Double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of toxin-producing Corynebacterium diphtheriae

An enzyme-linked immunosorbent assay for determining the toxigenicity of Corynebacterium diphtheriae is presented. The assay uses hyperimmune horse diphtheria antitoxin as a capture antibody and mouse monoclonal diphtheria antitoxin as a detecting antibody. Growth of bacteria and capture of diphtheria toxin by antitoxin are carried out in one step. Toxin produced by as little as 100 toxin-producing corynebacteria is detectable, corresponding to a sensitivity of 10 ng of diphtheria toxin per ml. Demonstration of toxin after incubation of the bacteria for 4.75 h, as well as after 18 h, was in accordance with the modified Elek gel diffusion method and the guinea pig inoculation test. However, heavy inocula incubated overnight produced significantly lower optical density than did diluted inocula; thus, the higher optical density was used as an indicator of toxin production. A decrease in optical density was also seen by shortening the incubation time. For laboratory safety, ethanol was added to the microtiter plate wells before washing out of the bacteria. This resulted in a further decrease in optical density. Using 4.75-h incubation time gave a single false-negative result. No false-positive results were ever seen. Incubation for 18 h is suitable for large-scale screening, and 4.75 h of incubation is suitable for rapid identification of toxin-producing C. diphtheriae.