Electrophoresis on a starch-supporting medium was used to fractionate sera containing human diphtheria antitoxin of the following varieties (a) precipitating antitoxin, (b) non-precipitating skin-sensitizing antitoxin, and (c) mixtures containing precipitating and skin-sensitizing antitoxins. Aliquots of the protein fractions thus separated were tested for activity using the rabbit toxin neutralization test, precipitin techniques, and passive transfer tests in human skin.
Non-precipitating, skin-sensitizing diphtheria antitoxin migrated largely as a fast moving gamma (γ1) globulin. Passive transfer studies of isolated antitoxic fractions showed that they were as potent as whole serum in the ability to cause immediate wheal reactions. These fractions were not precipitable using appropriate quantities of purified toxoid.
Precipitating diphtheria antitoxin migrated largely as a slow moving gamma (γ2) globulin. Isolated antitoxic fractions of appropriate strength obtained from representative sera were precipitable by toxin and were unable to cause immediate wheal reactions upon toxoid challenge in human recipients.
Mixtures of skin-sensitizing and precipitating antitoxins were separable by the technique of starch electrophoresis. The individual components removed from mixtures by this method retained the properties by which they could be characterized in whole serum.