Vol. 19, No. 3
INFECTION AND IMMUNITY, Mar. 1978, P. 961-965
0019-9567/78/0019-0961$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Immunodiffusion Method for Identification of Group A Streptococci I. M. LYAMPERT,* N. A. BORODIYUK, T. K. ASOSKOVA, AND I. I. RASSOKHINA
Laboratory of Streptococcal Infections, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Science, Moscow 123098, U.S.S.R. Received for publication 15 July 1977
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, linked (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 fi-
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. Data on sensitivity of the developed test compared with those of the CIEP method are presented. MATERIALS AND METHODS Antisera. Rabbits were immunized with group A streptococci (type 1, no. 2/49; a strain collection of streptococci belonging to group A and other groups was kindly supplied by J. Rotta, Prague, Czechoslovakia) that was killed by heating and treated with pepsin (18) or not treated. Some animals received an A-variant culture (27T 32/18; gift of M. McCarty) treated with pepsin. Rabbits underwent three cycles of immunization each lasting 4 weeks (three inoculations per week). During week 1 they were inoculated with 0.25 ml of the vaccine (2 x 109 microbial cells per ml). Within the next 3 weeks, the dose was increased to 0.5, 0.75, and 1.0 ml, respectively. HC1 extracts. HC1 extracts were prepared by the Lancefield method (13), as follows: 1012 microbial cells in 6 ml of 1/20 N HCl solution were heated in a boiling-water bath for 15 min. Strains. The following streptococcal strains were used for preparation of HCI extracts: 25 strains of 961
962
LYAMPERT ET AL.
group A, containing M-proteins and belonging to different types; 16 strains representative of other groups (B, C, D, E, G, H, K, L, M, N, 0, P, Q, R, S, T; Prague collection); and the A-variant strain. Also used were freshly isolated cultures: 81 belonging to group A, 106 of other groups (the grouping was accomplished by a convenient method), and 2 cultures of staphylococci. Polysaccharides and other streptococcal antigens. A-polysaccharide was prepared from a strain of streptococcus group A type 5. The A-variant strain was used for preparing V-polysaccharide and peptidoglycan. Both polysaccharides and peptidoglycan were prepared from cell walls by the formamide method (10). Several antigens were obtained from HCl extracts of a group A type 1 streptococcus by using preparative electrophoresis. The preparative electrophoresis was performed in 1% agar gel using Veronal-medinal buffer (ionic strength 0.025, pH 8.6) for 5 h at 300 mA, 160 to 180 V, potential gradient 5 to 6 V/cm (24). Type-specific proteins were obtained from the starting zone; non-type-specific protein antigens moved the same length as did serum albumin (15), whereas PGP had three times greater mobility (3); a fraction containing A-polysaccharide was obtained from the zone corresponding to pyronine mobility (1). From the same strain, E4 antigen was extracted with 0.85% NaCl, pH 5.9 (23). Also used in the study was a staphylococcal A-protein-containing preparation (kindly supplied by A. K. Akatov). Absorption of sera and inhibition of antibody. Cultures of A-variant and group A streptococci killed by heating and treated with pepsin (18) or staphylococcal cultures not treated with pepsin were used for absorption of sera (100 to 250 mg of lyophilized cells per ml). HCl extracts obtained from staphylococcal or group A and group L streptococcal cultures were added to some of the sera (0.2 ml of 5x concentrated HCl extract per ml). Specificity of reactions between A-polysaccharide and immune sera was controlled also by adding A- or V-polysaccharides (0.2 to 2.0 mg/ml), peptidoglycan (10 to 50 mg/ml), or synthetic N-acetylglucosamine (Biochemical Research Corp., Los Angeles, Calif.) (80 mg/ml) to the serum. Immunodiffusion techniques. Immunoelectrophoresis (IEP) (8) and a gel precipitation test (PT) (19) as a micromethod were performed with the modifications of Zilber and Abelev (24). Agar (1%; Difco Laboratories, Detroit, Mich.) in pH 8.6 Veronal-medinal buffer was used. IEP was run for 1 h at 100 V and 20 mA (potential gradient, 4 V/cm). PT was read after 2 and 24 h, and IEP was read after 24 h, after washing agar plates in 10% NaCl solution for 20 min. CIEP (5) was performed by using 1% ion-agar N 2 (Oxoid Ltd., London) prepared in Veronal-medinal buffer (pH 8.6) for 30 min at 20 mA and 100 V (potential gradient, 4 V/cm). All immunodiffusion experiments were run with 5x concentrated sera and nonconcentrated HCl extracts. In experiments comparing PT and CIEP, the formamide preparation of A-polysaccharide was used in a range of 3 to 100 jyg per ml (0.02 ml of solution in each well in PT, and 0.01 ml of solution in CIEP). The
INFECT. IMMUJN. results were read after 30 and 45 min as well as after 1, 2, 3, 4, 5 and 24 h.
RESULTS Examination of A-polysaccharide preparations in PT and IEP. A single precipitation line can be obtained in PT in as early as 2 h when A-polysaccharide (25 to 100 jig per ml) is tested against sera of rabbits immunized with group A streptococcal culture treated with pepsin. The reaction is completely abolished by absorption of the serum with group A streptococcal culture treated with pepsin as well as by inhibition with A-polysaccharide or 13-N-acetylglucosamine (Fig. 1). In contrast, absorption with A-variant culture and addition of V-polysaccharide or peptidoglycan do not exert any influence on the reaction between sera and Apolysaccharide. A-polysaccharides obtained by the formamide method and by preparative electrophoresis from HCO extracts were identical in PT. When tested
5589~~2
Br~r
FIG. 1. Reaction between A-polysaccharide and rabbit serum obtained by immunization with pepsintreated group A streptococci and absorbed with Avariant culture. (A) Central well, immune serum; peripheral wells (1 to 5), A -polysaccharide prepared by the formamide method with 6 to 100 pug/ml. Well 1, 100tpg/ml; Well 2, 5O,ug/ml; Well 3, 25,Lg/ml; Well 4, 12 jig/ml; Well 5, 6 pg/ml. (B) and (C) Central wells, A-polysaccharide prepared by the formamide method (25 Ag/ml); peripheral wells (1 and 2), immune serum before (B) and after (C) the addition of 83-N-
acetylglucosamine.
VOL. 19, 1978
.
METHOD FOR STREPTOCOCCAL IDENTIFICATION
by IEP, they produced a precipitation line in the zone of pyronine mobility in the system used. When the serum was placed in the well and Apolysaccharide was placed in the trough, the reaction was detected in the zone of immunoglobulin G mobility (Fig. 2). Determination of antibodies against Aand V-polysaccharides. Most sera after two cycles of immunization with group A streptococcal culture treated with pepsin and all sera after three cycles gave distinct reactions with A-polysaccharide at a concentration of 10 mg/ml. Such sera were designated as immune sera. If the pepsin treatment was omitted, no reactions with A-polysaccharide under similar conditions were observed with sera of cycle 2 and very rarely (3 out of 14) with sera of cycle 3. None of the 24 sera of nonimmunized rabbits revealed any positive reaction in PT with different concentrations of A-polysaccharide. No positive reactions were observed between unabsorbed immune sera and V-polysaccharide (5 mg to 25 ,ug/ml). At the same time, V-polysaccharide in these concentrations distinctly reacted with sera prepared by immunization with pepsin-treated A-variant cultures. Occurrence of antibodies against other streptococcal antigens. Immune sera did not react in PT with type-specific and non-type-specific protein antigens that gave positive reactions with sera against whole microbial cells. Antibodies to PGP and E4 antigen were present in some immune sera. The precipitation lines formed with these antigens could be washed out from the gel by immersion in 10% NaCl solution for 20 min. The antibodies to PGP and E4 antigen
963
were completely removed by absorption of immune sera with A-variant culture treated with pepsin. The absorption did not decrease markedly the intensity of reactions with A-polysaccharide. Examination of HCl extracts from group A streptococci. Using immune sera absorbed with A-variant culture, only one precipitation line was obtained with HC1 extracts prepared from 25 strains containing M-protein and belonging to different types (Prague collection), as well as with extracts from 81 freshly isolated group A streptococcal cultures. These reactions were visible as early as 2 h at room temperature and became more intensive by 24 h. The component of HCl extracts reacting with absorbed sera was identical to A-polysaccharide (Fig. 3). No reactions were observed in PT between normal rabbit sera and HCl extracts prepared from different cultures of group A. Testing of sera with HCI extracts from streptococci of other groups and staphylococci. Absorbed immune sera did not react with HCl extracts prepared from the collectional and freshly isolated streptococci not belonging to group A. The only exception was the group L strain, with which HCl extracts reacted positively in 3 out of 15 absorbed and 5 out of 24 nonabsorbed immune sera. These HCl extracts did not react with normal rabbit sera. One or two precipitation lines were observed between some immune sera and HCl extracts from staphylococci (eight nonabsorbed sera out of 40 and 1 absorbed serum out of 15). One of the staphylococcal antigens was identical to Aprotein. Both staphylococcal antigens were dif-
-~~~~
I
dw
o.
..,
w~~~~~-. e
.,
-===Z'=-,
!rn
aIs . ......... ..... c II
.. , ...oc
... ..=.
FIG. 2. IEP patterns obtained in studying A-polysaccharide with immune sera. (1) Well, A-polysaccharide preparation; troughs, immune serum. (2) Well, immune serum; troughs, A-polysaccharide preparations.
964
LYAMPERT ET AL.
A
(_ 1 &,
B
INFECT. IMMUJN.
l
2
e& 1
r- -,i- )
2
FIG. 3. Identity between the reacting substance in HCl extract of group A streptococci and A-polysaccharide prepared by the formamide method. Central wells, immune sera. Peripheral wells, (1) HCl extract of group A streptococci; (2) polysaccharide preparation (100 jig/ml). (A) Reaction of precipitation in 2 h. (B) Reaction ofprecipitation in 24 h.
ferent from the antigen of group L culture, which reacted with some immune sera. Absorption of sera with A-variant streptococci only weakened the reaction with HCl extract from L streptococci and did not affect the reactions with staphylococcal extracts. When the staphylococcals culture was used for absorption, reactions with the homologous extract were absent and, simultaneously, there was a reduction of the antibody titers against A-polysaccharide. Comparison of CIEP and PT in detecting A-polysaccharide. HC1 extracts of group A streptococci gave a single precipitation line in CIEP in 30 min if absorbed immune sera were used. Similar time was required to obtain reactions in CIEP with A-polysaccharide in concentrations of 25 to 100 ,ug/ml. With the same concentrations of A-polysaccharide, positive reactions in PT were obtained in 2 h. At the polysaccharide levels of 6 to 12 ,ug/ml, positive reactions were evident in 45 min for CIEP and in 3 to 4 h for PT. With still lower concentration (3 pg/ml), both methods produced a distinct reaction after only 24 h. DISCUSSION As the present study shows, a gel microprecipitation technique can be successfully used to identify group A streptococci. This technique is unquestionably more demonstrable and exact than precipitation in liquid medium. Sera of rabbits, immunized with pepsin-treated group A streptococcal culture (18), as a rule, contain high antibody levels against A-polysaccharide after absorption with the A-variant strain. This level
is sufficient for reaction with a large dose of Apolysaccharide and permits detection of very low doses (3 ug/ml). Only single rabbit sera obtained by immunization with intact microbial cells possessed such activity. The reaction of absorbed sera with A-polysaccharide is highly specific since it depends on antibodies against the terminal antigenic determinant containing N-acetylglucosamine. Addition of A-polysaccharide or synthetic N-acetylglucosamine abolished this reaction, although it was not inhibited by V-polysaccharide or peptidoglycan. Although it was shown that preparations of A-polysaccharide included molecules lacking the terminal determinant (1, 2), antisera obtained by immunization with pepsin-treated group A streptococci do not contain antibodies against V-polysaccharide. This excludes the possibility of cross-reactions in PT with streptococci of other groups due to the common rhamnose determinant. Cross-reactions with other groups, caused by other antigens, can also be excluded. Antibodies against the cell wall proteins were shown to be absent from immune sera prepared by the method used. The antibodies against PGP and E4 antigen could be completely eliminated by absorption with A-variant culture. As far as the rare cross-reactions with streptococci of group L are concerned (such reactions had been described earlier by Jelinkova et al., 9), the problem can be solved by proper selection of sera. In the same way, the rare reactions with staphylococcal antigens can be excluded. The latter reactions, most probably are due to the interaction between the A-protein of staphylococci and the Fc portion of immunoglobulins (7) and are not very frequent with rabbit sera (P. Christensen, personal communication). Similar reactions between the protein antigens of group A streptococci and the Fc portion of immunoglobulins (4) seem not to be detectable in the gel PT with HCl extracts and rabbit sera. This was supported by absence of any additional precipitation lines between immune sera and HCl extracts prepared from strains containing M proteins, as well as by negative results in tests with the fraction containing cell wall proteins. Thus, by immunization of rabbits with pepsintreated group A streptococci followed by absorption with the A-variant strain and selection of sera that do not react with HCl extracts of group L streptococci and staphylococci, it is possible to obtain a highly specific system suitable for microprecipitation in gel. Some authors (22) reported data showing that CIEP is a more sensitive method than PT for detecting streptococcal group A polysaccharide.
METHOD FOR STREPTOCOCCAL IDENTIFICATION
VOL. 19, 1978
According to the data obtained in our study, examination by PT of HCO extracts prepared from group A cultures or A-polysaccharide solutions (25 to 100 [Lg/ml) did not require a significantly longer time than did testing by CIEP, since the reactions in PT could be read in 2 h. It is possible that by optimizing the conditions (using different serum doses or other agar grades), smaller polysaccharide doses could be detected in CIEP. In this particular case, however, application of a more sensitive method seems not to be necessary, since all the examined HCO extracts prepared from group A streptococci contain sufficient polysaccharide levels and give clear-cut reactions in PT within a reasonably short time. Besides, the CIEP technique is undoubtedly more complicated than microprecipitation in gel. Together with the simplicity and possibility of obtaining results in a short time, the gel PT permits accurate identification of the reacting substance of HCO extracts and A-polysaccharide, if the latter is used as a control. The proposed test can, therefore, be considered a sufficiently sensitive, specific, and demonstrable tool for identification of group A streptococci. LITERATURE CITED 1. Borodiyuk, N. A., 0. P. Galachyants, and L. L. Rassokhina. 1974. Study of streptococcal group A polysaccharide by immunodiffusion methods [In Russian]. J. Microbiol. Epidemiol. Immunobiol. (USSR) 4:94-99. 2. Borodiyuk, N. A., 0. P. Galachyants, L. I. Rassokhina, and I. AL Lyampert. 1975. Preparation of sera for the detection of group A streptococci with the aid of gel
precipitation [In Russian]. J. Microbiol. Epidemiol. Immunobiol. (USSR) 8:62-67. 3. Borodiyuk, N. A., M. N. Smirnova, and M. K. Prishep. 1966. Study of antigenic substances of streptococcal allergen [In Russian]. J. Microbiol. Epidemiol. Immunobiol. 11:20-24. 4. Christensen, P., and V. A. Oxelius. 1974. Quantitation of the uptake of human IgG by some streptococci groups A, B, C and G. Acta Pathol. Microbiol. Scand. Sect. B. 82:475-483. 5. Dajani, A. S. 1973. Rapid identification of beta homolytic streptococci by counterimmunoelectrophoresis. J. Immunol. 110:1702-1705. 6. Edwards, E. A., and G. L. Larson. 1973. Serological grouping of hemolytic streptococci by counterimmunoelectrophoresis. Appl. Microbiol. 26:899-903.
965
7. Forsgren, A., and J. Sjoquist. 1967. Protein A from staphylococcus aureus. III. Reaction with rabbit gamma globulin. J. Immunol. 99:19-24. 8. Grabar, P., and P. Burtin. 1964. Immunoelectrophoretic analysis [In Russian], translated ed. Moscow. 9. Jelinkova, J., R. Bikova, and J. Rotta. 1967. Some peculiarities of relationship of group A and L streptococci. J. Hyg. Epidemiol. Microbiol. Immunol. 11:323-327. 10. Krause, R. M. 1967. Preparation of cell wall antigens from Gram-positive bacteria. Methods Immunol. Immunochem. 1:34-40. 11. Krause, R. M. 1972. The streptococcal cell: relationship of structure to function and pathogenesis, p. 3-18. In L. W. Wannameker and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., London. 12. Kronvall, G. 1973. A surface component in group A, C and G streptococci with non-immune reactivity for immunoglobulin G. J. Immunol. 111:1401-1406. 13. Lancefield, R. C. 1933. Serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 57:571-595. 14. Lancefield, R. C., and G. E. Perlmann. 1952. Preparation and properties of a protein (R-antigen) occurring in streptococci of group A, type 28 and certain of other serological groups. J. Exp. Med. 96:83-97. 15. Lyampert, L. M., S. G. Shuratova, V. V. Akimova, V. P. Buckhova, and V. Y. Kolesnikova. 1977. Common (non-type-specific) antigens of group A streptococci. Infect. Immun. 17:21-27. 16. McCarty, M. 1959. The occurrence of polyglycerophosphate as an antigenic component of various gram-positive bacterial species. J. Exp. Med. 109:361-378. 17. McCarty, M. 1971. The streptococcal cell wall. Harvey Lect. 65:73-96. 18. Osterland, C. K., E. J. Miller, W. W. Karakawa, and R. M. Krause. 1966. Characteristics of streptococcal group-specific antibody isolated from hyper-immune rabbits. J. Exp. Med. 123:599-614. 19. Ouchterlony, 0. 1962. Diffusion in gel methods for immunological analysis. Prog. Allergy 1:30-154. 20. Rotta, J. 1974. Streptococcus pyogenes. Scientific information. Sevac. Prague, Czechoslovakia. 21. Rotta, J., R. M. Krause, R. C. Lancefield, W. C. Everly and H. Lackland. 1971. New approaches for the laboratory recognition of M types of group A streptococci. J. Exp. Med. 134:1298-1315. 22. Wadstrom, T., C.-E. Nord, A. A. Lindberg, and R. Moilby. 1974. Rapid grouping of streptococci by immunoelectrophoresis. Med. Microbiol. Immunol. 159:191-200. 23. Wilson, A. T., and G. G. Wiley. 1963. The cellular antigens of group A streptococci: immunoelectrophoretic studies of the C, M, T, PGP, F and E4 antigens of serotype 17 streptococci. J. Exp. Med. 118:527-556. 24. Zilber, L A., and G. L. Abelev. 1962. Virology and Immunology of Cancer [In Russian]. Medicine, Moscow.
INFECTION AND IMMUNITY, Mar. 1978, P. 961-965
0019-9567/78/0019-0961$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Immunodiffusion Method for Identification of Group A Streptococci I. M. LYAMPERT,* N. A. BORODIYUK, T. K. ASOSKOVA, AND I. I. RASSOKHINA
Laboratory of Streptococcal Infections, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Science, Moscow 123098, U.S.S.R. Received for publication 15 July 1977
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, linked (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 fi-
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. Data on sensitivity of the developed test compared with those of the CIEP method are presented. MATERIALS AND METHODS Antisera. Rabbits were immunized with group A streptococci (type 1, no. 2/49; a strain collection of streptococci belonging to group A and other groups was kindly supplied by J. Rotta, Prague, Czechoslovakia) that was killed by heating and treated with pepsin (18) or not treated. Some animals received an A-variant culture (27T 32/18; gift of M. McCarty) treated with pepsin. Rabbits underwent three cycles of immunization each lasting 4 weeks (three inoculations per week). During week 1 they were inoculated with 0.25 ml of the vaccine (2 x 109 microbial cells per ml). Within the next 3 weeks, the dose was increased to 0.5, 0.75, and 1.0 ml, respectively. HC1 extracts. HC1 extracts were prepared by the Lancefield method (13), as follows: 1012 microbial cells in 6 ml of 1/20 N HCl solution were heated in a boiling-water bath for 15 min. Strains. The following streptococcal strains were used for preparation of HCI extracts: 25 strains of 961
962
LYAMPERT ET AL.
group A, containing M-proteins and belonging to different types; 16 strains representative of other groups (B, C, D, E, G, H, K, L, M, N, 0, P, Q, R, S, T; Prague collection); and the A-variant strain. Also used were freshly isolated cultures: 81 belonging to group A, 106 of other groups (the grouping was accomplished by a convenient method), and 2 cultures of staphylococci. Polysaccharides and other streptococcal antigens. A-polysaccharide was prepared from a strain of streptococcus group A type 5. The A-variant strain was used for preparing V-polysaccharide and peptidoglycan. Both polysaccharides and peptidoglycan were prepared from cell walls by the formamide method (10). Several antigens were obtained from HCl extracts of a group A type 1 streptococcus by using preparative electrophoresis. The preparative electrophoresis was performed in 1% agar gel using Veronal-medinal buffer (ionic strength 0.025, pH 8.6) for 5 h at 300 mA, 160 to 180 V, potential gradient 5 to 6 V/cm (24). Type-specific proteins were obtained from the starting zone; non-type-specific protein antigens moved the same length as did serum albumin (15), whereas PGP had three times greater mobility (3); a fraction containing A-polysaccharide was obtained from the zone corresponding to pyronine mobility (1). From the same strain, E4 antigen was extracted with 0.85% NaCl, pH 5.9 (23). Also used in the study was a staphylococcal A-protein-containing preparation (kindly supplied by A. K. Akatov). Absorption of sera and inhibition of antibody. Cultures of A-variant and group A streptococci killed by heating and treated with pepsin (18) or staphylococcal cultures not treated with pepsin were used for absorption of sera (100 to 250 mg of lyophilized cells per ml). HCl extracts obtained from staphylococcal or group A and group L streptococcal cultures were added to some of the sera (0.2 ml of 5x concentrated HCl extract per ml). Specificity of reactions between A-polysaccharide and immune sera was controlled also by adding A- or V-polysaccharides (0.2 to 2.0 mg/ml), peptidoglycan (10 to 50 mg/ml), or synthetic N-acetylglucosamine (Biochemical Research Corp., Los Angeles, Calif.) (80 mg/ml) to the serum. Immunodiffusion techniques. Immunoelectrophoresis (IEP) (8) and a gel precipitation test (PT) (19) as a micromethod were performed with the modifications of Zilber and Abelev (24). Agar (1%; Difco Laboratories, Detroit, Mich.) in pH 8.6 Veronal-medinal buffer was used. IEP was run for 1 h at 100 V and 20 mA (potential gradient, 4 V/cm). PT was read after 2 and 24 h, and IEP was read after 24 h, after washing agar plates in 10% NaCl solution for 20 min. CIEP (5) was performed by using 1% ion-agar N 2 (Oxoid Ltd., London) prepared in Veronal-medinal buffer (pH 8.6) for 30 min at 20 mA and 100 V (potential gradient, 4 V/cm). All immunodiffusion experiments were run with 5x concentrated sera and nonconcentrated HCl extracts. In experiments comparing PT and CIEP, the formamide preparation of A-polysaccharide was used in a range of 3 to 100 jyg per ml (0.02 ml of solution in each well in PT, and 0.01 ml of solution in CIEP). The
INFECT. IMMUJN. results were read after 30 and 45 min as well as after 1, 2, 3, 4, 5 and 24 h.
RESULTS Examination of A-polysaccharide preparations in PT and IEP. A single precipitation line can be obtained in PT in as early as 2 h when A-polysaccharide (25 to 100 jig per ml) is tested against sera of rabbits immunized with group A streptococcal culture treated with pepsin. The reaction is completely abolished by absorption of the serum with group A streptococcal culture treated with pepsin as well as by inhibition with A-polysaccharide or 13-N-acetylglucosamine (Fig. 1). In contrast, absorption with A-variant culture and addition of V-polysaccharide or peptidoglycan do not exert any influence on the reaction between sera and Apolysaccharide. A-polysaccharides obtained by the formamide method and by preparative electrophoresis from HCO extracts were identical in PT. When tested
5589~~2
Br~r
FIG. 1. Reaction between A-polysaccharide and rabbit serum obtained by immunization with pepsintreated group A streptococci and absorbed with Avariant culture. (A) Central well, immune serum; peripheral wells (1 to 5), A -polysaccharide prepared by the formamide method with 6 to 100 pug/ml. Well 1, 100tpg/ml; Well 2, 5O,ug/ml; Well 3, 25,Lg/ml; Well 4, 12 jig/ml; Well 5, 6 pg/ml. (B) and (C) Central wells, A-polysaccharide prepared by the formamide method (25 Ag/ml); peripheral wells (1 and 2), immune serum before (B) and after (C) the addition of 83-N-
acetylglucosamine.
VOL. 19, 1978
.
METHOD FOR STREPTOCOCCAL IDENTIFICATION
by IEP, they produced a precipitation line in the zone of pyronine mobility in the system used. When the serum was placed in the well and Apolysaccharide was placed in the trough, the reaction was detected in the zone of immunoglobulin G mobility (Fig. 2). Determination of antibodies against Aand V-polysaccharides. Most sera after two cycles of immunization with group A streptococcal culture treated with pepsin and all sera after three cycles gave distinct reactions with A-polysaccharide at a concentration of 10 mg/ml. Such sera were designated as immune sera. If the pepsin treatment was omitted, no reactions with A-polysaccharide under similar conditions were observed with sera of cycle 2 and very rarely (3 out of 14) with sera of cycle 3. None of the 24 sera of nonimmunized rabbits revealed any positive reaction in PT with different concentrations of A-polysaccharide. No positive reactions were observed between unabsorbed immune sera and V-polysaccharide (5 mg to 25 ,ug/ml). At the same time, V-polysaccharide in these concentrations distinctly reacted with sera prepared by immunization with pepsin-treated A-variant cultures. Occurrence of antibodies against other streptococcal antigens. Immune sera did not react in PT with type-specific and non-type-specific protein antigens that gave positive reactions with sera against whole microbial cells. Antibodies to PGP and E4 antigen were present in some immune sera. The precipitation lines formed with these antigens could be washed out from the gel by immersion in 10% NaCl solution for 20 min. The antibodies to PGP and E4 antigen
963
were completely removed by absorption of immune sera with A-variant culture treated with pepsin. The absorption did not decrease markedly the intensity of reactions with A-polysaccharide. Examination of HCl extracts from group A streptococci. Using immune sera absorbed with A-variant culture, only one precipitation line was obtained with HC1 extracts prepared from 25 strains containing M-protein and belonging to different types (Prague collection), as well as with extracts from 81 freshly isolated group A streptococcal cultures. These reactions were visible as early as 2 h at room temperature and became more intensive by 24 h. The component of HCl extracts reacting with absorbed sera was identical to A-polysaccharide (Fig. 3). No reactions were observed in PT between normal rabbit sera and HCl extracts prepared from different cultures of group A. Testing of sera with HCI extracts from streptococci of other groups and staphylococci. Absorbed immune sera did not react with HCl extracts prepared from the collectional and freshly isolated streptococci not belonging to group A. The only exception was the group L strain, with which HCl extracts reacted positively in 3 out of 15 absorbed and 5 out of 24 nonabsorbed immune sera. These HCl extracts did not react with normal rabbit sera. One or two precipitation lines were observed between some immune sera and HCl extracts from staphylococci (eight nonabsorbed sera out of 40 and 1 absorbed serum out of 15). One of the staphylococcal antigens was identical to Aprotein. Both staphylococcal antigens were dif-
-~~~~
I
dw
o.
..,
w~~~~~-. e
.,
-===Z'=-,
!rn
aIs . ......... ..... c II
.. , ...oc
... ..=.
FIG. 2. IEP patterns obtained in studying A-polysaccharide with immune sera. (1) Well, A-polysaccharide preparation; troughs, immune serum. (2) Well, immune serum; troughs, A-polysaccharide preparations.
964
LYAMPERT ET AL.
A
(_ 1 &,
B
INFECT. IMMUJN.
l
2
e& 1
r- -,i- )
2
FIG. 3. Identity between the reacting substance in HCl extract of group A streptococci and A-polysaccharide prepared by the formamide method. Central wells, immune sera. Peripheral wells, (1) HCl extract of group A streptococci; (2) polysaccharide preparation (100 jig/ml). (A) Reaction of precipitation in 2 h. (B) Reaction ofprecipitation in 24 h.
ferent from the antigen of group L culture, which reacted with some immune sera. Absorption of sera with A-variant streptococci only weakened the reaction with HCl extract from L streptococci and did not affect the reactions with staphylococcal extracts. When the staphylococcals culture was used for absorption, reactions with the homologous extract were absent and, simultaneously, there was a reduction of the antibody titers against A-polysaccharide. Comparison of CIEP and PT in detecting A-polysaccharide. HC1 extracts of group A streptococci gave a single precipitation line in CIEP in 30 min if absorbed immune sera were used. Similar time was required to obtain reactions in CIEP with A-polysaccharide in concentrations of 25 to 100 ,ug/ml. With the same concentrations of A-polysaccharide, positive reactions in PT were obtained in 2 h. At the polysaccharide levels of 6 to 12 ,ug/ml, positive reactions were evident in 45 min for CIEP and in 3 to 4 h for PT. With still lower concentration (3 pg/ml), both methods produced a distinct reaction after only 24 h. DISCUSSION As the present study shows, a gel microprecipitation technique can be successfully used to identify group A streptococci. This technique is unquestionably more demonstrable and exact than precipitation in liquid medium. Sera of rabbits, immunized with pepsin-treated group A streptococcal culture (18), as a rule, contain high antibody levels against A-polysaccharide after absorption with the A-variant strain. This level
is sufficient for reaction with a large dose of Apolysaccharide and permits detection of very low doses (3 ug/ml). Only single rabbit sera obtained by immunization with intact microbial cells possessed such activity. The reaction of absorbed sera with A-polysaccharide is highly specific since it depends on antibodies against the terminal antigenic determinant containing N-acetylglucosamine. Addition of A-polysaccharide or synthetic N-acetylglucosamine abolished this reaction, although it was not inhibited by V-polysaccharide or peptidoglycan. Although it was shown that preparations of A-polysaccharide included molecules lacking the terminal determinant (1, 2), antisera obtained by immunization with pepsin-treated group A streptococci do not contain antibodies against V-polysaccharide. This excludes the possibility of cross-reactions in PT with streptococci of other groups due to the common rhamnose determinant. Cross-reactions with other groups, caused by other antigens, can also be excluded. Antibodies against the cell wall proteins were shown to be absent from immune sera prepared by the method used. The antibodies against PGP and E4 antigen could be completely eliminated by absorption with A-variant culture. As far as the rare cross-reactions with streptococci of group L are concerned (such reactions had been described earlier by Jelinkova et al., 9), the problem can be solved by proper selection of sera. In the same way, the rare reactions with staphylococcal antigens can be excluded. The latter reactions, most probably are due to the interaction between the A-protein of staphylococci and the Fc portion of immunoglobulins (7) and are not very frequent with rabbit sera (P. Christensen, personal communication). Similar reactions between the protein antigens of group A streptococci and the Fc portion of immunoglobulins (4) seem not to be detectable in the gel PT with HCl extracts and rabbit sera. This was supported by absence of any additional precipitation lines between immune sera and HCl extracts prepared from strains containing M proteins, as well as by negative results in tests with the fraction containing cell wall proteins. Thus, by immunization of rabbits with pepsintreated group A streptococci followed by absorption with the A-variant strain and selection of sera that do not react with HCl extracts of group L streptococci and staphylococci, it is possible to obtain a highly specific system suitable for microprecipitation in gel. Some authors (22) reported data showing that CIEP is a more sensitive method than PT for detecting streptococcal group A polysaccharide.
METHOD FOR STREPTOCOCCAL IDENTIFICATION
VOL. 19, 1978
According to the data obtained in our study, examination by PT of HCO extracts prepared from group A cultures or A-polysaccharide solutions (25 to 100 [Lg/ml) did not require a significantly longer time than did testing by CIEP, since the reactions in PT could be read in 2 h. It is possible that by optimizing the conditions (using different serum doses or other agar grades), smaller polysaccharide doses could be detected in CIEP. In this particular case, however, application of a more sensitive method seems not to be necessary, since all the examined HCO extracts prepared from group A streptococci contain sufficient polysaccharide levels and give clear-cut reactions in PT within a reasonably short time. Besides, the CIEP technique is undoubtedly more complicated than microprecipitation in gel. Together with the simplicity and possibility of obtaining results in a short time, the gel PT permits accurate identification of the reacting substance of HCO extracts and A-polysaccharide, if the latter is used as a control. The proposed test can, therefore, be considered a sufficiently sensitive, specific, and demonstrable tool for identification of group A streptococci. LITERATURE CITED 1. Borodiyuk, N. A., 0. P. Galachyants, and L. L. Rassokhina. 1974. Study of streptococcal group A polysaccharide by immunodiffusion methods [In Russian]. J. Microbiol. Epidemiol. Immunobiol. (USSR) 4:94-99. 2. Borodiyuk, N. A., 0. P. Galachyants, L. I. Rassokhina, and I. AL Lyampert. 1975. Preparation of sera for the detection of group A streptococci with the aid of gel
precipitation [In Russian]. J. Microbiol. Epidemiol. Immunobiol. (USSR) 8:62-67. 3. Borodiyuk, N. A., M. N. Smirnova, and M. K. Prishep. 1966. Study of antigenic substances of streptococcal allergen [In Russian]. J. Microbiol. Epidemiol. Immunobiol. 11:20-24. 4. Christensen, P., and V. A. Oxelius. 1974. Quantitation of the uptake of human IgG by some streptococci groups A, B, C and G. Acta Pathol. Microbiol. Scand. Sect. B. 82:475-483. 5. Dajani, A. S. 1973. Rapid identification of beta homolytic streptococci by counterimmunoelectrophoresis. J. Immunol. 110:1702-1705. 6. Edwards, E. A., and G. L. Larson. 1973. Serological grouping of hemolytic streptococci by counterimmunoelectrophoresis. Appl. Microbiol. 26:899-903.
965
7. Forsgren, A., and J. Sjoquist. 1967. Protein A from staphylococcus aureus. III. Reaction with rabbit gamma globulin. J. Immunol. 99:19-24. 8. Grabar, P., and P. Burtin. 1964. Immunoelectrophoretic analysis [In Russian], translated ed. Moscow. 9. Jelinkova, J., R. Bikova, and J. Rotta. 1967. Some peculiarities of relationship of group A and L streptococci. J. Hyg. Epidemiol. Microbiol. Immunol. 11:323-327. 10. Krause, R. M. 1967. Preparation of cell wall antigens from Gram-positive bacteria. Methods Immunol. Immunochem. 1:34-40. 11. Krause, R. M. 1972. The streptococcal cell: relationship of structure to function and pathogenesis, p. 3-18. In L. W. Wannameker and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., London. 12. Kronvall, G. 1973. A surface component in group A, C and G streptococci with non-immune reactivity for immunoglobulin G. J. Immunol. 111:1401-1406. 13. Lancefield, R. C. 1933. Serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 57:571-595. 14. Lancefield, R. C., and G. E. Perlmann. 1952. Preparation and properties of a protein (R-antigen) occurring in streptococci of group A, type 28 and certain of other serological groups. J. Exp. Med. 96:83-97. 15. Lyampert, L. M., S. G. Shuratova, V. V. Akimova, V. P. Buckhova, and V. Y. Kolesnikova. 1977. Common (non-type-specific) antigens of group A streptococci. Infect. Immun. 17:21-27. 16. McCarty, M. 1959. The occurrence of polyglycerophosphate as an antigenic component of various gram-positive bacterial species. J. Exp. Med. 109:361-378. 17. McCarty, M. 1971. The streptococcal cell wall. Harvey Lect. 65:73-96. 18. Osterland, C. K., E. J. Miller, W. W. Karakawa, and R. M. Krause. 1966. Characteristics of streptococcal group-specific antibody isolated from hyper-immune rabbits. J. Exp. Med. 123:599-614. 19. Ouchterlony, 0. 1962. Diffusion in gel methods for immunological analysis. Prog. Allergy 1:30-154. 20. Rotta, J. 1974. Streptococcus pyogenes. Scientific information. Sevac. Prague, Czechoslovakia. 21. Rotta, J., R. M. Krause, R. C. Lancefield, W. C. Everly and H. Lackland. 1971. New approaches for the laboratory recognition of M types of group A streptococci. J. Exp. Med. 134:1298-1315. 22. Wadstrom, T., C.-E. Nord, A. A. Lindberg, and R. Moilby. 1974. Rapid grouping of streptococci by immunoelectrophoresis. Med. Microbiol. Immunol. 159:191-200. 23. Wilson, A. T., and G. G. Wiley. 1963. The cellular antigens of group A streptococci: immunoelectrophoretic studies of the C, M, T, PGP, F and E4 antigens of serotype 17 streptococci. J. Exp. Med. 118:527-556. 24. Zilber, L A., and G. L. Abelev. 1962. Virology and Immunology of Cancer [In Russian]. Medicine, Moscow.
