Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.
Abstract: Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3-100.0) and 99.5 (95% CI 98.8-99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3-100.0) and 99.8 (95% CI 99.2-100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the test population.
Copyright © 2019 Elsevier B.V. All rights reserved.
Publication Date: 2019-09-09 PubMed ID: 31513861PubMed Central: PMC8782155DOI: 10.1016/j.jviromet.2019.113731Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the use of two developed neutralizing monoclonal antibodies in a blocking format ELISA test for the detection of Hendra virus antibodies in various animal species. The study shows that the refined tests enable minimization of false positive reactions, providing accurate and effective results for diverse species.
Understanding the Research Context
- The research focuses on Hendra virus (HeV), a virus maintained in bat populations that has been associated with spillover events in horses, humans, and dogs. The virus has also been shown to infect other species in experimental studies, such as guinea pigs, cats, and ferrets.
- At the time of the study, currently available tests could not meet the criteria of being sensitive and specific, acting effectively across a range of species, without requiring the use of live virus.
Experiment Methodology
- The research evaluated the use of two neutralizing monoclonal antibodies (mAbs) in a blocking enzyme-linked immunosorbent assay (bELISA) format to detect serum antibodies against a recombinant expressed HeV G protein in several animal species.
- The two mAbs tested were the human mAb m102.4, which neutralizes both HeV and the closely related Nipah virus (NiV), and the mouse mAb 1.2, which neutralizes only HeV.
Results
- The research optimized Diagnostic Sensitivity (DSe) and Diagnostic Specificity (DSp) using individual thresholds for mAb 1.2 and m102.4.
- For mAb 1.2, a positive threshold of >33% inhibition yielded DSe and DSp values of 100% and 99.5% respectively.
- For mAb m102.4, a positive threshold of >49% inhibition delivered DSe and DSp values of 100 and 99.8 respectively.
- At these thresholds, the DSe was 100% for both tests relative to the virus neutralization test.
Conclusions and Significance
- The study showed that false positive reactions did not overlap across the assays. With sequential and selective application of these assays, false positive reactions can be identified, and the DSp can be brought close to 100% in the test population, enhancing the test’s accuracy.
- This research provides an improved methodology for detecting HeV, enabling better diagnostic capabilities for a range of animal species. As a result, monitoring and controlling disease spread can be achieved more effectively.
Cite This Article
APA
Di Rubbo A, McNabb L, Klein R, White JR, Colling A, Dimitrov DS, Broder CC, Middleton D, Lunt RA.
(2019).
Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.
J Virol Methods, 274, 113731.
https://doi.org/10.1016/j.jviromet.2019.113731 Publication
Researcher Affiliations
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia. Electronic address: antonio.dirubbo@csiro.au.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
- Center for Antibody Therapeutics, University of Pittsburgh, Pittsburgh, PA 15261, USA.
- Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
- CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC, Australia.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Antibodies, Neutralizing / blood
- Antibodies, Viral / blood
- Antigens, Viral / immunology
- Enzyme-Linked Immunosorbent Assay / methods
- Hendra Virus / immunology
- Henipavirus Infections / diagnosis
- Henipavirus Infections / veterinary
- Humans
- Sensitivity and Specificity
Grant Funding
- U01 AI077995 / NIAID NIH HHS
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