Varicella-zoster plaque assay and plaque reduction neutralization test by the immunoperoxidase technique

A new plaque assay for the quantitation of varicella-zoster virus and a plaque reduction neutralization test for the determination of neutralizing antibody titer have been developed using the indirect immunoperoxidase technique. As compared with the classical plaque assay using a solid overlay, the test gives earlier results since plaque counting can be performed on day 3 after the inoculation of cell cultures. In six patients with zoster infection, neutralizing antibody titers ranged from 1:20 to 1:40 before the onset of infection and reached high levels (1:320 to 1:5,120) during the convalescent phase of the disease. Complement-fixing (CF) titers were all negative (less than 1:8) in prezoster serum samples from the same patients and ranged from 1:128 to 1:2,048 in the convalescent-phase sera. In the two cases in which late serum samples were available, neutralizing antibody titers matched the preillness levels, whereas CF titers dropped to undetectable levels. Neither neutralizing nor CF antibody was detected in two sera from individuals with no history of varicella-zoster infection. No differences in virus titers or neutralizing antibody titers were observed between the immunoperoxidase and the classical plaque assays. The appropriate characterization of reagent specificity is required before routine application of the test.

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Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells

Journal of Clinical Microbiology ◽

10.1128/jcm.4.1.61-66.1976 ◽

1976 ◽

Vol 4 (1) ◽

pp. 61-66

Author(s):

N J Schmidt ◽

J Dennis ◽

E H Lennette

Keyword(s):

Human Cytomegalovirus ◽

Neutralization Test ◽

Plaque Assay ◽

Neutralizing Antibody ◽

Neutral Red ◽

Plaque Reduction Neutralization Test ◽

Cell Monolayers ◽

Human Sera ◽

Antibody Titers ◽

Infected Cells

Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.

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A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-19 Patients

10.1101/2021.01.19.21250137 ◽

2021 ◽

Author(s):

Yiqi Ruben Luo ◽

Cassandra Yun ◽

Indrani Chakraborty ◽

Alan H.B. Wu ◽

Kara L. Lynch

Keyword(s):

Symptom Onset ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Label Free ◽

Serum Samples ◽

Initial Rise ◽

Igg Avidity ◽

Antibody Titers

AbstractBackgroundThe laboratory-based methods to measure the SARS-CoV-2 humoral response include virus neutralization tests (VNTs) to determine antibody neutralization potency. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed.MethodsA surrogate virus neutralization test established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of RBD to ACE2 after neutralizing RBD with antibodies in serum.ResultsThe LF-sVNT neutralizing antibody titers (IC50) were determined from serum samples (n=246) from COVID-19 patients (n=113), as well as the IgG concentrations and the IgG avidity indices. Although there is variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there is an initial rise, plateau and then in some cases a gradual decline at later timepoints after 40 days post-symptom onset. The IgG avidity indices, in the same cases, plateau after the initial rise and did not show a decline.ConclusionsThe LF-sVNT can be a valuable tool in clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau and then remains constant up to 8 months post-infection. The decline of antibody neutralization potency can be attributed to the reduction in antibody quantity rather than the deterioration of antibody avidity, a measure of antibody quality.SummaryA surrogate virus neutralization test established on a label-free immunoassay platform (LF-sVNT). Using the LF-sVNT and other assays, 246 serum samples from 113 COVID-19 patients were measured. We observed the time course of antibody characteristics beyond 200 days post-symptom onset.

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Development of a Poliovirus Neutralization Test with Poliovirus Pseudovirus for Measurement of Neutralizing Antibody Titer in Human Serum

Clinical and Vaccine Immunology ◽

10.1128/cvi.05225-11 ◽

2011 ◽

Vol 18 (11) ◽

pp. 1889-1894 ◽

Cited By ~ 19

Author(s):

Minetaro Arita ◽

Masae Iwai ◽

Takaji Wakita ◽

Hiroyuki Shimizu

Keyword(s):

Human Serum ◽

Antibody Titer ◽

Neutralization Test ◽

Critical Role ◽

Neutralizing Antibody ◽

Polio Eradication ◽

Serum Samples ◽

Human Serum Samples ◽

Wide Range ◽

Antibody Titers

ABSTRACTIn the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV.

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Serum Neutralizing Activity against B.1.1.7, B.1.351, and P.1 SARS-CoV-2 Variants of Concern in Hospitalized COVID-19 Patients

Viruses ◽

10.3390/v13071347 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1347

Author(s):

Claudia Maria Trombetta ◽

Serena Marchi ◽

Simonetta Viviani ◽

Alessandro Manenti ◽

Linda Benincasa ◽

...

Keyword(s):

Natural Infection ◽

Neutralizing Antibody ◽

Original Strain ◽

Immune Protection ◽

Serum Samples ◽

Symptomatic Infection ◽

Neutralizing Activity ◽

The United Kingdom ◽

Antibody Titers ◽

Pandemic Wave

The recent spreading of new SARS-CoV-2 variants, carrying several mutations in the spike protein, could impact immune protection elicited by natural infection or conferred by vaccination. In this study, we evaluated the neutralizing activity against the viral variants that emerged in the United Kingdom (B.1.1.7), Brazil (P.1), and South Africa (B.1.351) in human serum samples from hospitalized patients infected by SARS-CoV-2 during the first pandemic wave in Italy in 2020. Of the patients studied, 59.5% showed a decrease (≥2 fold) in neutralizing antibody titer against B.1.1.7, 83.3% against P.1, and 90.5% against B.1.351 with respect to the original strain. The reduction in antibody titers against all analyzed variants, and in particular P.1 and B.1.351, suggests that previous symptomatic infection might be not fully protective against exposure to SARS-CoV-2 variants carrying a set of relevant spike mutations.

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Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19

Scientific Reports ◽

10.1038/s41598-021-96143-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hitoshi Kawasuji ◽

Yoshitomo Morinaga ◽

Hideki Tani ◽

Miyuki Kimura ◽

Hiroshi Yamada ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Pseudotyped Virus ◽

Serum Samples ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Time Points ◽

Moderate Disease ◽

Activity Dynamics

AbstractAdaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics remain largely unknown. The neutralizing antibody (NAb) levels in patients with coronavirus disease 2019 (COVID-19) are helpful for understanding the pathology. Using SARS-CoV-2 pseudotyped virus, serum sample neutralization values in symptomatic COVID-19 patients were measured using the chemiluminescence reduction neutralization test (CRNT). At least two sequential serum samples collected during hospitalization were analyzed to assess NAbs neutralizing activity dynamics at different time points. Of the 11 patients, four (36.4%), six (54.5%), and one (9.1%) had moderate, severe, and critical disease, respectively. Fifty percent neutralization (N50%-CRNT) was observed upon admission in 90.9% (10/11); all patients acquired neutralizing activity 2–12 days after onset. In patients with moderate disease, neutralization was observed at earliest within two days after symptom onset. In patients with severe-to-critical disease, neutralization activity increased, plateauing 9–16 days after onset. Neutralization activity on admission was significantly higher in patients with moderate disease than in patients with severe-to-critical disease (relative % of infectivity, 6.4% vs. 41.1%; P = .011). Neutralization activity on admission inversely correlated with disease severity. The rapid NAb response may play a crucial role in preventing the progression of COVID-19.

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Longitudinal Study of SARS-CoV-2 Antibody Characteristics Using Label-Free Immunoassays

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.063 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S32-S32

Author(s):

Y R Luo ◽

C Yun ◽

A H Wu ◽

K L Lynch ◽

I Chakraborty

Keyword(s):

Symptom Onset ◽

Neutralizing Antibody ◽

Label Free ◽

Serum Samples ◽

Neutralization Activity ◽

Antibody Avidity ◽

Avidity Assay ◽

Initial Rise ◽

Igg Avidity ◽

Antibody Titers

Abstract Introduction/Objective Since the start of the COVID-19 pandemic, much research has focused on the kinetics and magnitude of humoral immune response. With the advantages of monitoring real-time immunoreactions, label-free immunoassay (LFIA) is becoming a powerful tool in serology studies. We have developed LFIAs to measure SARS- CoV-2 antibody avidity and neutralization activity in a cohort of COVID-19 patients and determine if they correlate with antibody concentration. Serial serum samples collected from mild to severe COVID-19 patients were measured out to 8 months post-symptom onset to determine the durability of the neutralizing antibody response. Methods/Case Report Based on thin-film interferometry technology, we established a label-free IgG avidity assay and a label-free surrogate virus neutralization test (LF-sVNT). For measurement, sensing probes pre-coated with receptor-binding domain (RBD) of SARS-CoV-2 spike protein are applied to serum samples containing SARS-CoV-2 antibodies. The label-free IgG avidity assay measures the binding strength between RBD and IgG under urea dissociation. The LF-sVNT analyzes the binding ability of RBD to ACE2 after neutralizing RBD with antibodies. Results (if a Case Study enter NA) IgG avidity indices and neutralizing antibody titers (IC50) were determined from serum samples (n=246) from COVID-19 patients (n=113). IgG concentrations were measured using a fluorescent immunoassay. The neutralizing antibody titers showed a weak correlation with IgG concentrations and no correlation with IgG avidity indices. Over the time course up to 8 months post-symptom onset, IgG concentrations and neutralizing antibody titers presented similar trends: an initial rise, plateau and then in some cases a gradual decline after 40 days. The IgG avidity indices, in the same cases, plateaued after the initial rise. Conclusion The results demonstrated that LFIA could be used an excellent solution in the determination of SARS- CoV-2 antibody characteristics. The study found that IgG concentration and neutralizing antibody titer declined over time, while IgG avidity index remained constant after reaching a plateau. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.

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Antibody titers measured by commercial assays are correlated with neutralizing antibody titers calibrated by international standards

10.1101/2021.07.16.21260618 ◽

2021 ◽

Author(s):

Yu-An Kung ◽

Chung-Guei Huang ◽

Sheng-Yu Huang ◽

Kuan-Ting Liu ◽

Peng-Nien Huang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Development ◽

Geometric Mean ◽

Neutralizing Antibody ◽

Diagnostic Techniques ◽

International Standards ◽

World Health ◽

Serum Samples ◽

Antibody Titers ◽

Health Organization

The World Health Organization (WHO) has highlighted the importance of an international standard (IS) for SARS-CoV-2 neutralizing antibody titer detection, with the aim of calibrating different diagnostic techniques. In this study, IS was applied to calibrate neutralizing antibody titers (IU/mL) and binding antibody titers (BAU/mL) in response to SARS-CoV-2 vaccines. Serum samples were collected from participants receiving the Moderna (n = 20) and Pfizer (n = 20) vaccines at three time points: pre-vaccination, after one dose, and after two doses. We obtained geometric mean titers of 1404.16 and 928.75 IU/mL for neutralizing antibodies after two doses of the Moderna and Pfizer vaccines, respectively. These values provide an important baseline for vaccine development and the implementation of non-inferiority trials. We also compared three commercially available kits from Roche, Abbott, and MeDiPro for the detection of COVID-19 antibodies based on binding affinity to S1 and/or RBD. Our results demonstrated that antibody titers measured by commercial assays are highly correlated with neutralizing antibody titers calibrated by IS.

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Prevalence and Persistence of Maternal Dengue Neutralizing Antibodies in Infants From Central and Southern Thailand: A Retrospective Cohort Study

Asia Pacific Journal of Public Health ◽

10.1177/1010539519853396 ◽

2019 ◽

Vol 31 (4) ◽

pp. 288-295 ◽

Cited By ~ 1

Author(s):

Adrienne Guignard ◽

François Haguinet ◽

Stéphanie Wéry ◽

Phirangkul Kerdpanich

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Development ◽

Geometric Mean ◽

Neutralizing Antibody ◽

Maternal Antibodies ◽

Childhood Immunization ◽

Serum Samples ◽

Denv Serotypes ◽

Antibody Titers ◽

Antibody Kinetics

Understanding maternal dengue virus (DENV) neutralizing antibody kinetics in infants remains timely to develop a safe and effective childhood immunization. This retrospective study evaluated the prevalence and persistence of maternal antibody titers against DENV serotypes 1 to 4 in 139 Thai infants at 2, 6, and 7 months of age, using serum samples collected in a vaccination trial ( http://clinicaltrials.gov ; NCT00197275). Neutralizing antibodies against all 4 DENV serotypes were detected in 87.8% and 22.9% of infants at 2 and 7 months, respectively. At 2 months, DENV-4 neutralizing antibody geometric mean titers were notably lower (80) compared with DENV-1 to DENV-3 (277-471). Our results corroborate previous findings that DENV-1 to DENV-4 maternal antibodies persist at 7 months despite titers decrease from 2 months onwards. As persisting maternal antibodies may inhibit immune responses in DENV-vaccinated infants, a comprehensive understanding of DENV antibody kinetics is required in the perspective of vaccine development for infants.

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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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Serum Neutralization Assay Can Efficiently Replace Plaque Reduction Neutralization Test for Detection and Quantitation of West Nile Virus Antibodies in Human and Animal Serum Samples

Clinical and Vaccine Immunology ◽

10.1128/cvi.00426-14 ◽

2014 ◽

Vol 21 (10) ◽

pp. 1460-1462 ◽

Cited By ~ 27

Author(s):

Annapia Di Gennaro ◽

Alessio Lorusso ◽

Claudia Casaccia ◽

Annamaria Conte ◽

Federica Monaco ◽

...

Keyword(s):

West Nile Virus ◽

Neutralization Test ◽

Neutralization Assay ◽

Serum Samples ◽

West Nile ◽

Plaque Reduction Neutralization Test ◽

Serum Neutralization ◽

Human Sera

ABSTRACTA serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes.

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