CASTELLANI METHOD (A. Castellani, ital. doctor, sort. in 1878; synonym: method of adsorption, Castellani's reaction, method of adsorption of agglutinins according to Castellani) — the immunological reaction based on adsorption of agglutinins by microorganisms from antimicrobic serums. This reaction is offered Castellani in 1902. Is the cornerstone of this method the fact established still by P. Ehrlich and Yu. Morgenrot (1899), and also, Borde (1899) that ability selectively is inherent in each antigen to connect antibodies from the corresponding serums therefore the last lose ability to react with the antigen used for adsorption. This phenomenon of Castellani used for the purpose of removal from immune serums of the corresponding antibodies. It showed that at immunization of rabbits mix of 2 — 3 strains of bacteria of intestinal group forms agglutinins to each of them in the credits depending on amount of the entered material and time of capture of blood. At addition to such immune serum of one of strains there is an adsorption of the corresponding agglutinins while agglutinins to other strains remain.
If in the course of immunization of animals one strain heterological agglutinins to other strain appear, then adsorption of the received immune serum a homologous strain leads to loss of both homologous, and heterological agglutinins. Adsorption of such serum other strain, on the contrary, extracts agglutinins only to a hetero logical strain, agglutinins to a homologous strain remain.
To. the m is used for detection of antigenic similarity and distinction between microorganisms. Most widely it is applied to studying of antigenic properties of intestinal group of bacteria. It is used also for production of monospecific (monoreceptor) serums, i.e. containing only one type of agglutinins. Such serums are applied to diagnosis and typing of microorganisms.
In the All-Union center for escherichias of Research institute of vaccines and serums of I. I. Mechnikov of I. V. Golubeva with sotr. (1965) the indicative scheme for the choice of adsorbent at production of the OK-koli-syvorotok which are most exempted from heterological agglutinins is made. Technological process of preparation of such adsorbed serums much more becomes simpler during the use of the prepared in advance dry adsorbents of broad set.
The scheme of reaction of cross adsorption of agglutinins by Castellani's method is presented in the table. Immune serums, apparently from the table, contain antibodies both to somatic O-antigens, and to flagellar N-antigens.
For adsorption of immune serum strains of bacteria of intestinal group grow up in test tubes with a beef-extract broth during 3 hours at t ° 37 °. This broth use for crops in mattresses from 200 ml 1,5% of a beef-extract agar which place in the thermostat on 18 hours at t ° 37 °. Then condensate is merged and bring 9 ml of isotonic solution of sodium chloride to which carefully wash away the grown cultures in mattresses. The received suspensions of microorganisms spill on test tubes and boil in the water bath at t ° 100 ° within 1 hour for an inactivation of H-antigens. Immune serum in number of 0,2 ml is mixed from 9,8 ml of boiled culture. Mix leave at t ° 37 ° during 4 hours, and then at t ° 4 ° during the 18th hour. Make centrifuging at 6000 rpm within 55 min. for release from microorganisms of Nadosadochnuyu liquid, edges represents the adsorbed serum, in cultivation 1: 50 decant and filter via bacterial filters. Such serum is used for an agglutination test with the corresponding bacteria, previously having checked completeness of adsorption. If there was an incomplete release of serum from heterological antibodies, make its repeated saturation by adsorbent.
For adsorptions of flagellar N-agglutinins the microorganisms killed with formalin are used.
Success of adsorption of nonspecific antibodies depends on a number of factors: specificity of native serum, in a cut specific antibodies shall prevail considerably over nonspecific, selection and quality of adsorbent and conditions of adsorption.
The great influence on quality of adsorption is exerted by a right choice of a strain adsorbent. The culture shall be in a S-form (see. Dissociation of bacteria ). For detection of dissociation of cultures use of a heat test of microbic suspensions and microscopy of colonies in slantwise transmitted light can be recommended. As strains adsorbents it is necessary to use the cultures of microorganisms having ability to adsorb on itself hetero logical agglutinins, but not to exhaust serum from homologous agglutinins.
Specificity of the adsorbed serums much more increases that has essential value during the use them for diagnosis of infectious diseases. Castellani's reaction is the most rational method of receiving high-specific OK-koli-syvorotok.
This method was applied to receiving adsorbed luminescing OK - and N-koli-syvorotok. It is widely used for studying of antigenic properties of some other microorganisms, and also cells and fabrics it is normal also of pathology.
Table. The SCHEME of STATEMENT of REACTION of CROSS ADSORPTION of AGGLUTININS BY CASTELLANI'S METHOD
Bibliography: The multivolume guide to microbiology, clinic and epidemiology of infectious diseases, under the editorship of H. N. Zhukova-Verezhnikov, t. 3, page 598, M., 1964; Prick Scientific achievements in studying - infections, under the editorship of I. V. Golubeva, page 112, M., 1973; The Guide to microbiological diagnosis of infectious diseases, under the editorship of K. I. Matveev, M., 1973; Castellani A. Die Agglutination bei gemischter Infektion und die Diagnose der letzteren, Z. Hyg. Infekt. - Kr., Bd 40, S. 1, 1902; E h of of 1 i with h P. u. Morgenroth J. t } ber Haemolysine, Berl. klin. Wschr., S. 569, 1901.
G. M. Bochko, And. H. May.