Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay

Abstract

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.

Lateral flow immunoassay-based laboratory algorithm for rapid diagnosis of diphtheria

“…To better understand the discrepancy, the toxigenicity status of strain KL 1902 was studied by additional methods. This strain was found to be negative in ELISA 13 based on the same mAbs as LFIA. We also used the immunochromatographic strip (ICS) test 18 .…”

“…LFIA test strips were manufactured by Senova, Weimar, Germany, under the H2020-MSCA-IF-2018 (843405-DIFTERIA) research program using reagents previously used to develop ELISA for DT detection 13 . These included polyclonal goat anti-mouse IgG (control Ab) and a pair of mouse monoclonal antibodies to the diphtheria toxin B subunit (both IgG 1 ): anti-DT 675-3 (capture mAb) and anti-DT 676-3 labeled with colloidal gold (detection mAb).…”

“…To determine the presence and the level of DT production by C. diphtheriae and C. ulcerans , an immunoassay based on monoclonal antibodies (mAbs) has been designed 13 . A pair of mAbs specific to subunit B of DT was developed, which made it possible to detect DT in a sandwich enzyme-linked immunosorbent assay (ELISA) with a detection limit of DT less than 1 ng/mL.…”

Challenges of Diphtheria Toxin Detection

“…Immunoassay methods are based on the antigen–antibody reaction and are usually performed with mouse monoclonal antibodies (mAb) and, more rarely, equine polyclonal antibodies (pAb) directed against subunit A or B of DT. The limit of detection (LOD) for those methods is 0.1–4.0 ng/mL [ 34 ].…”

“…The enzyme-linked immunosorbent assay (ELISA) for DT determination was described in [ 48 ]. ELISA can be a qualitative or quantitative method, with quantitative immunoassay methods based on mAbs being easier to standardise [ 34 ]. The results of sandwich ELISA cover the results of other methods for DT detection, such as the Elek test or genetic identification of the tox gene, and are characterised by high sensitivity (approximately 99%) and specificity (100%).…”