Establishment of a rapid ELISPOT assay for influenza virus titration and neutralizing antibody detection

Abstract Background H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. Results The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. Conclusions In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

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Identification and Structural Characterization of a Broadly Neutralizing Antibody Targeting a Novel Conserved Epitope on the Influenza Virus H5N1 Hemagglutinin

Journal of Virology ◽

10.1128/jvi.02344-12 ◽

2012 ◽

Vol 87 (4) ◽

pp. 2215-2225 ◽

Cited By ~ 29

Author(s):

L. Du ◽

L. Jin ◽

G. Zhao ◽

S. Sun ◽

J. Li ◽

...

Keyword(s):

Influenza Virus ◽

Structural Characterization ◽

Neutralizing Antibody ◽

Virus H5n1 ◽

Broadly Neutralizing Antibody ◽

Antibody Targeting ◽

Conserved Epitope

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A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

10.1101/2021.02.25.432905 ◽

2021 ◽

Author(s):

Jenna J. Guthmiller ◽

Julianna Han ◽

Henry A. Utset ◽

Lei Li ◽

Linda Yu-Ling Lan ◽

...

Keyword(s):

Influenza Virus ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Human Memory ◽

Influenza Virus Vaccine ◽

Virus Infections ◽

Broadly Neutralizing Antibodies ◽

Cell Repertoire ◽

Influenza Virus Hemagglutinin

SummaryBroadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is “anchored” to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

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DEVELOPMENT OF AN IMPROVED EPSTEIN-BARR VIRUS (EBV) NEUTRALIZING ANTIBODY ASSAY TO FACILITATE DEVELOPMENT OF A PROPHYLACTIC GP350-SUBUNIT EBV VACCINE

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2020.016 ◽

2020 ◽

Vol 12 (1) ◽

pp. e2020016

Author(s):

Hui Liu ◽

Lorraine Gemmell ◽

Rui Lin ◽

Fengrong Zuo ◽

Henry H. Balfour ◽

...

Keyword(s):

High Throughput ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Epidemiologic Studies ◽

Antibody Detection ◽

Healthy Human ◽

Barr Virus ◽

Human Sera ◽

Epstein Barr ◽

Bioanalytical Assays

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, sensitive, and high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed and improved analytical tools for such a vaccine’s pre-clinical and clinical validation including a gp350-specific antibody detection assay and an EBV-GFP based neutralization assay for measuring EBV specific antibodies in human donors. The sensitivity of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay was further improved when guinea pig complement was supplemented using a panel of healthy human sera. Anti-gp350 antibody titers, which were evaluated using an anti-gp350 IgG ELISA assay optimized for capture and detection conditions, were moderately correlated to the FFA-based neutralization titers. Overall, these sensitive, and high-throughput bioanalytical assays are capable of characterizing the serologic response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

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Correlation of Cellular Immune Responses with Protection against Culture-Confirmed Influenza Virus in Young Children

Clinical and Vaccine Immunology ◽

10.1128/cvi.00397-07 ◽

2008 ◽

Vol 15 (7) ◽

pp. 1042-1053 ◽

Cited By ~ 202

Author(s):

Bruce D. Forrest ◽

Michael W. Pride ◽

Andrew J. Dunning ◽

Maria Rosario Z. Capeding ◽

Tawee Chotpitayasunondh ◽

...

Keyword(s):

Influenza Virus ◽

Young Children ◽

Immune Responses ◽

Virus Vaccine ◽

Elispot Assay ◽

Influenza Virus Vaccine ◽

The Philippines ◽

Influenza Viruses ◽

Ifn Γ

ABSTRACT The highly sensitive gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay permits the investigation of the role of cell-mediated immunity (CMI) in the protection of young children against influenza. Preliminary studies of young children confirmed that the IFN-γ ELISPOT assay was a more sensitive measure of influenza memory immune responses than serum antibody and that among seronegative children aged 6 to <36 months, an intranasal dose of 107 fluorescent focus units (FFU) of a live attenuated influenza virus vaccine (CAIV-T) elicited substantial CMI responses. A commercial inactivated influenza virus vaccine elicited CMI responses only in children with some previous exposure to related influenza viruses as determined by detectable antibody levels prevaccination. The role of CMI in actual protection against community-acquired, culture-confirmed clinical influenza by CAIV-T was investigated in a large randomized, double-blind, placebo-controlled dose-ranging efficacy trial with 2,172 children aged 6 to <36 months in the Philippines and Thailand. The estimated protection curve indicated that the majority of infants and young children with ≥100 spot-forming cells/106 peripheral blood mononuclear cells were protected against clinical influenza, establishing a possible target level of CMI for future influenza vaccine development. The ELISPOT assay for IFN-γ is a sensitive and reproducible measure of CMI and memory immune responses and contributes to establishing requirements for the future development of vaccines against influenza, especially those used for children.

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Chick embryo lethal orphan (CELO) virus as a possible contaminant of egg-grown virus vaccines

Journal of Hygiene ◽

10.1017/s0022172400041401 ◽

1969 ◽

Vol 67 (1) ◽

pp. 41-47 ◽

Cited By ~ 7

Author(s):

J. S. Oxford ◽

C. W. Potter

Keyword(s):

Influenza Virus ◽

British Empire ◽

Neutralizing Antibody ◽

Influenza Virus Vaccine ◽

Inactivation Kinetics ◽

Serum Dilution ◽

Human Sera ◽

Normal Human ◽

Celo Virus ◽

Kinetics Of

SUMMARYThe inactivation kinetics of CELO virus were studied in the presence of 1/4000 formaldehyde. Inactivation of the virus by formaldehyde at 4° C. was not complete after 14 days incubation. Formaldehyde inactivation at 36° C., however, was rapid and no virus was detected after 24 hr. incubation.Neutralizing antibody to CELO virus was detected in 20–88% of sera tested from five flocks of hens. This suggested dissemination of the virus in England and Scotland. However, no CELO virus neutralizing antibody at a serum dilution of 1/8 was detected in 142 normal human sera or in 229 sera from persons who had been immunized with egg grown, inactivated influenza virus vaccine.We would like to thank Professor C. H. Stuart-Harris and Drs J. E. Wilson, D. A. Martin and D. Breeze for their help and their criticisms of the manuscript.Dr G. C. Schild kindly supplied a number of the human sera used in the study. The study was financed in part by the National Fund for Research in Poliomyelitis and other Crippling Diseases and by the British Empire Cancer Campaign.

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