Jun 8, 1983 - tetramethyl-pentadecane (Pristane; Sigma) (20) at monthly intervals. At 1 week after the last Pristane injection, the mice were given 2 x 106 to 6 ...
Vol. 18, No. 3
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1983, p. 480-485
0095-1137/83/090480-06$02.00/0 Copyright C 1983, American Society for Microbiology
Monoclonal Antibodies Against Group- and Type-Specific Lipopolysaccharide Antigens of Vibrio cholerae 0:1 BJORN GUSTAFSSON* AND TORD HOLME Department of Bacteriology, Karolinska Institute, S-104 01 Stockholm, Sweden
Received 10 May 1983/Accepted 8 June 1983
Hybrid cell lines producing monoclonal antibodies against the 0-antigenic determinants of Vibrio cholerae 0:1 have been established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay inhibition experiments by using lipopolysaccharides from V. cholerae 0:1 strains and type strains of groups 0:2 and 0:21. The anti-A antibody was of the immunoglobulin M (IgM) class, whereas the anti-B and -C antibodies were IgG3. The antibodies had a good agglutinating capacity when tested against V. cholerae 0:1 strains in the slide agglutination test. The disease cholera is caused only by Vibrio cholerae strains belonging to the serogroup 0:1, characterized by the group-specific antigen A. This group is divided into two serotypes, Ogawa and Inaba, each characterized by a type-specific antigen (1, 2, 5, 10, 23). These antigens are thermostable polysaccharides, part of the cell wall lipopolysaccharide (LPS) of V. cholerae (7, 18, 25). During infection with V. cholerae, vibriocidal antibodies directed against the LPS determinants are produced (16, 17). As shown by Svennerholm and Holmgren (26), a vaccine containing LPS and an immunogenic subunit of the cholera toxin is more effective than the two components alone. It is of great importance to reveal the structure of the antigenic determinants of LPS from V. cholerae to provide a basis for the development of an efficient vaccine against cholera. This is true irrespective of whether this vaccine is a mixture of chemically defined components or a well-characterized, nonvirulent vaccine strain of V. cholerae. The structural analysis of the cell wall LPS of V. cholerae has been difficult owing to the extreme conditions needed for the degradation of the dominating part of the 0 antigen (12, 21). The side chain of the LPS is mainly composed of a homopolymer of perosamine (11, 21) with the amino groups acylated with 3-deoxy-L-glycerotetronic acid (11). The A-antigenic determinant may be situated on this part of the LPS, since it is identical in the two serotypes Ogawa and Inaba (11). We have also shown, with a monoclonal antibody directed against the core of the V. cholerae LPS, that core determinants are exposed in isolated LPS from a number of Ogawa and Inaba strains as well as from some non-0:1 V. cholerae strains (6). In addition, experiments with monoclonal antibodies ad480
sorbed to protein A-carrying staphylococci showed that core antigens were exposed on the surface of all V. cholerae strains tested (6, 13). To enable further studies on the chemical structure of the V. cholerae LPS, sufficient quantities of monospecific antibodies are needed. For this purpose, we have now developed hybrid cell lines that produce monoclonal antibodies against the antigenic determinants A, which is shared by the Ogawa and Inaba serotypes, B, which is Ogawa specific, and C, which is Inaba specific. MATERIALS AND METHODS Bacterial strains and cultivation. The strains used in this study, V. cholerae 34 Ogawa, 1451 Ogawa, 35 Inaba, 569B Inaba, NCTC 4711 (0:2), and 109-68 (0:21) were all provided by J. Holmgren, University of Goteborg, Sweden. All strains were cultivated in an aerated, stirred 12liter fermentor at 37°C and at a constant pH of 7.2. A tryptone-yeast extract medium (TY-2) (8) was used for all strains. Precultures were grown overnight in Erlenmeyer flasks on a rotary shaker at 37°C in the same medium with a reduced sugar content (5 g per liter). LPS preparation. LPS was extracted from the bacteria by the phenol-water method (28) and purified by high-speed centrifugation as described earlier (6). Protein content. The protein content of LPS preparations was measured by the method of Lowry et al.
(15).
Immunization. Female BALB/c mice, 6 to 10 weeks of age, were immunized for 4 to 9 weeks with Ogawa LPS and Inaba LPS according to the immunization schedule described earlier (6). Fusion and cloning. Hybridomas were established according to the protocol of Nowinski et al. (19) as described earlier (6) with the following modifications. The mouse myeloma SP2/0-Ag 14 (24; kindly provided by S. Hammarstrom, University of Stockholm, Sweden) was used for all fusions. Upon fusion, the initial
VOL. 18, 1983
MONOCLONAL ANTIBODIES AGAINST V. CHOLERAE LPS
selection of hybrids was performed by growing cells in microtiter plates (3042; Falcon Plastics, Oxnard, Calif.) on HAT (14) medium, consisting of RPMI 1640 medium (GIBCO Laboratories, Glasgow, Scotland) supplemented with 10%o fetal calf serum, L-glutamine (1 mM), penicillin (100 U/ml), streptomycin (100 ,ug/ml), hypoxanthine (0.1 mM; Sigma Chemical Co., St. Louis, Mo.), aminopterin (0.01 mM; Sigma), and thymidine (0.03 mM; Sigma). The microtiter plates were incubated in a tissue culture incubator at 37°C, 80% humidity and 5% CO2. After 2 weeks on HAT medium, with changes twice a week, supernatants from wells containing visible clones were assayed for antibody production in the microenzyme-linked immunosorbent assay (ELISA) method specific for cholera LPS. Hybrid cells producing antibodies of interest were diluted in HT medium (without aminopterin) with 15% fetal calf serum and antibiotics to a density of 500, 100, 50, 10, and 5 cells per ml and distributed in 0.1-ml samples into 96-well microtiter plates (3042; Falcon Plastics) with thymocytes at a concentration of 106 cells per ml as feeder cells. This cloning procedure was repeated until the clones of interest were found to be monoclonal by the methods described below. Antibody production in vitro. Monoclonal antibodies were produced in 250-ml tissue culture flasks (3075; Costar Data Packaging, Cambridge, Mass.) and in 4liter penicillin flasks (113.362; Kebo, Stockholm, Sweden) by growing cells in RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine (1 mM), and antibiotics as described above. A total of 50 to 90%o of cell medium was changed once or twice a week, depending on cell density. Antibody production in vivo. To obtain high yields of antibodies, the hybridomas were grown as ascites tumors in mice. Male BALB/c mice, about 10 weeks of age, were primed twice (0.1 and 0.5 ml) with 2,6,10,14tetramethyl-pentadecane (Pristane; Sigma) (20) at monthly intervals. At 1 week after the last Pristane injection, the mice were given 2 x 106 to 6 x 106 cells intraperitoneally. Detection of antibodies. Antibody production was measured by ELISA (3) performed in 96-well microtiter trays (Dynatech M 129 A; Flow Laboratories, Irwine, Scotland) (27) as described earlier (6). Briefly, 50 ,ul of cell culture supernatant or ascitic fluid was added to wells previously coated with LPS from V. cholerae 34 (Ogawa) and 35 (Inaba), respectively. The immune reaction was performed by adding 50 ,u1 of rabbit anti-mouse immunoglobulin (Dako, Copenhagen, Denmark), followed by incubation with 50 .l1 of horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin. As substrate, 100 ,ul of 3 mM 1,2phenylenediaminedihydrochloride (Fluka, Buchs, Switzerland) in 40 mM Tris-hydrochloride buffer (pH 7.6) was used. The optical density at 450 nm was measured in a Titertec Multiscan (Flow Laboratories) spectrophotometer. An optical density of 0.2 above background was considered a positive result. As a negative control, wells were coated with RPMI 1640 medium or ascites fluid from mice injected with cells of the myeloma SP2/0-Ag 14. ELISA inhibition. Inhibition of monoclonal antibodies with LPS preparations was performed as described earlier (6). Briefly, monoclonal antibodies were incubated with LPS in different concentrations in polysty-
481
rene tubes (Hegerplastics, Stallarholmen, Sweden) for 30 min at 22°C. The remaining antibodies were measured by ELISA as described above. The 50%o inhibitory concentration was defined as the concentration of LPS needed to obtain a 50% decrease in the optical density as compared with control tubes with no inhibitor added. Immunoglobulin class, subclass, and light chain. Immunoglobulin class, subclass, and light chain was determined by immunodiffusion by the method of Ouchterlony with 1% agarose (Marine Colloids, Rockland, Maine) in 10 mM phosphate buffer (pH 7.2) and specific rabbit antiserum to mouse immunoglobulin M (IgM), IgG (7S), IgGl, IgG2a, IgG2b, IgG3, K and X light chain (Bionetics Laboratory Products, Kensington, Md.). Isoelectric focusing. Antibodies were produced by growing hybrid cell lines in penicillin flasks. The antibodies were precipitated by the addition of saturated (NH4)2SO4 to a final concentration of 50% (9). The precipitate was dialyzed against 10 mM phosphatebuffered saline (PBS; pH 7.2) for 48 h with four changes of buffer. The H4 antibodies were further purified by passing the material through a column containing rabbit antimouse immunoglobulins (Dako) coupled to CNBractivated Sepliarose (Pharmacia Fine Chemicals, Uppsala, Sweden). The monoclonal antibodies were eluted with 6 M guanidine-hydrochloride (Sigma) and dialyzed overnight against PBS at 4°C. The antibodies H8 and C6 were purified by adding the (NH4)2SO4-precipitated antibodies to a protein ASepharose column (Pharmacia). The antibodies were eluted with 0.1 M acetate buffer in 0.15 M NaCl (pH 2.9). The pH of the eluted material was immediately adjusted to 7.0 by adding 2 M Tris base (Sigma). Antibody-containing fractions were dialyzed overnight against PBS at 4°C. Isoelectric focusing was performed in 0.5-mm thin-layer agarose gels containing 1% agarose IEF (Pharmacia) in double-distilled water and 0.8% ampholytes (pH 3.5 to 9.5; LKB-Produkter AB, Bromma, Sweden) (22) as described earlier (6) with the following modifications. H4 antibodies were subjected to isoelectric focusing by running the samples at 15 W for 30 min. The mouse immunoglobulin bands were fixed by soaking the gel with rabbit antimouse immunoglobulins (Dako) diluted 1:2 in PBS. H8 and C6 antibodies were subjected to isoelectric focusing by running the samples initially at 15 W. After 30 min, the voltage was increased to 1,800 V for an additional 30 min. Fixation of immunoglobulin bands was performed by soaking the gel with rabbit antimouse immunoglobulins diluted 1:5 in PBS.
RESULTS
Cloning. Three hybridomas producing specific antibodies against 0-antigenic determinants of V. cholerae 0:1 LPS were established. These clones were isolated from three different fusion experiments. Clone H4 originated from a mouse immunized for 5 weeks with LPS from V. cholerae 35 Inaba. Clone H8 was derived from a fusion of SP2/0 cells and spleen cells from a mouse immunized for 9 weeks with LPS from V. cholerae 34 Ogawa, whereas clone C6 was de-
482
GUSTAFSSON AND HOLME
rived from a mouse immunized for 4 weeks with LPS from the strain 569B Inaba. Isoelectric focusing. Purified monoclonal antibodies were tested by isoelectric focusing with agarose gels. H4 antibodies showed two distinct bands at pH 4.0 and 4.5, respectively. The band at pH 4.0 showed three minor side bands (Fig. 1A). H8 and C6 antibodies did not focus unless the voltage was increased to 1,800 V and the running time was prolonged to a total of 1 h. The H8 antibodies showed four bands in the region of pH 7.8 to 8.8 and several side bands between the major bands. C6 antibodies yielded six major bands at pH 8.0 to 9.0 (Fig. 1B). No bands were detected with cell culture supernatants from the myeloma SP2/0 subjected to the same purification as the hybridomas (Fig. 1). Class, subclass, and light chain. The results from double-diffusion experiments by the Ouchterlony technique are shown in Table 1. Antibody production in vitro. The clones producing monoclonal antibodies were cultivated in tissue culture flasks for more than 5 months, producing antibodies in titers from 128 to 1,024 as determined by ELISA (Table 1).
A
3H|t i
.
9+ I.
a i
i
75 -E I
5t 6 4+
l
2
i47t
FIG. 1. Isoelectric focusing followed by immunofixation of purified monoclonal antibodies. (A) Culture supernatant from SP2/0 myeloma cell line (lane 1) and monoclonal antibodies from H4 hybridoma (lane 2). (B) Culture supernatant from SP2/0 myeloma cell line (lane 1), monoclonal antibodies from H8 hybridoma (lane 2), and monoclonal antibodies from C6 hybridoma (lane 3).
J. CLIN. MICROBIOL.
TABLE 1. Characteristics of monoclonal antibodies and their titer in ELISA Monoclonal antibody
Class or subclass
Light chain
Specificity
Titer' In vitro
Ascites
H4 H8 C6
K A 256 8,000 IgM X B IgG3 1,024 32,000 K IgG3 128 8,000 C a Endpoint titers of cell cultures or ascites fluid in ELISA. Endpoint titers were defined as the highest dilution in a twofold serial dilution still giving an optical density at 450 nm of >0.2.
Antibody production in vivo. The monoclonal antibodies were also produced by growing cells as ascites in mice. Ascitic fluid developed usually within 2 weeks, and 2 to 10 ml could be withdrawn from the peritoneal cavity with a 0.9by 40-mm needle 2 to 4 times. The titers ranged between 8,000 and 32,000 (Table 1). ELISA-inhibition. Inhibition of the monoclonal antibodies was performed with LPS preparations from the V. cholerae strains 34, 1824, 1451, 35, 569B, NCTC 4711, and 109-68 in concentrations up to 5 mg/ml. The monoclonal antibodies produced by clone H4 were inhibited by LPS from Ogawa 34 and Inaba 35 but not by LPS from strain NCTC 4711 (0:2), indicating that these antibodies were specific for the A antigen, which is 0:1 specific, but shared by the Ogawa and Inaba serogroups (Fig. 2A). Antibodies produced by clone H8 were directed against the B determinant, as shown by the complete inhibition obtained by LPS from strain Ogawa 34 but not from strains Inaba 35 or NCTC 4711 (0:2) (Fig. 2B). Antibodies from clone C6 were specific, since they were inhibited by Inaba LPS only (Fig. 2C). The protein contents of the LPS preparations varied between 4 and 24%. To avoid misleading results due to the protein content, ELISA inhibition was also performed on heat-treated (30 min at 100°C) LPS. This experiment resulted in inhibition curves almost identical to those in Fig. 2. The specificity of the monoclonal antibodies was further studied by estimating the amount of LPS needed to inhibit the ELISA system to 50%. H4 antibodies were inhibited by different Ogawa LPS preparations as well as Inaba LPS preparations in amounts ranging from 0.10 to 0.44 mg/ml. H8 antibodies were inhibited by Ogawa LPS preparations in amounts ranging from 0.14 to 1.10 mg/ml. Inaba LPS from two different strains showed no inhibitory capacity in the concentrations tested (
