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Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies
Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly, Rabies lyssavirus-specific nAb titers need to be determined in...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9159627/ https://www.ncbi.nlm.nih.gov/pubmed/35536867 http://dx.doi.org/10.1371/journal.pntd.0010425 |
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author | Schöler, Lara Le-Trilling, Vu Thuy Khanh Dittmer, Ulf Fiedler, Melanie Trilling, Mirko |
author_facet | Schöler, Lara Le-Trilling, Vu Thuy Khanh Dittmer, Ulf Fiedler, Melanie Trilling, Mirko |
author_sort | Schöler, Lara |
collection | PubMed |
description | Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly, Rabies lyssavirus-specific nAb titers need to be determined in routine diagnostics to identify individuals being at risk of Rabies lyssavirus infections due to insufficient immunity. The current gold standard for the quantification of Rabies lyssavirus-specific nAbs is the rapid fluorescent focus inhibition test (RFFIT). However, RFFITs are expensive and labor-intensive since multiple microplate wells must be evaluated one-by-one by trained personnel through microscopic inspection, which limits the number of samples that can be processed. To overcome this disadvantage, we established a novel assay for Rabies lyssavirus-specific nAbs relying on an in-cell-ELISA (icELISA)-based neutralization test (icNT). The icNT differs from the RFFIT in the readout phase, and can be automatically quantified in minutes using broadly available microplate readers. During the establishment, icNT parameters such as antibody concentrations, permeabilization procedures, blocking reagents, infectious doses, and the duration of infection were optimized. Afterwards, a dose-dependent detection of Rabies lyssavirus neutralization was demonstrated using the WHO Standard Rabies Immunoglobulin reference. A panel of 200 sera with known RFFIT titers revealed very good sensitivity and specificity of the icNT. Furthermore, the icNT showed very good intra- and inter-assay precision. By recognizing Rabies lyssavirus-specific antigens, the assay can be applied immediately to automatically quantify the concentration of Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for antiviral compounds. |
format | Online Article Text |
id | pubmed-9159627 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-91596272022-06-02 Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies Schöler, Lara Le-Trilling, Vu Thuy Khanh Dittmer, Ulf Fiedler, Melanie Trilling, Mirko PLoS Negl Trop Dis Research Article Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly, Rabies lyssavirus-specific nAb titers need to be determined in routine diagnostics to identify individuals being at risk of Rabies lyssavirus infections due to insufficient immunity. The current gold standard for the quantification of Rabies lyssavirus-specific nAbs is the rapid fluorescent focus inhibition test (RFFIT). However, RFFITs are expensive and labor-intensive since multiple microplate wells must be evaluated one-by-one by trained personnel through microscopic inspection, which limits the number of samples that can be processed. To overcome this disadvantage, we established a novel assay for Rabies lyssavirus-specific nAbs relying on an in-cell-ELISA (icELISA)-based neutralization test (icNT). The icNT differs from the RFFIT in the readout phase, and can be automatically quantified in minutes using broadly available microplate readers. During the establishment, icNT parameters such as antibody concentrations, permeabilization procedures, blocking reagents, infectious doses, and the duration of infection were optimized. Afterwards, a dose-dependent detection of Rabies lyssavirus neutralization was demonstrated using the WHO Standard Rabies Immunoglobulin reference. A panel of 200 sera with known RFFIT titers revealed very good sensitivity and specificity of the icNT. Furthermore, the icNT showed very good intra- and inter-assay precision. By recognizing Rabies lyssavirus-specific antigens, the assay can be applied immediately to automatically quantify the concentration of Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for antiviral compounds. Public Library of Science 2022-05-10 /pmc/articles/PMC9159627/ /pubmed/35536867 http://dx.doi.org/10.1371/journal.pntd.0010425 Text en © 2022 Schöler et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Schöler, Lara Le-Trilling, Vu Thuy Khanh Dittmer, Ulf Fiedler, Melanie Trilling, Mirko Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title | Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title_full | Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title_fullStr | Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title_full_unstemmed | Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title_short | Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies |
title_sort | establishment and clinical validation of an in-cell-elisa-based assay for the rapid quantification of rabies lyssavirus neutralizing antibodies |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9159627/ https://www.ncbi.nlm.nih.gov/pubmed/35536867 http://dx.doi.org/10.1371/journal.pntd.0010425 |
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