coronavirus disease (COVID-19) and other contagious viruses (measles, rubella, chickenpox, mumps, and hepatitis B) to prevent nosocomial infection. However, some do not produce sufficient antibodies after vaccination (low responders). This study investigated changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody levels among HCWs after SARS-CoV-2 vaccination and assessed whether low responders produced adequate SARS-CoV-2 anti-spike and neutralizing antibodies. Methods We conducted a prospective cohort study of HCWs before and after vaccination with the BNT162b2 mRNA vaccine in a hospital in Tokyo, Japan. The HCWs received two doses of BNT162b2 vaccine, 3 weeks apart. Those whose antibody levels against previous antiviral vaccines did not reach protective antibody levels after receiving two doses were defined as low responders, whereas those who produced adequate antibodies were defined as normal responders. SARS-CoV-2 anti-spike antibodies were measured 11 times from before the first BNT162b2 vaccination to 5 months after the second vaccination. SARS-CoV-2 neutralizing antibody activity was measured twice in low responders, 1 week to 1 month and 5 months after the second vaccination. Results Fifty HCWs were included in the analytic cohort. After vaccination, SARS-CoV-2 anti-spike antibody was detectable in the samples from both responders at each timepoint, but the level was lower at 5 months than at 1 week after the second vaccination. Low responders had SARS-CoV-2 neutralizing antibody activity 1 week to 1 month after the second vaccination, which exceeded the positive threshold after 5 months. Conclusion After BNT162b2 vaccination, low responders acquired adequate SARS-CoV-2 anti-spike and SARS-CoV-2 neutralizing antibodies to prevent SARS-CoV-2. However, SARS-CoV-2 anti-spike antibody levels were lower at 5 months than at 1 week after the second dose of BNT162b2 vaccine in low and normal responders. Therefore, low responders should also receive a third dose of BNT162b2 vaccine.

scholarly journals A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

2020 ◽  
Author(s):  
Antonio E. Muruato ◽  
Camila R. Fontes-Garfias ◽  
Ping Ren ◽  
Mariano A. Garcia-Blanco ◽  
Vineet D. Menachery ◽  
...  
Keyword(s):  
High Throughput ◽  
Gold Standard ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Antibody Assay ◽  
Plasma Therapy ◽  
High Throughput Assay ◽  
Key Parameter

AbstractVirus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. Our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.

scholarly journals Predicting COVID-19 Vaccine Efficacy from Neutralizing Antibody Levels

2021 ◽  
Author(s):  
Majid R. Abedi ◽  
Samuel Dixon ◽  
Timothy Guyon ◽  
Serene Hsu ◽  
Aviva R. Jacobs ◽  
...  
Keyword(s):  
High Throughput ◽  
Vaccine Efficacy ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Neutralizing Antibody ◽  
Published Data ◽  
Real World Data ◽  
Symptomatic Infection ◽  
Viral Neutralization ◽  
Antibody Levels

Recent studies using data accrued from global SARS-CoV-2 vaccination efforts have demonstrated that breakthrough infections are correlated with levels of neutralizing antibodies. The decrease in neutralizing antibody titers of vaccinated individuals over time, combined with the emergence of more infectious variants of concern has resulted in waning vaccine efficacy against infection and a rise in breakthrough infections. Here we use a combination of neutralizing antibody measurements determined by a high throughput surrogate viral neutralization test (sVNT) together with published data from vaccine clinical trials and comparative plaque reduction neutralization test (PRNT) between SARS-CoV-2 variants to develop a model for vaccine efficacy (VE) against symptomatic infection. Vaccine efficacy estimates using this model show good concordance with real world data from the US and Israel. Our work demonstrates that appropriately calibrated neutralizing antibody measurements determined by high throughput sVNT can be used to provide a semi-quantitative estimate of protection against infection. Given the highly variable antibody levels among the vaccinated population, this model may be of use in identification of individuals with an elevated risk of breakthrough infections.

scholarly journals Neutralizing Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 in Coronavirus Disease 2019 Inpatients and Convalescent Patients

2020 ◽  
Vol 71 (10) ◽  
pp. 2688-2694 ◽  
Author(s):  
Xiaoli Wang ◽  
Xianghua Guo ◽  
Qianqian Xin ◽  
Yang Pan ◽  
Yaling Hu ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Antibody Level ◽  
Neutralizing Antibodies ◽  
Clinical Characteristics ◽  
Early Stage ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Estimating Equation ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay

Abstract Background Coronavirus disease 2019 (COVID-19) is a pandemic with no specific antiviral treatments or vaccines. There is an urgent need for exploring the neutralizing antibodies from patients with different clinical characteristics. Methods A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. Antibodies were determined with a modified cytopathogenic neutralization assay (NA) based on live severe acute respiratory syndrome coronavirus 2 and enzyme-linked immunosorbent assay (ELISA). The dynamics of neutralizing antibody levels at different time points with different clinical characteristics were analyzed. Results The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till days 41–53. The total geometric mean titer was 1:163.7 (95% confidence interval [CI], 128.5–208.6) by NA and 1:12 441.7 (95% CI, 9754.5–15 869.2) by ELISA. The antibody level by NA and ELISA peaked on days 31–40 since onset, and then decreased slightly. In multivariate generalized estimating equation analysis, patients aged 31–45, 46–60, and 61–84 years had a higher neutralizing antibody level than those aged 16–30 years (β = 1.0470, P = .0125; β = 1.0613, P = .0307; β = 1.3713, P = .0020). Patients with a worse clinical classification had a higher neutralizing antibody titer (β = 0.4639, P = .0227). Conclusions The neutralizing antibodies were detected even at the early stage of disease, and a significant response was shown in convalescent patients.

scholarly journals Robust Neutralizing Antibody Levels Detected after Either SARS-CoV-2 Vaccination or One Year after Infection

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2003
Author(s):  
Stefan Glöckner ◽  
Franziska Hornung ◽  
Michael Baier ◽  
Sebastian Weis ◽  
Mathias W. Pletz ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Booster Vaccination ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Neutralization Titer ◽  
Neutralization Capacity ◽  
A Cell ◽  
One Year ◽  
Study Participants ◽  
Antibody Levels

Humoral immunity after infection or after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been attributed a key part in mitigating the further transmission of the virus. In this study, we used a commercial anti-Spike immunoglobulin G (S-IgG) assay and developed a cell culture-based neutralization assay to understand the longitudinal course of neutralizing antibodies in both SARS-CoV2 infected or vaccinated individuals. We show that even more than one year after infection, about 78% of observed study participants remained seropositive concerning S-IgG antibodies. In addition, the serum of the individuals had stable neutralization capacity in a neutralization assay against a SARS-CoV-2 patient isolate from March 2020. We also examined volunteers after either homologous BNT162b2 prime-boost vaccination or heterologous AZD1222 prime/mRNA-based booster vaccination. Both the heterologous and the homologous vaccination regimens induced higher levels of neutralizing antibodies in healthy subjects when compared to subjects after a mild infection, showing the high effectiveness of available vaccines. In addition, we could demonstrate the reliability of S-IgG levels in predicting neutralization capacity, with 94.8% of seropositive samples showing a neutralization titer of ≥10, making it a viable yet cheap and easy-to-determine surrogate parameter for neutralization capacity.

scholarly journals Characterization of Alpha-ToxinhlaGene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

2014 ◽  
Vol 53 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Batu K. Sharma-Kuinkel ◽  
Yuling Wu ◽  
David E. Tabor ◽  
Hoyin Mok ◽  
Bret R. Sellman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Clinical Outcome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alpha Toxin ◽  
Content Type ◽  
Antibody Levels

Alpha-toxin is a majorStaphylococcus aureusvirulence factor. This study evaluated potential relationships betweenin vitroalpha-toxin expression ofS. aureusbloodstream isolates, anti-alpha-toxin antibody in serum of patients withS. aureusbacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwentspatyping andhlasequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encodedhla(197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%).In vitroalpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml;P< 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95;P< 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27;P< 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressingS. aureusisolates (P< 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis ofhlarevealed 12 distincthlagenotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinicalS. aureusisolates. Higherin vitroalpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producingS. aureusexhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

scholarly journals Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients

2020 ◽  
Vol 58 (12) ◽  
Author(s):  
Larry L. Luchsinger ◽  
Brett P. Ransegnola ◽  
Daniel K. Jin ◽  
Frauke Muecksch ◽  
Yiska Weisblum ◽  
...  
Keyword(s):  
New York ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Pseudotyped Virus ◽  
Antibody Activity ◽  
Plasma Samples ◽  
Serological Tests ◽  
Population Immunity ◽  
Therapeutic Trials ◽  
Antibody Levels

ABSTRACT The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

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 Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype B

2010 ◽  
Vol 17 (6) ◽  
pp. 930-936 ◽  
Author(s):  
Daniel M. Held ◽  
Amy C. Shurtleff ◽  
Scott Fields ◽  
Christopher Green ◽  
Julie Fong ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Neutralizing Antibodies ◽  
Botulinum Neurotoxin ◽  
Specific Binding ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Titer ◽  
Serotype B

ABSTRACT New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 107 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.

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