The in vitro determination of the D antigens of poliomyelitis viruses: I. The gel diffusion test

1. A technique for the measurement of poliomyelitis virus D antigens by means of gel diffusion tests against antisera obtained from hyperimmunized calves is described in detail.

2. The precision of the method was estimated by performing several replicate tests with three or four dilutions of concentrated antigen of each of the three types. The D antigen content, if measured by means of twelve precipitation hexagons in agar can be calculated with a precision of about 10 per cent (95 per cent confidence limits).

3. The calf sera gave no precipitation with C antigens in the concentrations suitable for measuring D antigens.

4. The local reference antigens, the D antigen concentrations of which were adjusted to equal that of the reference antigens used in the Glaxo Laboratories, contained C as well as D antigens. This was observed in gel diffusion tests with specific anti-C sera from guinea-pigs.

5. The guinea-pig anti-C sera showed a precipitation with the immune calf sera as well as with normal calf serum. This indicated the presence of anti-calf antibodies in the guinea-pig sera, presumably elicited by traces of calf proteins in the vaccines. These antibodies might give rise to non-specific precipitation if antisera used in the test for D antigen were obtained from animal species other than those used to provide components in the tissue culture medium from which the antigens were derived.

     

The antigenic structure of the rous sarcoma communication I. Detection of the specific rous sarcoma antigen by the precipitation test in agar

Summary With the aid of the precipitation reaction in agar and the ring-precipitation test the authors compared the antigenic composition of Rous sarcoma with that of the organs of healthy chickens (liver, kidney, spleen, subcutaneous cellular tissue, muscles, etc.), transplantable chicken sarcoma MKh-659 (primarily induced with methylcholanthrene), and normal chicken serum. Evidence was obtained that it is possible to detect the specific antigen in Rous sarcoma extracts only with the aid of precipitation in agar, while the ring precipitation reaction does not permit differentiation of this antigen.Division of Immunology and Oncology (Head-Active Member AMN SSSR L.A. Zil'ber) of the N. F. Gamalei Institute of Epidemiology and Microbiology (Director-Prof. S. N. Muromtsev)(Presented by Active Member AMN SSSR L. A. Zil'ber) Translated from Byulleten' Èksperimental'noi Biologii i Meditsiny, Vol 49, No. 6, pp. 79–84, June, 1960     

Spore antigens of Clostridium sporogenes.

By means of spore-agglutination of fluorescent-antibody techniques, three serological types were identified among 84 strains of Clostridium sporogenes. Four spore antigens were identified, designated A, B, C and D. A, B and C were specific for the respective types whilst D was a group antigen shared by strains of the three types. The spore antigens had corresponding somatic antigens; the type-specific somatic antigens were designated I, II, III, and the shared somatic antigen IV. The flagellar antigens were found to be type specific and were designated 1, 2 and 3; no common flagellar antigen was detected. The results of precipitation tests with spore extracts depended on the method of testing. By a capillary-tube ring method there were cross reactions among the three types of C. sporogenes, whilst by immunodiffusion in agar layers the reactions were generally type specific. In a disintegrated spore extract, two non-protein antigenic components, possibly polysaccharide, were detected by means of immunoelectrophoresis. This extract showed cross reaction with antisera to strains of all types of all types of C. sporogenes by the ring test and by immunodiffusion.     

The antigenic properties of the red cells in leukemia

Summary By their antigenic properties the erythrocytes of leukemic patients differ from those of healthy donors of the same blood group (by the ABO and Rh systems). These differences are revealed in the reaction of specific inhibition of precipitation in agar with the antiserum obtained by immunization of rabbits with the leukemic splenic tissue.By inhibiting precipitation through introduction of surplus erythrocytes of healthy donors into agar, the antiserum would not reveal their full range (incomplete inhibition) or, in reacting with them, would not form any lines of precipitation at all. It was against this background that the lines of precipitation formed in reaction of the antiserum with the erythrocytic antigens of leukemic patients were especially pronounced.In 262 tests out of 293 (in 89% of all cases) the erythrocytes of patients suffering from various forms of leukemia could be differentiated from those of healthy donors by their antigenic properties.Presented by Active Member Acad. Med. Sci. USSR, L. A. Zil'berThe subject matter of this article formed part of a lecture given at the 36th Plenum of the Central Order of Lenin Institute of Blood Transfusion on June 3. 1957.     

Quantitative study of immune horse serum antibody a gainst Clostridium oedematiens by means of precipitation on paper or paper-fixed antigen

Summary Reaction of flocculation between the antitoxin ofCl,oedematiens and the corresponding immune horse serum was studied with the aid of precipitation on paper. When toxin and immune serum were placed on the same place on the paper, a flocculate appears, which unlike all the rest of the proteins is not washed off the paper by the veronal buffer. The fiocculate is precipitated at the zone of considerabic surplus of antitoxin. The quantity of precipitate is increased in proportion to the quantity of the added antigen up to the time when the whole quantity of the antigen is precipitated. Then the precipitate begins to dissolve in the surplus of the antigen. Data which were obtained by the method of precipitation on paper correspond to the results of determination of the quantity of the antibodies in the same serum with the aid of the antigen fixed on paper through diazoconnection and the residual haloidalkylate. This points to the possibillity of replacement of the biological method of determination of the antigens ofCl.oedematiens by the immunochemical method.Presented by V.N. Orekhovich, Active Member Acad. Med. Sci. USSR     

Common antigens of streptococcal and non-streptococcal oral bacteria: immunochemical studies of extracellular and cell-wall-associated antigens from Streptococcus sanguis, Streptococcus mutans, Lactobacillus salivarius, and Actinomyces viscosus.

Soluble extracellular antigens (ESA) were prepared from the culture supernatant of exponential growing cells of Streptococcus sanguis OMZ 9 by a combination of ammonium sulfate precipitation and chromatography on a Bio-Gel P6 column. Soluble cell wall antigens (WEA) were obtained from the bacterial pellet by extraction with 1 M phosphate buffer (pH 6). Antisera against whole cells of S. sanguis and S. mutans of different serotypes, 10% trichloroacetic extracts of bacterial cell walls, dextran, ESA, and WEA were prepared by injecting the different antigens several times in rabbits. ESA and WEA were prepared from a representative strain of Bratthall's seven serological groups, Lactobacillus salivarius, and Actinomyces viscosus. All sera showed various agglutinin titers against heat-killed cells, and titers were generally higher with homologous cells. The comparison of the different antigens using agar gel diffusion and immunoelectrophoresis showed the presence of extracellular common antigens in both ESA and WEA between the different strains. Absorption of anti-ESA sera with WEA, and anti-WEA sera with ESA, showed the existence of a specific antigen common to all bacteria in each fraction. Enzymatic treatment of the antigen before immunodiffusion demonstrated the protein nature of the two antigens present in ESA and WEA.     

Specific precipitation reaction in agar by the ouchterlony method

Summary Reaction of specific precipitation in agar is one of the methods of immunological analysis which have future prospects.This method provides an opportunity to reveal the structure of complex combined antigens, to identify their separate components and to conduct a direct comparative analysis of various antigenic preparations. Special advantage of this method over other serological methods lies in the fact that it permits differentiation of the complex antigen, which is a molecule with two determinative groups from the combination or mixture of two antigens, each one of which represents a separate molecule.Presented by Active Member AMN SSSR L. A. Zilber     

Characterization of polysaccharide antigens of Streptococcus mutans B13 grown under various conditions.

Bacteria may respond to changes in their environment by varying the synthesis of surface components. This study examined the effects of various culture conditions on two wall polysaccharides of Streptococcus mutans B13 (serotype d): serotype d antigen, a galactose-glucose polymer, and RGP antigen, a rhamnose-glucose polymer. Cells were grown in a chemostat at various dilution rates (D) and pH values, including D = 0.05, 0.1, and 0.5 h-1 at pH 6.0 and D = 0.1 h-1 at pH 5.5, 6.0, 6.5, and 7.5. The cells were examined for protein and carbohydrate content by colorimetric assays and gas-liquid chromatography. Rantz-Randall extracts (120 degrees C, 30 min) and M-1 N-acetylmuramidase digests were prepared and examined for the presence of specific antigens by agar gel diffusion and quantitative precipitation assays. Cell preparations did not vary significantly with respect to total protein or carbohydrate content; however, cells grown at D = 0.1 h-1 and pH 7.5 had a significantly higher rhamnose content than did the other preparations. Rapidly growing cultures appeared to be more resistant to M-1 N-acetylmuramidase digestion than did slower-growing cultures. Agar gel diffusion studies demonstrated that both serotype d and RGP antigens were present in all samples, although significantly less RGP antigen was noted in the pH 7.5 culture. These observations were confirmed by quantitative precipitation assays. M-1 N-acetylmuramidase digests of the pH 7.5 culture were lacking in RGP precipitation activity although RGP inhibition activity was demonstrated. The data suggest that the cell content of serotype d antigen was relatively constant under the growth conditions tested, whereas the synthesis of RGP antigen was modified under conditions of high pH.     

Behavior of Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagglutination.

Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad. We propose to include this antigen into the international E. coli typing scheme. Ultrasonic extracts of field strains of E. coli possessing the K88ab, K88ac, or K88ad antigen and their E. coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C. By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis. When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic. The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E. coli K-12. Anodic and cathodic K88ac antigens could not be distinguished serologically. The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow. We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture. It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes.     

Evaluation of Bacillus anthracis extractable antigen for testing anthrax immunity

Three extractable Bacillus anthracis cell-wall-associated antigens were evaluated for potential use as skin testing agents, and as possible candidates for in-vitro diagnosis of anthrax immunity. Anthraxin and a partially purified extractable antigen (EAP) were produced from avirulent B. anthracis strain 34F2 (Sterne). The thermoextractable antigen used for the Ascoli reaction was obtained commercially. Guinea-pigs were immunised and boosted several times subcutaneously with the Sterne live veterinary anthrax vaccine. Four weeks after the last booster dose, animals were skin-tested with the three antigens. Serum antibody levels were also determined by ELISA, and the in-vitro T-cell response was evaluated by [3H]-thymidine incorporation. EAP was the most active antigen in both the serological and cellular reactions. EAP also elicited a distinct positive skin reaction in animals immunised with B. anthracis. The data obtained in this preliminary study indicated that extractable cell-wall antigens obtained from the vegetative form of B. anthracis may be used for skin tests and in-vitro testing of specific humoral and cell-mediated anthrax immunity.     

Limiting sensitivity of the method of precipitation in agar

Summary In order to increase the utilization of the method of precipitation in agar, a study was made of the relationship between its sensitivity and the absolute concentrations of antigens and their antibodies. The sensitivity of the precipitation reaction, as measured by either the relative or absolute quantities, may be increased by choice of the corresponding antigen and antibody concentration, and by the use of cadmium salts.(Presented by Active Member AMN SSSR L. A. Zil'ber) Translated from Byullenten' Éksperimental'noi Biologii i Meditsiny, Vol. 52, No. 12, pp. 107–111, December, 1961     

On the antigenic structure of rous sarcoma

Summary The following constituents were obtained in fractionating Rous' sarcoma: 1) supernatant fluid (60,000 g; 1.5–2 hours), which did not contain corpuscular biologically active virus; 2) doubly reprecipitated precipitate, provoking tumors in chickens in the 1500 titre. Purified specific precipitating antibodies were isolated from the precipitate formed by the specific Rous sarcoma antigen (supernatant fluid). Testing of these antibodies in reaction of precipitation in agar has demonstrated that they react with the supernatant fluid in the same way as with the Rous sarcoma extract and do not react with the doubly reprecipitated precipitate; they did not neutralize sarcoma virus. Consequently, specific precipitating antigen of Rous sarcoma is not a virus antigen and is evidently of tissue origin. Apart from the specific precipitating antibodies, the virus neutralizing antibodies were revealed in the Rous sarcoma antiserum. These antibodies neutralized the virus at the same titre, as the initial Rous sarcoma antisera. However, they did not react in precipitation reactions in agar with any of the fractions of the Rous sarcoma tissue.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 50, No. 7, pp. 70–73, July, 1960     

Analysis of antigen—antibody complexes by immunoelectrophoresis

Immune complexes composed of two insoluble and two soluble antigens and their corresponding antibodies of rabbit origin were separated by electrophoresis in agar gel performed at 56°. Antibodies could then be demonstrated in the cathodal part of the electrophoretic field; they formed precipitation lines with goat antiserum to rabbit serum. The separation of soluble antigens could be demonstrated by the precipitation reaction with their corresponding antisera.     

Some properties of low-molecular-weight tetanus antitoxin

After intravenous injection of tetanus antitoxin obtained by tryptic digestion of Diaferm-3 horse immunoglobulin, purification, and concentration of the active fragments, the antitoxin was excreted by rabbits 3 times faster than after injection of the original Diaferm-3 antitoxin. After injection of the fragmented antitoxin its excretion continued until the 6th days, whereas after injection of Diaferm-3 antitoxin its excretion continued until the 19th day; in the first case much less antitoxin was excreted than in the second (2% and 3.5% respectively). In both cases the antitoxin excreted in the urine consisted of monovalent Fab' fragments which caused delay of precipitation in the cross reaction in agar gel between tetanus toxoid and antitetanus serum. The Fab' gragment obtained by this method possessed anaphylactogenic properties.L'vov Medical Institute. Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 331–333, March, 1976.     

Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals.

Immunological tolerance is a state of unresponsiveness to foreign substances (antigens) which can develop in human and animal species as the result of continued exposure to antigens early in life. We utilized this principle for the preparation of antibodies against Clostridium botulinum type A toxin. By selective suppression of the immunological response of rabbits to unwanted antigens and subsequent immunization with a toxoid, we were able to produce a specific type A antitoxin without the need to purify the toxin. Despite cross-reactivity with C. botulinum type B, our type A antitoxin was otherwise specific since it did not react with culture filtrates of nontoxigenic variants of type B, any other C. botulinum type (C, D, E, F, and G), nor with 18 other Clostridium species, including Clostridium sporogenes. Using this antitoxin, we developed a sensitive enzyme-linked immunosorbent assay for detection of C. botulinum type A toxin.     

Experimental pulpal immunization. I. Route of application and demonstration of immune response.

The experimental exposure of the pulpal room of rabbit teeth made it possible to apply an ovalbumin agar gel deposit into the pulpal room and subsequently investigate the immune response. Diffusion of antigen from the agar into environment was proven. After 3 weekly applications 5 rabbits showed hemagglutinating anti-ovalbumin titers between 1:128 and 1:512, while 7 rabbits receiving 10 weekly applications had titers between 1:8000 and 1:128,000. Control rabbits which received the agar deposit alone showed no antibody response. Control rabbits which obtained 3 identical weekly doses of ovalbumin in PBS by subcutaneous injection showed an identical immune response as animals immunized via the pulpal room. Specificity of antibodies was ascertained by passive hemagglutination with control antigens and hemagglutination inhibition. Intradermal injection of ovalbumin in 3 rabbits which obtained pulpal immunization, induced an Arthus reaction. The precipitating property of ovalbumin antibodies and their identity with defined ovalbumin antisera was proven by gel diffusion. The results obtained show, that antigens present in the pulpal room provide a strong immunogenic stimulus.     

Preparation of rubella virus haemagglutinating antigen by polyethlene glycol precipitation.

Polyethylene glycol (PEG) was used for precipitation of rubella virus haemagglutinating (HA) antigen from the fluid phase of infected Vero cell cultures, maintained with or without calf serum. In both cases potent antigen preparation were obtained. When the antigen was precipitated from serum-containing medium, higher titres were obtained, provided that substrate or final product were treated with Tween 80 and ether. HA antigen obtained by PET precipitation was entirely valuable in the haemagglutination-inhibition test, as compared with antigens prepared by other methods.     

Immunological relationship among glycogen-protein complexes of different species

Glycogen-protein complexes from the muscles of Celerio euphorbiae and horse as well as from the livers of frog, carp and man were isolated and used as antigens. After immunization of rabbits antisera were obtained from which fractions of immunoglobulins were isolated. Antibodies were used in agar immunodiffusion tests and in quantitative reaction of immunoprecipitation with glycogens. The immunoprecipitation test proved the occurrence of quantitative differences in cross reaction of complexes with different origin antibodies, testifying to variability of their antigen properties. The immunodiffusion test allows to suppose the presence in antigens of at least two determinants of protein-carbohydratic character.     

A trial of using antibodies as carriers of alkylating agents. II. Evaluation of ability to form 32P-cyclophosphamide + immune antibody complexes with homologous antigen.

32P-cyclophosphamide was found to combine with gamma-globulin fractions of immune sera. Immune sera incubated with 32P-cyclophosphamide retained ability to react specifically with homologous antigen in vitro in the system: MN antigens of human erythrocytes + rabbit anti-MN antibody, and probably reacted selectively with target antigens in vivo in the system: antigens of guinea pig kidney tissue + rabbit antibodies against these antigens. Hemagglutination, passive hemagglutination and precipitation in agar gel tests were used in the experiments. Ability to combine of the immune antibody + 32P-cyclophosphamide complex with homologous antigens was evaluated by measurements of radioactivity of studied materials (erythrocyte agglutinates and organ homogenates). The results indicate feasibility of using immune antibodies as carriers of cytostatic agents.     

Biochemical characterization of tumor-specific cell surface antigens on avian oncornavirus transformed cells.

A sensitive indirect immune precipitation method coupled with polyacrylamide-gel electrophoresis was used to detect new antigens from avian oncornavirus-transformed chicken embryo cells (CEC). A total of four antigens were recognized by this method. Two of these antigens appeared to be identical to the gp85 and gp37 components of the virus envelope. The other two antigens were distinct from the viral envelope antigens on the basis of both size and antigenicity, and were group specific for the avian oncornavirus system. The larger nonvirion antigen was detectable on transformed CEC but absent from normal or productively infected nontransformed CEC and therefore was clearly tumor specific. Because of the weak detectability of the smaller nonvirion antigen, its tumor specificity could not be decided. The tumor-specific antigen was a glycoprotein of 100,000-dalton apparent molecular weight, whereas the smaller nonvirion antigen was 32,000 daltons and probably also a glycoprotein. The 100,000-dalton tumor-specific antigen was found on the cell surface and therefore represented a tumor-specific cell surface antigen (TSSA).