THE ASSAY OF DIPHTHERIA TOXIN USING MOUSE

Abstract
In the titration of Dlm of a diphtheria toxin it is necessary to use a large number of guinea pigs, for example, several tens as many guinea pigs for an accurate estimation. Therefore, it is natural that attempts have been made to find an alternative and less expensive method which would be of value, at least for a preliminary work.Jensen (1933) proposed a method by which a fairly precise estimation of Dlm was thought to be possible. His method was to compute Dlm from (DRM) m actually measured by the intracutaneous rabbit method, basing on the evidence obtained in his experiments that the Dlm/DRM ratio of diphtheria toxin of every kind was constant.Lately, Pope (1954) offered an opinion that the intracutaneous method was an alternative for the subcutaneous method in the titration of diphtheria toxin and was advantageous in the point that many titrations might be made on one animal.Gerwing (1957) recently proposed a more reasonable method of Drm titration, in which the parallel line assay method was introduced, saying that the minimal reacting dose was preferable to the Dlm to define a Schick test dose. However, they declared nothing as to the Dlm/DRM or Dlm/Drm ratio.A good number of experiments carried out in our laboratory with various kinds of diphtheria toxins have shown that the assumption that the Dlm/DRM or Dlm/Drm ratio of diphtheria toxin of every kind was constant did not hold in the majority of cases; particularly with fresh toxins, the Dlm/DRM ratio frequently being proved to be far from constant (Kurokawa, Kondo and Kondo, to be published) . Therefore, it is clear that the method proposed by Jensen cannot be used as a method estimating Dlm.As we were compelled to do a good number of fairly accurate titrations of Dlm in experiments pursuing the factor or factors concerning the Dlm/DRM ratio of diphtheria toxin (Kurokawa, Kondo and Kondo, to be published), an alternative method, less expensive and fairly precise, for the classical subcutaneous guinea pig method was looked for.Two methods which may meet this demand are found in the literatures.Frobisher and Parsons (Frobisher and Parsons, 1940; Frobisher, 1942) reported that mice inoculated intracerebrally with living culture of toxigenic diphtheria bacilli or toxic culture filtrates showed characteristic responses, which could be specifically neutralized with diphtheria antitoxin. According to their brief description, the minimal dose that killed almost all mice fairly corresponded with 1 guinea pig Dlm when the toxin was intracerebrally inoculated into mice. Shortly later, Suzuki (1944) confirmed Frobisher's report basing on his extensive experiments and showed that there was a certain relationship between Dlm and the minimal dose killing mice with certainty.Meanwhile, Hosoya and his collaborators (Hosoya, Ozawa and Tanaka, 1934; Ozawa, 1935) described that domestic fowls and 3 to 4 weeks old chicks of White Leghorn having received intramuscularly diphtheria toxin developed a characteristic paralysis of wings and of legs, and died for the most parts. Inoue (1936) confirmed the report of Hosoya and his collaborators and reported that approximately one week old chicks were the most susceptible to diphtheria toxin and that the paralysis developed in chicks with the toxin of one-several tensth of Dlm. Frobisher and his collaborators (Frobisher, 1940; Frobisher, Parsons and Tung, 1942), probably independently from Japanese authors' works, reported briefly that White Leghorn or Plymouth Rock chicks could be used for toxigenicity test of diphtheria bacillus. Branham and Wormald (1954) used the chicks in titration of diphtheria antitoxin.These methods may provide a new tool which enables the quantitative titration of diphtheria toxin, less expensively and fairly precisely.However, few report directed to this purpose using mice or chicks has yet appeared.