CHAPTER XXVIII.
RECEPTORS OF THE SECOND ORDER.
AGGLUTININS.
Charrin and Rogers appear to have been the first (1889) to observe the clumping together of bacteria (Pseudomonas pyocyanea) when mixed with the blood serum of an animal immunized against them. Gruber and Durham (1896) first used the term “agglutination” in this connection and called the substance in the blood-serum “agglutinin.” Widal (1896) showed the importance of the reaction for diagnosis by testing the blood serum of an infected person against a known culture (typhoid fever).
It is now a well-known phenomenon that the proper injection of cells of any kind foreign to a given animal will lead to the accumulation in the animal’s blood of substances which will cause a clumping together of the cells used when suspended in a suitable liquid. The cells settle out of such suspension much more rapidly than they would otherwise do. This clumping is spoken of as “agglutination” and the substances produced in the animal are called “agglutinins.” If blood cells are injected then “hemagglutinins” result: if bacterial cells “bacterial agglutinins” for the particular organism used as “glanders agglutinin” for Pfeifferella mallei, “abortion agglutinin” for Bacterium abortus, “typhoid agglutinin” for Bacterium typhosum, etc.
The phenomenon may be observed either under the microscope or in small test-tubes, that is, either microscopically or macroscopically.
In this case the cells introduced, or more properly, some substances within the cells, act as stimuli to the body cells of the animal injected to cause them to produce more of the specific cell receptors which respond to the stimulus. The substance within the introduced cell which acts as a stimulus (antigen) to the body cells is called an “agglutinogen.” That “agglutinogen” is present in the cell has been shown by injecting animals experimentally with extracts of cells (bacterial and other cells) and the blood serum of the animal injected showed the presence of agglutinin for the given cell. It will be noticed that the receptors which become the free agglutinins have at least two functions, hence at least two chemical groups. They must combine with the foreign cells and also bring about their clumping together, their agglutination. Hence it can be stated technically that an agglutinin possesses a haptophore group and an agglutinating group.
It is probable that the agglutination, the clumping, is a secondary phenomenon depending on the presence of certain salts and that the agglutinin acts on its antigen as an enzyme, possibly a “splitting” enzyme. This is analogous to what occurs in the curdling of milk by rennet and in the coagulation of blood. This probability is substantiated by the fact that suspensions of bacteria may be “agglutinated” by appropriate strengths of various acids.
The formation of agglutinin in the body for different bacteria does not as yet appear to be of any special significance in protecting the animal from the organism, since the bacteria are not killed, even though they are rendered non-motile, if of the class provided with flagella, and are clumped together. The fact that such bodies are formed, however, is of decided value in the diagnosis of disease, and also in the identification of unknown bacteria.
In many bacterial diseases, agglutinins for the particular organism are present in the blood serum of the affected animal. Consequently if the blood serum of the animal be mixed with a suspension of the organism supposed to be the cause of the disease and the latter be agglutinated, one is justified in considering it the causative agent, provided certain necessary conditions are fulfilled. In the first place it must be remembered that the blood of normal animals frequently contains agglutinins (“normal agglutinins”) for many different bacteria when mixed with them in full strength. Hence the serum must always be diluted with physiological salt solution (0.85 per cent.). Further, closely related bacteria may be agglutinated to some extent by the same serum. It is evident that if they are closely related, their protoplasm must contain some substances of the same kind to account for this relationship. Since some of these substances may be agglutinogens, their introduction into the animal body will give rise to agglutinins for the related cells, as well as for the cell introduced. The agglutinins for the cell introduced “chief agglutinins,” will be formed in larger quantity, since a given bacterial cell must contain more of its own agglutinogen than that of any other cell. By diluting the blood serum from the animal to be tested the agglutinins for the related organisms (so-called “coagglutinins” or “partial agglutinins”) will become so much diminished as to show no action, while the agglutinin for the specific organism is still present in an amount sufficient to cause its clumping. Agglutinins are specific for their particular agglutinogens, but since a given blood serum may contain many agglutinins, the serum’s specificity for a given bacterium can be determined only by diluting it until this bacterium alone is agglutinated. Hence the necessity of diluting the unknown serum in varying amounts when testing against several known bacteria to determine for which it is specific, i.e., which is the cause of the disease in the animal.
The agglutinins in the serum may be removed from it by treating it with a suspension of the cells for which agglutinins are present. If the “chief” cell is used all the agglutinins will be absorbed. If related cells are used, only the agglutinins for this particular kind are removed. These “absorption tests” furnish another means of determining specificity of serum, or rather of determining the “chief agglutinin” present.
Just as an unidentified disease in an animal may be determined by testing its serum as above described against known kinds of bacteria, so unknown bacteria isolated from an animal, from water, etc., may be identified by testing them against the blood sera of different animals, each of which has been properly inoculated with a different kind of known bacteria. If the unknown organism is agglutinated by the blood of one of the animals in high dilution, and not by the others, evidently the bacterium is the same as that with which the animal has been inoculated, or immunized, as is usually stated. This method of identifying cultures of bacteria is of wide application, but is used practically only in those cases where other methods of identification are not readily applied, and especially where other methods are not sufficient as in the “intestinal group” of organisms in human practice.
The diagnosis of disease in an animal by testing its serum is also a valuable and much used procedure. This is the method of the “Widal” or “Gruber-Widal” test for typhoid fever in man and is used in veterinary practice in testing for glanders, contagious abortion, etc. In some cases a dilution of the serum of from 20 to 50 times is sufficient for diagnosis (Malta fever), in most cases, however, 50 times is the lowest limit. Evidently the greater the dilution, that is, the higher the “titer,” the more specific is the reaction.
PRECIPITINS.
Since agglutinins act on bacteria, probably through the presence of substances within the bacterial cell, it is reasonable to expect that if these substances be dissolved out of the cell, there would be some reaction between their (colloidal) solution and the same serum. As a matter of fact Kraus (1897) showed that broth cultures freed from bacteria by porcelain filters do show a precipitate when mixed with the serum of an animal immunized against the particular bacterium and that the reaction is specific under proper conditions of dilution. It was not long after Kraus’s work until the experiments were tried of “immunizing” an animal not against a bacterium or its filtered culture, but against (colloidal) solutions of proteins, such as white of egg, casein of milk, proteins of meat and of blood serum, vegetable proteins, etc. It was ascertained that in all these cases the animal’s serum contains a substance which causes a precipitate with solutions of the protein used for immunization. The number of such precipitating serums that have been made experimentally is very large and it appears that protein from any source when properly introduced into the blood or tissues of an animal will cause the formation of a precipitating substance for its solutions. This substance is known, technically as a “precipitin.” The protein used as antigen to stimulate its formation, or some part of the protein molecule (haptophore group), which acts as stimulus to the cell is spoken of as a “precipitinogen,” both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific precipitation and agglutination are strictly analogous phenomena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on substances within cells which cells are in suspension in a fluid and cause the cells to settle out. Ehrlich’s theory of the formation of precipitins is similar to that of agglutinins, and need not be repeated. Substitute the corresponding words in the theory of formation of agglutinins as above given and the theory applies.
The precipitin reaction has not found much practical use in bacteriology largely because the “agglutination test” takes its place as simpler of performance and just as accurate. The reaction is, however, generally applicable to filtrates of bacterial cultures and could be used if needed. The so-called “mallease” reaction in glanders is an instance.
Precipitins find their greatest usefulness in legal medicine and in food adulteration work. As was noted above, if animals, rabbits for example, are immunized with the blood of another animal (human beings) precipitins are developed which are specific for the injected blood with proper dilution. This forms an extremely valuable means of determining the kind of blood present in a given spot shown by chemical and spectroscopic tests to be blood and has been adopted as a legal test in countries where such rules of procedure are applied. Similarly the test has been used to identify the different kinds of meat in sausage, and different kinds of milk in a mixture. An extract of the sausage is made and tested against the serum of an animal previously treated with extract of horse meat, or hog meat, or beef, etc., the specific precipitate occurring with the specific serum. Such reactions have been obtained where the protein to be tested was diluted 100,000 times and more. Biological relationships and differences have been detected by the reaction. Human immune serum shows no reaction with the blood of any animals except to a slight extent with that of various monkeys, most with the higher, very slight with the lower Old World and scarcely any with New World monkeys.
It is a fact of theoretical interest mainly that if agglutinins and precipitins themselves be injected into an animal they will act as antigens and cause the formation of antiagglutinins or antiprecipitins, which are therefore receptors of the first order since they simply combine with these immune bodies to neutralize their action, have only a combining or haptophore group. Also if agglutinins or precipitins be heated to the proper temperature they may retain their combining power but cause no agglutination or precipitation, i.e., they are converted into agglutinoid or precipitinoid respectively after the analogy of toxin and toxoid.
Precipitins like agglutinins possess at least two groups—a combining or haptophore group and a precipitating (sometimes called zymophore) group. Hence they are somewhat more complex than antitoxins or antienzymes which have a combining group only. For this reason Ehrlich classes agglutinins and precipitins as receptors of the second order.