INFUCTION AND IMMUNrry, Jan. 1976, p. 69-77 Copyright C) 1976 American Society for Microbiology
Vol. 13, No. 1
Printed in USA.
Immunochemical Analysis of Distinct Cellular Antigens Isolated from a Nontypable Bovine Strain of Group B Streptococci BRIAN TOOLE, JUDITH A. KANE,*
AND
WALTER W. KARAKAWA
Department of Biochemistry, Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 7 August 1975
Four immunologically distinct antigens were isolated from a bovine strain of B streptococci, designated 14 Mi. Chemical analysis indicated that two of the cell surface antigens consisted of glucose, galactose, and glucosamine, whereas the other cell surface antigen, an acidic protein, contained a predominance of aspartic acid, glutamic acid, and alanine. A cell wall-associated triheteroglycan consisting of galactose, glucose, and glucosamine was isolated from strain 14 Mi by 10% trichloroacetic acid. Immunochemical studies suggested that this glycan is type specific and consists of an immunodominant a-linked galactosyl-glucose disaccharide. group
Group B streptococci isolated from human infections can, in most instances, be classified into one of the five known serotypes on the basis of distinct type-specific capsular antigens (6, 14, 19, 20). In contrast, serotyping of organisms isolated from bovine mastitis has been inconsistent. This inconsistency has been attributed, in part, to the fact that these organisms were devoid of recognizable capsular polysaccharides (11). At the present time, the antigenic diversity of the bovine strains of group B streptococci has not been extensively studied (11). Because of the relative lack of knowledge of the antigenic structure of the cell surfaces of these organisms, studies have been initiated to determine whether or not it is possible to obtain chemically definable cell surface antigens or cell wall antigens that might be suitable for serotyping the "nontypable" bovine strains. In the present report, four cellular antigens were isolated from the prototypic bovine strain of group B streptococci, designated strain 14 Mi. The purification and immunochemical characterization of these antigens are described, and evidence is presented indicating that the cell wall-associated antigen, which consists of an immunodominant a-galactosyl-glucose disaccharide, may represent a possible type-specific antigen.
lanta, Ga. The bovine strains were obtained from R. Eberhardt, Veterinary Science Department, Pennsylvania State University. Antisera. Standard type-specific antisera were obtained from R. C. Lancefield. The remaining antisera were obtained from rabbits immunized with the bovine and Compton strains according to the procedures described by McCarty and Lancefield (8). Serological methods. Procedures for the qualitative and quantitative precipitin tests have been previously described (8). Double-diffusion analysis in agar was performed according to the method described by Tan and Kunkel (17). Immunoelectrophoresis was performed according to the method described by Kane et al. (2). Isolation of antigens. Strain 14 Mi was grown in 24 liters of Todd-Hewitt broth as previously described (2). Whole cells were recovered by centrifugation, and the packed cells were resuspended in pH 7.0 buffered saline and boiled for varying lengths of time (30 and 60 min). After each extraction, the cells were separated from the clear supernatant and reextracted. The supernatant fractions were dialyzed, lyophilized, and stored for future use. The pH 7.0extracted cells were then further extracted in pH 2.0 buffer for 20 min at 100 C. The supernatant was recovered from the insoluble fraction by centrifugation, dialyzed, and lyophilized. After pH 2.0 extraction, the cells were subjected to 10% trichloroacetic acid at 4 C for 24 h and the supernatant was recovered by centrifugation, dialyzed, and lyophilized (9). The whole cells that had been extracted extensively were then extracted with trichloroacetic acid at 90 C for 15 min and the soluble fraction was recovered, dialyzed, and lyophilized. Chromatography. Extracts obtained from the pH 7.0, pH 2.0, cold trichloroacetic acid, and hot trichloroacetic acid procedures were purified by diethylaminoethyl (DEAE)-cellulose chromatography according to the methods previously described (2). Im-
MATERIALS AND METHODS Strains of streptococci. Types Ia (090/14), Ib (H36B/60/1), Ic (A909/14), II (18RS21/45/1), and III (D136C) were kindly supplied by R. C. Lancefield, Rockefeller University. "Compton" strains SS 970 (X protein) and SS 971 (R protein) were obtained from H. W. Wilkinson, Center for Disease Control, At69
70
TOOLE, KANE, AND KARAKAWA
munologically active fractions eluted from the DEAE-cellulose column (30 by 2.5 cm) were further purified by Bio-Gel filtration as described by Kane et al. (2). Paper chromatography was performed in n-butyl alcohol-pyridine-water (6:4:3) by the multiple-ascent technique described by Pazur and Anderson (12). Purified carbohydrates were hydrolyzed in 0.1 or 2 N HCI at 100 C, and 5-I,u samples were spotted onto Whatman no. 1 paper, chromatograghed, and examined for the presence of reducing sugars by the alkaline silver nitrate method of Partridge (10). Analytical methods. Quantitative analysis of amino acids was performed by the method of Spackman et al. (15). Amino sugars were quantitated by the method of Weber and Winzler, which used a Technicon autoanalyzer (18). Hexoses and deoxy sugars were identified and quantitated with a Technicon autoanalyzer according to the method described by Kesler (4).
RESULTS Isolation of surface antigens from bovine strain 14 Mi. Lancefield and Freimer have shown that the conventional methods used in the isolation of streptococcal carbohydrates can partially degrade capsular antigens consisting of labile sialic acid moieties (7). In view of this observation, it was apparent that a prerequisite for the isolation of undegraded capsular antigens would involve the selection of appropriately mild procedures. In an attempt to minimize degradation of the surface antigens, unwashed whole cells of strain 14 Mi, which formed the capsular-rich diffuse-type growth in serum-soft agar, were extracted sequentially by a mild pH 7.0 buffer extraction procedure for periods of 30 and 60 min (3). The buffer-soluble fractions were separated from the insoluble cell residue by centrifugation, dialyzed against distilled water, and concentrated by flash evaporation. The concentrates were then subjected to DEAE-cellulose chromatography and eluted with increasing molarities of (NH4)2C03 buffer, pH 8.6. Three serologically active fractions were eluted with 0.075, 0.1, and 0.25 M (NH4)2C03 and were designated fractions A, B, and C, respectively. Double-diffusion analysis in agar indicated that these three fractions represent three distinct antigens as shown in Fig. 1. Although fractions A and B were shown to be immunologically distinct antigens, chemical analysis revealed the presence of similar constituents, namely glucose, galactose, and minimal amounts of glucosamine. It should also be emphasized that although both polymers were chemically similar to the acid-extracted type II capsule of group B streptococci isolated by Lancefield and Freimer (7), immunodiffusion studies indicated that these antigens were distinct from the type II capsule. Fraction C,
INFECT. IMMUN.
which represented the major constituent of the buffer extract, was shown to be an acidic protein. Chemical analysis of this protein (Table 1) indicated a high concentration of acidic and neutral amino acids, namely aspartic acid, glutamic acid, and alanine. The acidic nature of this polymer was verified by immunoelectrophoretic analysis in agar. On the basis of its inability to react with antisera rich in either X or R protein antibodies, it was suggested that the 14 Mi acidic protein was distinct from either the X or R proteins that are commonly associated with the group B organisms. These results indicate that upon mild extraction in pH 7.0 buffer at least three distinct surface antigens can be extracted from the whole cells of strain 14 Mi: a major acidic protein antigen and two distinct polysaccharide antigens. Attempts are now being made to elucidate the immunochemical features of these antigens. Isolation of a cell wall antigen from strain 14 Mi. Cells that were previously extracted with pH 7.0 buffer and subsequently with pH 2.0 buffer were extracted with 10% trichloroacetic acid for 24 h at 4 C or for 10 min at 90 C. After centrifugation, a clear supernatant fraction was separated from the cell residue after each of the extraction procedures, dialyzed, and lyophilized. The acid extract was then subjected to a Bio-Gel P-30 column (90 by 2.5 cm) and eluted with pH 7.0 phosphate buffer in 4-ml aliquots. Two fractions, designated fractions 1 and 2, were eluted from the column and chemically analyzed. Depicted in Fig. 2A and B are the results of the chromatographic analyses of fractions 1 and 2. Fraction 1, which was eluted at void volume, was shown to consist of galactose, glucose, and glucosamine in concentrations of 2.38 (42%), 1.56 (28%), and 1.52 (25%) ,umol/mg, respectively. Fraction 2, the retarded peak, consisted of rhamnose, galactose, and glucosamine in concentrations of 3.07 (51%), 0.51 (10%), and 0.64 (14%) ,mol/mg, respectively. Analysis of the two fractions by the capillary precipitin test and double-diffusion studies in agar indicated that fraction 1 represents a strain-specific antigen, whereas fraction II represents the group B-specific carbohydrate described by Curtis and Krause (1). Antigenic specificity of fraction 1. Chemical analysis indicated that fraction 1 was composed of the same constituents as the two antigens that were isolated from the pH 7.0 extract of strain 14 Mi. However, double-diffusion analysis in agar indicated that fraction 1 was immunologically distinct from these antigens. Fraction 1 (well 2) formed a precipitin band with its homologous antiserum (Fig. 3); however, this band did not merge with the precipitin bands
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
71
FIG. 1. Immunodiffusion reactions in agargel between fractions A, B, and C eluted from a DEAE-cellulose column and anti-strain 14 Mi serum. Well 1, Fraction A; well 2, fraction B; wells 3, 5, 6, fraction C; well 7, anti-strain 14 Mi serum.
formed by the group-specific carbohydrate (well 1) or by fraction A (well 3) and anti-14 Mi serum. The precipitin bands formed between well 4 or 5 and antiserum represent the acidic protein reaction. Additional immunological studies indicated that fraction 1 did not react with most of the available antisera to the various bovine strains. In an attempt to correlate this antigen with the five known type-specific antigens, precipitin tests were performed with type-specific antisera that were directed against the known group B streptococcal serotypes, Ia, Tb, Ic, II, and III. Fraction 1 gave negative reactions with four of the antisera and a weak reaction with type III (strain D136C) antiserum (Fig. 4). Double-diffusion analysis in agar using specific type Ill antiserum, anti-14 serum, type Ill antigen, and fraction 1 suggested that the observed cross-reactivity between fraction 1 and type III antiserum was possibly due to antibodies directed against a
TABLE 1. Chemical composition of the acidic protein isolated from the pH 7.0 extract of strain 14 Mi Components
j.mol/mg
Molar ratio
Lysine .............. Histidine ........... Arginine ............ Aspartic acid ........ Threonine .......... Serine .............. Glutamic acid ....... Proline ............. Glycine ............. Alanine ............. Half-cystine ......... Valine .............. Leucine ............. Tyrosine ............ Phenylalanine ...... Ammonia ...........
0.309 0.054 0.126 0.526 0.252 0.266 0.687 0.095 0.277 0.562
1.16
a
Less than 0.015
1.99 0.94 1.00 2.58 1.04 2.11
a
0.281 0.312 0.076 0.065 0.984
pmol/mg.
1.06 1.17
72
INFECT. IMMUN.
TOOLE, KANE, AND KARAKAWA P-30
eHZ
*umeudr
.5
30 so
5
25
ao
35
S P-30 RetoWe @2
I
I
Ba..,-o
0.1I
LO
XL5
d0
3.
4.0
45
FIG. 2. (A) Analysis of an acid hydrolysate offraction 1 that was eluted from a P.30 Bio-Gel column (90 by 2.5 cm) at void volume. (B) Analysis of an acid hydrolysate ofthe retarded fraction 2 that was eluted from a P30 Bio-Gel column. Hydrolysis of both fractions was carried out in 2 N HCI at 100 C for2 h. The sugars were identified by anion exchange chromatography on an automatic Technicon analyzer adapted for both neutral and amino sugars.
cellular constituent of the strain D136C that immunologically similar to fraction 1 and not due to antibodies with type III capsular specificity. Structural analysis of fraction 1 carbohydrate. To identify the nonreducing terminus of was
fraction 1 that functions as the immunodeterminant, the triheteroglycan was hydrolyzed under mild acid (0.1 N HCl) conditions for varying time intervals and the products of hydrolysis were analyzed by paper chromatography. Hydrolysis of the triheteroglycan for 30 min re-
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
73
FIG. 3. Immunodiffusion reactions in agar gel between the fractions isolated from the pH 7.0 extract and the fractions from the trichloroacetic acid extract and anti-strain 14 Mi serum. Well 1, Fraction 2 (groupspecific carbohydrate); well 2, fraction 1; well 3, fraction A (pH 7.0 carbohydrate); wells 4 and 5, fraction C (acidic protein); well 6, anti-14 Mi serum.
sulted in the selective cleavage of only galactose, which suggested that this monosaccharide may occupy a terminal position on the polymer. Quantitative precipitin analysis of the triheteroglycan treated with trifluoroacetic acid (TFA) for varying periods of time supported the notion that galactose represents the terminal immunodominant determinant (Fig. 5). Note that the quantitative precipitin reaction between the triheteroglycan treated with TFA for 2 h at 80 C and antiserum was approximately 50% less than that of the untreated control. Significantly, the dialysate of the 2- and 4-h TFAtreated antigens contained only galactose as determined by the paper chromatographic analyses. The terminus position of galactose on the polymer was further substantiated by enzymatic hydrolysis procedures (Fig. 6). Triheteroglycan treated with a-galactosidase yielded only galactose, whereas f-galactosidase and a-
and f-glucosidase had no appreciable effect upon the polysaccharide. Quantitative precipitin inhibition analysis also confirmed the notion that galactose residues occupy a terminal position on the nonreducing end of the polymer and, thus, constitute the immunodeterminant (Fig. 7). Nitrophenol-a-galactoside at low concentrations was a more effective inhibitor of the triheteroglycan precipitin reaction than was either nitrophenol-3-galactoside or n-galactose (Fig. 7). Melibiose, 6-0-a-D-galactopyranosyl-Dglucose, was shown to be slightly more effective as an inhibitor than was nitrophenol-a-galactoside. Additional inhibition studies indicated that stachyose, an a-n-galactosyl-a-n-galactosyl-a-D-glucosyl-,&--fructose, was less effective as an inhibitor than melibiose. These results, therefore, suggest that the immunodeterminant of the glycan may consist of a disaccharide of a-galactosyl-D-glucose. In an attempt to sub-
74
TOOLE, KANE, AND KARAKAWA
INFECT. IMMUN.
the serological activity was greatly diminished. The treated polymer was subsequently subjected to mild acid hydrolysis as described above, and the products of hydrolysis were analyzed by paper chromatography. After 10 min of hydrolysis in 0.1 N HCI at 100 C, glucose was the major sugar released from the polymer. These observations support the view that the terminal galactose units are a-linked to glucose residues and that the immunodominant determinant of the cell wall triheteroglycan is, in fact, an a-galactosyl-D-glucose disaccharide.
E 0
In 0
.a001 .0 .4
Anti- 14 Mi serum
m) Conc. of Antigen (Ag/mi)
FIG. 4. Quantitative precipitin reactions between fraction 1 and homologous anti-14 Mi serum, heterologous anti-9F serum, and anti-D136C serum which represents serum rich in anti-type III capsular antibodies.
..
kl,7 i
. -,
..,y
.i
ei #. . -". e.1 .,
1.1
1.0 0.9
0.8 E
00.7 . 0.6 0
CHO
0 a
.0
4 0.4
treated CHO
0.31
0.2 0.I treated CHO 40 60 20 Conc. of Antigen (,ug/mI)
FIG. 5. Quantitative precipitin reactions between untreated fraction 1, TFA-treated fraction 1, and anti-14 Mi serum.
stantiate the existence of an a-galactosyl-D-glucose disaccharide at the nonreducing terminus of the triheteroglycan, 10 mg of the antigen was treated with a-galactosidase for 48 h or until
0
a
-C 0 QI
FIG. 6. Paper chromatogram of fraction 1 treated with a-galactosidase.
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
75
DISCUSSION
conditions indicated that galactose was the initial sugar released from the antigen. With the The results described indicate that the proto- concomitant release of galactose residues, the typic bovine strain 14 Mi of group B strepto- immunological reactivity of the polymer was cocci, isolated from bovine mastitis in Central noticeably diminished. Second, the antigenic Pennsylvania, produces four distinct antigens, determinant of the polymer, namely the nonrednamely an acidic protein and three carbohy- ucing terminal galactose, was selectively drate antigens. The protein and two of the poly- cleaved off the polymer by a-galactosidase but saccharides can readily be extracted from un- not by f3galactosidase, again with a significant washed whole cells by boiling in pH 7.0 loss of immunological reactivity. This a-galactobuffered saline. The remaining polysaccharide sidase-treated polymer, when subjected to mild can be extracted with 10% trichloroacetic acid. acid hydrolysis, released a predominance of gluChemical analyses indicate that the protein an- cose residues. These results suggest that the tigen consists of a predominance of aspartic terminal galactose units are a-linked to glucose acid, glutamic acid, and alanine, whereas the units. Finally, melibiose, an a-1-6-galactosylcarbohydrate antigens, although immunologi- glucose disaccharide, was found to be the most cally distinct antigens, consist of the same con- effective inhibitor of the precipitin reaction bestituents, namely galactose, glucose, and gluco- tween fraction 1 and homologous antiserum, samine. Chemical evidence also indicates that whereas stachyose, a tetrasaccharide consistthe cell wall-associated carbohydrate antigen, ing of a-galactosyl-a-galactosyl-a-glucQsyl-jdesignated fraction 1 and extracted from the fructose, was less effective as an inhibitor. cells by trichloroacetic acid, consists of galacAt the present time, little is known about the tose, glucose, and glucosamine in a molar ratio immunochemical features of the cell surface of 1.5, 1.0, and 1.0, respectively. Immunochemi- triheteroglycans. Although both consist of the cal and enzymatic data suggest that a-linked same constituents as the type II capsular antigalactosyl-D-glucose disaccharide residues oc- gen described by Lancefield, they are shown to cupy the terminal positions on the side chains be immunologically distinct. Effort is now of the triheteroglycan. This notion is supported being made to determine the immunochemical by three types of evidence. First, timed hydroly- features of these antigens. sis of the polymer under mild acid (0.1 N HCl) Since the antigenic classification of group B
1001 90 80
Mlibiose
c
.° 70 c
60 I~
C U
50 I.
-
r~~~ P-NO^2 40-C"GlaCto"
P-NO2-p-1p-GaIactose D-Galactose
.
L
IL An vr
30
20[ l10 "0
2
4
6
8
10
12
14
Conc. of Inhibitor (mg/ml)
16
18
20
FIG. 7. Inhibition of the quantitative precipitin reaction between fraction 1 (triheteroglycan) and homoloantiserum by mono- and disaccharides consisting ofgalactose and substituted galactosides.
gous
76
TOOLE, KANE, AND KARAKAWA
streptococci is based on the occurrence of distinct type-specific cellular antigens (5), a successful scheme for typing these organisms is greatly dependent upon the availability of these antigens on the cell surface of the clinical isolates. Most human strains of group B streptococci can be classified into one of the five known serotypes on the basis of the presence of capsular antigens (6). Nontypable strains, although isolated from human sources, appear to be more common among the strains isolated from bovine mastitis. Stableforth, using precipitin and agglutination procedures, separated a group of bovine group B streptococci into 16 serotypes (16). Pattison et al., recognizing the presence of the cross-reactive X and R proteins on many strains, were able to group the bovine strains into four serotypes on the basis of precipitin reactions that used surface polysaccharides (11). However, 43 of 170 strains were still nontypable and therefore were thought to be devoid of capsular antigens. Subequently, Pattison et al., using the R and X proteins, demonstrated that these non-encapsulated strains could be serotyped on the basis of discriminate use of the X and R proteins (11). The results presented in this study suggest that the cell wall triheteroglycan with a galactosyl-glucose disaccharide as its immunodeterminant may be a type-specific antigen and can be used as a means of serotyping bovine strains devoid of polysaccharide capsules. This view is supported by a number of data. First, this antigen is not a surface antigen (capsule) since extraction with pH 7.0 was ineffective in releasing the antigen from the cells; however, trichloroacetic acid was very effective in extracting this glycan as well as the group-specific carbohydrate, a known cell wall component. Second, serological analysis with many bovine strains and the known group B serotypes indicated that this cell wall triheteroglycan is a common feature of only a few bovine strains. This antigen did give a weak cross-reaction with antiD136C (type III) serum; however, this reaction was not due to type Ill-specific capsular antigen of group B streptococci, but rather to a cellular component associated with strain D136 (type
m).
At the present time, relatively few antigens of the bovine strains of group B streptococci have been extensively studied, and as a result serotyping has been based entirely on the few known capsular substances that are frequently associated with human strains. Because of the lack of knowledge of the antigenic structure of these organisms, studies should be continued in an effort to develop a suitable scheme for sero-
INFECT. IMMUN.
typing of bovine strains on the basis of capsular or cell wall polysaccharides. The results presented indicate that it may be feasible to use cell wall polysaccharides as a means of assisting in the typing of bovine strains devoid of type-specific capsular materials. ACKNOWLEDGMENT This investigation was supported in part by Public Health Service grant A1-11598 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Curtis, S. N., and R. M. Krause. 1964. Antigenic relationship between groups B and G streptococci. J. Exp. Med. 120:629-637. 2. Kane, J. A., W. W. Karakawa, and J. H. Pazur. 1972. Glycans from streptococcal cell walls: structural features of a diheteroglycan isolated from the cell wall of Streptococcus bovis. J. Immunol. 108:1218-1226. 3. Kane, J. A., A. E. Rabkin, and W. W. Karakawa. 1975. Demonstration of the capsular antigens of bovine group B streptococci by the serum-soft agar method. J. Clin. Microbiol. 2:35-41. 4. Kesler, R. B. 1967. Rapid quantitative anion-exchange chromatography of carbohydrates. Anal. Chem. 39:1416-1422. 5. Lancefield, R. C. 1934. Serological differentiation of specific types of bovine hemolytic streptococci (group B). J. Exp. Med. 59:441-458. 6. Lancefield, R. C. 1972. Cellular antigens of group B streptococci, p. 57-64. In L. W. Wanamaker and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York. 7. Lancefield, R. C., and E. H. Freimer. 1966. Type-specific polysaccharide antigens of group B streptococci. J. Hyg. 64:191-203. 8. McCarty, M., and R. C. Lancefield. 1955. Variation in the group-specific carbohydrates of Group A streptococci. I. Immunochemical studies on the carbohydrate of variant strains. J. Exp. Med. 102:11-28. 9. Park, J. T., and R. Hancock. 1960. A fractionation procedure for studies of cell wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J. Gen. Microbiol. 22:249-297. 10. Partridge, S. M. 1948. Filter-paper chromatography of sugars. Biochem. J. 42:238-241. 11. Pattison, I. H., R. J. Matthews, and D. G. Howell. 1955. The type classification of group B streptococci with special reference to bovine strains apparently lacking in type polysaccharide. J. Pathol. Bacteriol. 69:5160. 12. Pazur, J. H., and J. S. Anderson. 1963. Thymidine triphosphate: a-D-galactose-1-phosphate thymidylyltransferase from Streptococcus faecalis grown on Dgalactose. J. Biol. Chem. 238:3155-3159. 13. Pazur, J. H., J. S. Anderson, and W. W. Karakawa. 1971. Glycans from streptococcal cell walls: immunological and chemical properties of a new diheteroglycan from Streptococcus faecalis. J. Biol. Chem. 246:1793-1798. 14. Romero, R., and H. W. Wilkinson. 1974. Identification of group B streptococci by immunofluorescence staining. Appl. Microbiol. 28:199-204. 15. Spackman, D. H., W. H. Stein, and S. Moore. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30:1190-1206. 16. Stableforth, A. W. 1932. Studies on bovine mastitis. VI. Serological characters of mastitis streptococci. J. Comp. Pathol. Ther. 45:185-190.
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CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
17. Tan, E. M., and H. G. Kunkel. 1966. Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J. Immunol. 96:464471. 18. Weber, P., and R. H. Winzler. 1969. Determination of hexosaminitols by ion-exchange chromatography and its application to alkali-labile glycosidic linkages in
77
glycoproteins. Arch. Biochem. Biophys. 129:534-538. 19. Wilkinson, J. W. 1975. Immunochemistry of purified polysaccharide type antigens of group B streptococci types Ia, Ib, and Ic. Infect. Immun. 11:845-852. 20. Wilkinson, H. W., and R. G. Eagon. 1971. Type-specific antigens of group B type Ic streptococci. Infect. Immun. 4:594-604.
Vol. 13, No. 1
Printed in USA.
Immunochemical Analysis of Distinct Cellular Antigens Isolated from a Nontypable Bovine Strain of Group B Streptococci BRIAN TOOLE, JUDITH A. KANE,*
AND
WALTER W. KARAKAWA
Department of Biochemistry, Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 7 August 1975
Four immunologically distinct antigens were isolated from a bovine strain of B streptococci, designated 14 Mi. Chemical analysis indicated that two of the cell surface antigens consisted of glucose, galactose, and glucosamine, whereas the other cell surface antigen, an acidic protein, contained a predominance of aspartic acid, glutamic acid, and alanine. A cell wall-associated triheteroglycan consisting of galactose, glucose, and glucosamine was isolated from strain 14 Mi by 10% trichloroacetic acid. Immunochemical studies suggested that this glycan is type specific and consists of an immunodominant a-linked galactosyl-glucose disaccharide. group
Group B streptococci isolated from human infections can, in most instances, be classified into one of the five known serotypes on the basis of distinct type-specific capsular antigens (6, 14, 19, 20). In contrast, serotyping of organisms isolated from bovine mastitis has been inconsistent. This inconsistency has been attributed, in part, to the fact that these organisms were devoid of recognizable capsular polysaccharides (11). At the present time, the antigenic diversity of the bovine strains of group B streptococci has not been extensively studied (11). Because of the relative lack of knowledge of the antigenic structure of the cell surfaces of these organisms, studies have been initiated to determine whether or not it is possible to obtain chemically definable cell surface antigens or cell wall antigens that might be suitable for serotyping the "nontypable" bovine strains. In the present report, four cellular antigens were isolated from the prototypic bovine strain of group B streptococci, designated strain 14 Mi. The purification and immunochemical characterization of these antigens are described, and evidence is presented indicating that the cell wall-associated antigen, which consists of an immunodominant a-galactosyl-glucose disaccharide, may represent a possible type-specific antigen.
lanta, Ga. The bovine strains were obtained from R. Eberhardt, Veterinary Science Department, Pennsylvania State University. Antisera. Standard type-specific antisera were obtained from R. C. Lancefield. The remaining antisera were obtained from rabbits immunized with the bovine and Compton strains according to the procedures described by McCarty and Lancefield (8). Serological methods. Procedures for the qualitative and quantitative precipitin tests have been previously described (8). Double-diffusion analysis in agar was performed according to the method described by Tan and Kunkel (17). Immunoelectrophoresis was performed according to the method described by Kane et al. (2). Isolation of antigens. Strain 14 Mi was grown in 24 liters of Todd-Hewitt broth as previously described (2). Whole cells were recovered by centrifugation, and the packed cells were resuspended in pH 7.0 buffered saline and boiled for varying lengths of time (30 and 60 min). After each extraction, the cells were separated from the clear supernatant and reextracted. The supernatant fractions were dialyzed, lyophilized, and stored for future use. The pH 7.0extracted cells were then further extracted in pH 2.0 buffer for 20 min at 100 C. The supernatant was recovered from the insoluble fraction by centrifugation, dialyzed, and lyophilized. After pH 2.0 extraction, the cells were subjected to 10% trichloroacetic acid at 4 C for 24 h and the supernatant was recovered by centrifugation, dialyzed, and lyophilized (9). The whole cells that had been extracted extensively were then extracted with trichloroacetic acid at 90 C for 15 min and the soluble fraction was recovered, dialyzed, and lyophilized. Chromatography. Extracts obtained from the pH 7.0, pH 2.0, cold trichloroacetic acid, and hot trichloroacetic acid procedures were purified by diethylaminoethyl (DEAE)-cellulose chromatography according to the methods previously described (2). Im-
MATERIALS AND METHODS Strains of streptococci. Types Ia (090/14), Ib (H36B/60/1), Ic (A909/14), II (18RS21/45/1), and III (D136C) were kindly supplied by R. C. Lancefield, Rockefeller University. "Compton" strains SS 970 (X protein) and SS 971 (R protein) were obtained from H. W. Wilkinson, Center for Disease Control, At69
70
TOOLE, KANE, AND KARAKAWA
munologically active fractions eluted from the DEAE-cellulose column (30 by 2.5 cm) were further purified by Bio-Gel filtration as described by Kane et al. (2). Paper chromatography was performed in n-butyl alcohol-pyridine-water (6:4:3) by the multiple-ascent technique described by Pazur and Anderson (12). Purified carbohydrates were hydrolyzed in 0.1 or 2 N HCI at 100 C, and 5-I,u samples were spotted onto Whatman no. 1 paper, chromatograghed, and examined for the presence of reducing sugars by the alkaline silver nitrate method of Partridge (10). Analytical methods. Quantitative analysis of amino acids was performed by the method of Spackman et al. (15). Amino sugars were quantitated by the method of Weber and Winzler, which used a Technicon autoanalyzer (18). Hexoses and deoxy sugars were identified and quantitated with a Technicon autoanalyzer according to the method described by Kesler (4).
RESULTS Isolation of surface antigens from bovine strain 14 Mi. Lancefield and Freimer have shown that the conventional methods used in the isolation of streptococcal carbohydrates can partially degrade capsular antigens consisting of labile sialic acid moieties (7). In view of this observation, it was apparent that a prerequisite for the isolation of undegraded capsular antigens would involve the selection of appropriately mild procedures. In an attempt to minimize degradation of the surface antigens, unwashed whole cells of strain 14 Mi, which formed the capsular-rich diffuse-type growth in serum-soft agar, were extracted sequentially by a mild pH 7.0 buffer extraction procedure for periods of 30 and 60 min (3). The buffer-soluble fractions were separated from the insoluble cell residue by centrifugation, dialyzed against distilled water, and concentrated by flash evaporation. The concentrates were then subjected to DEAE-cellulose chromatography and eluted with increasing molarities of (NH4)2C03 buffer, pH 8.6. Three serologically active fractions were eluted with 0.075, 0.1, and 0.25 M (NH4)2C03 and were designated fractions A, B, and C, respectively. Double-diffusion analysis in agar indicated that these three fractions represent three distinct antigens as shown in Fig. 1. Although fractions A and B were shown to be immunologically distinct antigens, chemical analysis revealed the presence of similar constituents, namely glucose, galactose, and minimal amounts of glucosamine. It should also be emphasized that although both polymers were chemically similar to the acid-extracted type II capsule of group B streptococci isolated by Lancefield and Freimer (7), immunodiffusion studies indicated that these antigens were distinct from the type II capsule. Fraction C,
INFECT. IMMUN.
which represented the major constituent of the buffer extract, was shown to be an acidic protein. Chemical analysis of this protein (Table 1) indicated a high concentration of acidic and neutral amino acids, namely aspartic acid, glutamic acid, and alanine. The acidic nature of this polymer was verified by immunoelectrophoretic analysis in agar. On the basis of its inability to react with antisera rich in either X or R protein antibodies, it was suggested that the 14 Mi acidic protein was distinct from either the X or R proteins that are commonly associated with the group B organisms. These results indicate that upon mild extraction in pH 7.0 buffer at least three distinct surface antigens can be extracted from the whole cells of strain 14 Mi: a major acidic protein antigen and two distinct polysaccharide antigens. Attempts are now being made to elucidate the immunochemical features of these antigens. Isolation of a cell wall antigen from strain 14 Mi. Cells that were previously extracted with pH 7.0 buffer and subsequently with pH 2.0 buffer were extracted with 10% trichloroacetic acid for 24 h at 4 C or for 10 min at 90 C. After centrifugation, a clear supernatant fraction was separated from the cell residue after each of the extraction procedures, dialyzed, and lyophilized. The acid extract was then subjected to a Bio-Gel P-30 column (90 by 2.5 cm) and eluted with pH 7.0 phosphate buffer in 4-ml aliquots. Two fractions, designated fractions 1 and 2, were eluted from the column and chemically analyzed. Depicted in Fig. 2A and B are the results of the chromatographic analyses of fractions 1 and 2. Fraction 1, which was eluted at void volume, was shown to consist of galactose, glucose, and glucosamine in concentrations of 2.38 (42%), 1.56 (28%), and 1.52 (25%) ,umol/mg, respectively. Fraction 2, the retarded peak, consisted of rhamnose, galactose, and glucosamine in concentrations of 3.07 (51%), 0.51 (10%), and 0.64 (14%) ,mol/mg, respectively. Analysis of the two fractions by the capillary precipitin test and double-diffusion studies in agar indicated that fraction 1 represents a strain-specific antigen, whereas fraction II represents the group B-specific carbohydrate described by Curtis and Krause (1). Antigenic specificity of fraction 1. Chemical analysis indicated that fraction 1 was composed of the same constituents as the two antigens that were isolated from the pH 7.0 extract of strain 14 Mi. However, double-diffusion analysis in agar indicated that fraction 1 was immunologically distinct from these antigens. Fraction 1 (well 2) formed a precipitin band with its homologous antiserum (Fig. 3); however, this band did not merge with the precipitin bands
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
71
FIG. 1. Immunodiffusion reactions in agargel between fractions A, B, and C eluted from a DEAE-cellulose column and anti-strain 14 Mi serum. Well 1, Fraction A; well 2, fraction B; wells 3, 5, 6, fraction C; well 7, anti-strain 14 Mi serum.
formed by the group-specific carbohydrate (well 1) or by fraction A (well 3) and anti-14 Mi serum. The precipitin bands formed between well 4 or 5 and antiserum represent the acidic protein reaction. Additional immunological studies indicated that fraction 1 did not react with most of the available antisera to the various bovine strains. In an attempt to correlate this antigen with the five known type-specific antigens, precipitin tests were performed with type-specific antisera that were directed against the known group B streptococcal serotypes, Ia, Tb, Ic, II, and III. Fraction 1 gave negative reactions with four of the antisera and a weak reaction with type III (strain D136C) antiserum (Fig. 4). Double-diffusion analysis in agar using specific type Ill antiserum, anti-14 serum, type Ill antigen, and fraction 1 suggested that the observed cross-reactivity between fraction 1 and type III antiserum was possibly due to antibodies directed against a
TABLE 1. Chemical composition of the acidic protein isolated from the pH 7.0 extract of strain 14 Mi Components
j.mol/mg
Molar ratio
Lysine .............. Histidine ........... Arginine ............ Aspartic acid ........ Threonine .......... Serine .............. Glutamic acid ....... Proline ............. Glycine ............. Alanine ............. Half-cystine ......... Valine .............. Leucine ............. Tyrosine ............ Phenylalanine ...... Ammonia ...........
0.309 0.054 0.126 0.526 0.252 0.266 0.687 0.095 0.277 0.562
1.16
a
Less than 0.015
1.99 0.94 1.00 2.58 1.04 2.11
a
0.281 0.312 0.076 0.065 0.984
pmol/mg.
1.06 1.17
72
INFECT. IMMUN.
TOOLE, KANE, AND KARAKAWA P-30
eHZ
*umeudr
.5
30 so
5
25
ao
35
S P-30 RetoWe @2
I
I
Ba..,-o
0.1I
LO
XL5
d0
3.
4.0
45
FIG. 2. (A) Analysis of an acid hydrolysate offraction 1 that was eluted from a P.30 Bio-Gel column (90 by 2.5 cm) at void volume. (B) Analysis of an acid hydrolysate ofthe retarded fraction 2 that was eluted from a P30 Bio-Gel column. Hydrolysis of both fractions was carried out in 2 N HCI at 100 C for2 h. The sugars were identified by anion exchange chromatography on an automatic Technicon analyzer adapted for both neutral and amino sugars.
cellular constituent of the strain D136C that immunologically similar to fraction 1 and not due to antibodies with type III capsular specificity. Structural analysis of fraction 1 carbohydrate. To identify the nonreducing terminus of was
fraction 1 that functions as the immunodeterminant, the triheteroglycan was hydrolyzed under mild acid (0.1 N HCl) conditions for varying time intervals and the products of hydrolysis were analyzed by paper chromatography. Hydrolysis of the triheteroglycan for 30 min re-
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
73
FIG. 3. Immunodiffusion reactions in agar gel between the fractions isolated from the pH 7.0 extract and the fractions from the trichloroacetic acid extract and anti-strain 14 Mi serum. Well 1, Fraction 2 (groupspecific carbohydrate); well 2, fraction 1; well 3, fraction A (pH 7.0 carbohydrate); wells 4 and 5, fraction C (acidic protein); well 6, anti-14 Mi serum.
sulted in the selective cleavage of only galactose, which suggested that this monosaccharide may occupy a terminal position on the polymer. Quantitative precipitin analysis of the triheteroglycan treated with trifluoroacetic acid (TFA) for varying periods of time supported the notion that galactose represents the terminal immunodominant determinant (Fig. 5). Note that the quantitative precipitin reaction between the triheteroglycan treated with TFA for 2 h at 80 C and antiserum was approximately 50% less than that of the untreated control. Significantly, the dialysate of the 2- and 4-h TFAtreated antigens contained only galactose as determined by the paper chromatographic analyses. The terminus position of galactose on the polymer was further substantiated by enzymatic hydrolysis procedures (Fig. 6). Triheteroglycan treated with a-galactosidase yielded only galactose, whereas f-galactosidase and a-
and f-glucosidase had no appreciable effect upon the polysaccharide. Quantitative precipitin inhibition analysis also confirmed the notion that galactose residues occupy a terminal position on the nonreducing end of the polymer and, thus, constitute the immunodeterminant (Fig. 7). Nitrophenol-a-galactoside at low concentrations was a more effective inhibitor of the triheteroglycan precipitin reaction than was either nitrophenol-3-galactoside or n-galactose (Fig. 7). Melibiose, 6-0-a-D-galactopyranosyl-Dglucose, was shown to be slightly more effective as an inhibitor than was nitrophenol-a-galactoside. Additional inhibition studies indicated that stachyose, an a-n-galactosyl-a-n-galactosyl-a-D-glucosyl-,&--fructose, was less effective as an inhibitor than melibiose. These results, therefore, suggest that the immunodeterminant of the glycan may consist of a disaccharide of a-galactosyl-D-glucose. In an attempt to sub-
74
TOOLE, KANE, AND KARAKAWA
INFECT. IMMUN.
the serological activity was greatly diminished. The treated polymer was subsequently subjected to mild acid hydrolysis as described above, and the products of hydrolysis were analyzed by paper chromatography. After 10 min of hydrolysis in 0.1 N HCI at 100 C, glucose was the major sugar released from the polymer. These observations support the view that the terminal galactose units are a-linked to glucose residues and that the immunodominant determinant of the cell wall triheteroglycan is, in fact, an a-galactosyl-D-glucose disaccharide.
E 0
In 0
.a001 .0 .4
Anti- 14 Mi serum
m) Conc. of Antigen (Ag/mi)
FIG. 4. Quantitative precipitin reactions between fraction 1 and homologous anti-14 Mi serum, heterologous anti-9F serum, and anti-D136C serum which represents serum rich in anti-type III capsular antibodies.
..
kl,7 i
. -,
..,y
.i
ei #. . -". e.1 .,
1.1
1.0 0.9
0.8 E
00.7 . 0.6 0
CHO
0 a
.0
4 0.4
treated CHO
0.31
0.2 0.I treated CHO 40 60 20 Conc. of Antigen (,ug/mI)
FIG. 5. Quantitative precipitin reactions between untreated fraction 1, TFA-treated fraction 1, and anti-14 Mi serum.
stantiate the existence of an a-galactosyl-D-glucose disaccharide at the nonreducing terminus of the triheteroglycan, 10 mg of the antigen was treated with a-galactosidase for 48 h or until
0
a
-C 0 QI
FIG. 6. Paper chromatogram of fraction 1 treated with a-galactosidase.
VOL. 13, 1976
CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
75
DISCUSSION
conditions indicated that galactose was the initial sugar released from the antigen. With the The results described indicate that the proto- concomitant release of galactose residues, the typic bovine strain 14 Mi of group B strepto- immunological reactivity of the polymer was cocci, isolated from bovine mastitis in Central noticeably diminished. Second, the antigenic Pennsylvania, produces four distinct antigens, determinant of the polymer, namely the nonrednamely an acidic protein and three carbohy- ucing terminal galactose, was selectively drate antigens. The protein and two of the poly- cleaved off the polymer by a-galactosidase but saccharides can readily be extracted from un- not by f3galactosidase, again with a significant washed whole cells by boiling in pH 7.0 loss of immunological reactivity. This a-galactobuffered saline. The remaining polysaccharide sidase-treated polymer, when subjected to mild can be extracted with 10% trichloroacetic acid. acid hydrolysis, released a predominance of gluChemical analyses indicate that the protein an- cose residues. These results suggest that the tigen consists of a predominance of aspartic terminal galactose units are a-linked to glucose acid, glutamic acid, and alanine, whereas the units. Finally, melibiose, an a-1-6-galactosylcarbohydrate antigens, although immunologi- glucose disaccharide, was found to be the most cally distinct antigens, consist of the same con- effective inhibitor of the precipitin reaction bestituents, namely galactose, glucose, and gluco- tween fraction 1 and homologous antiserum, samine. Chemical evidence also indicates that whereas stachyose, a tetrasaccharide consistthe cell wall-associated carbohydrate antigen, ing of a-galactosyl-a-galactosyl-a-glucQsyl-jdesignated fraction 1 and extracted from the fructose, was less effective as an inhibitor. cells by trichloroacetic acid, consists of galacAt the present time, little is known about the tose, glucose, and glucosamine in a molar ratio immunochemical features of the cell surface of 1.5, 1.0, and 1.0, respectively. Immunochemi- triheteroglycans. Although both consist of the cal and enzymatic data suggest that a-linked same constituents as the type II capsular antigalactosyl-D-glucose disaccharide residues oc- gen described by Lancefield, they are shown to cupy the terminal positions on the side chains be immunologically distinct. Effort is now of the triheteroglycan. This notion is supported being made to determine the immunochemical by three types of evidence. First, timed hydroly- features of these antigens. sis of the polymer under mild acid (0.1 N HCl) Since the antigenic classification of group B
1001 90 80
Mlibiose
c
.° 70 c
60 I~
C U
50 I.
-
r~~~ P-NO^2 40-C"GlaCto"
P-NO2-p-1p-GaIactose D-Galactose
.
L
IL An vr
30
20[ l10 "0
2
4
6
8
10
12
14
Conc. of Inhibitor (mg/ml)
16
18
20
FIG. 7. Inhibition of the quantitative precipitin reaction between fraction 1 (triheteroglycan) and homoloantiserum by mono- and disaccharides consisting ofgalactose and substituted galactosides.
gous
76
TOOLE, KANE, AND KARAKAWA
streptococci is based on the occurrence of distinct type-specific cellular antigens (5), a successful scheme for typing these organisms is greatly dependent upon the availability of these antigens on the cell surface of the clinical isolates. Most human strains of group B streptococci can be classified into one of the five known serotypes on the basis of the presence of capsular antigens (6). Nontypable strains, although isolated from human sources, appear to be more common among the strains isolated from bovine mastitis. Stableforth, using precipitin and agglutination procedures, separated a group of bovine group B streptococci into 16 serotypes (16). Pattison et al., recognizing the presence of the cross-reactive X and R proteins on many strains, were able to group the bovine strains into four serotypes on the basis of precipitin reactions that used surface polysaccharides (11). However, 43 of 170 strains were still nontypable and therefore were thought to be devoid of capsular antigens. Subequently, Pattison et al., using the R and X proteins, demonstrated that these non-encapsulated strains could be serotyped on the basis of discriminate use of the X and R proteins (11). The results presented in this study suggest that the cell wall triheteroglycan with a galactosyl-glucose disaccharide as its immunodeterminant may be a type-specific antigen and can be used as a means of serotyping bovine strains devoid of polysaccharide capsules. This view is supported by a number of data. First, this antigen is not a surface antigen (capsule) since extraction with pH 7.0 was ineffective in releasing the antigen from the cells; however, trichloroacetic acid was very effective in extracting this glycan as well as the group-specific carbohydrate, a known cell wall component. Second, serological analysis with many bovine strains and the known group B serotypes indicated that this cell wall triheteroglycan is a common feature of only a few bovine strains. This antigen did give a weak cross-reaction with antiD136C (type III) serum; however, this reaction was not due to type Ill-specific capsular antigen of group B streptococci, but rather to a cellular component associated with strain D136 (type
m).
At the present time, relatively few antigens of the bovine strains of group B streptococci have been extensively studied, and as a result serotyping has been based entirely on the few known capsular substances that are frequently associated with human strains. Because of the lack of knowledge of the antigenic structure of these organisms, studies should be continued in an effort to develop a suitable scheme for sero-
INFECT. IMMUN.
typing of bovine strains on the basis of capsular or cell wall polysaccharides. The results presented indicate that it may be feasible to use cell wall polysaccharides as a means of assisting in the typing of bovine strains devoid of type-specific capsular materials. ACKNOWLEDGMENT This investigation was supported in part by Public Health Service grant A1-11598 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Curtis, S. N., and R. M. Krause. 1964. Antigenic relationship between groups B and G streptococci. J. Exp. Med. 120:629-637. 2. Kane, J. A., W. W. Karakawa, and J. H. Pazur. 1972. Glycans from streptococcal cell walls: structural features of a diheteroglycan isolated from the cell wall of Streptococcus bovis. J. Immunol. 108:1218-1226. 3. Kane, J. A., A. E. Rabkin, and W. W. Karakawa. 1975. Demonstration of the capsular antigens of bovine group B streptococci by the serum-soft agar method. J. Clin. Microbiol. 2:35-41. 4. Kesler, R. B. 1967. Rapid quantitative anion-exchange chromatography of carbohydrates. Anal. Chem. 39:1416-1422. 5. Lancefield, R. C. 1934. Serological differentiation of specific types of bovine hemolytic streptococci (group B). J. Exp. Med. 59:441-458. 6. Lancefield, R. C. 1972. Cellular antigens of group B streptococci, p. 57-64. In L. W. Wanamaker and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York. 7. Lancefield, R. C., and E. H. Freimer. 1966. Type-specific polysaccharide antigens of group B streptococci. J. Hyg. 64:191-203. 8. McCarty, M., and R. C. Lancefield. 1955. Variation in the group-specific carbohydrates of Group A streptococci. I. Immunochemical studies on the carbohydrate of variant strains. J. Exp. Med. 102:11-28. 9. Park, J. T., and R. Hancock. 1960. A fractionation procedure for studies of cell wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J. Gen. Microbiol. 22:249-297. 10. Partridge, S. M. 1948. Filter-paper chromatography of sugars. Biochem. J. 42:238-241. 11. Pattison, I. H., R. J. Matthews, and D. G. Howell. 1955. The type classification of group B streptococci with special reference to bovine strains apparently lacking in type polysaccharide. J. Pathol. Bacteriol. 69:5160. 12. Pazur, J. H., and J. S. Anderson. 1963. Thymidine triphosphate: a-D-galactose-1-phosphate thymidylyltransferase from Streptococcus faecalis grown on Dgalactose. J. Biol. Chem. 238:3155-3159. 13. Pazur, J. H., J. S. Anderson, and W. W. Karakawa. 1971. Glycans from streptococcal cell walls: immunological and chemical properties of a new diheteroglycan from Streptococcus faecalis. J. Biol. Chem. 246:1793-1798. 14. Romero, R., and H. W. Wilkinson. 1974. Identification of group B streptococci by immunofluorescence staining. Appl. Microbiol. 28:199-204. 15. Spackman, D. H., W. H. Stein, and S. Moore. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30:1190-1206. 16. Stableforth, A. W. 1932. Studies on bovine mastitis. VI. Serological characters of mastitis streptococci. J. Comp. Pathol. Ther. 45:185-190.
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CELLULAR ANTIGENS OF GROUP B STREPTOCOCCI
17. Tan, E. M., and H. G. Kunkel. 1966. Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J. Immunol. 96:464471. 18. Weber, P., and R. H. Winzler. 1969. Determination of hexosaminitols by ion-exchange chromatography and its application to alkali-labile glycosidic linkages in
77
glycoproteins. Arch. Biochem. Biophys. 129:534-538. 19. Wilkinson, J. W. 1975. Immunochemistry of purified polysaccharide type antigens of group B streptococci types Ia, Ib, and Ic. Infect. Immun. 11:845-852. 20. Wilkinson, H. W., and R. G. Eagon. 1971. Type-specific antigens of group B type Ic streptococci. Infect. Immun. 4:594-604.
