Ehrlich, harmonizes with the facts established with regard to toxic action and the behaviour of antitoxins, and may now be regarded as established. His view as to the dual composition of the toxin molecule has already been mentioned, and it is evident that if the haptophorous or combining group has its affinity satisfied by union with antitoxin, the toxin will no longer combine with living cells, and will thus be rendered harmless. One other important fact in support of what has been stated is that a toxin may have its toxic action diminished, and may still require the same amount of antitoxin as previously for neutralization. This is readily intelligible on the supposition that the toxophorous group is more labile than the haptophorous. There is, however, still dispute with regard to the exact nature of the union of toxin and antitoxin. Ehrlich's view is that the two substances form a firm combination like a strong acid and a base. He found, however, that if he took the largest amount of toxin which was just neutralized by a given amount of antitoxin, much more than a single dose of toxin had to be added before a single dose was left free. For example, if 100 doses of toxin were neutralized by a unit of antitoxin (v. supra) it might be that 125 doses would need to be added to the same amount of antitoxin before the mixture produced a fatal result when it was injected. This result, which is usually known now as the "Ehrlich phenomenon," was explained by him on the supposition that the "toxin" does not represent molecules which are all the same, but contains molecules of different degrees of combining affinity and of toxic action. Accordingly, the most actively toxic molecules will be neutralized first, and those which are left over, that is, uncombined with antitoxin, will have a weaker toxic action. This view has been assailed by Thorvald Madsen and S. A. Arrhenius, who hold that the union of toxin and antitoxin is comparatively loose, and belongs to the class of reversible actions, being comparable in fact with the union of a weak acid and base. If such were the condition there would always be a certain amount both of free toxin and of free antitoxin in the mixture, and in this case also considerably more than a dose of toxin would have to be added to a "neutral mixture" before the amount of free toxin was increased by a dose, that is, before the mixture became lethal. It may be stated that while in certain instances the union of toxin and antitoxin may be reversible, all the facts established cannot be explained on this simple hypothesis of reversible action. Still another view, advocated by Bordet, is that the union of toxin and antitoxin is rather of physical than of strictly chemical nature, and represents an interaction of colloidal substances, a sort of molecular deposition by which the smaller toxin molecule becomes entangled in the larger molecule of antitoxin. Sufficient has been said to show that the subject is one of great intricacy, and no simple statement with regard to it is as yet possible. We are probably safe in saying, however, that the molecules of a toxin are not identical but vary in the degree of their combining affinities, and also in their toxic action, and that, while in some cases the combination of anti-substances has been shown to be reversible, we are far from being able to say that this is a general law.
The origin of antitoxin is of course merely a part of the general Formation of antitoxin. question regarding the production of anti-substances in general, as these all combine in the same way with their homologous substances and have the same character of specificity. As, however, most of the work has been done with regard to antitoxin production we may consider here the theoretical aspect of the subject. There are three chief possibilities: (a) that the antitoxin is a modification of the toxin; (b) that it is a substance normally present, but produced in excess under stimulation of the toxin; (c) that it is an entirely new product. The first of these, which would imply a process of a very remarkable nature, is disproved by what is observed after bleeding an animal whose blood contains antitoxin. In such a case it has been shown that, without the introduction of fresh toxin, new antitoxin appears, and therefore must be produced by the living tissues. The second theory is the more probable a priori, and if established removes the necessity for the third. It is strongly supported by Ehrlich, who, in his so-called "side-chain" (Seitenkette) theory, explains antitoxin production as an instance of regeneration after loss. Living protoplasm, or in other words a biogen molecule, is regarded as consisting of a central atom group (Leistungskern), related to which are numerous secondary atom groups or side-chains, with unsatisfied chemical affinities. "Side-chain" theory. The side-chains constitute the means by which other molecules are added to the living molecule, e.g. in the process of nutrition. It is by means of such side-chains that toxin molecules are attached to the protoplasm, so that the living molecules are brought under the action of the toxophorous groups of the toxins. In antitoxin production this combination takes place, though not in sufficient amount to produce serious toxic symptoms. It is further supposed that the combination being of somewhat firm character, the side-chains thus combined are lost for the purposes of the cell and are therefore thrown off. By the introduction of fresh toxin the process is repeated and the regeneration of side-chains is increased. Ultimately the regeneration becomes an over-regeneration and free side-chains produced in excess are set free and appear in the blood as antitoxin molecules. In other words the substances, which when forming part of the cells fix the toxin to the cells, constitute antitoxin molecules when free in the serum. This theory, though not yet established, certainly affords the most satisfactory explanation at present available. In support of it there is the remarkable fact, discovered by A. Wassermann and Takaki in the case of tetanus, that there do exist in the nervous system molecules with combining affinity for the tetanus toxin. If, for example, the brain and spinal cord removed from an animal be bruised and brought into contact with tetanus toxin, a certain amount of the toxicity disappears, as shown by injecting the mixture into another animal. Further, these molecules in the nervous system present the same susceptibility to heat and other physical agencies as does tetanus antitoxin. There is therefore strong evidence that antitoxin molecules do exist as part of the living substance of nerve cells. It has, moreover, been found that the serum of various animals has a certain amount of antitoxic action, and thus the basis for antitoxin production, according to Ehrlich's theory, is afforded. The theory also supplies the explanation of the power which an animal possesses of producing various antitoxins, since this depends ultimately upon susceptibility to toxic action. The explanation is thus carried back to the complicated constitution of biogen molecules in various living cells of the body. It may be added that in the case of all the other kinds of anti-substances, which are produced by a corresponding reaction, we have examples of the existence of traces of them in the blood serum under normal conditions. We are, accordingly, justified in definitely concluding that their appearance in large amount in the blood, as the result of active immunization, represents an increased production of molecules which are already present in the body, either in a free condition in its fluids or as constituent elements of its cells.
In preparing anti-bacterial sera the lines of procedure correspond Anti-bacterial serum. to those followed in the case of antitoxins, but the bacteria themselves in the living or dead condition or their maceration products are always used in the injections. Sometimes dead bacteria, living virulent bacteria, and living supervirulent bacteria, are used in succession, the object being to arrive ultimately at a high dosage, though the details vary in different instances. The serum of an animal thus actively immunized has powerful protective properties towards another animal, the amount necessary for protection being sometimes almost inconceivably small. As a rule it has no action on the corresponding toxin, i.e. is not antitoxic. In addition to the protective action, such a serum may possess activities which can be demonstrated outside the body. Of these the most important are (a) bacteriolytic or lysogenic action, (b) agglutinative action, and (c) opsonic action.
The first of these, lysogenic or bacteriolytic action, consists in (a) Lysogenic action.
the production of a change in the corresponding bacterium whereby it becomes granular, swells up and ultimately may undergo dissolution. Pfeiffer was the first to show that this occurred when the bacterium was injected into the peritoneal cavity of the animal immunized against it, and also when a little of the serum of such an animal was injected with the bacterium into the peritoneum of a fresh, i.e. non-immunized animal. Metchnikoff and Bordet subsequently devised means by which a similar change could be produced in vitro, and analysed the conditions necessary for its occurrence. It has been completely established that in this phenomenon of lysogenesis there are two substances concerned, one specially developed or developed in excess, and the other present in normal serum. The former (Immunkörper of Ehrlich, substance sensibilisatrice of Bordet) is the more stable, resisting a temperature of 60° C., and though giving the specific character to the reaction cannot act alone. The latter is ferment-like and much more labile than the former, being readily destroyed at 60° C. It may be added that the protective power is not lost by exposure to the temperature mentioned, this apparently depending upon a specific anti-substance. Furthermore, lysogenic action is not confined to the case of bacteria but obtains also with other organized structures, e.g. red corpuscles (Bordet, Ehrlich and Morgenroth), leucocytes and spermatozoa (Metchnikoff). That is to say, if an animal be treated with injections of these bodies, its serum acquires the power of dissolving or of producing some disintegrative effect in them. The development of the immune body with specific combining affinity thus presents an analogy to antitoxin production, the difference being that in lysogenesis another substance is necessary to complete the process. It can be shown that in many cases when bacteria are injected the serum of the treated animal has no bacteriolytic effect, and still an immune body is present, which leads to the fixation of complement; in this case bacteriolysis does not occur, because the organism is not susceptible to the action of the complement. In all cases the important action is the binding of complement to the bacterium by means of the corresponding immune body; whether or not death of the bacterium occurs, will depend upon its susceptibility to the action of the particular complement, the latter acting like a toxin or digestive ferment. It is to be noted that in the process of immunization complement does not increase in amount; accordingly the immune serum comes to contain immune body much in excess of the amount of complement necessary to complete its action. An important point with regard to the therapeutic application of an anti-bacterial serum, is that when the serum is kept in vitro the complement rapidly disappears, and accordingly the complement necessary for the production of the bactericidal action must be supplied by the blood of the patient treated. This latter complement may not suit the immune body, that is, may not be fixed to the bacterium by means of it, or if the latter event does occur, may fail to bring about the death of the bacteria. These circumstances serve, in part at least, to explain the fact that the success attending the use of anti-bacterial sera has been much inferior to that in the case of antitoxic sera.
Another property which may be possessed by an anti-bacterial (b) Agglutination. serum is that of agglutination. By this is meant the aggregation into clumps of the bacteria uniformly distributed in an indifferent fluid; if the bacterium is motile its movement is arrested during the process. The process is of course observed by means of the microscope, but the clumps soon settle in the fluid and ultimately form a sediment, leaving the upper part clear. This change, visible to the naked eye, is called sedimentation. B. J. A. Charrin and G. E. H. Roger first showed in the case of B. pyocyaneus that when a small quantity of the homologous serum (i.e. the serum of an animal immunized against the bacterium) was added to a fluid culture of this bacillus, growth formed a sediment instead of a uniform turbidity. Gruber and Durham showed that sedimentation occurred when a small quantity of the homologous serum was added to an emulsion of the bacterium in a small test-tube, and found that this obtained in all cases where Pfeiffer's lysogenic action could be demonstrated. Shortly afterwards Widal and also Grünbaum showed that the serum of patients suffering from typhoid fever, even at an early stage of the disease, agglutinated the typhoid bacillus—a fact which laid the foundation of serum diagnosis. A similar phenomenon has been demonstrated in the case of Malta fever, cholera, plague, infection with B. coli, "meat-poisoning" due to Gärtner's bacillus, and various other infections. As regards the mode of action of agglutinins, Gruber and Durham considered that it consists in a change in the envelopes of the bacteria, by which they swell up and become adhesive. The view has various facts in its support, but F. Kruse and C. Nicolle have found that if a bacterial culture be filtered germ-free, an agglutinating serum still produces some change in it, so that particles suspended in it become gathered into clumps. E. Duclaux, for this reason, considers that agglutinins are coagulative ferments.
The phenomenon of agglutination depends essentially on the union of molecules in the bacteria—the agglutinogens—with the corresponding agglutinins, but another essential is the presence of a certain amount of salts in the fluid, as it can be shown that when agglutinated masses of bacteria are washed salt-free the clumps become resolved. The fact that agglutinins appear in the body at an early stage in a disease has been taken by some observers as indicating that they have nothing to do with immunity, their development being spoken of as a reaction of infection. This conclusion is not justified, as we must suppose that the process of immunization begins to be developed at an early period in the disease, that it gradually increases, and ultimately results in cure. It should also be stated that agglutinins are used up in the process of agglutination, apparently combining with some element of the bacterial structure. In view of all the facts it must be admitted that the agglutinins and immune bodies are the result of corresponding reactive processes, and are probably related to one another. The development of all antagonistic substances which confer the special character on antimicrobic sera, as well as antitoxins, may be expressed as the formation of bodies with specific combining affinity for the organic substance introduced into the system—toxin, bacterium, red corpuscle, &c., as the case may be. The bacterium, being a complex organic substance, may thus give rise to more than one antagonistic or combining substance.
By opsonic action is meant the effect which a serum has on (c) Opsonic action. bacteria in making them more susceptible to phagocytosis by the white corpuscles of the blood (q.v.). Such an effect may be demonstrated outside the body by making a suitable mixture of (a) a suspension of the particular bacterium, (b) the serum to be tested, and (c) leucocytes of a normal animal or person. The mixture is placed in a thin capillary tube and incubated at 37° C. for half an hour; a film preparation is then made from it on a glass slide, stained by a suitable method and then examined microscopically. The number of bacteria contained within a number of, say fifty, leucocytes can be counted and the average taken. In estimating the opsonic power of the serum in cases of disease a control with normal serum is made at the same time and under precisely the same conditions. The average number of bacteria contained within leucocytes in the case tested, divided by the number given by the normal serum, is called the phagocytic index. Wright and Douglas showed that under these conditions phagocytosis might occur when a small quantity of normal serum was present, whereas it was absent when normal salt solution was substituted for the serum; the latter thus contained substances which made the organisms susceptible to the action of the phagocytosis. They further showed that this substance acted by combining with the organisms and apparently producing some alteration in them; on the other hand it had no direct action on the leucocytes. This opsonin of normal serum is very labile, being rapidly destroyed at 55° C.; that is, a serum heated at this temperature has practically no greater effect in aiding phagocytosis than normal salt solution has. Various observers had previously found that the serum of an animal immunized against
a particular bacterium had a special action in bringing about phagocytosis of that organism, and it had been found that this property was retained when the serum was heated at 55° C. It is now generally admitted that at least two distinct classes of substances are concerned in opsonic action, that thermostable immune opsonins are developed as a result of active immunization and these possess the specific properties of anti-substances in general, that is, act only on the corresponding bacterium. On the contrary the labile opsonins of normal serum have a comparatively general action on different organisms. It is quite evident that the specific immune-opsonins may play a very important part in the phenomena of immunity, as by their means the organisms are taken up more actively by the phagocytic cells, and thereafter may undergo rapid disintegration.
The opsonic action of the serum has been employed by Sir A. Wright and his co-workers to control the treatment of bacterial infections by vaccines; that is, by injections of varying amounts of a dead culture of the corresponding bacterium. The object in such treatment is to raise the opsonic index of the serum, this being taken as an indication of increased immunity. The effect of the injection of a small quantity of vaccine is usually to produce an increase in the opsonic index within a few days. If then an additional quantity of vaccine be injected there occurs a fall in the opsonic index (negative phase) which, however, is followed later by a rise to a higher level than before. If the amounts of vaccine used and the times of the injection are suitably chosen, there may thus be produced by a series of steps a rise of the opsonic index to a high level. One of the chief objects in registering the opsonic power in such cases is to avoid the introduction of additional vaccine when the opsonic index is low, that is, during the negative phase, as if this were done a further diminution of the opsonic action might result. The principle in such treatment by means of vaccines is to stimulate the general production of anti-substances throughout the body, so that these may be carried to the sites of bacterial growth, and aid the destruction of the organisms by means of the cells of the tissues. A large number of favourable results obtained by such treatment controlled by the observation of the opsonic index have already been published, but it would be unwise at present to offer a decided opinion as to the ultimate value of the method.