Flocculation

FLOCCULATION, an precipitation reaction occurring between a toxin and its specific antitoxic serum. F. represents a particular case of the immunological precipitation reaction. In 1923, F. was proposed by Ramon for the titration of the antitoxic strength of sera and the antigenic strength of toxins. The reaction consists in that upon mixing the toxin with the corresponding immune serum, a turbidity occurs in a series of test tubes. First in one of them, and then in the other nearest to it, a floccular precipitate (floculate) precipitates. This series of test tubes that became turbid and precipitated is called the "zone of flocculation," and the test tube in which the floculate precipitates first is called the test tube of "initial" flocculation. The precipitate falls under the condition of complete neutralization of the toxin by the antitoxin. The initial F. usually corresponds to that limiting dose of antitoxic serum which is still capable of neutralizing a given amount of toxin. Therefore, by the initial F., one can calculate the antitoxic titer of the serum. On the other hand, knowing how many antitoxic units are contained in the taken dose of serum, one can, by the initial F., determine the antigenic strength of the toxin. The setting up of the F. reaction is performed as follows: certain equal amounts of toxin and decreasing amounts of antitoxic serum are poured into a series of test tubes. The test tubes are shaken, placed on a water bath at 40-45°, and the order and time of appearance of the turbidity first, and then of the flocculate, are observed. There are several modifications in the setting up of the F. reaction, differing only in the amount of ingredients (Ramon, Glenny). According to Ramon, the toxin is taken in a volume of 20 cm³, and the serum is added in an undiluted state, the volumes of the mixtures not being equalized; Glenny, however, takes the toxin in a volume of 1-2 cm³, and the serum, diluted from 1:5 to 1:40 (strong sera), is dosed with a micropipette or a microsyringe. Scholz, in order to save toxin and sera, uses toxin in a volume of 2 cm³ and dilutes the serum so that the same volume of mixture is always preserved upon its addition. This methodology, however, is complex and cumbersome; it requires the special selection of standard toxins and the production of a large number of serum dilutions. Therefore, in modern practice it has been abandoned. The method of Dyer stands apart, according to which the toxin is poured into the test tubes in increasing, and the serum in decreasing amounts, so that the contents of the test tubes are equal in volume (4 cm³). The latter method found application in the titration of antibacterial sera, especially under the condition that their titer is unknown. With this method, it is possible to quickly find the zone of F. - In addition to observing the appearance of the initial F., the time that passes from the moment of mixing the ingredients to the appearance of this F. (Kf) is also noted. According to Ramon's view, which is shared by other researchers, the time index (Kf) can serve for determining the avidity of sera. The greater the avidity of the serum, the faster the reaction proceeds. Data from numerous observations speak to the sufficient practical usefulness of the F. method; however, cases of the absence of this phenomenon in some toxins are described, as well as cases of non-coincidence of antitoxic titers of sera determined by flocculation and on animals (Glenny, Moloney, Sordelli and Serpa). The cause of these phenomena is not yet clear. The second complicating moment of the reaction must be considered the appearance sometimes of several zones of F. (Glenny, Schmidt and Scholz, Ramon, etc.). The cause of the appearance of these zones is also not yet clear. Glenny considers only the zone of neutralization of the toxin by the antitoxin as specific. He gives no explanation for the other zone, considering it a non-specific phenomenon. Schmidt and Scholz, like Glenny, consider only the first zone as specific, but they link the second zone to the interaction between the precipitin of the serum and the precipitinogen of the toxin. - All the above circumstances force one to regard F. as an auxiliary method, allowing a reduction in the number of experimental animals. Regarding the essence of F., several hypotheses exist. Thus, Ramon, Schmidt and Scholz, Glenny consider that in this reaction the toxin and antitoxin combine; this combination precipitates together with the protein that adsorbs it. Others (Zinger, Bronfenbrenner) identify this reaction with the precipitation reaction; Moerch expresses the assumption of the existence in the serum of the so-called "flocudine" and in the toxin of "floculinogen" as separate substances, interacting with each other and precipitating in this reaction. This brief list of hypotheses speaks to the fact that we are still far from understanding the essence of the F. reaction, the practical significance of which, however, is so indisputable that it has found widespread application in the practice of institutes producing serum preparations. Lit.: Arkhipov K. and Novgorodskaya E., On the question of flocculation, Microbiol. Zhurn., t. IV, vyp. 1, 1927; Eliseevskiy M., Experience in studying the flocculating, resp. precipitating properties of antibacterial sera, ibid., t. XIII, vyp. 1, 1931; Zdravodskiy P. and Khalapina K., Active immunization with diphtheria anatoxin, Tr. of the Azerbaijan Institute of Microbiology and Hygiene, vyp. 2-3, Baku, 1925-26; Levin Ya. and Feigin S., On the question of the flocculating properties of antiscarlet fever serum, Zhurn. of epidemiol. and microbiol., 1932, No. 9-10; Khalapina K. and Asrieva T., On the flocculating ability of scarlatinal streptococcus filtrate and its significance, Microbiol. zhurn., t. VII, vyp. 2, 1928; Dyer R., Application of the Ramon flocculation principle to the titration of scarlet fever streptococcus toxin and antitoxin, Publ. health rep., v. XL, 1925; Glenny A. and Okell C., The titration of diphtheria toxin and antitoxin by flocculation methods, Journ. of path. a. bacteriol., v. XXVII, 1924; Moerch J., La reaction de Ramon, Compt. rend, de Soc. de biol., v. CVIII, 1931; Moloney P. and Weid C., Diphtheria toxin-antitoxin flocculation, Journ. of path. a. bacteriol., v. XXVIII, 1925; Ramon G., Floculation dans un melange de toxine-antitoxine diphteriques, Ann. de l'inst Pasteur, v. XXXVII, 1923; Schmidt H., Zur Kenntnis der Natur der Diphtherie-Toxin-Antitoxin-Flockung, Zeitschr. f. Immunitatsf. u. exp. Therap., B. XLVIII, 1928; Schmidt H. u. Scholz W., Studien zur Kenntnis der Eigenschaften von Diphtherie-Toxin-Antitoxin-Gemische, Arch. f. Hyg., B. XCV, 1925; Scholz W., Ober die Brauchbarkeit der Flokkungsreaktion zur Auswertung antitoxischer Sera, Zentralbl. f. Bakt., Orig., B. XCI, 1923; Sordelli A. et Serpa R., Titrage du serum antidiphterique par la methode de Ramon, Compt. rend, de Soc. de biol., v. XCI, 1954.

V. Krestovnikov.