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Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay

Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the vi...

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Autores principales: Simonova, Maria A., Melnikov, Vyacheslav G., Lakhtina, Olga E., Komaleva, Ravilya L., Berger, Anja, Sing, Andreas, Zavriev, Sergey K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9498204/
https://www.ncbi.nlm.nih.gov/pubmed/36140605
http://dx.doi.org/10.3390/diagnostics12092204
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author Simonova, Maria A.
Melnikov, Vyacheslav G.
Lakhtina, Olga E.
Komaleva, Ravilya L.
Berger, Anja
Sing, Andreas
Zavriev, Sergey K.
author_facet Simonova, Maria A.
Melnikov, Vyacheslav G.
Lakhtina, Olga E.
Komaleva, Ravilya L.
Berger, Anja
Sing, Andreas
Zavriev, Sergey K.
author_sort Simonova, Maria A.
collection PubMed
description Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria.
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spelling pubmed-94982042022-09-23 Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay Simonova, Maria A. Melnikov, Vyacheslav G. Lakhtina, Olga E. Komaleva, Ravilya L. Berger, Anja Sing, Andreas Zavriev, Sergey K. Diagnostics (Basel) Article Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria. MDPI 2022-09-11 /pmc/articles/PMC9498204/ /pubmed/36140605 http://dx.doi.org/10.3390/diagnostics12092204 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Simonova, Maria A.
Melnikov, Vyacheslav G.
Lakhtina, Olga E.
Komaleva, Ravilya L.
Berger, Anja
Sing, Andreas
Zavriev, Sergey K.
Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title_full Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title_fullStr Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title_full_unstemmed Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title_short Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
title_sort determination of diphtheria toxin in bacterial cultures by enzyme immunoassay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9498204/
https://www.ncbi.nlm.nih.gov/pubmed/36140605
http://dx.doi.org/10.3390/diagnostics12092204
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