Until recently diphtheria " toxin " was supposed to be a single definite substance and to have a definite toxicity in animals and neutralizing power for antitoxin. A fatal dose of toxin, without regard to the conditions under which it was produced or preserved, was supposed to require always the same quantity of antitoxin to neutralize it. Ehrlich's researches have completely done away with this theory, and have substituted for it one which assumes the toxin to be only at its origin a single definite chemical compound with definite physiological and antitoxic properties. According to Ehrlich the toxin is an unstable substance which readily loses its toxicity, while at the same time its affinity for antitoxin may be either increased or decreased. Its neutralization by antitoxin he considers to be due to a chemical union between the toxin and the antitoxin.
The results of our experiments as detailed in this paper are fully in accord with those published by Ehrlich, as to the varying neutralizing value of a minimal fatal dose of " toxin "; they, however, go further and indicate roughly a general law in accordance with which these changes occur.
The neutralizing value of a fatal dose of toxin is at its lowest in the culture fluid when the first considerable amounts of toxin have been produced. After a short period, during which the quantity of toxin in the fluid is increasing, the neutralizing value of the fatal dose begins to increase, at first rapidly, then more slowly.
While the culture is still in vigorous growth and new toxin is being produced, the neutralizing value of the fatal dose fluctuates somewhat, but with a generally upward tendency. After the cessation of toxin production the neutralizing value of the fatal dose increases steadily until it becomes five to ten times its original amount.
In our experiments the greatest value for L+ was 126, the least 27. As at 6 hours L+ was only 72 and at 28 hours only 91, we doubt whether L+ ever reaches above 150, and therefore hardly expect Ehrlich's figures of 200 to be realized. When we seek to analyze the above-described process, we find certain facts which seem partly to explain it. Experiments have shown that filtered toxin, preserved for any length of time in conditions under which access of air occurs, gradually loses in both its toxicity and neutralizing power, and that it loses more rapidly in the former property than in the later. Thus, while the fatal dose of a toxin preserved for one year rose from .01 cc. to .55 cc., it lost only half as much in neutralizing value, one unit neutralizing at first 1 cc., at the end of the year 25 cc. These processes take place more rapidly at room temperature than in the ice chest, and in the incubator than in the room.
In the fluid holding the living bacilli we have, therefore, after the first few hours of toxin formation, a double process going on, one of deterioration in the toxin already accumulated, which tends to increase the neutralizing value of the fatal dose, the other of new toxin formation, which probably tends to diminish the neutralizing value. The chemical changes produced by the growth of the bacilli in the bouillon tend to aid one or the other of these processes and so to make from hour to hour slight changes in the value of the fatal dose. Later, with the period of cessation of toxin production, the gradual deterioration of the toxicity alone continues, and the fatal dose gradually and steadily increases in its neutralizing value.
Ehrlich's theories, as to the splitting up of " toxin " into toxoids having little or no toxicity but on the average full neutralizing power for antitoxin, have not in our opinion been substantiated by the results of these experiments. The difference between the amount of toxin mixed with a unit of antitoxin which causes the first symptoms and that causing death upon the fourth day would be, it is true, explained by his theory, but the failure of this difference to be greater where, by his theories, epitoxoids should be in great abundance prevents our acceptance of his views. The fact of the greater neutralization value of a fatal dose of a deteriorated toxin would be accounted for on his protoxoid theory. This, however, is not proof of its correctness, as other theories, such as the production by the diphtheria bacillus of two or more closely allied toxins, similar to the allied alkaloids produced by plants, would equally account for it, if we supposed the one which had the greater neutralization value was more resistant to destruction than the other. We only advance this theory to call attention to the fact that many theories can on paper explain a process without necessarily being thereby established.
Even if his theories prove partially correct, we feel certain that his formula for standardizing toxins is founded upon error and cannot be employed for the purpose intended by him.
While we do not believe, therefore, that he has changed the principles of testing antitoxin, yet we believe he has contributed greatly to uniformity in results by calling attention to the necessity of selecting a suitable toxin and by employing and distributing an antitoxin as a standard to test toxins by. In this way smaller testing stations can make their results correspond with those of the central station.
In spite of the great variations in the neutralizing value of a fatal dose in different toxins, we do not believe there has been any such great difference in the toxins used by the different stations for testing purposes. Most laboratories have taken the culture fluid at about the time of its greatest toxicity, and the neutralizing value of a fatal dose of this toxin would seldom vary more than 10 per cent above or below the standard now adopted in Germany by the government testing station, this latter being presumably as close as possible to that used to establish the original Behring-EhrIich unit.
Where error has been made, it has usually been by taking too old culture fluids, which would cause the antitoxin strength of samples tested to be estimated below and not above its real value. Culture 8, which is used not only by us but by many other laboratories in the United States and Europe, fortunately produces on the 6th day, the time at which the culture is usually removed, a toxin which grades Elirlich's antitoxin within five per cent of the strength given by him.
We believe that by using such a bacillus, we can, after gaining a fuller knowledge of its characteristics, obtain a toxin of a known and suitable neutralizing value, and thus always correctly standardize an antitoxic serum. Meanwhile a fairly permanent antitoxin, such as Ehrlich provides, is of immense value in insuring a uniform though not necessarily correct standard among the different testing stations and in allowing of comparison between them.