TOXINS AND ANTITOXINS. Mitchell and Reichart (at the University of Pennsyl vania) first demonstrated that the poisonous constituents of snake venom are proteins, or closely related bodies. This statement did not meet with favor in Europe, especially in Ger many, at the time, but further studies have confirmed it and it is now a well-established fact that some of the most powerful poisons known are proteins, or at least so closely asso ciated with proteins that all attempts to obtain them free from proteins up to the present time have failed. Such bodies have been found in animal, vegetable and bacterial life, but are limited to certain genera and species. In 1887 Sewall (at the University of Michigan) re ported a research which should be recorded as the first step in the discovery of methods of securing immunity to the action of these pro tein poisons, now known as toxins. Sewall studied the effects of the venom of the rattle snake on pigeons. Having ascertained the minimum fatal dose of this poison he at first administered less than this quantity, and, by gradually increasing the dose, established an immunity which enabled the pigeons to bear without apparent harm many times the mini mum fatal quantity. That this investigator at the time appreciated the possible bearing and significance on protection against disease of his results is shown by the following quotation from his report: uThis work was undertaken with the hope that it might form a worthy con tribution to the theory of prophylaxis. I have assumed an analogy between the venom of the poisonous serpent and the ptomaine produced under the influence of bacterial In 1891 Ehrlich, in a similar manner, succeeded in establishing in animals a high degree of im munity against two of the most potent protein vegetable poisons known — ricin, from the castor bean, and abrin, from the jequirity bean. One gram of ricin is sufficient to kill one and one-half million guinea pigs, and the potency of abrin is about one-half that of ricin. To these poisons immunity, as Ehrlich demonstrated, is easily established by feeding the animals by the mouth upon small and gradually augmented doses. While these vegetable products differ from snake venom in being absorbable from the alimentary canal, the resemblance in potency and in the production of immunity is striking. Moreover, like snake venoms these are pro tein poisons. Ehrlich made two important ad vances over preceding workers. He found that the immunity produced with these substances is specific. An animal immunized to ricin has no immunity to abrin, and vice versa. It was later found that this specificity holds good with all poisons of this class. In the second place, Ehrlich found that the blood serum of the im munized animal contains the immunizing agent, and that transference of this serum to a fresh non-treated animal confers a passive immunity upon the recipient. Moreover, this transferred immunity is quantitative. If the first or actively treated animal has been given an immunity of 10 times the fatal dose, and no more, then one tenth of its blood will be required to give the fresh animal immunity to one minimum lethal dose, while if the actively treated animal has been immunized to 100 fatal doses, one-hun dredth of its blood serum will immunize a fresh animal to one fatal dote, and one-tenth of its serum to 10 fatal doses. Furthermore, he found that the immunity of an actively treated mother may be transferred to the nurs ing young through the milk. Roux and Yersin of the Pasteur Institute found that cultures of the diphtheria bacillus, especially cultures four or five weeks old, when freed from the living organism by filtration through porcelain, con tain a poison similar in many respects to snake venom and the vegetable products ricin and abrin. The bacteria — free cultures — when injected into guinea pigs even in minute doses, killed the animals in the same time and with the same symptoms and lesions that result from inoculation with the living organism. Von Behring later immunized larger animals, first goats, then horses, to filtered diphtheria cul tures, and demonstrated that the blood of ani mals thus immunized has both protective and curative value in the treatment of The toxin of diphtheria is prepared by grow ing a virulent culture of the diphtheria bacillus at for two weeks or more and then re moving the bacteria by filtration. In other words, diphtheria toxin is an old filtered cul ture of the diphtheria bacillus. The potency of the toxin solution depends upon many con ditioni, all of which must be considered when one attempts to secure a highly active product. The medium generally employed for the growth of the diphtheria bacillus consists of beef tea containing 1 per cent of sodium chloride, from 1 to 3 per cent of peptone, and made feebly alkaline with a solution of sodium carbonate. This medium is placed in glass flasks, each of which should not be more than one-third full in order that there may be a large surface exposed to air which favors the growth of the bacillus and the production of toxin. The flasks are inoculated by floating
small masses of diphtheria bacillus growths, taken from agar tubes, on the surface of the beef tea in the flasks. After having been thus inoculated, the flasks are kept at 37°C. for about 14 days, when they are filtered and the filtrate constitutes the diphtheria toxin. The toxin solution quickly loses its potency when freely exposed to air and light, but when properly protected it remains without material deterioration for months, and under most favorable conditions, for years. Protection is secured by covering the toxin solution kept in dark bottles with a layer of toluol. In this way both air and light are excluded. Pro tection is made more complete by keeping the bottles in a dark room, the temperature of which does not rise above 15°C. When por tions of the solution are to be used, with drawal is made by sterilized pipettes intro duced through the layer of toluol. Under these precautions, the toxin solution may be kept without marked loss in toxicity for two years or even longer. Before the toxin pre pared as stated above is used in the prepara tion of antitoxin, its strength must he deter mined. This is done by ascertaining the mini mum amount of it necessary to kill a guinea pig of from 200 to 300 gram-weight within from three to five days. This amount is known as the minimum lethal dose. In most laboratories engaged in the preparation of diphtheria anti toxin, the toxin is not regarded as sufficiently potent unless the minimum lethal dose does not exceed 0.02 cubic centimeter. However, it is not always easy to secure a preparation of such high potency, and weaker solutions may be used. Occasion ally a much stronger product is obtained, and when this happens the preparation is greatly prized and carefully kept under the conditions mentioned above. Horses free from disease are carefully selected by skilled veterinarians. Usually they are submitted to a malein test to be sure that they are free from glanders. Dur ing the procedure the animals should be care fully guarded against wounds however slight, since these may serve as ports of entry for tetanus infection. Into these horses the toxin solution is injected, at first in small amounts. The first dose is usually followed by a tran sient disturbance in health. This may be slight, and indeed may not be in evidence at all. However, often after the first dose the ani mal's coat roughens, the appetite is impaired, and it may show some elevation of temperature. When there is complete return to health, usually after three or four days, a second in jection of the toxin is made. It is safer to make the second injection no larger than the first. Indeed, the practice is to make the second injection slightly less than the first. In this case there is usually no recognizable dis turbance in the well-being of the animal. After the third or fourth treatment the quantity of toxin used can be rapidly increased and it soon develops that the animal bears without apparent effect many times the amount which if used without previous treatment would have caused death. After this condition of im munity has been secured, a portion of blood is drawn from a vessel in the neck of the horse, allowed to coagulate, and the separated serum tested for its antitoxic strength. This process is known as the standardization of the antitoxin. The procedure consists in ascer taining how much of the blood serum of the treated horse is necessary to neutralize 100 minimum lethal doses of the toxin. The serum of the horse and the toxin are mixed in vary ing proportions in vitro, and the mixture in jected into guinea pigs of from 200 to 300 grams weight. The amount of toxin taken in these mixtures represents 100 minimum lethal doses, and the minimum amount of serum which must be added to this so that the mix ture will have no effect on the guinea pig is known as an immunity unit. It will be seen from what has been said that the filtered culture of the diphtheria bacillus constitutes the toxin, while the blood serum of the immunized horse constitutes the antitoxin. Both of these are standardized, and one neu tralizes the other quite as effectively as acid neutralizes alkali. Diphtheria antitoxin, which is the blood serum of an immunized horse, is put on the market in tubes fitted with hypo dermic needles and ready for use without transfer. This avoids the possibility of con tamination after the preparation leaves the laboratory of the manufacturer. Such con tainers are labeled showing the number of im munity units contained, and this is wholly with out reference to the amount of fluid in the tube. When such a preparation is labeled u5,000 Immunity Units') it is to be understood that all the fluid in that tube, whether it be 5 cubic centimeters or more, contains enough antitoxin to neutralize 5,000 times 100 minimum lethal doses of the toxin as tested on a guinea pig. The practice of medical men most skilful in treating diphtheria is to use an antitoxin of the highest obtainable potency, and doses of from 20,000 to 40,000 immunity units are now commonly employed.