Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria

Abstract

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.

Cutaneous diphtheria complicated oncologic reconstruction surgery in osteosarcoma

“…29 Enzyme Immunoassay (EIA), using monoclonal antibody to fragment A of the exotoxin, is highly accurate and could improve diagnosis in false-negative the Elek and RT-PCR test. 30 Since the differential diagnosis of malignant skin lesions such as squamous cell carcinoma (SCC) should be considered for non-healing chronic ulcers, the histopathologic examination is encouraged for definite diagnosis and rule outing non-infectious etiologies.…”

Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay

“…ELISA testing was performed between February 2021 and January 2022. For ELISA testing bacterial strains were cultured on Elek broth [ 6 , 7 ] for 6 h at 37 °C, after which the bacterial cells were removed by filtration through a 0.22-µm-pore-size membrane (Merck Millipore, Burlington, MA, USA). The culture supernatants were stored at −20 °C prior to analysis in the ELISA.…”

“…ELISA remains a very robust and reliable method for the detection of various protein analytes. Previously, enzyme-linked immunosorbent and immunochromatographic methods for detecting DT were developed, the sensitivity of which varied from 0.1 to 4 ng/mL [ 5 , 6 , 7 ]. These tests were based on a sandwich immunoassay with equine polyclonal antibodies as binding antibodies, and mouse monoclonal antibodies (mAbs) as detecting antibodies.…”

Challenges of Diphtheria Toxin Detection

“…EIA is also characterised by a very high diagnostic sensitivity (LOD is 0.1 ng/mL). In a comparative study of 220 toxigenic Corynebacterium isolates, the results obtained by the EIA method correlate 100% with the results obtained by the Elek test [ 51 ]. Engler et al also reported 100% consistency of the results of both methods [ 51 ].…”