Paul Ehrlich’s Side-Chain Antibody Theory (1900) Part 2: The Complement System

For of course it would be absurd to imagine that the mechanical diagrams have any representation in the world of fact. They are figments of the imagination, and may serve some useful purpose as picture books serve in teaching a child the alphabet. But as the time comes when the child puts aside the blocks and takes in hand the pen, so pathologists must ultimately lay aside the crude mechanism of haptophores and amboceptors and learn to deal with the phenomena of immunity in terms of the protein molecule and the chemical atom.

Williams and Beveridge, “Mechanism of Immunization” (cit. n. 27), pp. 623-624.

In part one of Paul Ehrlich’s address to the Royal Society, we looked at the outline for his “Side-Chain” theory of immunity. Ehrlich laid out the main components of his vision such as the side-chains, the haptophore group, the toxophore group, the antigen-antibody relationship, the lock and key mechanism, and the “tentacles” aiding in digestion. He built his concepts off of Emil Von Behring’s diphtheria toxin research and the suggestion of something present in the blood. From there, Ehrlich abandoned the law of parsimony and used his “lively imagination” to dream up what he considered to be the most plausible and easy explanation for immunity.

This last part of Ehrlich’s presentation to the Royal Society centered around the idea of the complement system. In short, this is a part of the immune system that complements the ability of antibodies to clear out the damaged cells and the inhabiting microbes through inflammation in order to attack the pathogen and eliminate it from the body. Here is a brief overview of Ehrlich’s role in the creation of this theory:

The complement system: history, pathways, cascade and inhibitors

“Paul Ehrlich described the side-chain theory of antibody formation, especially the mechanisms of antibody neutralisation by toxins that induced bacterial lysis with the help of complement (which has replaced the historical term alexin). According to his theory, the immune cells contained receptors that could recognise antigens, and following immunisation, these receptors multiplied and were shed into the circulation as ‘amboceptors’ (now called antibodies). These antibodies attached not only to specific antigens but also to a heat-labile antimicrobial component called ‘complement’ [8, 9]. Ehrlich’s theory proposed that the antibody and complement combined to form a complex enzyme capable of attacking and killing cells and micro-organisms. In the ensuing years, this concept had a protagonist in the form of Bordet who argued that the antigen-antibody union was reversible, contradicting Ehrlich’s view that the antigen-antibody union was a firm and based on stereo chemical specificity [10]. Ehrlich’s concept emphasised the presence of multiple antigens and complements in the serum, while Bordet’s view revolved around a ‘single complement’ component that bound non-specifically to the antigen.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956958/

It looked as if there was a war brewing in the early 20th century between Paul Ehrlich and Jules Bordet’s competing antibody theories. While Ehrlich envisioned antibodies and their complements that were specific to the antigen, Bordet viewed the relationship as non-specific. Fortunately, as virology and Immunology love to have their cake and eat it too, both Ehrlich and Bordet were deemed correct when it was “understood” that the complements can be both specific and non-specific, thus averting the war:

“Ehrlich believed that each antigen-specific amboceptor had its own specific complement, while Bordet believed that there is only one type of complement. In the early 20th century, this controversy was resolved when it was understood that complement can act in combination with specific antibodies, or on its own in a non-specific way.”

https://www.bionity.com/en/encyclopedia/Complement_system.html

Below are the rest of the highlights from the remainder of Ehrlich’s report on his theoretical investigation into immunity which details the complement system. I provided some additional insight afterwards regarding how this theory was utilized and the controversy surrounding Ehrlich’s “beautfil drawings:”

On Immunity with Special Reference to Cell Life.

“I have now laid before you the fundamental facts which up to the present constitute our knowledge in the field pertaining to immunity,
and which can be most easily and successfully explained through the agency of “the side-chain theory.” I wish in a few words to dispel some erroneous ideas which have been advanced in opposition to this theory.

Roux has shown that very small quantities of tetanus toxine, if injected directly into the brain, cause the death of the animal. Roux assumes that such an occurrence is not compatible with my theory. Roux is of opinion that according to my theory the brain must be quite immune against tetanus toxine, as the toxophile side-chains of the brain-cells must be identical with the antitoxine, and therefore must exercise an immediate protective action. Experiment showing quite the reverse, the theory is overthrown.

Roux came to this incorrect conception through an erroneous conception of antitoxine. The toxophile side-chains of the brain cells draw directly to themselves the toxine molecules, and, according to my theory, are thus a necessary preliminary condition of the illness. The toxophile groups are therefore really inducers of the action of the poison, and not its preventives.

Those toxophile groups which, like the antitoxines present in the serum, are able to lay hold of toxine immediately on its entry into the blood, and so to divert it from organs essential to life, can alone be regarded as being possessed of any antitoxic action in the true sense of the word. I may be allowed to call to mind Weigert’s excellent simile of iron and the lightning conductor. Iron attracts electricity, and is therefore used as a lightning conductor. Great masses of iron present in buildings give rise to, or increase, the risk of their being struck by lightning, and the metal only becomes protective against lightning when it is so employed that the electricity is conducted away outside the building. It would never occur to anyone to speak of great masses of iron machinery present in buildings as if they were lightning conductors. It is equally unreasonable to speak of
the antitoxic property of the brain cortex, in which the toxophile groups are present in great quantity, but also retain their relations with the nerve-cells. When this really considerable misunderstanding is eliminated from Roux’s results these become entirely confirmatory of my views, and it is difficult to understand how, subsequent to Weigert having placed the matter in so clear a light, the beautiful experiments of Roux can be utilised by another eminent authority as a means of combating my theory.

Much more complex than in the cases hitherto discussed are the conditions when, instead of the relatively simple metabolic products of microbes, the living micro-organisms themselves come to be considered, as in immunisation against cholera, typhoid, anthrax, swine fever, and many other infectious diseases. There then come into existence alongside of the antitoxines, produced as a result of the action of the toxines, manifold other reaction products. This is because the bacterium is a highly complicated living cell, of which the solution in the organism yields a great number of bodies of different nature, in consequence of which a multitude of “Antikorper” are called into existence. Thus we see, as a result of the injection of bacterial cultures, that there arise alongside of the specific bacteriolysines, which dissolve the bacteria, other products, as, for example, “coagulines” (Kraus, Bordet), i.e., substances which are able to cause the precipitation of certain albuminous bodies contained in the culture fluid injected; also the so-much discussed “agglutinines”
(Durham, Gruber, Pfeiffer), the antiferments (von Dungern), and no doubt many other bodies which we have not yet recognised.

It is by no means unlikely that each of these reaction products finds its origin in special cells of the body; on the other hand, it is quite likely that the formation of any single one of these bodies is not of itself sufficient to confer immunity. Thus in case of the introduction of bacteria into the body we have to do with a many-sided production of different forms of “Antikorper,” each of which is directed only against one definite quality or metabolic product of the bacterial cell. Accordingly, in recent times, the practice of using for the production of immunisation definite toxic bodies isolated from the bacterial cells has been more and more given up, and for this purpose it is now regarded as important to employ the bacterial cells as intact as possible. The beautiful results obtained for plague by Haffkine, and quite recently by Wright in your own country for typhoid fever, have been arrived at in this way.

The most interesting and important substances arising during such an immunising process are without doubt the bacteriolysines, in the investigation of which Pfeiffer has done such yeoman’s service. How really wonderful it is that after the introduction of the cholera-vibrio into the animal body a substance is formed endowed with the power of dissolving the cholera vibrio, and that vibrio only!

This seemingly purposeful and novel phenomenon seems at first sight to have nothing to do with those forces which are normally at the disposal of the organism. It was of the greatest importance to explain the origin of these substances from the standpoint of cellular physiology. The solution offered very considerable difficulties, and was first attained when instead of bacteriolysines, hsemolysines came to be employed in experiments. Hsemolysines are peculiar toxic bodies, which destroy red blood corpuscles by dissolving them. Hsemolysines may occur in a normal blood when they exercise a solvent action on the red blood corpuscles of other species, or they may be artificially produced, in which case, after an animal has undergone a process of immunisation against the blood corpuscles of another species, there appear in the serum hsemolysines which destroy the kind of blood corpuscles employed in the production of the immunity. In their essential characters they are absolutely comparable with the bacteriolysines: but they possess over them the great advantage that they admit of being employed in test-tube experiments, and thus afford opportunity for exact quantitative work altogether independent of the variability of the animal body.

Belfanti and Carbone first discovered the remarkable fact that horses which have been treated with the blood corpuscles of rabbits contain in their serum constituents which are poisonous for the rabbit, and for the rabbit only. While the serum of the normal horse, to the quantity of 60 c.c., could be intravenously injected without harm to the rabbit, a very few c.c. of serum from horses previously so treated with rabbit’s blood, proved fatal.

Bordet showed shortly thereafter, that in the case quoted there was present in the serum a specific hsemolysine which dissolved the blood corpuscles of the rabbit. He also proved that these hsemolysines as had already been shown by Buchner and Daremberg in the case of similarly acting bodies which are present in normal blood lost their solvent property on being maintained during half an hour at a temperature of 55° C. Bordet added, further, the new fact, that the blood-solvent property of these sera which had been deprived of solvent power by heat, the solvent action could be restored if certain normal sera were added to them.

By this important observation an exact analogy was established with the facts of bacteriolysis as elicited by the work of Pfeiffer, Metchnikoff, and Bordet. In the work on the Pfeiffer phenomenon of bacteriolysis, it had already been ascertained that the solution of bacteria by specific bacteriolysines was brought about by the combined action of two different bodies: one which was specific, arose during the immunisation and was stable; and another, a very unstable body, which was present in normal serum.

In collaboration with Dr. Morgenroth, I have sought in regard to this question, for which haemolysis offered prospects favourable to experimentation, to make clear the mechanism concerned in the action of these two components—the stable, which may be designated “immune body,” and the unstable, which may be designated “complement”—which, acting together, effect the solution of the red blood corpuscles. For this purpose, in the first place, solutions containing either only the “immune body” or only the “complement” were brought in contact with suitable blood corpuscles, and after separation of the fluid and the corpuscles by centrifugalising, we investigated whether these substances had been taken up by the red blood corpuscles or remained behind in the fluid. The proof of its location in the one position or in the other was readily forthcoming, since to restore to the hsemolysine its former activity, it was only necessary to add to the “immune body” a fresh supply of “complement,” or to the “complement” a fresh supply of “immune body,” in order that the presence of the hsemolysine in its integrity might be shown by the occurrence of solution of the blood-cells.

The experiments proved that, after centrifugalising, the “immune body” is quantitatively bound to the red blood corpuscles, and that the “complement,” on the contrary, remains entirely behind in the fluid. The presence of the two components in contact with blood corpuscles only occasions the solution of these at higher temperatures, and not at 0° C. And an active haemolytic serum (with “immune body” and “complement” both present) having been placed in contact with red blood corpuscles and maintained for a while at 0° C., it was found after centrifugalising that, under these circumstances also, the “immune body” had united with the red blood corpuscles, but that the “complement” remained in the serum. This experiment showed that both components must, at a temperature of 0° C., have existed alongside of one another in a free condition.

But when analogous experiments were undertaken at a higher temperature it was found that both components were retained in the sediment.

These facts can only be explained by making certain assumptions regarding the constitution of the two components, i.e., of the “immune body” and the “complement.” In the first place, two haptophore groups must be ascribed to the “immune body,” one having a great affinity for a corresponding haptophore group of the red blood corpuscles and with which at lower temperatures it quickly unites, and another haptophore group of a lesser chemical affinity, which at a higher temperature becomes united with the “complement” present in the serum. Therefore, at the higher temperature, the red blood corpuscles will draw to themselves those molecules of the “immune body” which in the fluid have previously become united with the “complement.” In this case the “immune body” represents in a measure the connecting chain which binds the complement to the red blood corpuscles, and so brings them under its deleterious influence. Since under the influence of the “complement”—at least, in the case of the bacteria— appearances are to be observed (for example, in the Pfeiffer phenomenon) which must be regarded as analogous to digestion, we shall not seriously err if we ascribe to this “complement” a ferment-like character.

It is obvious that when the normal serum of one animal possesses haemolytic action on the blood of another, the component of the hsemolysine which here unites with the red blood corpuscle and forms the connecting link between it and the “complement” which is essential to the occurrence of solution, cannot, in the absence of any preceding process of immunisation, be designated “immune body.” In its
characteristics and action, however, it only differs from this in occurring naturally, and may well be designated “intermediate body” (Zwischenkorper). It may here be stated that the constitution of a haemolysine is graphically represented in fig. 7, Plate 7.

Very important for the conclusion that only with the assistance of the
“intermediate body” or of the “immune body” can the “complement,” which leads to the solution, become united with the blood corpuscle, is the following experiment. The serum of the dog has very considerable solvent action upon guinea-pig’s blood, but loses this property if warmed. If dog’s serum, thus rendered inactive by warming, is brought into contact with suspended corpuscles of guinea-pig’s blood, these are not dissolved; but, if to such a mixture there be also added guinea-pig serum, i.e., the serum normal to these red blood corpuscles,
the erythrocytes are at once dissolved. Here the only explanation is that the “intermediate body,” which possesses a specific affinity for guinea-pig erythrocytes, and is present in the inactive dog’s serum, is able to seize on one of the many “complements” present in guinea-pig’s serum, with the result that the “complement” which cannot normally attach itself to the corpuscles, comes now to exercise its destructive influence.

We see at the same time from this experiment that the hsemolysines occurring naturally, obey the same laws as those produced through the process of immunising. In fact, for them also, in a great number of instances, precisely similar behaviour has been demonstrated.

The character of the specific union made it possible to find solutions for a number of important questions. In the first place, regarding the multiplicity of the heemolysines, which occur normally in serum, it is well known that numerous sera are able to dissolve blood corpuscles of different species. For example, serum of the dog dissolves blood corpuscles of the rabbit, guinea-pig, rat, goat, sheep, &c. The complex nature of these haemolysines has been already indicated.

Another question arises whether in a serum that is capable of such manifold action there is present one single haemolysine that destroys different red blood-cells, or whether a whole series of hsemolysines come into action, of which one is adapted to guinea-pig blood, another to rabbit blood, &c. The solution of this question may be approached in another way. The serum may be rendered inactive by heat, and then placed in contact with red blood corpuscles of a given kind. Then, supposing, for example, that rabbit blood has been employed, it is found that if the fluid is freed from the erythrocytes by centrifugalisation and the “complement” afterwards added, it is no longer in a position to dissolve rabbit blood, but has not suffered any impairment of its action on other kinds.

By this method of elective absorption it is proved that the normally occurring hsemolysines which chain the blood corpuscles of the rabbit to themselves, are specifically adapted to this purpose. If with suitable adjustment of conditions similar experiments be conducted with other kinds of blood, results are obtained which force us to the conviction that in such a serum acting on various kinds of blood there are present absolutely different “intermediate bodies” (analogues of the “immune bodies”), of which each one is specific for one kind of blood, i.e., one is adapted for rabbit’s blood, a second for calf’s blood, &c. Dr. Morgenroth and I have in some cases, indeed, succeeded in proving that the “complements” which are adapted to fit themselves to these “intermediate bodies,” and occur in normal sera, differ among themselves. If we reflect that in normal blood, in addition to these different hsemolysines, there are besides a long series of analogous bodies, agglutinines of very different kinds, bacteriolysines, enzymes, anti-enzymes, we are brought more and more to the conviction that the blood serum is the carrier of substances innumerable as yet little known or conceived of.

Having obtained a precise conception of the method of action of the lysines of the serum—of the hsemolysines, and thereby also of the bacteriolysines—it becomes possible for us to attempt to solve the mystery of the origin of these bodies. I have in the beginning of this lecture fully developed the “side-chain theory,” according to which the antitoxines are merely certain of the protoplasm “side-chains,” which have been produced in excess and pushed off into the blood.

The toxines, as secretion products of cells, are in all likelihood still relatively uncomplicated bodies; at least, by comparison with the primary arid complex albumins of which the living cell is composed. If a cell of the organism has, with the assistance of an appropriate “side-chain,” fixed to itself a giant molecule, as the proteid molecule really is, then, with the fixation of this molecule, there, is provided one of the conditions essential for the cell nourishment. Such giant molecules cannot at first be utilised by the cells, and are only made available when, by means of a ferment-like process, they are split into smaller fragments. This will be very effectually attained if, figuratively speaking, the “tentacle” or grappling arm of the protoplasm possesses a second haptophore group adapted to take to itself ferment-like material out of the blood fluid. Through such complex organisation, by which the “tentacle” acts also as the bearer of a ferment-functioning group, this group is brought into close relation with the prey destined to be digested and assimilated.

For such appropriate arrangements, in which the “tentacular” apparatus also exercises a digestive function—if it be permissible to pass from the abstract to the concrete—we find analogies in the different forms of insectivorous plants. Thus it has been known since the famous researches of Darwin that the tentacles of Drosera secrete a proteid-digesting fluid.

If we now recognise that the different lysines only arise through absorption of highly complex cell material—such as red blood corpuscles or bacteria—then the explanation, in accordance with what I have said, is that there are present in the organism “side-chains” of a special nature, so constituted that they are endowed not only with an atomic group by virtue of the affinities of which they are enabled to pick up material, but also with a second atomic group, which, being ferment-loving in its nature, brings about the digestion of the material taken up. Should the pushing-off of these “side-chains” be forced, as it were, by immunisation, then the “side-chains” thus set free must possess both groups, and will therefore in their characteristics entirely correspond to what we have placed beyond doubt as regards the “immune-body” of the hsemolysine.

In this manner is simply and naturally explained the astonishingly specialised arrangement that, through the introduction of a definite bacterium into the body, something is produced which is endowed with the power of destroying by solution the bacterium which was administered and no other. This contrivance of the organism is to be regarded as nothing more than a repetition of a process of normal cell-life, and the outcome of primitive wisdom on the part of the protoplasm.

In conclusion, I wish hastily to touch on only a few points. First, to direct attention to the fact that the immunising sera produced by the
administration of bacteria are sometimes limited in their operation to certain animal species, and are much more inconstant in their action than are the antitoxines. Sobernheim, in the laboratory of C. Fraenkel, found that the anthrax serum obtained by immunising German marmots (Hamster) protected this species, even in small doses; but was absolutely without action for rabbits. Kitt had a precisely similar experience with symptomatic anthrax. This circumstance is easy to understand, if the complex nature of the lysines be borne in mind. The lysine, be it bacteriolysine or hsemolysine ( i.e. “immune body + “complement”), possesses altogether three haptophore groups, of which two belong to the “immune-body” and one to the “complement.” Each one of these haptophore groups can be bound by an appropriate “anti-group.” Three anti-groups are thus conceivable, any one of which, by uniting with one of the haptophore groups of the lysine, can frustrate the action of the lysine. To my mind, of these three possible “Antikorper,” that one which can lay hold of the haptophore group of the “complement,” and so prevent this from uniting with the “immune body,” is the most important. Dr. Morgenroth and I have experimentally succeeded in producing such bodies by processes of immunisation, and in proving that they unite with the “complement” (anticomplement).

Dr. Neisser at the Steglitz Institute sought to find an explanation of Sobernheim’s experiments. He was able to determine that anthrax serum failed in mice, even if great quantities of fresh sheep’s serum (i.e., containing excess of “complement”) were at the same time introduced. The failure in this case appears to be due, on the one hand, to the destruction, in the body of the mouse, of the “complement” present in the sheep’s serum, and, on the other hand, to the fact that the “immune body” yielded, by the sheep does not find in mouse serum an appropriate new “complement.”

From this it appears, that in the therapeutic application of anti-bacterial sera to man, therapeutical success is only to be attained if we use either a bacteriolysine with a “complement” which is stable in man (“homostabile complement”), or at least a bacteriolysine, the “immune body” of which finds in human serum an appropriate “complement.” The latter condition will be the more readily fulfilled the nearer the species employed in the immunisation process is to man. Perhaps the non-success which as yet has attended the employment of typhoid and cholera serum will be converted into the contrary if the serum be derived from apes and not taken from species so distantly removed from man as the horse, goat, or dog. However this may be, the question of the provision of the appropriate “complement” will come more and more into the foreground, for it really represents the centre round which the practical advancement of bacterial immunity must turn.

A second and at present much-discussed question is the immunising of the organism against elements standing biologically much higher in the scale than erythrocytes and much less foreign to the body than those exceedingly lowly organisms, the bacteria. I refer here to the production of “Antikorper” against cells of the higher animal organisation, e.y., ciliated epithelium (v. Dungern), spermatozoa (Landsteiner, Metchnikoff, Moxter), kidney cells, and leucocytes. These “Antikorper” are also of a complex nature. They obey the already described law of elective absorption, and their origin is in keeping with the “side-chain” theory. It is to be hoped that such immunisations as these, which are of great theoretical interest, may also come to be available for therapeutic application. The idea has already been mooted by v. Dungern, of attacking epithelial new formations, particularly carcinoma, by means of specific “antiepithelial sera,” and Metchnikoff’ has expressed the somewhat bold hope of being able to delay old age by means of a serum directed against phagocytes
(macrophages). But even if in the immediate future no great practical success is attained, we must remember that we are only at the very beginning of a rational investigation of properties of cells which hitherto have been far too lightly regarded.

The sifting of the material obtained by observation is rendered more difficult by the occurrence under normal conditions of a great number of quite unlooked for bodies furnished with haptophore groups and arising from diverse organs, and which we may designate collectively as haptines. It is to be expected that the study of these haptines will not only throw light on the more minute details of cellular metabolism, but also prove fruitful in the fields of pathology and therapeutics. By the fact that we can cause the individual haptines of the cells to pass out into the blood serum by a process of specific immunisation, it becomes possible in the test-tube to analyse more accurately the mode of operation of their binding groups than is possible in the case of the complicated conditions which present themselves in the animal body. The importance, for the study of immunity, of considering the circumstances from a purely cellular standpoint is evident from all that I have said.

I trust, my lords and gentlemen, that from what I have said you may have obtained the impression, to allude again to my quotation from Bacon, that we no longer find ourselves lost on a boundless sea, but that we have already caught a distinct glimpse of the land which we hope, nay, which we expect, will yield rich treasures for biology and therapeutics.

I desire to express my indebtedness to Dr. E. F. Bashford, McCosh Scholar of the University of Edinburgh, now working with me in my Institute, for his kindness in undertaking the translation of my lecture into English, a task to which he has devoted much time and trouble.

https://doi.org/10.1098/rspl.1899.0121

This process of binding serum complement was yet another indirect method used in an attempt to prove antibodies exist. If a reaction occurs, it is assumed the antibody-antigen exist in the mixture. The complement system described by Ehrlich formed the basis for the complement fixation test which has been used as an indirect method to discover “novel viruses” and/or to determine someone positive for a known “virus.” You can find this test used in numerous virology papers as “evidence” that a new “virus” exists as well as whether or not it is related to other “viruses.” Here is a brief description of what this test entails:

Complement Fixation

“Complement fixation is a classic method for demonstrating the presence of antibody in patient serum. The complement fixation test consists of two components. The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum. When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause the red blood cells to lyse.

The second component is a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement. These two components of the complement fixation method are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from the patient serum and known antigens, it is available to bind to the indicator system of sheep cells and anti-sheep antibody. Lysis of the indicator sheep red blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test. If the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis.”

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/12%3A_Immunology_Applications/12.2%3A_Immunoassays_for_Disease/12.2G%3A_Complement_Fixation

If you are wondering how the combining of human, sheep, and guinea pig blood proves not only the presence of antibodies but also a “virus,” you are not alone. This lab-created concoction has no relevance to reality whatsoever. There are even some noted drawbacks for the complement fixation test beyond the combining of human blood with that from sheep/guinea pigs:

Disadvantages of Complement Fixation Test

1. Not sensitive – cannot be used for immunity screening.
2. Time-consuming.
3. Often non-specific e.g. cross-reactivity between Herpes Simplex Virus and Voricella Zoster Virus.

Complement Fixation Test- Steps, Advantages and Disadvantages

As can be seen, the complement fixation test, based on the imaginary story as proposed by Ehrlich, is not sensitive nor is it specific and often leads to cross-reactivity with other “viruses.” Sensitivity and specificity are the two most important criteria in determining whether a test is accurate or not:

What are sensitivity and specificity?

“Whenever we create a test to screen for a disease, to detect an abnormality or to measure a physiological parameter such as blood pressure (BP), we must determine how valid that test is—does it measure what it sets out to measure accurately? There are lots of factors that combine to describe how valid a test is: sensitivity and specificity are two such factors. We often think of sensitivity and specificity as being ways to indicate the accuracy of the test or measure.

In the clinical setting, screening is used to decide which patients are more likely to have a condition. There is often a ‘gold-standard’ screening test—one that is considered the best to use because it is the most accurate. The gold standard test, when compared with other options, is most likely to correctly identify people with the disease (it is specific), and correctly identify those who do not have the disease (it is sensitive). When a test has a sensitivity of 0.8 or 80% it can correctly identify 80% of people who have the disease, but it misses 20%. This smaller group of people have the disease, but the test failed to detect them—this is known as a false negative. A test that has an 80% specificity can correctly identify 80% of people in a group that do not have a disease, but it will misidentify 20% of people. That group of 20% will be identified as having the disease when they do not, this is known as a false positive. See box 1 for definitions of common terms used when describing sensitivity and specificity.”

https://ebn.bmj.com/content/23/1/2

How can a test be used when it is neither sensitive nor specific? Obviously, without these two components, the complement fixation test is not an accurate meaaure of anything whatsoever. The reason for this lack of accuracy is that in order to know which antibody reacts and is specific for a “virus” so that a test can be used to be able to detect it, one would have to have first purified/isolated an actual “virus” directly from a human sample. Without this, sensitivity and specificity can not be concluded. This applies to antibodies themselves as before antibody tests could be considered accurate, antibodies must also be purified and isolated from human fluids. As this has never been done for either “viruses” nor antibodies, the sensitivity and specificity will remain unknown and the tests will remain inaccurate.

This is the problem with basing tests on theoretical concepts and experimental reactions. To date, the antibody-antigen relationship is still nothing but an unproven theory conjured up in the mind of a man with a “lively imagination.” The existence of these invisible entities is still in doubt as they have never been properly purified nor isolated. Ehrlich did nothing more than create a story about what he believed occurred inside of lab-created chemical reactions, which even he himself apparently did not believe. This was brilliantly summarized in the highlights from the following 1993 paper:

Ehrlich’s “Beautiful Pictures” and the Controversial Beginnings of Immunological Imagery

ARE “ANTIBODIES” MATERIAL SUBSTANCES?

“The term antibody today refers to discrete biochemical entities present in the blood. The belief that antibodies are such entities is held not only by scientists and physicians, but also by the lay public, who learns about “antibodies,” if not in school, then at least in the course of routine medical practices such as vaccination and, increasingly, through the so-called popularization of science. In addition, some people will readily associate the term antibody with the characteristic Y-shaped structure found not only in textbooks and specialized scientific articles but also, more recently, in advertisements and even as the logos of pharmaceutical and biotechnology companies. In order to understand the beginnings of immunological imagery at the turn of the century, we have to discard our present-day notions about the reality and structure of antibodies.

Indeed, it is relatively easy to slide into anachronism. For instance, it is now commonplace to claim that in 1890 Emil von Behring and Shibasaburo Kitasato discovered “antitoxins” and further to describe this discovery as one of the inaugurating events of humoral immunology, and thus of antibody research. Even such a sophisticated historian as Arthur Silverstein speaks of this landmark contribution as the “discovery of antitoxic antibodies.” However, as Jean Lindenmann has pointed out, Behring and Kitasato carefully avoided the use of the term antitoxins, which they considered inappropriate, precisely because they regarded their findings as pointing to the action and properties of sera, and not to the presence of a substance. The contemporaries of Behring and Kitasato did not miss this distinction. In a letter to an Australian colleague written circa December 1898, Ehrlich noted that “two years ago Behring still believed that antitoxins should be conceptualized as forces rather than chemical substances.”

Following Behring and Kitasato’s contribution, scientists engaged in a number of discussions concerning the action of immune sera. While some researchers had reservations about what they saw as simplistic explanations of immune phenomena, others jumped to the conclusion that “antitoxins”-that is, specific, discrete substances-accounted for the property of certain immune sera to neutralize the effects of diphtheria and tetanus toxins. Along the same line, “agglutinins” and “precipitins” were deemed responsible, respectively, for agglutination and precipitation reactions, and “cytotoxins” for the lysis of cells, such as red blood cells and bacteria-in which case one spoke, respectively, of “hemolysins” and “bacteriolysins.”

The term antibody, used for the first time by Ehrlich in 1891 in a somewhat vague sense, was thus used concurrently with a variety of other terms that referred to specific immune reactions observed under laboratory conditions. Antibodies was by no means an unproblematic general term covering the more specialized ones, since the question of the possible (chemical) identity of these various entities and events was left open. In fact, while it could be argued that the term antibodies had, by the beginning of the 1900s, acquired a generic meaning, this was true only in a superficial sense. As late as 1929 Harry G. Wells, a defender of the “unitarian hypothesis” according to which “antibodies” should be considered as a single entity, independent of the variations in the experimental procedures by which they were “recognized,” could still lament that the hypothesis “currently accepted as if it were an established fact” was that the various precipitins, agglutinins, antitoxins, and so on were indeed “different and distinct substances.”

In addition to “experiment-laden” terms, “theory-laden” terms also circulated. For instance, following experiments examining the properties of immune sera heated to 55° C, cytotoxins were deemed to comprise two components. The first one, relatively heat resistant, was called by Ehrlich “immune body” (Immunkorper), “amboceptor” (Ambozeptor), or “intermediate body” (Zwischenkorper), while Bordet called it “sensitizing substance” (substance sensibilisatrice). Other researchers resorted to such terms as “copula” or “desmon.” The second, non-heat-resistant component of cytotoxins was called “complement” by Ehrlich, “alexin” by Bordet, who adopted the Greek-derived term (aleksein = protecting substance) proposed in 1889 by Hans Buchner, and “cytase” by other researchers. Lexicologists have noted that during periods characterized by the emergence of new referents, many terms compete for the status of designator. Thus, the presence of a plurality of terms is indicative of the presence of objects and practices that have yet to be stabilized. In our case, the different terms were “theory laden” both because they implied different mechanisms of action of the putative substances that they named and because they ascribed a different ontological status to those same substances. To speak of “intermediate body” or “amboceptor” was to postulate the existence of a chemical substance, composed of distinct chemical groups, that would literally insert itself, as a bridge, between the cell and the complement, thus allowing the complement to lyse the cell. To speak of “sensitizing substance” was to refer to a more diffuse model of action, metaphorically equated to the action of dyes on tissues, whereby no specific atom groups were entrusted with the capacity of establishing links with the cells or the alexin (Ehrlich’s complement). In particular, Bordet compared the action of the substance sensibilisatrice to the action of “certain fixing agents or mordants which confer to certain substances (or to cells, as is the case in histological techniques) the property to absorb colors they previously refused.”‘l

The debate over the existence, nature, and-properties of “antibodies” lasted for decades. Very few people shared the extreme position of Felix Le Dantec, who denied the physical existence of what he called “phenomenines”-that is, mythical substances that, like Moliere’s “vertu dormitive,” were being used tautologically to account for empirically observed phenomena:

While making fun of the physicians of his time, our great Moliere had foreseen Ehrlich’s system. Ehrlich has added nothing to the explanation of the imaginary invalid. Rather than saying that chloral causes sleep because it contains a soporific power (“vertu dormitive”), we would today say, according to the German scholar, that chloral contains a soporine (“dormitine”); well, it is the same thing! . . . Here then is a therapeutic serum which produces, when inoculated into the rabbit, a given phenomenon. How will you explain this particular activity? It is very simple: put on your glasses and your doctor’s cap and gravely say: “The serum produces this phenomenon because it contains a phenomenine which has that power.” Nobody will laugh. Point out timidly that the same serum has a different action on the snake; that it produces a second phenomenon; that will be because it contains a second phenomenine. If the same substance produces a thousand different phenomena in a thousand different species, then it contains a thousand different phenomenines; and there you go! Why had we not thought of this earlier? Henceforth we have the explanation for everything!

While Le Dantec’s remarks in this passage were addressed exclusively to Ehrlich, since they figured in one of the many anti-German pamphlets published by French scientists during World War I, they could equally have been directed at the “French” scientist Bordet, as indeed they had been in a prewar pamphlet. Moreover, other researchers, while not sharing Le Dantec’s extreme position, did share his hostility to “terminology-based” explanations. Henry Dean, for instance, noted that “agglutinins, precipitins, amboceptors are mere words, and a passive belief in the existence of such bodies tends to impede rather than advance our understanding of what is actually taking place”; he added that “ignorance, however aptly veiled in an attractive terminology, remains ignorance.”

So, despite various professions of faith in the material nature of “antibodies,” their ontological status remained uncertain, a situation ascribed by some scientists to the failure to purify chemically the elusive entities and thus to ascertain whether they were indeed material substances. This, of course, begged the question, because in order to base an argument on the possible chemical purification of antibodies one had first to assume that antibodies were indeed discrete chemical substances, which is precisely what Ehrlich’s opponents contested. In a 1902 letter nominating Ehrlich for the Nobel Prize, Bernhard Naunyn noted that the German researcher could be credited with the introduction of a stereochemical approach into biology and could thus be compared with the great scientists (August Kekule, Adolf von Baeyer) who had done the same in their own disciplines; but he added that Ehrlich’s contribution should still be regarded as tentative or premature, since the isolation and purification of the relevant substances (namely, “antibodies”) would long remain a chimera. Those same substances were treated, in a 1910 German textbook, as didactic devices:

In order to learn the nature of these antibodies attempts have been made to isolate them chemically. Thus far all such trials have been unsuccessful. It is even uncertain whether these so-called antibodies are definite chemical entities. Only the effects of the serum as a whole are known, and the ingredients in it to which these activities are attributed are thought of as antibodies. For didactic purposes antibodies, as antitoxins, agglutinins,
etc., will be spoken of in this book when the antitoxin or agglutinating properties, exclusively, are meant.

Julius Citron’s 1910 statement was echoed nineteen years later by Wells, according to whom:

We attribute this altered reactivity [of sera] to the presence of “antibodies,” despite the fact that we have absolutely no knowledge of what these antibodies may be, or even that they exist as material objects. Like the enzymes, we recognize them by what they do without discovering just what they are. We do not know whether they are specific molecular aggregates or merely physical forces dependent on altered surface energy of the same substances present in the blood before the process of immunization was begun.

REPRESENTATIONAL PRACTICES AND THE ORIGINS OF EHRLICH’S IMAGERY

“Early twentieth-century serological manuals, such as the one published in France by Paul-Felix Armand-Delille in 1911, resorted to three kinds of illustrations, corresponding to three types of representational practices. A first kind referred to the macroscopic observation of the behavior of blood in test tubes following manipulation with immune sera (Figure 3). In the case of hemolytic sera, a plate would
typically represent a series of test tubes containing a red liquid (blood): in some tubes the liquid would appear to be opaque, while in others-where, according to the caption, hemolysis had taken place-the liquid would appear to be transparent. In yet other test tubes, described as having undergone centrifugation, a red pellet could be observed, which would be lacking in the case of hemolysis. A second kind of illustration aimed at showing microscopic pictures of bacterial cultures mixed with immune sera (Figure 4). According to the descriptions accompanying the figures, bacteria, immobilized and agglutinated by the immune serum, could be seen. Finally, a third kind of illustration, derived from Ehrlich’s imagery, depicted the interactions of the invisible entities allegedly causing the macroscopic and microscopic phenomena represented in the previous two kinds of illustration (Figure 5). Notice that all three kinds of illustration function by contrast: they presuppose a comparison between a “before” and an “after,” which corresponds to the order in which they are supposed to be read, from left to right or from top to bottom.

This third kind of illustration belongs to what has been termed the “domain of invisible specimen behavior” or the “molecular realm,” a virtual space devoted to molecular events, construed as lying along a visibility continuum stretching from microscopic to macroscopic laboratory bench operations. There is sharp distinction between the first two types of illustrations and the third. Even though scientists might, and indeed often do, entertain a realistic, mirror-of-nature conception of the first two types of illustration, this distinction does not lie so much in the fact that they are held to be more “real” than the third type. Although, from the scientists’ point of view, the reality issue can indeed be relevant and, as we shall see, controversial, it is, so to speak, derivative. The primary distinction lies, rather, in the fact that the first two types of illustration deal with representations of elements that can in principle, as well as in practice (depending on the availability of the necessary instruments, reagents, and so on), be seen or made visible, while the third type is representative of elements that are, at a given time, by definition invisible. Lest the reader believe that, in establishing this distinction, we have abandoned our participant-centered perspective, consider Figure 6, taken from a 1930 article, in which a “threshold of visibility” is presented as an explicit dimension of immunological practice and simultaneously situated along a visibility-invisibility continuum. Moreover, Ehrlich himself, in his Croonian Lecture, had hinted at the distinction between a visible and an in-principle invisible domain when he pointed out that “we may regard the cell quite apart from its familiar morphological aspects, and contemplate its constitution from the purely chemical standpoint.” As we shall see, it is precisely in the establishment of a “domain of invisible specimen behavior” for immunology that Ehrlich’s controversial contribution lay.”

“It is a telling indication of the disregard in which visual elements were held that the second possibility-images as heuristic, in a strong, constitutive sense-was used by Ehrlich’s opponents as an argument against his theories: yes, images were constitutive of Ehrlich’s science, and that is why his science was flawed. As Bordet was to argue: “By its abusive use of quite puerile graphical representations, which simply translate the exterior aspects of phenomena without at all penetrating their intimacy, Ehrlich’s theory has created a deceiving taste for facile but illusory explanations.” Ehrlich’s reaction to these accusations was, to a large extent, to distance himself in principle from a literal interpretation of his images while in practice making liberal use of them in both his articles and his experimental work. In other words, the argument that the diagrams were fictions could be (and was) interpreted in two ways: the drawings were not a faithful image of reality, and thus they should be discarded because they were fundamentally misleading; while the drawings did not correspond to anything “out there,” immune reactions happened as if the various entities they portrayed did actually exist, and thus the diagrams were an important heuristic tool. Ehrlich’s attitude approximated the latter interpretation; it is well summarized by a statement he reportedly made to Karl Fliigge: “Those stupid people think that I really do represent the things to myself in that way.”

DOI:10.1086/356636

In Summary:

• Ehrlich felt immunity could be most easily and successfully explained through the agency of “the side-chain theory” (haptophore, toxophore, side-chains, complement, etc…obviously he did not believe in Occam’s Razor which states the simplest explanation is preferred)
• Roux showed that very small quantities of tetanus toxine, if injected directly into the brain, causes the death of the animal which was not compatible with Ehrlich’s theory
• Ehrlich claimed that Roux came to this incorrect conception through an erroneous conception of antitoxine
• He stated that the toxophile groups are therefore really inducers of the action of the poison, and not its preventive
• Yet Ehrlich immediately contradicted himself by claiming that those toxophile groups which, like the antitoxines present in the serum, are able to lay hold of toxine immediately on its entry into the blood, and so to divert it from organs essential to life, can alone be regarded as being possessed of any antitoxic action in the true sense of the word
• Upon immunisation, many other reaction products come into existence alongside of the antitoxines which are produced as a result of the action of the toxines
• As a result of the injection of bacterial cultures, there arise:
  1. Specific bacteriolysines, which dissolve the bacteria
  2. Other products such as “coagulines,” substances which are able to cause the precipitation of certain albuminous bodies contained in the culture fluid injected
  3. The much discussed “agglutinines”
  4. The antiferments
  5. And no doubt many other bodies which have not yet recognised
• He believed it was quite likely that the formation of any single one of these bodies is not of itself sufficient to confer immunity
• Thus in case of the introduction of bacteria into the body they have to do with a many-sided production of different forms of “Antikorper,” (i.e. antibodies) each of which is directed only against one definite quality or metabolic product of the bacterial cell
• The practice of using for the production of immunisation definite toxic bodies isolated from the bacterial cells had been more and more given up, and for this purpose it was regarded as important to employ the bacterial cells as intact as possible
• Ehrlich stated that there are two different bodies acting in combination for bacteriolysines (the rupture of a bacterial cell by antibodies): a stable one brought about by immunization and an unstable one already present in the blood
• The stable element was called the “immune body” while the unstable element was referred to as the “complement”
• After centrifugation, the “immune body” stays with the red blood cells while the “complement” is left behind
• Through his experiments, Ehrlich determined that temperature had an effect on whether or not the substances were left behind after centrifugation
• Ehrlich claimed that these facts can only be explained by making certain assumptions regarding the constitution of the two components, i.e., of the “immune body” and the “complement”
• When the blood of a non-immunized animal has haemolytic action on the blood of another animal, this can not be considered the action of the “immune body”
• Ehrlich termed this phenomena the “intermediate body” even though it had exactly the same action and characteristics of the “immune body” and the only difference was it occurred naturally
• He stated that the process of hsemolysines (lysis of the red blood cells) is the same when it occurs naturally or through immunizations
• The question arose whether in a serum that is capable of such manifold action if there is present one single haemolysine that destroys different red blood-cells, or whether a whole series of hsemolysines come into action, of which one is adapted to guinea-pig blood, another to rabbit blood, &c
• Ehrlich stated that if experiments are carried out on various blood types, and there are multiple “intermediate bodies” specific for each kind of blood (rabbits blood, calf blood, Guinea pig blood, etc.), then he succeeded in proving that there are different complements that fit these “intermediate bodies”
• His experiments brought more and more to the conviction that the blood serum is the carrier of substances innumerable as yet little known or conceived of
• Having obtained a precise conception of the method of action of the lysines of the serum—of the hsemolysines, and thereby also of the bacteriolysines—Ehrlich felt it became possible for them to attempt to solve the mystery of the origin of these bodies
• The toxines, as secretion products of cells, were in all likelihood still relatively uncomplicated bodies
• Ehrlich believed that this will be very effectually attained if, figuratively speaking, the “tentacle” or grappling arm of the protoplasm possesses a second haptophore group adapted to take to itself ferment-like material out of the blood fluid
• Through such complex organisation, by which the “tentacle” acts also as the bearer of a ferment-functioning group, this group is brought into close relation with the prey destined to be digested and assimilated
• This tentacle business where it aids in the process of digestion/fermentation to eliminate toxins is a perfect example of Ehrlich’s “lively imagination” for which he was criticized for
• In order to add proof for his tentacle creation, Ehrlich decided to pass from the abstract to the concrete—to find analogies in the different forms of insectivorous plants
• He claimed that there are present in the organism “side-chains” of a special nature that are endowed not only with an atomic group by virtue of the affinities of which they are enabled to pick up material, but also with a second atomic group, which, being ferment-loving in its nature, brings about the digestion of the material taken up
• He stated that should the pushing-off of these “side-chains” be forced, as it were, by immunisation, then the “side-chains” thus set free must possess both groups
• Ehrlich felt that the inconsistencies of immunising sera of animals in different experiments could be easily be explained by his theory
• Ehrlich’s “easy” explanation:
  1. This circumstance was easy to understand, if the complex nature of the lysines be borne in mind
  2. The lysine, be it bacteriolysine or hsemolysine ( i.e. “immune body + “complement”), possessed altogether three haptophore groups, of which two belong to the “immune-body” and one to the “complement”
  3. Each one of these haptophore groups can be bound by an appropriate “anti-group”
  4. Three anti-groups are thus conceivable, any one of which, by uniting with one of the haptophore groups of the lysine, can frustrate the action of the lysine
  5. To his mind, of these three possible “Antikorper,” that one which can lay hold of the haptophore group of the “complement,” and so prevent this from uniting with the “immune body,” was the most important
• Thus, the “easy” explanation involves the immune body, the complement, three haptophore groups, and three conceivable anti-groups (antibodies)…Occam’s Razor anyone?
• Dr. Neisser at the Steglitz Institute was able to determine that anthrax serum failed in mice, even if great quantities of fresh sheep’s serum (i.e., containing excess of “complement”) were at the same time introduced
• The failure in this case appeared to be due, on the one hand, to the destruction, in the body of the mouse, of the “complement” present in the sheep’s serum, and, on the other hand, to the fact that the “immune body” yielded, by the sheep does not find in mouse serum an appropriate new “complement” (in other words, it is explained by Ehrlich’s lively imagination but not by any observed processes)
• In the therapeutic application of anti-bacterial sera to man, therapeutical success is only to be attained if they use either a bacteriolysine with a “complement” which is stable in man (“homostabile complement”), or at least a bacteriolysine, the “immune body” of which finds in human serum an appropriate “complement”
• The latter condition would be the more readily fulfilled the nearer the species employed in the immunisation process is to man
• Ehrlich felt that perhaps the non-success for typhoid and cholera serum would be fixed if the serum was derived from apes and not taken from species so distantly removed from man as the horse, goat, or dog (why not take from a human instead of an animal…?)
• According to Ehrlich, the “Antikorper” (antibodies) are of a complex nature
• He claimed his unseen theoretical creations obey the already described law of elective absorption, and the origin of his imaginary substances is in keeping with his own “side-chain” theory (go figure that his dreamt up entities fit in with his dreamt up theory)
• It was hoped that immunisations with antibodies, which were of great theoretical interest, may come to be available for therapeutic application
• The sifting of the material obtained by observation was rendered more difficult by the occurrence under normal conditions of a great number of quite unlooked for bodies furnished with haptophore groups and arising from diverse organs, and which he designated collectively as haptines (i.e. he found numerous other unknown substances and threw them all under the same category he created)

• Complement fixation is a classic method for demonstrating the presence of antibody in patient serum
• The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum
• When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells
• The second component is a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement
• If the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis
• Disadvantages of complement fixation tests:
  1. Not sensitive – cannot be used for immunity screening.
  2. Time-consuming.
  3. Often non-specific e.g. cross-reactivity between Herpes Simplex “Virus” and Voricella Zoster “Virus”
• As can be seen, the complement fixation test is neither sensitive nor specific
• Sensitivity and specificity are the ways to indicate the accuracy of the test or measure
• The gold standard test, when compared with other options, is most likely to correctly identify people:
  1. With the disease (it is specific)
  2. Those who do not have the disease (it is sensitive)

• The term antibody today refers to discrete biochemical entities present in the blood
• The belief that antibodies are such entities is held not only by scientists and physicians, but also by the lay public, who learns about “antibodies,” if not in school, then at least in the course of routine medical practices such as vaccination and, increasingly, through the so-called popularization of science
• In order to understand the beginnings of immunological imagery at the turn of the century, we have to discard our present-day notions about the reality and structure of antibodies
• Behring and Kitasato carefully avoided the use of the term antitoxins, which they considered inappropriate, precisely because they regarded their findings as pointing to the action and properties of sera, and not to the presence of a substance
• In a letter to an Australian colleague written circa December 1898, Ehrlich noted that “two years ago Behring still believed that antitoxins should be conceptualized as forces rather than chemical substances.”
• The term antibody, used for the first time by Ehrlich in 1891 in a somewhat vague sense, was thus used concurrently with a variety of other terms that referred to specific immune reactions observed under laboratory conditions
• As late as 1929 Harry G. Wells, a defender of the “unitarian hypothesis” according to which “antibodies” should be considered as a single entity, independent of the variations in the experimental procedures by which they were “recognized,” could still lament that the hypothesis “currently accepted as if it were an established fact” was that the various precipitins, agglutinins, antitoxins, and so on were indeed “different and distinct substances.”
• The different terms for “antibodies” were “theory laden” both because they implied different mechanisms of action of the putative substances that they named and because they ascribed a different ontological status to those same substances
• According to Felix Le Dantec, ‘Ehrlich has added nothing to the explanation of the imaginary invalid.”
• Henry Dean noted that “agglutinins, precipitins, amboceptors are mere words, and a passive belief in the existence of such bodies tends to impede rather than advance our understanding of what is actually taking place”; he added that “ignorance, however aptly veiled in an attractive terminology, remains ignorance.”
• Despite various professions of faith in the material nature of “antibodies,” their ontological status remained uncertain, a situation ascribed by some scientists to the failure to purify chemically the elusive entities and thus to ascertain whether they were indeed material substances
• This, of course, begged the question, because in order to base an argument on the possible chemical purification of antibodies one had first to assume that antibodies were indeed discrete chemical substances, which is precisely what Ehrlich’s opponents contested
• In a 1902 letter nominating Ehrlich for the Nobel Prize, Bernhard Naunyn stated Ehrlich’s contribution should still be regarded as tentative or premature, since the isolation and purification of the relevant substances (namely, “antibodies”) would long remain a chimera
• In a 1910 German textbook, it was stated: “In order to learn the nature of these antibodies attempts have been made to isolate them chemically. Thus far all such trials have been unsuccessful. It is even uncertain whether these so-called antibodies are definite chemical entities. Only the effects of the serum as a whole are known, and the ingredients in it to which these activities are attributed are thought of as antibodies.”
• Accirding to Wells in 1929: “We attribute this altered reactivity [of sera] to the presence of “antibodies,” despite the fact that we have absolutely no knowledge of what these antibodies may be, or even that they exist as material objects. Like the enzymes, we recognize them by what they do without discovering just what they are.”
• Ehrlich’s imagery depicted the interactions of the invisible entities allegedly causing the macroscopic and microscopic phenomena
• His imagery belonged to what has been termed the “domain of invisible specimen behavior” or the “molecular realm,” a virtual space devoted to molecular events, construed as lying along a visibility continuum stretching from microscopic to macroscopic laboratory bench operations
• The images are representative of elements that are, at a given time, by definition invisible
• It is precisely in the establishment of a “domain of invisible specimen behavior” for immunology that Ehrlich’s controversial contribution lay
• The argument that Ehrlich’s diagrams were fictions could be (and was) interpreted in two ways: the drawings were not a faithful image of reality, and thus they should bediscarded because they were fundamentally misleading; while the drawings did not correspond to anything “out there,” immune reactions happened as if the various entities they portrayed did actually exist, and thus the diagrams were an important heuristic tool
• Ehrlich’s attitude approximated the latter interpretation; it is well summarized by a statement he reportedly made to Karl Fliigge: “Those stupid people think that I really do represent the things to myself in that way.”

Ehrlich provided a theory that weaved together various disparate elements like the best fiction writers often do. He defined the concepts of antibody, antigen, the complement system, and provided a framework for how immunity could work. This led to indirect non-specific and non-sensitive complement fixation tests (among others) used as evidence for the existence of “viruses” and/or as proof that the vaccines are effective against them. The problem is that Ehrlich’s theories were just mere words with nothing physical backing them. He first assumed any such entities existed in the blood based on lab-created chemical reactions and then developed a framwork around his imagination. He created concepts and ideas for that which he could not physically see. His creations remain in the “domain of the invisible.” They belong to the realm of fantasy and fairy tales.

As Henry Dean stated: “Ignorance, however aptly veiled in an attractive terminology, remains ignorance.”