Two Octaves down

From looking up the basics of complement fixation, it turns out that it had other names. It was called complement deviation: instead of complement landing on cells and lysing them, its path was deviated to some other end. Just like complement fixation: before it can land on cells and lyse them, complement is fixed in place and stays in solution. This is all very straightforward.

It was also called “The Bordet-Gengou Reaction”. That sounds more interesting.

* * *

The best source for info on the early history of immunology is the book Immunology: Pasteur’s Heritage, edited by Pierre-André Cazenave and published in 1991 by Wiley. I have not read this book, but I will bet you $12 that it’s the best source for info on the early history of immunology. The passages available in Google Books are great.

Anyway, according to Wikipedia, complement itself was first called “alexin“, around 1890 by researcher Hans Ernst August Buchner, who observed that serum contained a substance that could lyse bacteria in the absence of cells. Richard Pfeiffer observed a similar phenomenon. Also around 1890, the word “antibody” (technically “Antikörper” because they were all publishing in German) was coined, by Paul Ehrlich whose laboratory was more focused on the interactions of individual molecules (in this case antibodies, which he saw could neutralize the harmful effects of toxins) than cells. All of these researchers were sort of rivals to the organization of Élie Metchnikoff at the Institut Pasteur in Paris, which was focused on the newly discovered phagocytic cells that he thought were responsible for all aspects of immune protection, more or less.

Jules Bordet

Belgian researcher Jules Bordet was an apprentice to Metchnikoff. And Octave Gengou was Bordet’s brother-in-law and close colleague. At some point in the late 1890s (I can’t tell which paper it was; all their papers were in Annales de l’Institut Pasteur, and most of them are just called “Recherches sur la coagulation du sang” with vague subtitles), Bordet showed that “alexine” was made up of two parts: a heat-stable part, which just bound up things like toxins and bacteria; and a part that could easily be destroyed by heat. Without this “heat-labile” element, the bacteria didn’t get lysed.

This led the Ehrlich school to divide its “Antikörper” concept into two parts: Amboceptor (heat-stable), and Komplement (heat-labile). Meanwhile the Bordet/Metchnikoff school divided it into substance sensibilitrice (sensitizing substance) and alexine (bacteria-lysing substance). Bordet showed that Amboceptor/sensibilitrice/antibody against a certain substance would only be present in the blood of people immunized with that substance; while just about any blood contained Komplement/alexine/complement which could lyse anything.

* * *

What about Gengou?

It looks like Bordet and Gengou start being paired together on these papers in 1901, which was also the year that Bordet was invited to be the director of the newly founded Institut Pasteur. Not the real one… the Institut Pasteur du Brabant in Brussels. Which was the only “Institut Pasteur” not to be an offshoot of the original one in France; this one was just called that because Madame Pasteur gave it her blessing. In the 1980s it moved away from the beautiful building on Parc Léopold, which now houses the Bavarian Representation to the EU (pdf), and was eventually merged with the Belgian government’s Scientific Institute of Public Health.

Jules Bordet and Octave Gengou are credited with discovering complement fixation. Earlier they had seen that the amboceptor/complement mixture that destroys bacteria (bacteriolysis) can also destroy red blood cells (hemolysis), provided the RBCs were from a different species or a different blood group (yes, they basically also discovered blood groups).

But if you take some serum with a lot of amboceptor and complement in it, and do a bacteriolysis… all the complement should get used up. Then you mix it with sheep red blood cells, and they don’t get lysed. What you just did was prove that the serum contained antibody against that particular bacteria. You can see how this would be useful. This is the Bordet-Gengou reaction.

* * *

What about the Nobel Prize?

Bordet and Gengou also discovered the bacterium that causes pertussis, which is now called Bordetella. That looks less silly than Gengouella, but the latter actually sounds better, to the cultured and discriminating ear, They also developed Bordet-Gengou agar, the ideal substance for growing Bordetella pertussis and distinguishing it from other small Gram-negative coccobacilli, In 1919, the first Nobel Prize ceremony in five years was held, and the prize in medicine and physiology was given to Bordet, aged 49, for achievements summarized most briefly as:

• Complement
• Complement fixation
• Pertussis bacterium

Two of those are accomplishments shared with Gengou. And both of those were procedures named after both Bordet and Gengou: in addition to the Bordet-Gengou complement phenomenon, the isolation of Bordetella pertussis depended on culturing it on what is known as Bordet-Gengou agar). And yet, only one person got the prize.

Octave Gengar

Granted, the prize had always been given to a single person: with two exceptions. One of them was in 1908, when it went to both Pasteur and Metchnikoff. Not to do that might have caused an outbreak of bad feelings to rival the Schism of 1054. I suppose Gengou would not mind the solitary recognition of his great collaborator.

Really, the biggest injustice is not the lack of a Nobel Prize, or the zero Google hits for the word “Gengouella”, but my inability to find any photos of him online.

* * *

The name “Octave” rang a bell. I don’t know if I’d heard of anyone by that name until this year. Who was the other one? Quickly the memory became clear: Octave Chanute.

Octave Chanute

From flyingmachines.org:

Octave Alexandre Chanut*  , a native of France, was retired from a distinguished engineering career and living in Chicago, Illinois, when he began to pursue his life-long interest in aeronautics. His experiments with “gliding machines” began in 1896 and were conducted at Miller Beach and Dune Park, Indiana, on the southern shore of Lake Michigan.

…

Octave Chanute went on to be the main enthusiast for the Wright Brothers during their early aerial trials, encouraging them and supplying them with the latest aerial information. By 1900 Chanute had become the center point for various aerial experimenters in Europe and the U.S. His 1894 book “Progress in Flying Machines” was a landmark volume and was the book recommended to Wilbur Wright by the Smithsonian Institution in 1899.

One of the messages I got from the displays at the Wright Brothers National Memorial, on North Carolina’s Outer Banks, was that the brothers cared deeply about impressing Octave Chanute, whose correspondence served as a nexus for information exchange between different experimentalists in heavier-than-air flight. Chanute was unquestionably the nearest “giant” on whose shoulders they stood. But I had never heard of him until this year. Maybe you had.

Takeaway message: If your name is “Octave”, “Staff”, “Rest”, “Flat” or another musical term, your contributions may be overlooked by the eye of history. You may want to change your name. Make a note of it. But don’t make “Note” your name.

* * *

Sources:

• Bordet biographical sketch from the Institut Pasteur
• Bordet biographical sketch from the Institut Jules Bordet
• Bordet biographical sketch from the American Association of Immunologists
• That book I mentioned earlier
• Another book via Google Books fragments

Yeouch!

From Geo. H. Chapman (1928), The isolation and estimation of Clostridium welchii, J. Bacteriol. 16(1): 49-56:

Holy cats! At least he’s had a year of safety with the new jar. And I hope it was safe for many years after that.

What is an amboceptor?

Amboceptors get their name…

Or I could say “Amboceptor gets its name…” — it’s one of those words that can be an object, or a substance.

Amboceptor gets its name from the Latin root “ambi-“, because it’s a receptor for two things.

An amboceptor is something that attaches to an antigen, and then attaches to complement molecules. When this happens on a cell surface, the complement pokes holes in the cell to damage it. When it happens in solution, it blocks the complement from attaching to cells.

Isn’t that… an antibody? An amboceptor is an antibody.

Yes, but we didn’t always know that.

* * *

We knew there were amboceptors. We knew there were antibodies. There could have been amboceptors that weren’t antibodies.

Amboceptor, as a substance, is critical in the complement fixation assay for serum antibody. This is a once-ubiquitous, now-too-complicated-to-be-worth-doing process of diagnosing diseases like syphilis by looking for (anti-syphilis) antibodies in a patient’s serum. It was a fixture in American life, under the monicker of the “Wassermann Test”, which countless practitioners trusted to confirm a diagnosis of syphilis, or even to diagnose syphilis in the absence of other evidence. You know how nowadays we have urban legends about babies named things like “Urine” and “Female” based on hospital mix-ups and parental idiocy? That list used to include “Positive Wassermann Johnson”.

How real?

Probably the best description of complement fixation on the internet is provided here by an organization that is always at the vanguard of science and health policy, the Texas state government. I’ll try to summarize in terms that would be familiar to blog readers from 1920.

• Sheep corpuscles (red blood cells) are mixed with amboceptor. This sensitizes them to the complement.
• If the sensitized corpuscles are then mixed with complement, the complement binds them and starts poking holes in the corpuscles, which is called “lysing” them.
• The solution turns pink as the contents of corpuscles spill out. This is called “laking”. Or it was back then, anyway.
• Serum is a solution containing amboceptor as well as many other things. If you take serum from a syphilitic patient, and mix it with syphilis antigen, the amboceptor should bind the antigen and form a complex in solution.
• If you mix this with complement, the antigen-amboceptor complexes should bind up the complement before it can attach to the corpuscles and lyse them.
• If you take serum from a non-syphilitic patient, the amboceptor will NOT bind the syphilis antigen, and therefore it will not bind the complement. The complement will not bind to amboceptor alone, only to complexes. So the complement is still available for lysing and laking.

As you can imagine, this is a touchy procedure prone to error.

First of all, what about the complement that’s already in the human serum? You have to inactivate it, so you can then see how the rest of the serum reacts to externally introduced complement that you bought from a supplier. How do you inactivate it? Complement is protein and you heat it to denature it. You heat it at 56 degrees Celsius. Exactly 56 degrees Celsius. For exactly 30 minutes. At 54 degrees, you won’t denature enough complement. At 58 degrees, you’ll also denature antibodies. If you do it for 60 minutes, you’ll also denature antibodies.

But that’s assuming your serum was isolated from the blood in a timely fashion, and kept at room temperature for 24 hours or less. Or maybe it’ll work after 48 hours. What if it’s been refrigerated? That has some effect on the protein stability, maybe they will start to aggregate and become useless. What if it hasn’t sat at room temperature for long enough? Blood needs to sit for an hour or so before you start processing it to remove the corpuscles and platelets, after all. How perfect does the procedure need to be? All you are showing is that there is something in the serum that blocks complement from depositing on the sheep cells. What if that something isn’t amboceptor?

And what if the serum contains something that’ll lyse the sheep corpuscles on its own? After all, sheep are not human and some people may have a natural antisheep hemolysin, which is a word used for amboceptors or other substances that lyse corpuscles. How do you correct for that? Well, you have to include a control with no complement, to figure out the background amount of lysing that goes on. Then you can dilute the serum properly. But if you dilute the serum, you make the procedure less sensitive. Some laboratories will do this, some will not.

And how much complement should you add to the serum? If you add too much, you get false negatives. Someone with early-stage syphilis may have a low level of amboceptor. If there’s too much complement, you’ll soak up all the amboceptor and still have plenty of complement left over. But it’s possible to add too little complement, too. With too little, you get ambiguous cases, where you’re not sure if you see laking or not. What if you don’t see laking in any of your samples? You need a positive control. Does that mean you need to have fresh serum on hand at all times from a known syphilitic? Oh dear.

And what should the antigen be, that can pick out syphilis amboceptor from all the other amboceptor in the serum? There’s no way of growing the syphilis spirochetes in your laboratory. Extract of syphilitic liver was used originally by Wassermann himself. Usually you would exploit the unusual lipids found in the spirochetes, by using some uninfected tissue, like some sort of heart or liver extract, with added cholesterin (a.k.a. cholesterol). I prefer Noguchi’s acetone-insoluble fraction of the alcoholic extract of fresh beef heart, or as I call it, NAIFAEFBH. In 1941 Pangborn will revolutionize syphilis diagnosis by standardizing this antigen as a certain combination of cardiolipin, cholesterol, and lecithin, all taken from beef heart. This will still be used in the 21st century [Castro et al. (2000), Clin Diagn Lab Immunol 7(4): 658-661].

And what about the Hecht-Weinberg modification, particularly the procedure of Gradwohl? A lot of people are not comfortable with adding external complement to the serum, instead trusting that the natural amount of complement in the serum is appropriate for the process. This means you wouldn’t heat-inactivate at all. This means other controls will have to be done. And the complement may end up getting inactivated anyway, based on natural half-life, exposure to light, etc.

* * *

And on, and on, and on. Most of the contemporary articles about these issues are 100% baffling to a 21st-century reader, but one of the least baffling is an exchange in the November and December 1914 issues of The Lancet-Clinic. No, not The Lancet. The Lancet-Clinic, which was published in Cincinnati until going out of business in 1916. Unfortunately it isn’t indexed in Pubmed or Google Scholar, but random Google searches for “Wassermann test” and “amboceptor” quickly found it in Googlle Books.

Go to your bookshelf, the one that groans under the weight of bound volumes of turn-of-the-century Ohio medical journals. Open up The Lancet-Clinic, Vol. CXII, issue 26, and turn to pages 536-539, where Albert Faller, M.D. issues forth a torrent of verbiage on all the problems he sees with the Wassermann test, and finishes by extolling the virtues of Gradwohl’s Hecht-Weinberg modification, which turns many negative results into positives. This is then followed by harrumphing from one Dr. Berghausen, who sees no reason why heating would ever cause any harm to anything, and opines that physicians should stop being afraid of reporting negative results just because they happen to conflict with the fact that a patient seems to have syphilis. Dr. E.A. North then muses that perhaps all this could be cleared up if people would mix their samples thoroughly, and Faller rounds out the exchange by saying that far from being “tired of condemning individuals as sufferers of syphilis”, he simply does not trust that a patient who goes from positive Wassermann to negative Wassermann has actually been cured. And he does trust when that happens with the Hecht-Weinberg. Finally, in the December issue [Lancet-Clinic CXII(27): 624], the great man himself, Rutherford Birchard Hayes Gradwohl, writes in to further heap scorn on the deplorable Berghausen, saying that “I wish to state as one who has been instrumental in urging this test before the American profession, no such ambitions have ever surged through my serological breast.”

* * *

The Wassermann test was a serious concern. For decades, in most U.S. states, people were required to be tested for various diseases, particularly STIs, particularly syphilis. Here in Pennsylvania we waited until 1997 to repeal the requirement, which had begun as part of a nationwide campaign by New Deal-era Surgeon General and future Pittsburgh public health supremo Thomas Parran. But what does it mean to have a negative Wassermann?

What if you have no signs of the disease, you have no family history of it, but you have a positive Wassermann? Does it mean anything? How can you not worry? What if I had a positive reading earlier, and now I’m negative? Am I cured, thanks to the new sulfonamide drugs that work so much better than salvarsan? Is it really possible to be cured? And doesn’t this just measure antibodies? I have antibodies against just about every infection I’ve ever suffered. That doesn’t mean I’m still an infection risk.

How to get reliable Wassermann results was a perennial topic in the literature for decades. Because it was such a devastating diagnosis. And for practical reasons, since so many of the tests had to be carried out and they were so cumbersome.

A sample:

• Noguchi, H. and J. Bronfenbrenner (1911). The Comparative Merits of Various Complements and Amboceptors in the Serum Diagnosis of Syphilis. J Exp Med 13(1): 78-91.
• Van Saun, A. I. and M. K. Preston (1918). Comparative Wassermann Tests with Two Antigens. Am J Public Health (NY) 8(2): 146-148.
• Lewis, P. A. and H. S. Newcomer (1919). Observations on the Wassermann Reaction: A Comparison of the New System of Noguchi with That Using Cholesterolized Antigen According to McIntosh and Fildes. J Exp Med 29(4): 351-359.
• Browning, C. H. and E. L. Kennaway (1920). Suggestions for a New Criterion of a Positive Wassermann Reaction Based on an Analysis of 2334 Quantitative Tests. J Hyg (Lond) 19(1): 87-106.
• Mackie, T. J. and C. C. Rowland (1920). The Value of Simultaneous Testing for the Wassermann Reaction, with Two Different Antigens and the “Ice-Box Method”. Br J Exp Pathol 1(5): 219-224.
• D.Aunoy, R. (1921). Comparative Study of the Wassermann and Sachs-Georgi Reactions. J Med Res 42(4): 339-347.
• Bigger, J. W. (1921). The Reliability of the Wassermann Test as performed by different Pathologists. J Hyg (Lond) 20(4): 383-389.
• Famulener, L. W. and J. A. W. Hewitt (1922). Studies on the Serodiagnosis of Syphilis: I. The Hecht-Weinberg-Gradwohl Test. J Inf Dis 31(3): 285-290.
• Shepardson, R. T. (1922). Preliminary Report on an Investigation of the “Provocative Wassermann” Controlled by the Ice-Box Method. Cal State Med J 20(3): 80-83.
• Dulaney, A. D. (1923). The Wassermann and Kahn Precipitation Tests Compared in 900 Cases. Am J Public Health (NY) 13(6): 472-474.
• Osmond, T. E. and D. McClean (1924). A Comparison of the Kahn and Wassermann Tests on 500 Serums. Br Med J 1(3301): 617-618.
• Ruediger, E. H. (1924). A Plea in Favor of a Standardized Wassermann Test. Cal West Med 22(11): 548-553.
• Malcolm, M. (1924). A Comparison of the Kahn Test with the Wassermann Test. Can Med Assoc J 14(3): 222-224.
• Wyler, E. J. (1927). A Note on Two Factors Affecting the Sero-diagnosis of Syphilis. Br J Vener Dis 3(4): 320-325.
• Green, F. (1929). The Kahn Test as Compared with a Standard Wassermann Reaction. Can Med Assoc J 20(1): 26-29.
• Ferguson, J. H. and E. C. Greenfield (1929). Value of the Hinton Test in the Serum Diagnosis of Syphilis: In Comparison with the Khan and Wassermann Tests. Br Med J 1(3558): 492-494.
• Evans, N. (1930) Kahn Precipitation Test for Syphilis: As Used in Conjunction with the Wassermann Test. Cal West Med 32(1): 24-26.
• Eagle, H. (1931). Studies in the Serology of Syphilis: IV. A More Sensitive Reaction for Use in the Wassermann Reaction. J Exp Med 53(5): 605-614.
• Chambers, S. O. (1932). The Kahn Precipitation Test: Compared with the Kolmer Modification of the Wassermann Test in Untreated Primary Darkfield Positive Seronegative Syphilis. Cal West Med 37(3): 153-155.
• Barritt, M. M. and A. O. Ross (1939). A Comparison of the Wassermann and Meinicke (M.K.R. II) tests in the Serological Diagnosis of Syphilis. Br J Vener Dis 15(3): 183-202.
• Richardson, G. M. (1940). The Specificity of the Bordet-Wassermann Reaction: Preliminary Note on an Improved Method. Br J Vener Dis 16(3-4): 166-185.
• Rickword Lane, C. (1944). Comparison of the Laughlen Reaction for Syphilis with the Wassermann and Kahn Reactions. Br J Vener Dis 20(2): 78-81.
• Kolmer, J. A. (1944). The Problem of Falsely Doubtful and Positive Reactions in the Serology of Syphilis. Am J Public Health Nations Health 34(5): 510-525.
• McMenemey, W. H. and W. H. Whitehead (1949). Ford Robertson and Colquhoun Modification of the Meinicke Clarification Reaction Compared with the Harrison-Wyler Wassermann and the Standard Kahn Reactions. Br J Vener Dis 25(3): 147-154.
• Osmond, T. E. (1950). Comparison of the Wassermann and Kahn Reactions. Br Med J 1(4652): 524.
• Bekierkunst, A. and F. Milgrom (1950). Complement-fixation Reactions with Cardiolipin Antigen Compared with Kahn Reactions. Bull World Health Organ 2(4): 687-688.
• Orpwood Price, I. N. and A. E. Wilkinson (1952). Comparative Serum Testing with Cardiolipin and Crude Heart Extract Wassermann Antigens. Br J Vener Dis 28(1): 16-19.
• Kahn, R. L. (1972). Syphilis Serology with Lipoidal Antigen: The Meaning of Positive Reactions. J Natl Med Assoc 64(2): 117-passim.

In addition to any number of more technical papers with titles like “The Amount of Hemolysin Absorbed By Sheep Corpuscles”, published in the American Journal of Syphilis.

The situation was summed up well by Knox College art benefactor L. W. Famulener and Julia A. W. Hewitt (1922), in the atypically lucid introduction to their typically inconclusive study.

It is quite difficult to delimit the borderline between the nonsyphilitic and the syphilitic person. Supersensitive laboratory tests on nonsyphilitic serums* in certain cases may give results which pass over to the syphilitic side. This is especially true when the worker is in pursuit of those who clearly show syphilitic conditions, but whose serums fail to give a positive reaction with the usual methods. The nonreacting syphilitic may not even carry “syphilitic fixing bodies” in his serum. It is well known that there is a decided quantitative difference existing between the different positive serums. If these “fixing bodies” are metabolites, they may arise anew, or may be normal substance markedly increased in amount during the course of the disease. In the latter case, it is quite conceivable that certain nonsyphilitic persons may naturally have an abnormally large content of these substances in their blood, while, on the contrary, known syphilitic persons may fail to elaborate these bodies and show only a very low content, even below the average norm for the healthy person. Therefore memers of either group may be found on opposite sides of an arbitrarily established borderline, where clinically they do not belong. …

As to the criteria which should determine the syphilitic from the nonsyphilitic persons, no common agreement exists. By cooperative studies among clinicians, pathologists and serologists, progress may be made toward that ultimate end. In the absence of a standard method for the serum diagnosis of syphilis, a multitude of modifications of the original Wassermann technic** have come into existence. Many of these modifications are erroneous in conception, even conflicting with the established laws of serology, consequently leading to false results.

And the number of modifications would only increase, until the complement-fixation process was given up as inherently unstable and imprecise, the flintlock musket of the serologist’s arsenal.

* Nowadays we say “sera”. We must be more pretentious nowadays.

** Nowadays we spell it “technique”. Pretentious?

* * *

So anyway. Why don’t we use the word “amboceptor” anymore? Is it still used in some circles? No, I don’t think so. A Pubmed search for “amboceptor” [All Fields] found 59 results. The last one was in 1991, and they were distributed fairly evenly over the previous 87 years. Which is pretty amazing when you consider how many more articles are indexed from 1991 than 1904. And in fact, most of the results since 1965 are either translated out of German, Czech, or Bulgarian, or are in odd journals like Developments in Biological Standardization and Bibliotheca Haematologica. The last appearance in an abstract is in a German-language paper entitled “Effect of different media on long-term cultivation of human synovial macrophages”, from the now-retired Eckhard Stofft of the University of Mainz, a specialist in tendon and ligament pathology.

Scientometric metadata

* * *

“In the extensive experiments and observations during the past twenty years, the greater amount of effort has been expended in the consideration of the manner of the production of immune bodies upon the part of the host, together with quantitative and qualitative methods for the determination of such bodies. Thus specific substances have been recognized, to which the names antitoxin, agglutinin, precipitin, amboceptor, opsonin, etc., have been given.”

– Duval, C.W. and F.B. Gurd (1911). Experimental Immunity with Reference to the Bacillus of Leprosy. Part I: A Study of the Factors Determining Infection in Animals. J Exp Med 14(2): 181-195.

Antitoxin: still used. Although all antitoxins are antibodies, the word is still medically useful.

Agglutinin: still used, but usually we say “hemagglutinin”. Not all hemagglutinins are antibodies.

Precipitin: still used. Although all precipitins are antibodies, the precipitin test, which is even older than the complement fixation test, is still used.

Opsonin: still used. A very useful word. And not all opsonins are antibodies.

Amboceptor: obsolete.