Sandwich-dot immunobinding assay (Sandwich-DIA), a new immunological method for the detection of diphtheria toxin

In this paper we describe a new assay for diphtheria toxin in bacterial cultures, based on a sandwich-dot immunobinding method. This method uses horse polyclonal diphtheria antitoxin as the coating antibody and mouse monoclonal diphtheria antitoxin as the detecting antibody. The sensitivity of this new method is within a range of 5-10 ng toxin per ml in Corynebacterium diphtheriae cultures. We did not observe any false-positive reactions. As a solid phase we used nitrocellulose disks which bind protein more strongly than the commonly used plastic solid phase. Results with strong toxin producers of C. diphtheriae can be obtained as soon as 11 h after starting a bacterial test culture. To detect weak toxin producers, the cultivation period must be extended to about 18 h and the results can be obtained after about 24 h. The method presented in this paper is simpler, faster and more reliable than currently used methods.