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

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.

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

Journal of Clinical Virology ◽

10.1016/j.jcv.2021.104984 ◽

2021 ◽

pp. 104984

Author(s):

Bastian Fischer ◽

Christoph Lichtenberg ◽

Lisa Müller ◽

Jörg Timm ◽

Johannes Fischer ◽

...

Keyword(s):

Neutralizing Antibody ◽

Plasma Samples ◽

Antibody Titers ◽

Combined Strategy

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Effect of a booster-dose of rabies vaccine on the duration of virus neutralizing antibody titers in bovines

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86821998000400006 ◽

1998 ◽

Vol 31 (4) ◽

pp. 367-371 ◽

Cited By ~ 5

Author(s):

Avelino Albas ◽

Paulo Eduardo Pardo ◽

Albério Antonio Barros Gomes ◽

Fernanda Bernardi ◽

Fumio Honma Ito

Keyword(s):

Immune Response ◽

Neutralizing Antibodies ◽

Booster Dose ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Rabies Vaccine ◽

Western Region ◽

Humoral Immune ◽

Paulo State ◽

Antibody Titers

Humoral immune response using inactivated rabies vaccine was studied in 35 nelore cross-bred bovines of western region of São Paulo state. Ninety days after vaccination, 13 (92.8%) animals presented titers 30.5IU/ml, through mouse neutralization test. After 180 days, 9 (64.3%) sera showed titers 30.5IU/ml, after 270 days, only one (7.1%) showed a titer of 0.51IU/ml, and after 360 days, all animals showed titers < 0.5IU/ml. Group of animals receiving booster dose 30 days after vaccination presented, two months after, all with titers > 0.5IU/ml. At 180 days, 17 (80.9%) sera presented titers > 0.5IU/ml; at 270 days, 15 (71.4%), with titers 30.5IU/ml and at 360 days, 4 (19.0%), with titers 30.5IU/ml. Booster-dose ensured high levels of neutralizing antibodies for at least three months, and 240 days after revaccination, 71.4% of animals were found with titers 30.5IU/ml.

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Evaluation of high-throughput SARS-CoV-2 serological assays in a longitudinal cohort of mild COVID-19 patients: sensitivity, specificity and association with virus neutralization test

10.1101/2020.09.30.20194290 ◽

2020 ◽

Author(s):

Antonin Bal ◽

Bruno Pozzetto ◽

Mary-Anne Trabaud ◽

Vanessa Escuret ◽

Muriel Rabilloud ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Healthcare Workers ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Serological Tests ◽

Course Of Disease ◽

Antibody Titers ◽

Sensitivity Specificity

BackgroundThe association between SARS-CoV-2 commercial serological assays and virus neutralization test (VNT) has been poorly explored in mild COVID-19 patients.MethodsA total of 439 serum specimens were longitudinally collected from 76 healthcare workers with RT-PCR-confirmed COVID-19. The sensitivity (determined weekly) of nine commercial serological assays were evaluated. Specificity was assessed using 69 pre-pandemic sera. Correlation, agreement and concordance with the VNT were also assessed on a subset of 170 samples. Area under the ROC curve (AUC) was estimated at several neutralizing antibody titers.ResultsThe Wantai Total Ab assay targeting the receptor binding domain (RBD) within the S protein presented the best sensitivity at different times during the course of disease. The specificity was greater than 95% for all tests except for the Euroimmun IgA assay. The overall agreement with the presence of neutralizing antibodies ranged from 62.2% (95%CI; 56.0-68.1) for bioMérieux IgM to 91.2% (87.0-94.2) for Siemens. The lowest negative percent agreement (NPA) was found with the Wantai Total Ab assay (NPA 33% (21.1-48.3)). The NPA for other total Ab or IgG assays targeting the S or the RBD was 80.7% (66.7-89.7), 90.3 (78.1-96.1) and 96.8% (86.8-99.3) for Siemens, bioMérieux IgG and DiaSorin, respectively. None of commercial assays have sufficient performance to detect a neutralizing titer of 80 (AUC<0.76).ConclusionsAlthough some assays presented a better agreement with VNT than others, the present findings emphasize that commercialized serological tests including those targeting the RBD cannot substitute a VNT for the assessment of functional antibody response.

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The feasibility of convalescent plasma therapy in severe COVID- 19 patients: a pilot study

10.1101/2020.03.16.20036145 ◽

2020 ◽

Cited By ~ 18

Author(s):

Kai Duan ◽

Bende Liu ◽

Cesheng Li ◽

Huajun Zhang ◽

Ting Yu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Clinical Symptoms ◽

Antiviral Agents ◽

Neutralizing Antibody ◽

Optimal Dose ◽

Plasma Therapy ◽

Reactive Protein ◽

Antibody Titers ◽

Novel Coronavirus ◽

High Level

AbstractCurrently, there are no approved specific antiviral agents for 2019 novel coronavirus disease (COVID-19). In this study, ten severe patients confirmed by real-time viral RNA test were enrolled prospectively. One dose of 200 mL convalescent plasma (CP) derived from recently recovered donors with the neutralizing antibody titers above 1:640 was transfused to the patients as an addition to maximal supportive care and antiviral agents. The primary endpoint was the safety of CP transfusion. The second endpoints were the improvement of clinical symptoms and laboratory parameters within 3 days after CP transfusion. The median time from onset of illness to CP transfusion was 16.5 days. After CP transfusion, the level of neutralizing antibody increased rapidly up to 1:640 in five cases, while that of the other four cases maintained at a high level (1:640). The clinical symptoms were significantly improved along with increase of oxyhemoglobin saturation within 3 days. Several parameters tended to improve as compared to pre-transfusion, including increased lymphocyte counts (0.65×109/L vs. 0.76×109/L) and decreased C-reactive protein (55.98 mg/L vs. 18.13 mg/L). Radiological examinations showed varying degrees of absorption of lung lesionswithin 7 days. The viral load was undetectable after transfusion in seven patients who had previous viremia. No severe adverse effects were observed. This study showed CP therapy was welltolerated and could potentially improve the clinical outcomes through neutralizing viremia in severe COVID-19 cases. The optimal dose and time point, as well as the clinical benefit of CP therapy, needs further investigation in larger well-controlled trials.Significance StatementCOVID-19 is currently a big threat to global health. However, no specific antiviral agents are available for its treatment. In this work, we explored the feasibility of convalescent plasma (CP) transfusion to rescue severe patients. The results from 10 severe adult cases showed that one dose (200 mL) of CP was welltolerated and could significantly increase or maintain the neutralizing antibodies at a high level, leading to disappearance of viremia in 7 days. Meanwhile, clinical symptoms and paraclinical criteria rapidly improved within 3 days. Radiological examination showed varying degrees of absorption of lung lesions within 7 days. These results indicate that CP can serve as a promising rescue option for severe COVID-19 while the randomized trial is warranted.

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Evaluation of high-throughput SARS-CoV-2 serological assays in a longitudinal cohort of patients with mild COVID-19: clinical sensitivity, specificity and association with virus neutralization test

Clinical Chemistry ◽

10.1093/clinchem/hvaa336 ◽

2021 ◽

Author(s):

Antonin Bal ◽

Bruno Pozzetto ◽

Mary-Anne Trabaud ◽

Vanessa Escuret ◽

Muriel Rabilloud ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Serological Tests ◽

Course Of Disease ◽

Clinical Sensitivity ◽

Antibody Titers ◽

Sensitivity Specificity

Abstract Background The association between SARS-CoV-2 commercial serological assays and virus neutralization test (VNT) has been poorly explored in mild patients with COVID-19. Methods 439 serum specimens were longitudinally collected from 76 healthcare workers with RT-PCR-confirmed COVID-19. The clinical sensitivity (determined weekly) of nine commercial serological assays were evaluated. Clinical specificity was assessed using 69 pre-pandemic sera. Correlation, agreement and concordance with the VNT were also assessed on a subset of 170 samples. Area under the ROC curve (AUC) was estimated at 2 neutralizing antibody titers. Results The Wantai Total Ab assay targeting the receptor binding domain (RBD) within the S protein presented the best sensitivity at different times during the course of disease. The clinical specificity was greater than 95% for all tests except for the Euroimmun IgA assay. The overall agreement with the presence of neutralizing antibodies ranged from 62.2% (95%CI; 56.0-68.1) for bioMérieux IgM to 91.2% (87.0-94.2) for Siemens. The lowest negative percent agreement (NPA) was found with the Wantai Total Ab assay (NPA 33% (21.1-48.3)). The NPA for other total Ab or IgG assays targeting the S or the RBD was 80.7% (66.7-89.7), 90.3 (78.1-96.1) and 96.8% (86.8-99.3) for Siemens, bioMérieux IgG and DiaSorin, respectively. None of commercial assays have sufficient performance to detect a neutralizing titer of 80 (AUC<0.76). Conclusions Although some assays show a better agreement with VNT than others, the present findings emphasize that commercialized serological tests including those targeting the RBD cannot substitute a VNT for the assessment of functional antibody response.

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Presence and short-term persistence of SARS-CoV-2 neutralizing antibodies in COVID-19 convalescent plasma donors.

10.1101/2020.09.01.20185942 ◽

2020 ◽

Author(s):

Kyle Annen ◽

Thomas E Morrison ◽

Melkon G DomBourian ◽

Mary K McCarthy ◽

Leah Huey ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Considerable Variability ◽

Binding Assays ◽

Short Term ◽

Live Virus ◽

Donor Population ◽

Antibody Titers ◽

Focus Reduction

In March 2020, the FDA approved the use of COVID-19 convalescent plasma (CCP) as an investigational new drug for treatment of COVID-19. Since then, collection of CCP from COVID-19 recovered patients has been implemented in several donor centers across the country. Childrens Hospital Colorado rapidly put into practice a CCP collection protocol, necessitating the development and implementation of assays to evaluate SARS-CoV-2 antibodies in CCP units. We evaluated 87 separate units of CCP collected from 36 donors over two to four sequential donations using both antigen-binding assays for SARS-CoV-2 nucleoprotein and spike antigens, and a live virus focus reduction neutralization test (FRNT50). Our data shows that the majority of donors (83 percent) had a FRNT50 titer of 1/80 or greater, and 61 percent had a titer greater than or equal to 1/160, which meet the FDA criteria for acceptable CCP units. Additionally, our data indicates that analysis of antibodies to a single SARS-CoV-2 antigen is likely to miss a percentage of seroconverters. These individuals, however, tend to have neutralizing antibody titers of less than 1/80. Of note, there was considerable variability in the short term, sustained antibody response, measured by neutralizing antibody titers, among our donor population.

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

Vaccines ◽

10.3390/vaccines8010120 ◽

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.

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Peptide enzyme‐linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine

BMC Veterinary Research ◽

10.1186/s12917-021-02757-5 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Qi Wu ◽

Zhixian Lin ◽

Jinsen Wu ◽

Kun Qian ◽

Hongxia Shao ◽

...

Keyword(s):

Immune Response ◽

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Vital Role ◽

Enzyme Linked Immunosorbent Assay ◽

Infectious Bronchitis ◽

Different Types ◽

Antibody Titers

Abstract Background Infectious bronchitis virus (IBV), a coronavirus, is one of the most important poultry pathogens worldwide due to its multiple serotypes and poor cross-protection. Vaccination plays a vital role in controlling the disease. The efficacy of vaccination in chicken flocks can be evaluated by detecting neutralizing antibodies with the neutralization test. However there are no simple and rapid methods for detecting the neutralizing antibodies. Results In this study, a peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine was developed. The pELISA could indirect evaluate neutralizing antibody titers against different types of IBV in all tested sera. The titers measured with the pELISA had a coefficient of 0.83 for neutralizing antibody titers. Conclusions The pELISA could detect antibodies against different types of IBV in all tested sera. The pELISA has the potential to evaluate samples for IBV-specific neutralizing antibodies and surveillance the infection of IBV.

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Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera

mBio ◽

10.1128/mbio.02492-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kasopefoluwa Y. Oguntuyo ◽

Christian S. Stevens ◽

Chuan Tien Hung ◽

Satoshi Ikegame ◽

Joshua A. Acklin ◽

...

Keyword(s):

High Throughput Screening ◽

Neutralizing Antibodies ◽

Particle System ◽

Neutralizing Antibody ◽

Virus Neutralization ◽

Enzyme Linked Immunosorbent Assay ◽

Strong Positive Correlation ◽

Plasma Therapy ◽

Live Virus ◽

Convalescent Phase

ABSTRACT The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

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Overview of Neutralizing Antibodies and Their Potential in COVID-19

Vaccines ◽

10.3390/vaccines9121376 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1376

Author(s):

José Javier Morales-Núñez ◽

José Francisco Muñoz-Valle ◽

Paola Carolina Torres-Hernández ◽

Jorge Hernández-Bello

Keyword(s):

Neutralizing Antibodies ◽

Selection Criteria ◽

Herd Immunity ◽

Neutralizing Antibody ◽

Mechanisms Of Action ◽

Major Focus ◽

Protective Response ◽

Plasma Therapy ◽

Therapeutic Development

The antibody response to respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a major focus of COVID-19 research due to its clinical relevance and importance in vaccine and therapeutic development. Neutralizing antibody (NAb) evaluations are useful for the determination of individual or herd immunity against SARS-CoV-2, vaccine efficacy, and humoral protective response longevity, as well as supporting donor selection criteria for convalescent plasma therapy. In the current manuscript, we review the essential concepts of NAbs, examining their concept, mechanisms of action, production, and the techniques used for their detection; as well as presenting an overview of the clinical use of antibodies in COVID-19.

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