APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/90/072193-07$02.00/0
JUlY 1990, p. 2193-2199
Vol. 56, No. 7
Copyright © 1990, American Society for Microbiology
Monoclonal Antibodies against the Ruminal Bacterium Selenomonas ruminantium JOHN D. BROOKER* AND BARBARA STOKES Department of Animal Sciences, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064, Australia Received 5 January 1990/Accepted 21 April 1990
Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 107 cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.
Serological methods for the identification and quantification of microorganisms are well established, particularly for the study of medically important organisms. Both polyclonal antibodies and monoclonal antibodies (MAbs) that react with surface antigens on live organisms have been used in quantitative solid-phase enzyme-linked immunosorbent assay (ELISA) procedures and for studies of cell surface structures (6). With the advantages of sensitivity and specificity, immunoassays form the basis of many bacterial identification procedures and microbial population studies. These methods may also have applications in the study of natural microbial ecosystems that exist, for instance, in the rumen, where mixed populations of bacteria interact in a complex and continuously changing environment (4). The characterization of ruminal microbial isolates may also be facilitated by MAb reagants. The characterization and classification of ruminal bacterial species have been based largely on morphologic and metabolic descriptions (4). However, the accuracy of these procedures has recently been questioned because species assignment based on these parameters differed from that suggested by genotype analyses through DNA sequencing (21). Unambiguous genotyping through DNA-DNA hybridization, DNA fingerprinting, and nucleic acid sequencing has also been responsible for new insights into phylogenetic relationships between strains within apparently homogeneous species (21). However, although exquisitely specific, nucleic acid-based procedures may not necessarily be the most appropriate for rapid quantification of bacterial populations or for screening a range of unknown isolates for identification purposes. Difficulties with plant contamination of ruminal samples and restricted sensitivity of unamplified gene probes (1) suggest that a serological approach may be more appropriate. The potential of immunological methods, including immunofluorescence microscopy (2), for the serological analysis of ruminal microorganisms has been investigated in several laboratories. The relationships between different morphological types of ruminal bacteria observed in vivo and in pure *
cultures in vitro have been established (7, 8), and two different species within the genus Ruminococcus have been identified serologically. Diurnal variation in ruminal numbers of cellulolytic bacteria has also been established by using fluorescent antisera (9). Polyclonal antisera and MAbs have been used to establish antigenic relationships between methanogenic archaebacteria (14), and MAbs raised against Butyrivibrio species have been investigated as potential probes for bacterial quantification in pure culture and ruminal samples. In the latter work, at least three different types of Butyrivibrio species were identified on the basis of differential reactivity. This result together with recent evidence of genetic diversity within an apparently homogeneous species, Selenomonas ruminantium (Zhang Ning, personal communication), invites questions about the validity of serologybased bacterial quantification procedures in which mixed populations of unknown proportions are present in crude ruminal samples. In this report, we describe the isolation of several MAbs against surface determinants on two different strains of S. ruminantium and investigate antibody specificity and quantitative cross-reactivity between these and other strains. The characterization of antigens by immunoblotting is described. MATERIALS AND METHODS
Materials. Brain heart infusion broth was obtained from Oxoid, multiwell polyvinyl microtiter ELISA plates were from Costar, goat anti-mouse immunoglobulin G (heavy plus light chain)-alkaline phosphatase conjugate was from Promega Biotec, anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate was from Sigma Chemical Co., and nitrocellulose membrane was from Schleicher & Schuell, Inc. Bacterial strains and culture conditions. Strains HD4 and MP72 of S. ruminantium were grown, anaerobically, in Hungate-type tubes containing either LMG medium (when the bacteria were to be used for immunization) or brain heart infusion broth. LMG medium was composed of 0.72% (wtlvol) DL-lactic acid (sodium salt), 0.2% mannitol, and 0.5% glycerol in a minimal broth (19). Cells were diluted in
Corresponding author. 2193
2194
BROOKER AND STOKES (a)
APPL. ENVIRON. MICROBIOL.
HD4
(b)
MP72
2.0
2.0E
r_
1 .5
LO) 0 a
1.0
a
.0 n0-o Q
0.5
0.0
1
2 3 4 5 6 7 8 910111213 Monoclonal antibodies
1 2 3 4 5 6 7 8 9 101 11213
Monoclonal antibodies
FIG. 1. ELISA screening of MAbs. Negative controls were prepared by using B. ruminicola (two strains), E. coli, M. elsdenii, and Streptococcus sp. Results are expressed as absorbance at 405 nm. (a) Strain HD4 cells; (b) strain MP72 cells.
phosphate-buffered saline (PBS), pH 7.0, for immunizations and ELISA screening procedures. Preparation and screening of MAbs. Bacteria for immunization protocols were diluted in PBS to 108/ml and heated for 5 min at 90°C. Mice (BALB/c) were immunized initially with heat-killed bacterial suspensions in Freund complete adjuvant (15) and again 4 weeks later with bacteria suspended in Freund incomplete adjuvant. Serum antibody titers were measured after a further 2 weeks, and seropositive animals were sacrificed for the isolation of spleen cells. MAbs were prepared by standard procedures (11) by using a mouse myeloma cell line, P3-NS1/1-Ag4-1. For screening, Costar flexible vinyl multiwell ELISA plates were coated with 0.1 ml of bacterial suspensions at a density of 108 cells per ml and assayed by using standard procedures (7). Negative controls included myeloma cell supernatant, without bacteria and without antibody reactions. MAb-containing culture samples were diluted fourfold and assayed by ELISA by using alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (heavy plus light chain) as the second antibody. Assay results were read in a Titertek ELISA plate reader at 405 nm. Ascites preparations of MAbs were obtained by intraperitoneal injection of 108 hybridoma cells into BALB/c mice that had been primed for 10 days with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Sigma). Immunoblotting. Cells (10-ml cultures) were centrifuged and washed with PBS. Cell pellets were freeze-dried and then ruptured by grinding with a small glass pestle in the presence of acid-washed sand. Cell wall extracts were obtained by suspending the ruptured cell pellet in 2 ml of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5, followed by treatment with 5 ig each of DNase I (Sigma) and pancreatic RNase (Sigma). The extract was centrifuged at 10,000 x g for 1 min, and the supernatant was centrifuged at 100,000 x g for 60 min. The pelleted material was suspended in 50 mM Tris-1 mM EDTA-1% sodium dodecyl sulfate, and protein content was determined by using Bradfords reagent (5). Samples containing up to 10 pLg of protein were fractionated (12) and immunoblotted by following a standard procedure (22). When periodic acid treatment (16) of gels was required, nitrocellulose membranes were washed in transfer buffer following the normal blocking step and then soaked in 20% (wt/vol) periodic acid solution in water for 10 min. The membranes were washed three times in transfer buffer to neutralize the periodic acid, followed by normal immunoblot development. Silver staining of gels was by the method of Morrisey (17).
Immunofluorescence microscopy. Bacterial cultures were centrifuged, and the cells were washed and suspended at 108/ml in PBS. A portion (100 ,ul) of the bacterial suspension was incubated with a one-half dilution of MAb, followed by anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate (Sigma). After being washed, cells were suspended in PBS and spread on slides for oil immersion fluorescence microscopy (2). Untreated cells were examined under bright-field phase-contrast microscopy. RESULTS
Specificity of MAbs. MAbs were prepared against S. ruminantium HD4 and purified by at least two cloning steps; these antibodies reacted strongly with intact S. ruminantium HD4 cells in an ELISA (Fig. la) but did not react significantly (an absorbance value less than 0.25) with control bacterial species tested, including Bacteroides ruminicola (two strains), Escherichia coli, Megasphaera elsdenii, and Streptococcus sp. Several MAbs appeared to be strain specific, reacting only with S. ruminantium HD4 and not with strain MP72; other MAbs from this preparation reacted with both strains (Fig. lb). One of the strain-specific MAbs, MAb2, was tested by immunofluorescence microscopy by using pure cultures of S. ruminantium, strains HD4 and MP72; E. coli and B. ruminicola were used as nonspecific controls. Strain HD4 cells were strongly fluorescent after reaction with MAb2 and fluorescein isothiocyanate-conjugated second antibody (Fig. 2), but there was little or no reaction above autofluorescence background with strain MP72, E. coli, or B. ruminicola. In a separate experiment, MAbs were also prepared against S. ruminantium MP72. One of these antibodies, MAbl4, was tested for its reactivity with three strains of S. ruminantium; E. coli and B. ruminicola were used as controls. The results (Fig. 3) show that although MAb14 was Selenomonas species specific, its reaction with different strains was not identical. Strain M7 reacted 50% less than strain MP72. This was not due to differences in attachment of cells to the microtiter plate because, when the same number of cells (107) was added to a test plate and stained with Coomassie blue and the absorbance was measured at 620 nm as described by Hazlewood et al. (7), there was no difference between cells of strains M7 and MP72 (result not shown). More likely, the effect was probably due to differences in the stage of growth of the cultures, because experiments to examine the effect of bacterial growth on antibody reactivity (Table 1) showed that with 107 cells per well, at
-St'Lf-sf>asWwi\,v*;'%p_Li_.:
0099-2240/90/072193-07$02.00/0
JUlY 1990, p. 2193-2199
Vol. 56, No. 7
Copyright © 1990, American Society for Microbiology
Monoclonal Antibodies against the Ruminal Bacterium Selenomonas ruminantium JOHN D. BROOKER* AND BARBARA STOKES Department of Animal Sciences, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064, Australia Received 5 January 1990/Accepted 21 April 1990
Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 107 cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.
Serological methods for the identification and quantification of microorganisms are well established, particularly for the study of medically important organisms. Both polyclonal antibodies and monoclonal antibodies (MAbs) that react with surface antigens on live organisms have been used in quantitative solid-phase enzyme-linked immunosorbent assay (ELISA) procedures and for studies of cell surface structures (6). With the advantages of sensitivity and specificity, immunoassays form the basis of many bacterial identification procedures and microbial population studies. These methods may also have applications in the study of natural microbial ecosystems that exist, for instance, in the rumen, where mixed populations of bacteria interact in a complex and continuously changing environment (4). The characterization of ruminal microbial isolates may also be facilitated by MAb reagants. The characterization and classification of ruminal bacterial species have been based largely on morphologic and metabolic descriptions (4). However, the accuracy of these procedures has recently been questioned because species assignment based on these parameters differed from that suggested by genotype analyses through DNA sequencing (21). Unambiguous genotyping through DNA-DNA hybridization, DNA fingerprinting, and nucleic acid sequencing has also been responsible for new insights into phylogenetic relationships between strains within apparently homogeneous species (21). However, although exquisitely specific, nucleic acid-based procedures may not necessarily be the most appropriate for rapid quantification of bacterial populations or for screening a range of unknown isolates for identification purposes. Difficulties with plant contamination of ruminal samples and restricted sensitivity of unamplified gene probes (1) suggest that a serological approach may be more appropriate. The potential of immunological methods, including immunofluorescence microscopy (2), for the serological analysis of ruminal microorganisms has been investigated in several laboratories. The relationships between different morphological types of ruminal bacteria observed in vivo and in pure *
cultures in vitro have been established (7, 8), and two different species within the genus Ruminococcus have been identified serologically. Diurnal variation in ruminal numbers of cellulolytic bacteria has also been established by using fluorescent antisera (9). Polyclonal antisera and MAbs have been used to establish antigenic relationships between methanogenic archaebacteria (14), and MAbs raised against Butyrivibrio species have been investigated as potential probes for bacterial quantification in pure culture and ruminal samples. In the latter work, at least three different types of Butyrivibrio species were identified on the basis of differential reactivity. This result together with recent evidence of genetic diversity within an apparently homogeneous species, Selenomonas ruminantium (Zhang Ning, personal communication), invites questions about the validity of serologybased bacterial quantification procedures in which mixed populations of unknown proportions are present in crude ruminal samples. In this report, we describe the isolation of several MAbs against surface determinants on two different strains of S. ruminantium and investigate antibody specificity and quantitative cross-reactivity between these and other strains. The characterization of antigens by immunoblotting is described. MATERIALS AND METHODS
Materials. Brain heart infusion broth was obtained from Oxoid, multiwell polyvinyl microtiter ELISA plates were from Costar, goat anti-mouse immunoglobulin G (heavy plus light chain)-alkaline phosphatase conjugate was from Promega Biotec, anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate was from Sigma Chemical Co., and nitrocellulose membrane was from Schleicher & Schuell, Inc. Bacterial strains and culture conditions. Strains HD4 and MP72 of S. ruminantium were grown, anaerobically, in Hungate-type tubes containing either LMG medium (when the bacteria were to be used for immunization) or brain heart infusion broth. LMG medium was composed of 0.72% (wtlvol) DL-lactic acid (sodium salt), 0.2% mannitol, and 0.5% glycerol in a minimal broth (19). Cells were diluted in
Corresponding author. 2193
2194
BROOKER AND STOKES (a)
APPL. ENVIRON. MICROBIOL.
HD4
(b)
MP72
2.0
2.0E
r_
1 .5
LO) 0 a
1.0
a
.0 n0-o Q
0.5
0.0
1
2 3 4 5 6 7 8 910111213 Monoclonal antibodies
1 2 3 4 5 6 7 8 9 101 11213
Monoclonal antibodies
FIG. 1. ELISA screening of MAbs. Negative controls were prepared by using B. ruminicola (two strains), E. coli, M. elsdenii, and Streptococcus sp. Results are expressed as absorbance at 405 nm. (a) Strain HD4 cells; (b) strain MP72 cells.
phosphate-buffered saline (PBS), pH 7.0, for immunizations and ELISA screening procedures. Preparation and screening of MAbs. Bacteria for immunization protocols were diluted in PBS to 108/ml and heated for 5 min at 90°C. Mice (BALB/c) were immunized initially with heat-killed bacterial suspensions in Freund complete adjuvant (15) and again 4 weeks later with bacteria suspended in Freund incomplete adjuvant. Serum antibody titers were measured after a further 2 weeks, and seropositive animals were sacrificed for the isolation of spleen cells. MAbs were prepared by standard procedures (11) by using a mouse myeloma cell line, P3-NS1/1-Ag4-1. For screening, Costar flexible vinyl multiwell ELISA plates were coated with 0.1 ml of bacterial suspensions at a density of 108 cells per ml and assayed by using standard procedures (7). Negative controls included myeloma cell supernatant, without bacteria and without antibody reactions. MAb-containing culture samples were diluted fourfold and assayed by ELISA by using alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (heavy plus light chain) as the second antibody. Assay results were read in a Titertek ELISA plate reader at 405 nm. Ascites preparations of MAbs were obtained by intraperitoneal injection of 108 hybridoma cells into BALB/c mice that had been primed for 10 days with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Sigma). Immunoblotting. Cells (10-ml cultures) were centrifuged and washed with PBS. Cell pellets were freeze-dried and then ruptured by grinding with a small glass pestle in the presence of acid-washed sand. Cell wall extracts were obtained by suspending the ruptured cell pellet in 2 ml of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5, followed by treatment with 5 ig each of DNase I (Sigma) and pancreatic RNase (Sigma). The extract was centrifuged at 10,000 x g for 1 min, and the supernatant was centrifuged at 100,000 x g for 60 min. The pelleted material was suspended in 50 mM Tris-1 mM EDTA-1% sodium dodecyl sulfate, and protein content was determined by using Bradfords reagent (5). Samples containing up to 10 pLg of protein were fractionated (12) and immunoblotted by following a standard procedure (22). When periodic acid treatment (16) of gels was required, nitrocellulose membranes were washed in transfer buffer following the normal blocking step and then soaked in 20% (wt/vol) periodic acid solution in water for 10 min. The membranes were washed three times in transfer buffer to neutralize the periodic acid, followed by normal immunoblot development. Silver staining of gels was by the method of Morrisey (17).
Immunofluorescence microscopy. Bacterial cultures were centrifuged, and the cells were washed and suspended at 108/ml in PBS. A portion (100 ,ul) of the bacterial suspension was incubated with a one-half dilution of MAb, followed by anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate (Sigma). After being washed, cells were suspended in PBS and spread on slides for oil immersion fluorescence microscopy (2). Untreated cells were examined under bright-field phase-contrast microscopy. RESULTS
Specificity of MAbs. MAbs were prepared against S. ruminantium HD4 and purified by at least two cloning steps; these antibodies reacted strongly with intact S. ruminantium HD4 cells in an ELISA (Fig. la) but did not react significantly (an absorbance value less than 0.25) with control bacterial species tested, including Bacteroides ruminicola (two strains), Escherichia coli, Megasphaera elsdenii, and Streptococcus sp. Several MAbs appeared to be strain specific, reacting only with S. ruminantium HD4 and not with strain MP72; other MAbs from this preparation reacted with both strains (Fig. lb). One of the strain-specific MAbs, MAb2, was tested by immunofluorescence microscopy by using pure cultures of S. ruminantium, strains HD4 and MP72; E. coli and B. ruminicola were used as nonspecific controls. Strain HD4 cells were strongly fluorescent after reaction with MAb2 and fluorescein isothiocyanate-conjugated second antibody (Fig. 2), but there was little or no reaction above autofluorescence background with strain MP72, E. coli, or B. ruminicola. In a separate experiment, MAbs were also prepared against S. ruminantium MP72. One of these antibodies, MAbl4, was tested for its reactivity with three strains of S. ruminantium; E. coli and B. ruminicola were used as controls. The results (Fig. 3) show that although MAb14 was Selenomonas species specific, its reaction with different strains was not identical. Strain M7 reacted 50% less than strain MP72. This was not due to differences in attachment of cells to the microtiter plate because, when the same number of cells (107) was added to a test plate and stained with Coomassie blue and the absorbance was measured at 620 nm as described by Hazlewood et al. (7), there was no difference between cells of strains M7 and MP72 (result not shown). More likely, the effect was probably due to differences in the stage of growth of the cultures, because experiments to examine the effect of bacterial growth on antibody reactivity (Table 1) showed that with 107 cells per well, at
-St'Lf-sf>asWwi\,v*;'%p_Li_.:
