Immunodiffusion method for identification of group A streptococci

Abstract

The development of a method suitable for identification of group A streptococci by microprecipitation in gels is described. The method is based on preparation of specific sera containing high antibody levels against the antigenic determinant characteristic of group A streptococcal polysaccharide. In a comparative study with a counterimmunoelectrophoresis method, the proposed test proved to be specific, easily read, and less complicated. Results were obtained in 2 h. Determination of groups to which hemolytic streptococci belong is still made by the Lancefield method of precipitation in liquid medium (13,20). To make the determination more convenient, attempts to utilize immunodiffusion techniques, including the very sensitive counterimmunoelectrophoresis (CIEP) method (5,6,22), have been made. No special investigations can be found, however, aimed at presenting evidence that the reactions obtained are due to antibodies against the polysaccharide determinants specific for particular groups. Besides, immunodiffusion with sera prepared against whole microbial cells reveals cross-reactions between streptococci of different groups (22).The specific antigenic determinant of group A streptococcal polysaccharide (A-polysaccharide) is known to be the terminal group of its side chains, represented by N-acetylglucosamine, filinked (11,17). A-polysaccharide also contains a determinant, associated with rhamnose oligosaccharides, which is shared by streptococci not belonging to group A (11).A-polysaccharide gave good reactions in the gel microprecipitation test. But along with the reactions due to antibodies against the specific terminal determinant, reactions with the rhamnose determinants were also detectable. The latter reactions were found with sera of animals immunized with A-variant streptococcus (1, 2). It is known that the polysaccharide of this strain (V-polysaccharide) lacks the terminal determinants, but contains the rhamnose groups (17). Observation of the reactions between A-polysaccharide and antisera containing antibodies to Vpolysaccharide, as well as inhibition of the reactions by V-polysaccharide, indicates that Apolysaccharide preparations contain a proportion of molecules without the terminal determinant (1, 2). This may be one of the reasons for the cross-reactions between streptococci of different groups.Cross-reactions are possible also because of polyglycerophosphate (PGP) (3, 16) and the socalled non-type-specific antigens (14, 15) present in HCl extracts of streptococci belonging to group A and other groups.One should also eliminate the possibility of nonspecific reactions, since the cell wall proteins of group A streptococci, just like A-protein of staphylococci, are capable of reacting with the Fc portion of immunoglobulins (4, 12).The present investigation was undertaken to develop a procedure for the identification of group A streptococci by a gel microprecipitation test based on the use of specific antisera with high antibody levels against the terminal antigenic determinant of A-polysaccharide. ...