Quantitation of Clostridium perfringens Type A Enterotoxin by Electroimmunodiffusion
Charles L. Duncan, Eileen B. Somers
Conditions for quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion are described. As little as 0.01 jig of enterotoxin could be detected. Electroimmunodiffusion was more sensitive than either single gel diffusion or quantitation based on erythemal activity of the toxin in guinea pig skin. Clostridium perfringens type A enterotoxin can be readily detected on the basis of its biological activity. The toxin induces fluid accumulation in ligated ileal loops of
... s and lambs (1, 4), is lethal in mice (3, 7), and produces erythema in the skin of guinea pigs or rabbits (2, 7). For quantitative purposes, the assay based on erythemal activity is more sensitive than the loop technique or the measurement of lethality in mice (3, 7). All of these techniques suffer from the variations in sensitivity of the animals being used, the requirement for sufficient controls to eliminate the activity of other toxins or nonspecific responses, and the cost of the animals involved. This study was initiated to determine the feasibility of using immunochemical procedures for specific enterotoxin quantitation. The technique of electroimmunodiffusion was chosen for investigation. This technique has been used for accurate and sensitive quantitation of a variety of proteins including immunoglobulins and albumin (5) and type A botulinum toxin (6). The procedure involves use of a high-voltage electrical field to induce rapid migration of a protein sample from a well into gel containing antibody prepared against the protein. A cone of protein antigen is precipitated along the path of antigen migration wherever antigen-antibody equivalence is reached. The cone length is proportional to the amount of protein in the sample. Conditions to be considered in adapting this method to the measurement of any protein soluble in low-ionic-strength electrolyte solutions have been discussed (5). The results reported here demonstrate the usefulness of the method for quantitating C. perfringens type A enterotoxin. The method is also compared with quantitation of enterotoxin by single gel diffusion and by the skin test.