Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay
Since diphtheria toxin (DT) is the main virulence factor of <i>Corynebacterium diphtheriae</i> and <i>C. ulcerans</i>, 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 produc...
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MDPI AG
2022-09-01
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| Series: | Diagnostics |
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| author | Maria A. Simonova Vyacheslav G. Melnikov Olga E. Lakhtina Ravilya L. Komaleva Anja Berger Andreas Sing Sergey K. Zavriev |
| author_facet | Maria A. Simonova Vyacheslav G. Melnikov Olga E. Lakhtina Ravilya L. Komaleva Anja Berger Andreas Sing Sergey K. Zavriev |
| author_sort | Maria A. Simonova |
| collection | DOAJ |
| description | Since diphtheria toxin (DT) is the main virulence factor of <i>Corynebacterium diphtheriae</i> and <i>C. ulcerans</i>, 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 <i>tox</i> 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 <i>C. ulcerans</i>, on average, produce 10 times less DT than <i>C. diphtheriae.</i> 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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| id | doaj.art-49dffbf006c6421e8ab5835c5517a331 |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-10T00:16:52Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-49dffbf006c6421e8ab5835c5517a3312023-11-23T15:50:25ZengMDPI AGDiagnostics2075-44182022-09-01129220410.3390/diagnostics12092204Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme ImmunoassayMaria A. Simonova0Vyacheslav G. Melnikov1Olga E. Lakhtina2Ravilya L. Komaleva3Anja Berger4Andreas Sing5Sergey K. Zavriev6Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaNational Conciliary Laboratory on Diphtheria, Veterinaerstrasse 2, 85764 Oberschleissheim, GermanyShemyakin-Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaNational Conciliary Laboratory on Diphtheria, Veterinaerstrasse 2, 85764 Oberschleissheim, GermanyNational Conciliary Laboratory on Diphtheria, Veterinaerstrasse 2, 85764 Oberschleissheim, GermanyShemyakin-Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaSince diphtheria toxin (DT) is the main virulence factor of <i>Corynebacterium diphtheriae</i> and <i>C. ulcerans</i>, 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 <i>tox</i> 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 <i>C. ulcerans</i>, on average, produce 10 times less DT than <i>C. diphtheriae.</i> 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.https://www.mdpi.com/2075-4418/12/9/2204<i>Corynebacterium diphtheriae</i><i>Corynebacterium ulcerans</i>diphtheria toxinELISAmonoclonal antibodieslevel of toxin production |
| spellingShingle | Maria A. Simonova Vyacheslav G. Melnikov Olga E. Lakhtina Ravilya L. Komaleva Anja Berger Andreas Sing Sergey K. Zavriev Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay Diagnostics <i>Corynebacterium diphtheriae</i> <i>Corynebacterium ulcerans</i> diphtheria toxin ELISA monoclonal antibodies level of toxin production |
| 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 | <i>Corynebacterium diphtheriae</i> <i>Corynebacterium ulcerans</i> diphtheria toxin ELISA monoclonal antibodies level of toxin production |
| url | https://www.mdpi.com/2075-4418/12/9/2204 |
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