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...
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ftdoajarticles:oai:doaj.org/article:0f5a8368962e4141b07effeb27913ff5 2023-05-15T15:12:15+02:00 Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies. Lara Schöler Vu Thuy Khanh Le-Trilling Ulf Dittmer Melanie Fiedler Mirko Trilling 2022-05-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0010425 https://doaj.org/article/0f5a8368962e4141b07effeb27913ff5 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0010425 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010425 https://doaj.org/article/0f5a8368962e4141b07effeb27913ff5 PLoS Neglected Tropical Diseases, Vol 16, Iss 5, p e0010425 (2022) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2022 ftdoajarticles https://doi.org/10.1371/journal.pntd.0010425 2022-12-31T02:25:15Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 16 5 e0010425 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Lara Schöler Vu Thuy Khanh Le-Trilling Ulf Dittmer Melanie Fiedler Mirko Trilling Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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 |
Article in Journal/Newspaper |
author |
Lara Schöler Vu Thuy Khanh Le-Trilling Ulf Dittmer Melanie Fiedler Mirko Trilling |
author_facet |
Lara Schöler Vu Thuy Khanh Le-Trilling Ulf Dittmer Melanie Fiedler Mirko Trilling |
author_sort |
Lara Schöler |
title |
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_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_sort |
establishment and clinical validation of an in-cell-elisa-based assay for the rapid quantification of rabies lyssavirus neutralizing antibodies. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
https://doi.org/10.1371/journal.pntd.0010425 https://doaj.org/article/0f5a8368962e4141b07effeb27913ff5 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 16, Iss 5, p e0010425 (2022) |
op_relation |
https://doi.org/10.1371/journal.pntd.0010425 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010425 https://doaj.org/article/0f5a8368962e4141b07effeb27913ff5 |
op_doi |
https://doi.org/10.1371/journal.pntd.0010425 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
16 |
container_issue |
5 |
container_start_page |
e0010425 |
_version_ |
1766342953515614208 |