An investigation of the antigenic composition of Shigella flexneri types 1b and 3
Miller, John T
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The purpose of this investigation was to study the antigenic structure of Shigeila flexneri types 1b and 3. A second aim was to study the number of molecular entities involved in the serologic cross-reactions between these types. The presence of antigen and antibody was detected by placing them in opposing wells of an agar-gel plate prepared according to the method of Ouchterlony (Acta Path. Microbiol., 26:567, 1949). The reactants diffused into the agar and where they met in equivalent concentrations a precipitation zone was formed. Antiserum was prepared in rabbits by immunization with whole, live organisms. Saline suspensions of weighed quantities of acetone-killed organisms were digested with trypsin and the supernatants used as antigenic extracts. Antiserum was absorbed directly in the agar-gel by placing antigenic extracts in the agar under the antiserum well. Specific antigens were absorbed directly from antigenic extracts in a similar manner, by placing antibody under the antigen well. The data were interpreted according to Wilson and Pringle (J. Immun., 75:460, 1955). They described the ways in which the serologic relationships of various antigens can be established. If two antigens are similar, then their precipitation zones will completely coalesce. However, if two antigens are similar but not identical, their zones coalesce but also one zone crosses the other zone to form a spur. If two antigens are completely dissimilar, their zones will cross without coalescing. The antibody response of rabbits to different strains of the same type of Sh. flexneri was uniform. The component antigens of type 1b or 3 Sh. flexneri antigenic extracts did not vary among strains of the same type. The homologous reaction of type 1b Sh. flexneri antigen and antibody was determined using antigenic extracts made from either 20 or 30 mg/ml suspensions of type 1b cells. These extracts were allowed to react with type 1b antiserum and both extracts formed three precipitation zones. These zones were considered to represent the major antigen-antibody systems of type 1b, although the other antigens are present in this organism. A type 1a antigenic extract made from a 20 mg/ml cell suspension and a type 1b antigenic extract from a 30 mg/ml cell suspension were allowed to react with type 1b antiserum. Whereas the homologous reaction formed three precipitation zones, the heterologous reaction formed only a single zone showing a reaction of complete identity with one zone of the homologous reaction. This cross-reaction was also studied using type 1a and 1b antiserum and type 1b antigenic extract. The results so obtained were the same as those presented above. From this it was concluded that of the three antigen-antibody systems detected im the homologous reaction, one was present in both types 1a and 1b, presumably due to the type specific I antigen. Type 1b and 3 antigenic extracts prepared from 30 mg/ml suspensions of the appropriate cells were permitted to react with type 1b antiserum. The homologous reaction formed three precipitation zones and the heterologous reaction formed only one zone. This zone showed a reaction of complete identity with one zone of the homologous reaction. Using type 1b and 3 antisera and type 1b antigenic extract, similar results were obtained. From this it was concluded that one of the three antigen-antibody systems detected in the homologous reaction was shared by types 1b and 3 Sh. flexneri, presumably due to the group specific 6 antigen. It was shown by direct absorption in agar of specific antibodies from antiserum that the antibody directed against the antigen shared by types 1a and 1b could not be removed by type 3 antigenic extract. Type 1a antigenic extract would only remove the antibody directed towards it from type 1b antiserum and left the antibodies directed towards the other two antigens. Experiments employing the direct absorption of specific antigens from antigenic extracts in agar confirmed the results obtained by serum absorption. It was concluded that the type specific I antigen antibody system was shared by types 1a and 1b, and the group specific 6 antigen-antibody system was shared by types 1b and 3. Of the two antigens not shared by types 1a and 1b only the 6 group antigen was found in types 1b and 3. The antigen-antibody system unique to type 1b probably represented the "s" antigen. The homologous reaction of type 3 Sh. flexneri antigen and antibody was studied using antigenic extracts made from either 20 or 30 mg/ml cell suspensions. They were allowed to react with type 3 antiserum and three precipitation zones were formed. These zones were considered to represent the major antigen-antibody systems, although other antigens are present in this type. Type 1b and 3 antigenic extracts, prepared from 30 mg/ml suspensions of the appropriate type cells were allowed to react with type 3 antiserum. The homologous reaction produced three precipitation zones, whereas heterologous reaction resulted in two precipitation zones which exhibited reactions of identity with two of the zones formed in the homologous reaction. It was concluded that two of the three antigen-antibody systems detected in the homologous reaction were shared by types 1b and 3. By means of the direct absorption of specific antibodies from antiserum, it was shown that the antibodies directed towards the cross-reacting antigens could be removed from an antiserum and the antibody directed towards the unique antibody left. The cross-reacting antigens could also be absorbed from type 3 antigenic extracts when type 1b antiserum was placed in the agar beneath the antigen well. From these experimental findings it was concluded that the type specific III antigen-antibody system was the system not shared by types 1b and 3. The two antigen-antibody systems shared by types 1b and 3 were concluded to represent two of the three group antigens, 4, 6, or 9. From the results of the experiments involving the specific absorption of antigens or antibodies of either types 1b or 3, it was concluded that the type specific antigens diffused through the agar as molecular entities separate from the group antigens. The relatiopship of this conclusion to other investigators' findings concerning the antigenic structure of Sh. flexneri was discussed.
Thesis (M.A.)--Boston University
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