APMIS 100: 57-62, 1992
Characterization of the a antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody LARS BEVANGER, OLE-JAN IVERSEN and AUGUSTA IRENE NAESS Department of Microbiology, Faculty of Medicine, Regional Hospital, University of Trondheim, Trondheim, Norway
Bevanger, L., Iversen, 0-J. & Naess, A. I. Characterization of the a antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody. APMIS ZOO: 57-62, 1992. A murine monoclonal antibody raised against the a antigen of the group B streptococcal c proteins was analysed by immunofluorescence and whole-cell ELISA against a collection of 22 c proteinproducing GBS. All the strains showing fluorescence and reactivity in ELISA turned out to be a antigen-carrying strains as defined by polyclonal rabbit antisera, while none of the strains producing only the fl antigen was positive. Western blot analyses of the a antigen released into the culture medium of growing bacteria suggest that the a antigen is present as distinct proteins of variable molecular weights. The upper limit of the molecular weights varies considerably from one strain to another, from approximately 200 kD to 70 kD. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD throughout the gel. Some strains gave rise to 15-16 bands, while others gave rise to only one or two bands. The present investigation suggests that a antigens include several, probably identical, repeating subunits of approximately 10 kD.The epitope recognized by the MAb seems to be located on a 10-kD fragment, and in addition, it appears to be surface located, making the MAb a suitable tool in serodiagnostic work.
Key words: Group B streptococci; c proteins; a antigen; monoclonal antibodies. Lars Bevanger, Department of Microbiology, Regional Hospital, University of Trondheim, N-7006 Trondheim, Norway.
The c proteins of GBS originally described by Wilkilason et al. (15) include the a and the p antigen (5). The two antigens exist independently of each other as demonstrated by the Occurrence of strains carrying only one of the antigens (3, 8). The a-antigen component is trypsin-resistant and pepsin-sensitive, while the antigen is susceptible to both trypsin and pepsin digestion. Various techniques have been used to extract the c proteins from the cells, including the use of hot HCI, detergents, trypsination (3), and recently mutanolysin (12). The proteins are also present in the broth of growing bacteria Received March 7, 1991. Accepted May 30, 1991,
(8), presumably in a less degraded form than the preparations obtained with harsh extraction methods like hot HCI. Experimental data suggest that the c proteins are surface localized on filamentous projections external to the cell wall (14). Attempts to purify the c proteins using various extraction methods suggest that both the a and the flantigen exhibit heterogeneity in molecular size ranging from 10-20 kD to 130 kD or more (3, 8, 13). Studies have indicated that the c-protein antigens may interfere with the resistance to opsonization and killing of GBS (8), and in vivo studies have shown a protective effect of anti-c-protein antibodies when the animals were preimmunized (9) or passively protected by injection of hyperimmune rabbit antisera (3, 6, 8, 11). 57
BEVANGER et al.
Recently, two additional c-protein antigens were described by Brady et al. (7), and named gamma and delta, respectively. In this study murine monoclonal antibodies were raised against the c proteins and used for the characterization of the a antigen. MATERIALS AND METHODS Bacteria The following GBS type strains were used: strain A909, type Ia/c a+P+; strain 335, type Ia/c, a+ p-; strain 70339, type Ia/c, a- p+ (4). Other strains were selected from a collection of clinical isolates preserved in our laboratory. The bacteria were cultured in a modified Todd-Hewitt broth and harvested as described (5). For the fluorescent antibody testing, bacteria grown overnight on bovine blood agar were used. Antigen preparation The “crude” a-antigen preparation was obtained from broth supernatants by precipitation with trichloro-acetic acid (TCA) as described (5). Preparation of immunosorbent-purified a and p antigens used in ELISA for screening of the hybridomas has been described elsewhere (4). Monoclonal antibodies (MAbs) For the preparation of MAbs, BALBic mice were immunized with whole-cell suspensions of heat-killed bacteria from the type Ia/c strain A909. The mice were immunized by intraperitoneal injection twice and finally one intravenous injection. The procedure used for vaccination, fusion of spleen cells with myeloma cells and cultivation, was essentially as described elsewhere (1). The culture supernatants from the hybridomas were screened in an indirect ELISA with a antigen from the strains A909 and 335, and with p antigen from strain 70339. A selected number of positive cultures were cloned by limiting dilution in 96well tissue culture plates with a feeder layer of spleen cells from normal BALB/c mice. Eight to ten days later, the cultures were once again screened by ELISA and recloned. Ascites was produced by i.p. injection of one selected a clone and one p clone in BALB/c mice. Debris and fat were removed from the ascitic fluids and antibodies purified by ion-exchange chromatography as described (1). Rabbit antibodies Rabbit antisera used in this study were produced by injections of immunosorbent-purified antigens; the a antigen from culture supernatants of strain 335, and HC1-extracted p antigen from strain 70339 (2,4). The a antigen was also purified by immunosorbent chromatography using MAb against the a antigen
58
(see below). Antibodies to the immunosorbent-purified a antigen (a’) were produced by multiple intradermal injections of the a’ antigen (200 pl) in complete Freund’s adjuvant (200 pl), followed by a booster after four weeks of a‘ in Freund’s incomplete adjuvant. The animal was bled two weeks later. ELISA Microtitration plates were coated overnight at 20°C with “crude” a antigen, or immunosorbentpurified a- or p antigen in 0.1 carbonate buffer pH 9.6. For the whole-cell ELISA, washed bacteria in coating buffer were allowed to dry in the wells at 37°C. The blocking procedure, washing, administration of culture supernatants from hybridomas, immunoglobulins from ascitic fluids, antisera, monoclonal immunoglobulins, and peroxidase-conjugated anti-immunoglobulins were as described (1). O-phenylene-diamine was added as substrate, and the reaction stopped by adding 2 M H,SO4. Immunoglobulin isotype of the MAb was determined using wells coated with immunosorbent-purified a antigen. Immunoglobulins bound were detected by peroxidase-conjugated goat anti-mouse IgG 1, IgG2a, IgG2b, IgG3, IgA or IgM, kappa light chain or lambda light chain (Nordic Immunological Labs, Tilburg, The Netherlands). SDS-PAGE and immunoblot SDS-PAGE was performed according to Laemmli (lo), using 10% polyacrylamide gel. Samples and standard proteins (SDS-PAGE standard low- and high-molecular-weight, Bio-Rad Laboratories, Richmond, CA) were run simultaneously. The polypeptides were transferred electrophoretically onto nitrocellulose membranes (Immobilon-P transfer membranes, pore size 0.45 pm, Millipore Corporation, Bedford, MA.), using a transblotting chamber containing 20% (vol/vol) methanol, 25 mM Tris-HC1, and 192 mM glycine buffer (pH 8.3) (1). The nitrocellulose membrane was washed in aqua dest. for 10 min and soaked in a blocking buffer pH 8.0 (50 mM Tris-HC1, 2 mM CaCl,, 80 mM NaCl) containing 5% (wt/vol) skim milk powder (Molico instant, Nestle) and 0.2% Noniodet P-40 (Bethesda Research La.) for one h. The membranes were cut into strips and incubated with rabbit anti-a serum (1 : 1000) or MAb (1:200) in blocking buffer for two h under moderate agitation. After washing in TEN-buffer pH 7.4 (50 mM Tris-HC1, 5 mM EDTA, 0.15 M NaCl, 0.05% Tween 20) the strips were incubated with peroxidaseconjugated goat anti-rabbit or rabbit anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark) in blocking buffer for two h followed by washing three times in TEN. Finally, substrate (0-phenylenediamine in 0.1 M citrate-phosphate buffer pH 5.0) was added (1). Immunosorbent chromatography The MAb was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden).
THE a ANTIGEN OF THE C PROTEINS OF GROUP B STREPTOCOCCI
The column was prepared as recommended by the manufacturer. Crude supernatant or a antigen concentrated from the supernatant by TCA precipitation was applied to the column. The column was washed with phosphate-buffered saline, pH 7.2 (PBS), and eluted by a 0.1 M glycine-HC1 buffer, pH 2.6, with 0.5 M NaCl. For some experiments, a’ antigen was coupled to cyanogen bromide-activated Sepharose and used for the isolation of rabbit anti-a antibodies. The immunosorbent chromatography was performed as described above. Indirect immunofluorescent antibody test (IFAT) Smears on glass slides were prepared from bacteria suspended in PBS, airdried and gently heat fixed. The slides were incubated with rabbit anti-a or anti-P serum or MAb (30 min; 20°C), washed in three different baths of PBS, followed by FITC-conjugated antirabbit or anti-mouse immunoglobulins (Dakopatts). The slides were read with a Nikon epifluorescence microscope.
RESULTS Selection of the anti-a monoclonal antibody Culture supernatants from cultivated hybridomas were tested for antibodies against purified a antigens from the GBS strains A909 and 335, respectively, and purified p antigen from strain 70339 in an indirect ELISA. One monoclonal antibody, 3 1 -5C7D2E5, which recognized an epitope in the a-antigen preparations from strain A909 as well as from strain 335, was selected for this study. The MAb was identified as an IgGl/kappa antibody by indirect ELISA using isotype and light chainspecific second antibodies. The clone was grown as ascitic tumours in mice, and the end-point titre of the ascitic fluid in an indirect ELISA was 128,000. The MAb was analysed by IFAT and by a whole-cell ELISA using a collection of 22 c protein-producing GBS. Seventeen of the strains were positive in IFAT and the same strains showed a positive reaction in ELISA (Table 1). All these strains turned out to be a-carrying strains as defined by polyclonal rabbit antisera, while the five strains carrying only /3 antigen were negative in immunofluorescence and in ELISA (Table 1). Analyses of the a antigen Western blot analyses of the a antigen from strain A909- and from strain 335 bacteria using
TABLE 1. Reactivity of MA6 31-5C7D2E.5 tested in a whole cell ELISA and IFAT against a collection of 23 group B streptococcal strains Serotype No. of strains reacting designationwith MAb 31-5C7D2E5 c-protein No. of in anti een strains ELISA IFAT 1 0 0 Ia Ialc-a 3 3 3 Ialc-P 2 0 0 Ialc-ap 1 1 1 Iblc-a 1 1 1 Iblc-ap 3 3 3 IIlc-ap 3 3 3 111I c-a p 1 1 1 IVlc-a 4 4 4 IVIC-p 1 0 0 NTIc-ap 1 1 1 NTIc-P 2 0 0 Total no. 23 17 17
the MAb and a rabbit anti-a serum as detecting antibodies are shown in Fig. 1. The immunoblots suggest that the a antigen is present as distinct proteins of variable molecular weights (mol. wts.), with an upper limit of about 200 kD and 120 kD for strains 335 and A909, respectively. The immunoblot patterns obtained with nine other @-producing strains are shown in Fig. 2. The upper limit of the mol. wts. varied considerably from one strain to another. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD
Fig. I . Western blot analyses of the “crude” a-antigen preparation obtained in culture supernatants from strain A909 (lanes 3 and 5 ) , and strain 335 (lanes 4 and 6). The blots were developed with MAb (lanes 3 and 4) and with rabbit anti-a serum (lanes 5 and 6). Mol. wt. standards in lanes 1 and 2 are stained with Coomassie brilliant blue.
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throughout the gel. Strain 335 gave rise to 15-16 bands, strain A909-eight bands; in other strains only one or two bands were detected. The staining intensity became weaker with decreasing mol. wts. of the bands. When the blots developed by the MAb are compared with the blots developed with rabbit antiserum, some bands with a molecular weight below 50 kD are detected by the polyclonal antiserum only (Fig. 1). Isolation of the a’ antigen The a antigen from strain 335 was purified by immunosorbent chromatography on a column carrying the MAb and applied in Western blot analyses (Fig. 3). The antigen (a‘ antigen) still appeared as several bands with regularly spaced intervals of approximately 10 kD. However, the low-molecular-weight antigens recognized by the polyclonal anti-a serum in a “crude” antigen preparation (Fig. 1) were not seen in the a’ antigen preparation (Fig. 3). Rabbit antibodies recognizing the a’ antigen The rabbit antibodies recognizing the a’ antigen were prepared by immunosorbent chromatography on a column carrying the a’ antigen. These antibodies were used in a Western blot analysis with “crude” a as antigen to investigate whether any of the low-molecular-weight antigens detected by the rabbit anti-a serum are integral parts of the a’ antigen (Fig. 4). One additional band of approximately 40 kD was recognized by the rabbit antibodies when compared to the bands obtained by the MAb. The 40-kD component was also detected when serum from a rabbit immunized with the a’ anti-
60
Fig. 3. Western blot analyses of a’ antigen (see text) from strain 335 (lanes 3 and 5) and “crude” a antigen from strain 335 (lanes 4 and 6). The blots were developed with rabbit anti-a serum (lanes 3 and 4) and MAb (lanes 5 and 6). Mol. wt. standards stained with Coomassie brilliant blue in lanes 1 and 2.
gen was applied in Western blot against the “crude” a antigen (data not shown). This suggests that the 40-kD band represents an integral part of the a antigen recognized by the monoclonal antibody, although the MAb epitope is not detected on this fragment. 6
DISCUSSION A murine monoclonal antibody, 3 1-5C7D2E5, is shown to recognize an epitope in the a-antigen preparations from strain A909 and from strain 335 in an indirect ELISA. The epitope is demonstrated in 100% of the a antigen-positive strains studied both in ELISA and in indirect immunofluorescent analyses, while none out of five p-
THE a ANTIGEN OF THE C PROTEINS OF GROUP B STREFTOCOCCI
carrying strains reacted with the MAb. This positive correlation between the MAb and the polyclonal rabbit anti-a serum suggests that the MAb is specific for the a antigen. Furthermore, it indicates that the MAb is directed against a common epitope on the a antigens from various strains, and that this epitope is surface exposed. Western blot analyses of the a antigen with the MAb as well as with the rabbit anti-a serum show a ladder-like immunoblot pattern with differences in mol. wts. of the neighbouring bands estimated to be approximately 10 kD (Fig. 1). Thus, the a antigen is present as proteins with different molecular masses, and the differences are presumably due to the incorporation of various numbers of, probably identical, subunits with mol. wts. of 10 kD. The immunoblot patterns obtained with a antigen from culture medium and antigens extracted by SDS from whole bacterial cells were identical (data not shown). Together with the fact that we were never able to detect free 10-kD subunits of the a antigen, we conclude that the heterogeneity of the a antigen within one strain is hardly caused by enzymatic degradation. One possible explanation could be that there are several initiation sites for the transcription of the a-antigen gene. The maximum number of 10-kD subunits in the a-antigen preparations investigated was 15-16, and was found in strain 335. In other
strains the number of 10-kD subunits was restricted to one or two (Fig. 2). For every strain, except strain A909, the strongest band on the immunoblot was the one with the highest molecular weight. With strain A909, two strong bands appeared, one with an apparent molecular weight of 120 kD and one about 70 kD. A possible explanation for this finding could be that strain A909 has two separate genes for the a antigen. No a antigens with subunit mol. wts. below 50 kD were recognized by the MAb (Fig. 3). However, when the rabbit antibodies purified by immunoadsorption to the a‘ antigen were used in immunoblot on a “crude” a-antigen preparation, an additional fragment of approximately 40 kD was detected. Accordingly, we conclude that the 40-kD component constitutes a part of the a antigen, and that the epitope recognized by the MAb is not located on this subunit. The difference in molecular mass between the 50- and the 40-kD band is 10 kD, and based on the immunoblot analyses it seems reasonable to suggest that the MAb epitope is located on this 10 kD fragment. The epitope recognized by the MAb is located at the surface of the bacterium as judged by the indirect immunofluorescent assay, making the MAb a suitable tool in serodiagnostic work.
REFERENCES 1. Bergh, K.. Iversen, 0-J. & Aasbakk, K.: Mono-
-
kD, 99 66
11
45
-
31
-
21
-
Fig. 4. Western blot analyses of “crude” a antigen from strain 335 (lanes 3, 5, and 7) and strain A909 (lanes 4, 6, and 8). The blots were developed with MAb (lanes 3 and 4), with rabbit anti-a serum (lanes 5 and 6). and with rabbit anti-a antibodies recognizing the a’ antigen (see text) (lanes 7 and 8). Mol. wt. standards stained with Coomassie brilliant blue in lanes 1 and 2.
2.
3. 4.
5.
6.
clonal antibodies against the major zymosan-induced chemotactic factor in rabbit serum. J. Immunol. Methods. 108: 179-187, 1988. Bevanger, L.: Ibc proteins as serotype markers of group B streptococci. Acta. path. microbiol. immunol. scand. Sect. B, 91: 231-234, 1983. Bevanger, L.: The Ibc proteins. In: Antibiot. Chemother., vol. 35, (Karger, Basel 1985), pp. 101-113. Bevanger, L.: The Ibc proteins of group B streptococci: Isolation of the a and p antigens by immunosorbent chromatography and test for human serum antibodies against the two antigens. Acta. path. microbiol. immunol. scand. Sect. B, 93: 113-1 19, 1985. Bevanger, L. & Madand, J. A.: Complete and incomplete Ibc protein fraction in group B streptococci. Atca. path. microbiol. scand. Sect. B, 87: 51-54, 1979. Bevanger, L. & N m s . A. I.: Mouse-protective
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7.
8. 9.
10.
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antibodies against the Ibc proteins of group B streptococci. Atca. path. microbiol. immunol. scand. Sect. B, 93: 121-124, 1985. Brady, L. J., Daphtary, U. D., Ayob, E. M. & Boyle, M . D.: Two novel antigens associated with group B streptococci identified by a rapid twostage radioimmunoassay. J. Infect. Dis. 158: 965-972, 1988. Ferrieri, I?: Surface-localized protein antigens of group B streptococci. Reviews of Infect. Dis. 10: Suppl. 2, 363-366, 1988. Itoh, T,Yan, X-J., Nakano, H. & Yoshioka, M.: Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants. Microbiol. Immunol. 30: 297-305, 1986. Laemmli, U. K.: Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685, 1970.
1 1 . Lancefield, R. C., McCarty, M. & Everly, W N.: Multiple mouse-protective antibodies directed against group B &reptococci. J. Exp. Med. 142: 165-179, 1975. 12. Madofl; L. C., Michel, J. L. & Kasper, D. L.: A monoclonal antibody identifies a protective Cprotein alpha-antigen epitope in group B streptococci. Infect. Immun. 59: 204210, 1991. 13. Russell-Jones, G. J. & Gotschlich, E. C.: Identification of protein antigens of group B streptococci, with special reference to the Ibc antigens. J. Exp. Med. 160: 1476-1484, 1984. 14. Wagner, B,, Wagner, M . , Kubin, K & Ryc, M.: Immunoelectron microscopic study of the location of group-specific and protein type-specific antigens of group B streptococci. J. Gen. Microbiol. 118: 95-105, 1980. 15. Wilkinson, H . W & Eagon, R. G.: Type-specific antigens of group B type Ic streptococci. Infect. Immun. 4: 596-604, 1971.
Characterization of the a antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody LARS BEVANGER, OLE-JAN IVERSEN and AUGUSTA IRENE NAESS Department of Microbiology, Faculty of Medicine, Regional Hospital, University of Trondheim, Trondheim, Norway
Bevanger, L., Iversen, 0-J. & Naess, A. I. Characterization of the a antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody. APMIS ZOO: 57-62, 1992. A murine monoclonal antibody raised against the a antigen of the group B streptococcal c proteins was analysed by immunofluorescence and whole-cell ELISA against a collection of 22 c proteinproducing GBS. All the strains showing fluorescence and reactivity in ELISA turned out to be a antigen-carrying strains as defined by polyclonal rabbit antisera, while none of the strains producing only the fl antigen was positive. Western blot analyses of the a antigen released into the culture medium of growing bacteria suggest that the a antigen is present as distinct proteins of variable molecular weights. The upper limit of the molecular weights varies considerably from one strain to another, from approximately 200 kD to 70 kD. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD throughout the gel. Some strains gave rise to 15-16 bands, while others gave rise to only one or two bands. The present investigation suggests that a antigens include several, probably identical, repeating subunits of approximately 10 kD.The epitope recognized by the MAb seems to be located on a 10-kD fragment, and in addition, it appears to be surface located, making the MAb a suitable tool in serodiagnostic work.
Key words: Group B streptococci; c proteins; a antigen; monoclonal antibodies. Lars Bevanger, Department of Microbiology, Regional Hospital, University of Trondheim, N-7006 Trondheim, Norway.
The c proteins of GBS originally described by Wilkilason et al. (15) include the a and the p antigen (5). The two antigens exist independently of each other as demonstrated by the Occurrence of strains carrying only one of the antigens (3, 8). The a-antigen component is trypsin-resistant and pepsin-sensitive, while the antigen is susceptible to both trypsin and pepsin digestion. Various techniques have been used to extract the c proteins from the cells, including the use of hot HCI, detergents, trypsination (3), and recently mutanolysin (12). The proteins are also present in the broth of growing bacteria Received March 7, 1991. Accepted May 30, 1991,
(8), presumably in a less degraded form than the preparations obtained with harsh extraction methods like hot HCI. Experimental data suggest that the c proteins are surface localized on filamentous projections external to the cell wall (14). Attempts to purify the c proteins using various extraction methods suggest that both the a and the flantigen exhibit heterogeneity in molecular size ranging from 10-20 kD to 130 kD or more (3, 8, 13). Studies have indicated that the c-protein antigens may interfere with the resistance to opsonization and killing of GBS (8), and in vivo studies have shown a protective effect of anti-c-protein antibodies when the animals were preimmunized (9) or passively protected by injection of hyperimmune rabbit antisera (3, 6, 8, 11). 57
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Recently, two additional c-protein antigens were described by Brady et al. (7), and named gamma and delta, respectively. In this study murine monoclonal antibodies were raised against the c proteins and used for the characterization of the a antigen. MATERIALS AND METHODS Bacteria The following GBS type strains were used: strain A909, type Ia/c a+P+; strain 335, type Ia/c, a+ p-; strain 70339, type Ia/c, a- p+ (4). Other strains were selected from a collection of clinical isolates preserved in our laboratory. The bacteria were cultured in a modified Todd-Hewitt broth and harvested as described (5). For the fluorescent antibody testing, bacteria grown overnight on bovine blood agar were used. Antigen preparation The “crude” a-antigen preparation was obtained from broth supernatants by precipitation with trichloro-acetic acid (TCA) as described (5). Preparation of immunosorbent-purified a and p antigens used in ELISA for screening of the hybridomas has been described elsewhere (4). Monoclonal antibodies (MAbs) For the preparation of MAbs, BALBic mice were immunized with whole-cell suspensions of heat-killed bacteria from the type Ia/c strain A909. The mice were immunized by intraperitoneal injection twice and finally one intravenous injection. The procedure used for vaccination, fusion of spleen cells with myeloma cells and cultivation, was essentially as described elsewhere (1). The culture supernatants from the hybridomas were screened in an indirect ELISA with a antigen from the strains A909 and 335, and with p antigen from strain 70339. A selected number of positive cultures were cloned by limiting dilution in 96well tissue culture plates with a feeder layer of spleen cells from normal BALB/c mice. Eight to ten days later, the cultures were once again screened by ELISA and recloned. Ascites was produced by i.p. injection of one selected a clone and one p clone in BALB/c mice. Debris and fat were removed from the ascitic fluids and antibodies purified by ion-exchange chromatography as described (1). Rabbit antibodies Rabbit antisera used in this study were produced by injections of immunosorbent-purified antigens; the a antigen from culture supernatants of strain 335, and HC1-extracted p antigen from strain 70339 (2,4). The a antigen was also purified by immunosorbent chromatography using MAb against the a antigen
58
(see below). Antibodies to the immunosorbent-purified a antigen (a’) were produced by multiple intradermal injections of the a’ antigen (200 pl) in complete Freund’s adjuvant (200 pl), followed by a booster after four weeks of a‘ in Freund’s incomplete adjuvant. The animal was bled two weeks later. ELISA Microtitration plates were coated overnight at 20°C with “crude” a antigen, or immunosorbentpurified a- or p antigen in 0.1 carbonate buffer pH 9.6. For the whole-cell ELISA, washed bacteria in coating buffer were allowed to dry in the wells at 37°C. The blocking procedure, washing, administration of culture supernatants from hybridomas, immunoglobulins from ascitic fluids, antisera, monoclonal immunoglobulins, and peroxidase-conjugated anti-immunoglobulins were as described (1). O-phenylene-diamine was added as substrate, and the reaction stopped by adding 2 M H,SO4. Immunoglobulin isotype of the MAb was determined using wells coated with immunosorbent-purified a antigen. Immunoglobulins bound were detected by peroxidase-conjugated goat anti-mouse IgG 1, IgG2a, IgG2b, IgG3, IgA or IgM, kappa light chain or lambda light chain (Nordic Immunological Labs, Tilburg, The Netherlands). SDS-PAGE and immunoblot SDS-PAGE was performed according to Laemmli (lo), using 10% polyacrylamide gel. Samples and standard proteins (SDS-PAGE standard low- and high-molecular-weight, Bio-Rad Laboratories, Richmond, CA) were run simultaneously. The polypeptides were transferred electrophoretically onto nitrocellulose membranes (Immobilon-P transfer membranes, pore size 0.45 pm, Millipore Corporation, Bedford, MA.), using a transblotting chamber containing 20% (vol/vol) methanol, 25 mM Tris-HC1, and 192 mM glycine buffer (pH 8.3) (1). The nitrocellulose membrane was washed in aqua dest. for 10 min and soaked in a blocking buffer pH 8.0 (50 mM Tris-HC1, 2 mM CaCl,, 80 mM NaCl) containing 5% (wt/vol) skim milk powder (Molico instant, Nestle) and 0.2% Noniodet P-40 (Bethesda Research La.) for one h. The membranes were cut into strips and incubated with rabbit anti-a serum (1 : 1000) or MAb (1:200) in blocking buffer for two h under moderate agitation. After washing in TEN-buffer pH 7.4 (50 mM Tris-HC1, 5 mM EDTA, 0.15 M NaCl, 0.05% Tween 20) the strips were incubated with peroxidaseconjugated goat anti-rabbit or rabbit anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark) in blocking buffer for two h followed by washing three times in TEN. Finally, substrate (0-phenylenediamine in 0.1 M citrate-phosphate buffer pH 5.0) was added (1). Immunosorbent chromatography The MAb was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden).
THE a ANTIGEN OF THE C PROTEINS OF GROUP B STREPTOCOCCI
The column was prepared as recommended by the manufacturer. Crude supernatant or a antigen concentrated from the supernatant by TCA precipitation was applied to the column. The column was washed with phosphate-buffered saline, pH 7.2 (PBS), and eluted by a 0.1 M glycine-HC1 buffer, pH 2.6, with 0.5 M NaCl. For some experiments, a’ antigen was coupled to cyanogen bromide-activated Sepharose and used for the isolation of rabbit anti-a antibodies. The immunosorbent chromatography was performed as described above. Indirect immunofluorescent antibody test (IFAT) Smears on glass slides were prepared from bacteria suspended in PBS, airdried and gently heat fixed. The slides were incubated with rabbit anti-a or anti-P serum or MAb (30 min; 20°C), washed in three different baths of PBS, followed by FITC-conjugated antirabbit or anti-mouse immunoglobulins (Dakopatts). The slides were read with a Nikon epifluorescence microscope.
RESULTS Selection of the anti-a monoclonal antibody Culture supernatants from cultivated hybridomas were tested for antibodies against purified a antigens from the GBS strains A909 and 335, respectively, and purified p antigen from strain 70339 in an indirect ELISA. One monoclonal antibody, 3 1 -5C7D2E5, which recognized an epitope in the a-antigen preparations from strain A909 as well as from strain 335, was selected for this study. The MAb was identified as an IgGl/kappa antibody by indirect ELISA using isotype and light chainspecific second antibodies. The clone was grown as ascitic tumours in mice, and the end-point titre of the ascitic fluid in an indirect ELISA was 128,000. The MAb was analysed by IFAT and by a whole-cell ELISA using a collection of 22 c protein-producing GBS. Seventeen of the strains were positive in IFAT and the same strains showed a positive reaction in ELISA (Table 1). All these strains turned out to be a-carrying strains as defined by polyclonal rabbit antisera, while the five strains carrying only /3 antigen were negative in immunofluorescence and in ELISA (Table 1). Analyses of the a antigen Western blot analyses of the a antigen from strain A909- and from strain 335 bacteria using
TABLE 1. Reactivity of MA6 31-5C7D2E.5 tested in a whole cell ELISA and IFAT against a collection of 23 group B streptococcal strains Serotype No. of strains reacting designationwith MAb 31-5C7D2E5 c-protein No. of in anti een strains ELISA IFAT 1 0 0 Ia Ialc-a 3 3 3 Ialc-P 2 0 0 Ialc-ap 1 1 1 Iblc-a 1 1 1 Iblc-ap 3 3 3 IIlc-ap 3 3 3 111I c-a p 1 1 1 IVlc-a 4 4 4 IVIC-p 1 0 0 NTIc-ap 1 1 1 NTIc-P 2 0 0 Total no. 23 17 17
the MAb and a rabbit anti-a serum as detecting antibodies are shown in Fig. 1. The immunoblots suggest that the a antigen is present as distinct proteins of variable molecular weights (mol. wts.), with an upper limit of about 200 kD and 120 kD for strains 335 and A909, respectively. The immunoblot patterns obtained with nine other @-producing strains are shown in Fig. 2. The upper limit of the mol. wts. varied considerably from one strain to another. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD
Fig. I . Western blot analyses of the “crude” a-antigen preparation obtained in culture supernatants from strain A909 (lanes 3 and 5 ) , and strain 335 (lanes 4 and 6). The blots were developed with MAb (lanes 3 and 4) and with rabbit anti-a serum (lanes 5 and 6). Mol. wt. standards in lanes 1 and 2 are stained with Coomassie brilliant blue.
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throughout the gel. Strain 335 gave rise to 15-16 bands, strain A909-eight bands; in other strains only one or two bands were detected. The staining intensity became weaker with decreasing mol. wts. of the bands. When the blots developed by the MAb are compared with the blots developed with rabbit antiserum, some bands with a molecular weight below 50 kD are detected by the polyclonal antiserum only (Fig. 1). Isolation of the a’ antigen The a antigen from strain 335 was purified by immunosorbent chromatography on a column carrying the MAb and applied in Western blot analyses (Fig. 3). The antigen (a‘ antigen) still appeared as several bands with regularly spaced intervals of approximately 10 kD. However, the low-molecular-weight antigens recognized by the polyclonal anti-a serum in a “crude” antigen preparation (Fig. 1) were not seen in the a’ antigen preparation (Fig. 3). Rabbit antibodies recognizing the a’ antigen The rabbit antibodies recognizing the a’ antigen were prepared by immunosorbent chromatography on a column carrying the a’ antigen. These antibodies were used in a Western blot analysis with “crude” a as antigen to investigate whether any of the low-molecular-weight antigens detected by the rabbit anti-a serum are integral parts of the a’ antigen (Fig. 4). One additional band of approximately 40 kD was recognized by the rabbit antibodies when compared to the bands obtained by the MAb. The 40-kD component was also detected when serum from a rabbit immunized with the a’ anti-
60
Fig. 3. Western blot analyses of a’ antigen (see text) from strain 335 (lanes 3 and 5) and “crude” a antigen from strain 335 (lanes 4 and 6). The blots were developed with rabbit anti-a serum (lanes 3 and 4) and MAb (lanes 5 and 6). Mol. wt. standards stained with Coomassie brilliant blue in lanes 1 and 2.
gen was applied in Western blot against the “crude” a antigen (data not shown). This suggests that the 40-kD band represents an integral part of the a antigen recognized by the monoclonal antibody, although the MAb epitope is not detected on this fragment. 6
DISCUSSION A murine monoclonal antibody, 3 1-5C7D2E5, is shown to recognize an epitope in the a-antigen preparations from strain A909 and from strain 335 in an indirect ELISA. The epitope is demonstrated in 100% of the a antigen-positive strains studied both in ELISA and in indirect immunofluorescent analyses, while none out of five p-
THE a ANTIGEN OF THE C PROTEINS OF GROUP B STREFTOCOCCI
carrying strains reacted with the MAb. This positive correlation between the MAb and the polyclonal rabbit anti-a serum suggests that the MAb is specific for the a antigen. Furthermore, it indicates that the MAb is directed against a common epitope on the a antigens from various strains, and that this epitope is surface exposed. Western blot analyses of the a antigen with the MAb as well as with the rabbit anti-a serum show a ladder-like immunoblot pattern with differences in mol. wts. of the neighbouring bands estimated to be approximately 10 kD (Fig. 1). Thus, the a antigen is present as proteins with different molecular masses, and the differences are presumably due to the incorporation of various numbers of, probably identical, subunits with mol. wts. of 10 kD. The immunoblot patterns obtained with a antigen from culture medium and antigens extracted by SDS from whole bacterial cells were identical (data not shown). Together with the fact that we were never able to detect free 10-kD subunits of the a antigen, we conclude that the heterogeneity of the a antigen within one strain is hardly caused by enzymatic degradation. One possible explanation could be that there are several initiation sites for the transcription of the a-antigen gene. The maximum number of 10-kD subunits in the a-antigen preparations investigated was 15-16, and was found in strain 335. In other
strains the number of 10-kD subunits was restricted to one or two (Fig. 2). For every strain, except strain A909, the strongest band on the immunoblot was the one with the highest molecular weight. With strain A909, two strong bands appeared, one with an apparent molecular weight of 120 kD and one about 70 kD. A possible explanation for this finding could be that strain A909 has two separate genes for the a antigen. No a antigens with subunit mol. wts. below 50 kD were recognized by the MAb (Fig. 3). However, when the rabbit antibodies purified by immunoadsorption to the a‘ antigen were used in immunoblot on a “crude” a-antigen preparation, an additional fragment of approximately 40 kD was detected. Accordingly, we conclude that the 40-kD component constitutes a part of the a antigen, and that the epitope recognized by the MAb is not located on this subunit. The difference in molecular mass between the 50- and the 40-kD band is 10 kD, and based on the immunoblot analyses it seems reasonable to suggest that the MAb epitope is located on this 10 kD fragment. The epitope recognized by the MAb is located at the surface of the bacterium as judged by the indirect immunofluorescent assay, making the MAb a suitable tool in serodiagnostic work.
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kD, 99 66
11
45
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31
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21
-
Fig. 4. Western blot analyses of “crude” a antigen from strain 335 (lanes 3, 5, and 7) and strain A909 (lanes 4, 6, and 8). The blots were developed with MAb (lanes 3 and 4), with rabbit anti-a serum (lanes 5 and 6). and with rabbit anti-a antibodies recognizing the a’ antigen (see text) (lanes 7 and 8). Mol. wt. standards stained with Coomassie brilliant blue in lanes 1 and 2.
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1 1 . Lancefield, R. C., McCarty, M. & Everly, W N.: Multiple mouse-protective antibodies directed against group B &reptococci. J. Exp. Med. 142: 165-179, 1975. 12. Madofl; L. C., Michel, J. L. & Kasper, D. L.: A monoclonal antibody identifies a protective Cprotein alpha-antigen epitope in group B streptococci. Infect. Immun. 59: 204210, 1991. 13. Russell-Jones, G. J. & Gotschlich, E. C.: Identification of protein antigens of group B streptococci, with special reference to the Ibc antigens. J. Exp. Med. 160: 1476-1484, 1984. 14. Wagner, B,, Wagner, M . , Kubin, K & Ryc, M.: Immunoelectron microscopic study of the location of group-specific and protein type-specific antigens of group B streptococci. J. Gen. Microbiol. 118: 95-105, 1980. 15. Wilkinson, H . W & Eagon, R. G.: Type-specific antigens of group B type Ic streptococci. Infect. Immun. 4: 596-604, 1971.
