The general characteristics of toxins have been described ([Chapter XII]). It has been stated that they are more or less specific in their action on cells. In order to affect a cell it is evident that a toxin must enter into chemical combination with it. This implies that the toxin molecule possesses a chemical group which can combine with a receptor of the cell. This group is called the haptophore or combining group. The toxic or injurious portion of the toxin molecule is likewise spoken of as the toxophore group. When a toxin is introduced into the body its haptophore group combines with suitable receptors in different cells of the body. If not too much of the toxin is given, instead of injuring, it acts as a chemical stimulus to the cell in the manner already described. The cell in response produces more of the specific thing, which in this instance is more receptors which can combine with the toxin, i.e., with its haptophore group. If the stimulus is kept up, more and more of these receptors are produced until an excess for the cell accumulates, which excess is excreted from the individual cell and becomes free in the blood. These free receptors have, of course, the capacity to combine with toxin through its haptophore group. When the toxin is combined with these free receptors, it cannot combine with any other receptors, e.g., those in another cell and hence cannot injure another cell. These free receptors constitute, in this case, antitoxin, so-called because they can combine with toxin and hence neutralize it. Antitoxins are specific—that is, an antitoxin which will combine with the toxin of Clostridium tetani will not combine with that of Corynebacterium diphtheriæ or of Clostridium botulinum, or of any other toxin, vegetable or animal.
When a toxin is kept in solution for some time or when it is heated above a certain temperature (different for each toxin) it loses its poisonous character. It may be shown, however, that it is still capable of uniting with antitoxin, and preventing the latter from uniting with a fresh toxin. This confirms the hypothesis that a toxin molecule has at least two groups: a combining or haptophore, and a poisoning or toxophore group. A toxin which has lost its poisonous property, its toxophore group, is spoken of as a toxoid. The theory of antitoxin formation is further supported by the fact that the proper introduction of toxoid, the haptophore group, and hence the real stimulus, can cause the production of antitoxin to a certain extent at least.
The close relationship between toxins and enzymes has already been pointed out. This is still further illustrated by the fact that when enzymes are properly introduced into the tissues of an animal there is formed in the animal an antienzyme specific for the enzyme in question which can prevent its action. The structure of enzymes, as composed of a haptophore, or uniting, and a zymophore or digesting (or other activity) group, is similar to that of toxins, and enzymoids or enzymes which can combine with the substance acted on but not affect it further, have been demonstrated.
These free cell receptors, antitoxins or antienzymes, which are produced in the body by the proper introduction of toxins or enzymes, respectively, have the function of combining with these bodies but no other action. As was pointed out above, this is sufficient to neutralize the toxin or enzyme and prevent any injurious effect since they can unite with nothing else. Since these receptors are the simplest type which has been studied as yet, they are spoken of by Ehrlich as receptors of the first order. Other antibodies which are likewise free receptors of the first, order and have the function of combining only have been prepared and will be referred to in their proper connection. They are mainly of theoretical interest.
Ehrlich did a large part of his work on toxins and antitoxins with ricin, the toxin of the castor-oil bean, abrin, from the jequirity bean, robin from the locust tree, and with the toxins and antitoxins for diphtheria and tetanus. Antitoxins have been prepared experimentally for a large number of both animal and vegetable poisons, including a number for bacterial toxins. The only ones which, as yet, are of much practical importance are antivenin for snake poison, (not a true toxin, however, see [p. 275]), antipollenin (supposed to be for the toxin of hay fever) and the antitoxins for the true bacterial toxins of Corynebacterium diphtheriæ and Clostridium tetani.
The method of preparing antitoxins is essentially the same in all cases, though differing in minor details. For commercial purposes large animals are selected, usually horses, so that the yield of serum may be large. The animals must, of course, be vigorous, free from all infectious disease. The first injection given is either a relatively small amount of a solution of toxin or of a mixture of toxin and antitoxin. The animal shows more or less reaction, increased temperature, pulse and respiration and frequently an edema at the point of injection, unless this is made intravenously. After several days to a week or more, when the animal has recovered from the first injection, a second stronger dose is given, usually with less reaction. Increasingly large doses are given at proper intervals until the animal may take several hundred times the amount which would have been fatal if given at first. The process of immunizing a horse for diphtheria or tetanus toxin usually takes several months. Variations in time and in yield of antitoxin are individual and not predictable in any given case.
After several injections a few hundred cubic centimeters of blood are withdrawn from the jugular vein and serum from this is tested for the amount of antitoxin it contains. When the amount is found sufficiently large (250 “units” at least for diphtheria per cc.)[24] then the maximum amount of blood is collected from the jugular with sterile trocar and cannula. The serum from this blood with the addition of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes “antidiphtheritic serum” or “antitetanic serum,” etc. All sera which are put on the market must conform to definite standards of strength expressed in “units” as determined by the U. S. Hygienic Laboratory. In reality a “unit” of diphtheria antitoxin in the United States is an amount equivalent to 1 cc. of a given solution of a standard diphtheria antitoxin which is kept at the above-mentioned laboratory. This statement, of course, gives no definite idea as to the amount of antitoxin actually in a “unit.” Specifically stated, a “unit” of antitoxin contains approximately the amount which would protect a 250 gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or protect 100 guinea-pigs weighing 250 grams each from one minimum lethal dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is the least amount that will kill a guinea-pig of the size mentioned within four days. Since toxins on standing change into toxoids to a great extent, the amount, of antitoxin in a “unit,” though protecting against 100 M. L. D., in reality would protect against about 200 M. L. D. of toxin containing no toxoid.
The official unit for tetanus antitoxin is somewhat different, since it is standardized against a standard toxin which is likewise kept at the Hygienic Laboratory. The unit is defined as “ten times the amount of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours against the standard test dose” of the standard toxin. The standard test dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To express it another way, one could say that a “unit” of tetanus antitoxin would protect one thousand 350 g. guinea-pigs from 1 M. L. D. each of standard tetanus toxin.
Various methods have been devised for increasing the amount of antitoxin in 1 cc. of solution by precipitating out portions of the blood-serum proteins and at the same time concentrating the antitoxin in smaller volume. It is not considered necessary in a work of this character to enter into these details nor to discuss the process of standardizing antitoxin so that the exact amount of “units” per cc. may be known.