Evaluation of single- and dual antigen delayed fluorescence immunoassay in comparison to an ELISA and the in vivo toxin neutralisation test for detection of diphtheria toxin antibodies

An evaluation of the delayed fluorescence immunoassay (Delfia) against an ELISA method for determination of diphtheria antitoxin levels in serum was performed. The Delfia was also validated in the in vivo toxin neutralisation test (Txn) in rabbits. Two variants of the Delfia were studied, a single-antigen Delfia (sDelfia) with only the diphtheria toxin included and a dual-antigen Delfia (dDelfia) with tetanus toxoid included for simultaneous detection of antibodies against two antigens. The diphtheria antitoxin cut-off levels in the sDelfia and the dDelfia were 0.004 and 0.002 AU/ml, respectively, which is lower than the internationally accepted level showing any protection against diphtheria (0.01 IU/ml). Both Delfia variants showed good correlation with the ELISA procedure above the ELISA cut-off level of 0.02 AU/ml. Results from samples assayed in the in vivo Txn assay indicated that the low antitoxin levels detected by the Delfia were valid. These results show that the Delfia could be considered as an in vitro reference method for detection of diphtheria antitoxin in seroepidemiological surveys and vaccine studies.