APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, June 1992, p. 1924-1929
Vol. 58, No. 6
0099-2240/92/061924-06$02.00/0 Copyright X3 1992, American Society for Microbiology
Monoclonal Antibody Specific for Listeria monocytogenes Associated with a 66-Kilodalton Cell Surface Antigent ARUN K. BHUNIA* AND MICHAEL G. JOHNSON
Department of Food Science and University of Arkansas Biotechnology Center, University of Arkansas, 272 Young Avenue, Fayetteville, Arkansas 72703 Received 25 October 1991/Accepted 6 April 1992
A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass GI antibody with K light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzymelinked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pl value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pl values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.
Among the species of the genus Listeria, Listeria monocytogenes is known to be pathogenic for humans. L. monocytogenes outbreaks due to contaminated foods emphasize the need to develop a rapid detection system (13, 30). Several culture and biochemical methods have been developed and used successfully to recover and confirm L. monocytogenes (10, 23, 25). However, culture methods are a lengthy process requiring 2 to 5 days or more. Recently, we have reported a microcolony immunoblot method to detect and enumerate very low numbers (8 to 10 CFU/g or CFU/ml) of L. monocytogenes in foods in 20 to 24 h (4). Specific DNA/RNA probes (11, 20, 34) and DNA amplification with polymerase chain reaction (2) for detection of L. monocytogenes are very promising. Enzyme immunoassays based on several monoclonal antibodies (MAbs) have been developed in an effort to detect L. monocytogenes in food and clinical samples (3, 13, 14, 26, 27). However, the MAbs reported so far are not specific for L. monocytogenes. These MAbs either react with all species of Listeria (8, 14, 24, 26, 35) or react with L. monocytogenes but show cross-reactions with L. welshimeri and L. innocua (32). Recently, we reported a MAb, CllE9, which reacts with L. monocytogenes and cross-reacts only with L. innocua (3). While these antibodies may be very useful for detecting Listeria spp. in food for quality assurance of the product, they cannot specifically identify or confirm the presence of L. monocytogenes in foods in which other nonpathogenic Listeria species are also present and possibly predominant. Therefore, there is a great deal of interest in developing MAbs which are specific for L. monocytogenes. The main objective of this study was to develop a MAb specific for L. monocytogenes. A secondary objective was to develop a MAb that would react with a stably expressed cell antigen present on the surfaces of L. monocytogenes cells. We describe herein an approach to the successful devel*
opment and characterization of a MAb, EM-7G1, specific for an L. monocytogenes cell surface protein. In addition, a MAb specific for all Listeria spp., EM-6E11, which reacted with two other cell antigens of the genus Listeria was partially characterized. MATERUILS AND METHODS Cultures and media. Listeria species and other grampositive and gram-negative cultures were maintained in tryptic soy agar slants containing 0.5% yeast extract (TSAYE; Difco Laboratories). The fresh cultures for all the experiments were obtained by inoculating slant cultures into tryptic soy broth containing 0.5% yeast extract (TSB-YE) at 37°C for 16 to 18 h. The heat-killed protein A-negative Staphylococcus aureus strain (SANSORBIN) was obtained from Calbiochem Co., La Jolla, Calif. L. monocytogenes Pedr is a pediocin AcH-resistant isolate (6). L. monocytogenes transposon mutants Tn590, Tn476, TnS85, TnS43, Tn104035, Tn571, Tn433, Tn434, Tn534, Tn570, and Tn524 with defective hemolysin genes were obtained from R. Buchanan, U.S. Department of Agriculture, Agriculture Research Service, Philadelphia, Pa. (7). Preparation of Listeria surface proteins. The crude cell surface proteins (CCSP) of Listeria species were extracted with 4 M guanidine-HCl, pH 7.0, as described by Bhunia et al. (3). The protein extracts were dialyzed extensively against deionized water and lyophilized for future use. Protein concentrations were determined by using the BioRad protein assay (Bio-Rad Laboratories, Richmond, Calif.). Peptide map analysis of the 66-kDa protein. The 66-kDa protein bands of L. monocytogenes and L. innocua CCSP were collected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide) (SDS-PAGE) (33), and peptide mapping by limited proteolysis was performed as described by Hames (16). After electrophoresis, the gel was stained briefly (about 15 min) in 0.1% Coomassie blue R 250 and destained. The 66-kDa protein bands were identified and excised with a razor blade and equilibrated with 10 ml of
Corresponding author.
t Published with the approval of the director of the Arkansas
Agricultural Experiment Station.
1924
VC)L. 58, 1992
Tris-SDS buffer (0.125 M Tris-HCI, 0.1% SDS, and 20% glycerol [PH 6.8]) for 30 min. The gel slices were then placed carefully into the wells of a second SDS-12% PAGE gel and overlaid with 20 RI of Tris-SDS buffer containing 0.32 pg of y-chymotrypsin (Sigma) and 0.001% bromophenol blue. The electrophoresis (10 mA) was continued until the tracking dye had traversed about two-thirds of the stacking gel and was discontinued for 20 min to allow partial enzymatic digestion. The electrophoresis was then continued at 35 mA. The gel was then either stained with Coomassie blue or transblotted to an Immobilon P membrane (Millipore Corp., Bedford, Mass.) and immunoprobed with MAb C11E9 (3, 5). Antigen preparation and mouse immunization. L. monocytogenes V7 cells were grown and heat killed (80°C for 20 min) as previously described (3). For each immunization, 0.5 ml of the cell suspension was reacted at 37°C for 1 h with 0.05 ml of purified MAb C11E9 (immunoglobulin G2b [IgG2b]) as described by Bhunia et al. (3). The cell suspension was centrifuged (8,000 x g for 4 min), washed to remove unbound antibodies, resuspended with 0.5 ml of PBS, and injected intraperitoneally into two six-week-old BALB/c mice (0.25 ml per mouse). The immunization was continued for 6 weeks with one injection per week. At least 6 days before splenocytes were harvested for fusion, the animals were immunized daily with the same antigen. Hybridoma production. The hybridoma cells were produced by using equal numbers of murine myeloma P3/Nsl/ 1-Ag4-1 (NS1) cells and spleen cells from immunized mice (21). Selected hybridoma cultures were cloned by limiting dilution, and selected clones were inoculated intraperitoneally into pristane-primed IRCF-1 mice (Simonsen Lab, Inc., Gilroy, Calif.) for ascites fluid production as described before (3, 32). MAb purification and isotyping. The MAb in ascites fluid was partially purified by ammonium sulfate precipitation (19). Immunoglobulin isotyping was accomplished in microtiter plates which had been previously coated with heatkilled L. monocytogenes V7 cells. After the addition of MAb (1:2,000) to the plate, immunoglobulin subclass and heavyand light-chain types were determined with the Bio-Rad Mouse Typer. ELISA. Antibody activity in hybridoma culture supernatants was tested by enzyme-linked immunosorbent assay (ELISA) with phosphate-buffered saline (PBS)-washed, heat-killed cells of L. monocytogenes V7 (108 cells per well) in 0.05 M carbonate coating buffer, pH 9.6, to coat microtiter plates for 12 to 14 h at 4°C. ELISAs were run and read as previously described (3). In a separate ELISA experiment, microtiter plates were coated with PBS-washed live cells of Listeria spp. and other gram-positive and gram-negative organisms (approximately 108 cells per well). The partially purified MAb, EM-7G1 or EM-6E11 (1:2,000 dilution), was reacted with bacterial cells (100 RI per well), and the antibody reaction was detected by using peroxidase-conjugated secondary antibody and substrate as mentioned before (3). Dot and colony blotting. The reactivities of MAbs EM-7G1 and EM-6E11 against different Listeria spp. and other grampositive and gram-negative organisms were tested in dot and colony immunoblots (3). Briefly, the test organisms were either adsorbed onto Immobilon P membranes (Millipore Corp.) with a dot blot manifold apparatus (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) or stabbed onto TSA-YE plates with an inoculation needle before being transferred onto Immobilon P membrane. The membranes containing cells were air dried and soaked in 30 ml of
L. MONOCYTOGENES-SPECIFIC MAb
1925
methanol containing H,02 (1%) for 30 min at 25°C to remove endogenous bacterial peroxidases (18). The membranes were blocked with 2% bovine serum albumin for 1 h and probed with MAb EM-7G1 (1:2,000 dilution) for 1 h. The blots were washed three times for 15-min periods with PBS-Tween and reacted with goat anti-mouse peroxidaseconjugated IgG (heavy and light chain) for 1 h. The blots were washed again as described above and reacted with 20 ml of substrate solution containing 10 mg of 3,3'-diaminobenzidine tetrahydrochloride, 20 ml of 0.05 M Tris (pH 7.6), 1 ml of 8% nickel chloride, and 0.1 ml of 30% H202 (Sigma). The color reaction was stopped by washing the membranes in water. PAGE and immunoblotting. Lyophilized CCSP samples from Listeria spp. were analyzed by SDS-PAGE (12% polyacrylamide) by the procedure described by Smith (33). A 2-mg amount of CCSP from each culture was dissolved in 1 ml of sample solvent (5) and loaded onto each of several wells for SDS-12% PAGE. At the end of electrophoresis (20 mA, 3.5 h), the gel was either stained with Coomassie blue
R250 or transblotted to Immobilon P membrane with the Bio-Rad transblot apparatus. The blotted membranes were immunoprobed with either MAb EM-7G1 or MAb EM-6E11 as described for dot blotting with the modification that an alkaline phosphatase-conjugated IgG (heavy and light chain) was used as the secondary antibody. The substrate mixture contained 0.33 mg of nitroblue tetrazolium per ml and 0.165 mg of 5-bromo-4-chloro-3-indolylphosphate per ml of alkaline phosphatase buffer, pH 9.6 (100 mM Tris, 100 mM NaCl, 5 mM MgCl,). IEF and immunoblotting. The CCSP from L. monocytogenes cells were focused in an 8% acrylamide gel containing ampholyte (pH 3 to 10) according to the Pharmacia isoelectric focusing (IEF) manual (Pharmacia Fine Chemical, Uppsala, Sweden) for 3 h at 2 W (constant power) (22). The proteins from IEF gel were transblotted to Immobilon P membrane and immunoprobed with either MAb EM-7G1 or MAb EM-6E11. Surface proteolysis. Surface proteolysis of L. monocytogenes V7 and Scott A cells was performed as described by Olson et al. (28). Trypsin or protease (pronase E) at a concentration of 200 pg/ml (both from Sigma) was added to 100 ml of PBS-washed cells and incubated in shaker incubators at 37°C for 1 h. Trypsin and protease reactions were terminated by the addition of either trypsin inhibitor (200 ,ug/ml) or EDTA (10 mM/ml) (Sigma), and the cells were sedimented at 15,300 x g at 10°C, washed once with PBS, and resuspended with 100 ml of PBS. Aliquots (0.5 ml each) of each of the two enzyme treatments along with untreated control cells were tested by dot blotting. The remaining cell suspensions were subjected to protein extraction by the method described above, and the extracts were analyzed by SDS-12% PAGE and by immunoblotting with MAb EM-7G1 and compared with untreated protein extracts. The enzymetreated cells were also checked by phase-contrast microscopy for motility and cell lysis and also streaked on TSA-YE agar plates to assess viability. RESULTS ChymoPeptide map analysis and antigen preparation. from L. monocytotrypsin treatment of the 66-kDa proteins genes and L. innoclua generated several protein fragments and L. ranging from 20 to 66 kDa for both L. monocytogenes not blue Coomassie shown). innocua upon staining (data Immunoblotting with MAb C11E9 showed that most of the
APPL. ENVIRON. MICROBIOL.
BHUNIA AND JOHNSON
1926
seeligeri, L. welshimeri, L. grayi, L. murrayi, and L. ivanovii. MAbs EM-7G1 and EM-2C1 reacted only with L. monocytogenes, whereas MAbs EM-7H10 and EM-6E11 reacted with all the species of Listeria. Therefore, EM-7G1 and EM-6E11 were selected and cloned by limiting dilution, and the MAbs from the two clones described above were used for further studies. Partially purified MAbs EM-7G1 and EM-6E11 from ascites fluid contained about 12 and 15 mg of protein per ml and belonged to immunoglobulin subclasses Gl and G2b, respectively, with light chains (Table 1). ELISA and dot and colony blots. ELISA and dot and colony blotting tests of MAbs EM-7G1 and EM-6E11 against several Listeria spp. along with several other gram-positive and gram-negative organisms indicated that EM-7G1 reacted with all the strains of L. monocytogenes tested, of which 11 strains were human isolates (20), 11 were animal or food isolates (20, 31), 10 were transposon mutants (7), and 1 was a pediocin-resistant isolate (6). EM-7G1 did not react with six strains of L. innocua, with two strains of each L. welshimeri, or with L. seeligeri, L. ivanovii, L. murrayi, L. grayi (31, 32). Similarly, EM-7G1 did not react with any other gram-positive test organisms (namely, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Leuconostoc oenos) or with gram-negative bacteria (including Pseudomonas putrefacens, Pseudomonas fluorescens, Escherichia coli and Salmonella typhimurium). In contrast, EM-6E11 reacted with all the species of Listeria and did not show reaction with any gram-positive or gram-negative organisms mentioned above. However, both MAbs gave a weak nonspecific cross-reaction with S. aureus because of the presence of surface protein A, whereas a protein A-negative S. aureus strain (SANSORBIN) did not show any reaction. PAGE and immunoblotting. SDS-PAGE (12% acrylamide under reducing conditions) analysis of CCSP from L. monocytogenes V7, Scott A, F4262, F1057, or Tn543 and from L. innocua LA-1 cells indicated the presence of several protein bands ranging from 10 to 105 kDa upon Coomassie blue staining. Immunoblot results indicated that MAb EM-7G1 reacted with proteins of 66 kDa from all the tested strains of L. monocytogenes (Fig. 2B, lanes 1 to 5) but not with CCSP from L. innocua (Fig. 2B, lane 6) or other Listeria spp. (data not presented). In contrast, SDS-PAGE (under both reducing and nonreducing conditions) and immunoblotting with MAb EM-6E11 of CCSP from L. monocytogenes V7, L. innocua LA-1, L. welshimeri ATCC 35897, L. ivanovii KC1714, L. seeligeri LA-15, L. grayi ATCC 19120, and L. K
FIG. 1. Western blot of the 66-kDa protein from L. monocyto(lanes 1 and 2) and L. innocua (lanes 3 and 4) after limited proteolysis with chymotrypsin. The enzyme-digested protein fragments (lanes 2 and 4) along with untreated protein bands (lanes 1 and 3) were then probed with MAb C11E9. Several of the protein fragments from L. monocytogenes (lane 2) gave a strong reaction with C11E9. In contrast, the undigested 66-kDa protein from L. innocua (lane 4) gave a weak reaction. Lane M contained fast-greenstained molecular mass standards (MW-SDS-7; Sigma).
genes
protein fragments from the 66-kDa protein of L. monocytothe antibody (Fig. 1, lane 2), whereas fragments from the same protein of L. innocua reacted with MAb C11E9 and only the undigested 66-kDa protein band did react (Fig. 1, lane 4). This result indicated that there were differences in the spatial arrangement of the C11E9-reactive epitopes in the 66-kDa proteins of L. monocytogenes versus L. innocua. According to this model, it was possible to mask the epitopes which the 66-kDa proteins of both L. monocytogenes and L. innocua organisms have in common while leaving some epitopes, unique to L. monocytogenes, unreacted and therefore available to serve as immunogens. We tested this hypothesis by reacting MAb C11E9 with heat-killed L. monocytogenes cells and then injecting these treated cells into mice. MAbs. Several hybridomas were generated after two fusions. Five hybridomas were selected on the basis of their reaction with heat-killed cells of L. monocytogenes in ELISA. The MAbs from EM-7G1, 2C1, 7H10, 6E11, and 1A5 were further tested in ELISA against live cells of several strains of L. monocytogenes, L. innocua, L.
genes reacted with none of the protein
TABLE 1. Reactivities of MAbs from selected hybridoma clones with different Listeria spp. in ELISAa
Reactivity" with clone (immunoglobulin subclass')
Organism
L. L. L. L. L. L. L.
monocytogenes innocua
seeligeri welshimen grayi murrayi ivanovii a
b
c
EM-7G1 (IgGl)
EM-2C1 (IgG2a)
EM-7H10 (IgGl)
EM-6E11 (IgG2b)
EM-lA5 (IgGl)
9/9 0/6 0/2 0/2 0/2 0/1 0/1
9/9 0/6 0/2 0/2 0/2 0/1 0/1
9/9 6/6 2/2 2/2 1/1 1/1 1/1
9/9 6/6 2/2 2/2 1/1 1/1 1/1
9/9 4/6 0/2 2/2 0/1 0/1 1/1
ELISA values of .0.2 were considered positive, and values of
AND
ENVIRONMENTAL MICROBIOLOGY, June 1992, p. 1924-1929
Vol. 58, No. 6
0099-2240/92/061924-06$02.00/0 Copyright X3 1992, American Society for Microbiology
Monoclonal Antibody Specific for Listeria monocytogenes Associated with a 66-Kilodalton Cell Surface Antigent ARUN K. BHUNIA* AND MICHAEL G. JOHNSON
Department of Food Science and University of Arkansas Biotechnology Center, University of Arkansas, 272 Young Avenue, Fayetteville, Arkansas 72703 Received 25 October 1991/Accepted 6 April 1992
A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass GI antibody with K light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzymelinked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pl value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pl values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.
Among the species of the genus Listeria, Listeria monocytogenes is known to be pathogenic for humans. L. monocytogenes outbreaks due to contaminated foods emphasize the need to develop a rapid detection system (13, 30). Several culture and biochemical methods have been developed and used successfully to recover and confirm L. monocytogenes (10, 23, 25). However, culture methods are a lengthy process requiring 2 to 5 days or more. Recently, we have reported a microcolony immunoblot method to detect and enumerate very low numbers (8 to 10 CFU/g or CFU/ml) of L. monocytogenes in foods in 20 to 24 h (4). Specific DNA/RNA probes (11, 20, 34) and DNA amplification with polymerase chain reaction (2) for detection of L. monocytogenes are very promising. Enzyme immunoassays based on several monoclonal antibodies (MAbs) have been developed in an effort to detect L. monocytogenes in food and clinical samples (3, 13, 14, 26, 27). However, the MAbs reported so far are not specific for L. monocytogenes. These MAbs either react with all species of Listeria (8, 14, 24, 26, 35) or react with L. monocytogenes but show cross-reactions with L. welshimeri and L. innocua (32). Recently, we reported a MAb, CllE9, which reacts with L. monocytogenes and cross-reacts only with L. innocua (3). While these antibodies may be very useful for detecting Listeria spp. in food for quality assurance of the product, they cannot specifically identify or confirm the presence of L. monocytogenes in foods in which other nonpathogenic Listeria species are also present and possibly predominant. Therefore, there is a great deal of interest in developing MAbs which are specific for L. monocytogenes. The main objective of this study was to develop a MAb specific for L. monocytogenes. A secondary objective was to develop a MAb that would react with a stably expressed cell antigen present on the surfaces of L. monocytogenes cells. We describe herein an approach to the successful devel*
opment and characterization of a MAb, EM-7G1, specific for an L. monocytogenes cell surface protein. In addition, a MAb specific for all Listeria spp., EM-6E11, which reacted with two other cell antigens of the genus Listeria was partially characterized. MATERUILS AND METHODS Cultures and media. Listeria species and other grampositive and gram-negative cultures were maintained in tryptic soy agar slants containing 0.5% yeast extract (TSAYE; Difco Laboratories). The fresh cultures for all the experiments were obtained by inoculating slant cultures into tryptic soy broth containing 0.5% yeast extract (TSB-YE) at 37°C for 16 to 18 h. The heat-killed protein A-negative Staphylococcus aureus strain (SANSORBIN) was obtained from Calbiochem Co., La Jolla, Calif. L. monocytogenes Pedr is a pediocin AcH-resistant isolate (6). L. monocytogenes transposon mutants Tn590, Tn476, TnS85, TnS43, Tn104035, Tn571, Tn433, Tn434, Tn534, Tn570, and Tn524 with defective hemolysin genes were obtained from R. Buchanan, U.S. Department of Agriculture, Agriculture Research Service, Philadelphia, Pa. (7). Preparation of Listeria surface proteins. The crude cell surface proteins (CCSP) of Listeria species were extracted with 4 M guanidine-HCl, pH 7.0, as described by Bhunia et al. (3). The protein extracts were dialyzed extensively against deionized water and lyophilized for future use. Protein concentrations were determined by using the BioRad protein assay (Bio-Rad Laboratories, Richmond, Calif.). Peptide map analysis of the 66-kDa protein. The 66-kDa protein bands of L. monocytogenes and L. innocua CCSP were collected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide) (SDS-PAGE) (33), and peptide mapping by limited proteolysis was performed as described by Hames (16). After electrophoresis, the gel was stained briefly (about 15 min) in 0.1% Coomassie blue R 250 and destained. The 66-kDa protein bands were identified and excised with a razor blade and equilibrated with 10 ml of
Corresponding author.
t Published with the approval of the director of the Arkansas
Agricultural Experiment Station.
1924
VC)L. 58, 1992
Tris-SDS buffer (0.125 M Tris-HCI, 0.1% SDS, and 20% glycerol [PH 6.8]) for 30 min. The gel slices were then placed carefully into the wells of a second SDS-12% PAGE gel and overlaid with 20 RI of Tris-SDS buffer containing 0.32 pg of y-chymotrypsin (Sigma) and 0.001% bromophenol blue. The electrophoresis (10 mA) was continued until the tracking dye had traversed about two-thirds of the stacking gel and was discontinued for 20 min to allow partial enzymatic digestion. The electrophoresis was then continued at 35 mA. The gel was then either stained with Coomassie blue or transblotted to an Immobilon P membrane (Millipore Corp., Bedford, Mass.) and immunoprobed with MAb C11E9 (3, 5). Antigen preparation and mouse immunization. L. monocytogenes V7 cells were grown and heat killed (80°C for 20 min) as previously described (3). For each immunization, 0.5 ml of the cell suspension was reacted at 37°C for 1 h with 0.05 ml of purified MAb C11E9 (immunoglobulin G2b [IgG2b]) as described by Bhunia et al. (3). The cell suspension was centrifuged (8,000 x g for 4 min), washed to remove unbound antibodies, resuspended with 0.5 ml of PBS, and injected intraperitoneally into two six-week-old BALB/c mice (0.25 ml per mouse). The immunization was continued for 6 weeks with one injection per week. At least 6 days before splenocytes were harvested for fusion, the animals were immunized daily with the same antigen. Hybridoma production. The hybridoma cells were produced by using equal numbers of murine myeloma P3/Nsl/ 1-Ag4-1 (NS1) cells and spleen cells from immunized mice (21). Selected hybridoma cultures were cloned by limiting dilution, and selected clones were inoculated intraperitoneally into pristane-primed IRCF-1 mice (Simonsen Lab, Inc., Gilroy, Calif.) for ascites fluid production as described before (3, 32). MAb purification and isotyping. The MAb in ascites fluid was partially purified by ammonium sulfate precipitation (19). Immunoglobulin isotyping was accomplished in microtiter plates which had been previously coated with heatkilled L. monocytogenes V7 cells. After the addition of MAb (1:2,000) to the plate, immunoglobulin subclass and heavyand light-chain types were determined with the Bio-Rad Mouse Typer. ELISA. Antibody activity in hybridoma culture supernatants was tested by enzyme-linked immunosorbent assay (ELISA) with phosphate-buffered saline (PBS)-washed, heat-killed cells of L. monocytogenes V7 (108 cells per well) in 0.05 M carbonate coating buffer, pH 9.6, to coat microtiter plates for 12 to 14 h at 4°C. ELISAs were run and read as previously described (3). In a separate ELISA experiment, microtiter plates were coated with PBS-washed live cells of Listeria spp. and other gram-positive and gram-negative organisms (approximately 108 cells per well). The partially purified MAb, EM-7G1 or EM-6E11 (1:2,000 dilution), was reacted with bacterial cells (100 RI per well), and the antibody reaction was detected by using peroxidase-conjugated secondary antibody and substrate as mentioned before (3). Dot and colony blotting. The reactivities of MAbs EM-7G1 and EM-6E11 against different Listeria spp. and other grampositive and gram-negative organisms were tested in dot and colony immunoblots (3). Briefly, the test organisms were either adsorbed onto Immobilon P membranes (Millipore Corp.) with a dot blot manifold apparatus (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) or stabbed onto TSA-YE plates with an inoculation needle before being transferred onto Immobilon P membrane. The membranes containing cells were air dried and soaked in 30 ml of
L. MONOCYTOGENES-SPECIFIC MAb
1925
methanol containing H,02 (1%) for 30 min at 25°C to remove endogenous bacterial peroxidases (18). The membranes were blocked with 2% bovine serum albumin for 1 h and probed with MAb EM-7G1 (1:2,000 dilution) for 1 h. The blots were washed three times for 15-min periods with PBS-Tween and reacted with goat anti-mouse peroxidaseconjugated IgG (heavy and light chain) for 1 h. The blots were washed again as described above and reacted with 20 ml of substrate solution containing 10 mg of 3,3'-diaminobenzidine tetrahydrochloride, 20 ml of 0.05 M Tris (pH 7.6), 1 ml of 8% nickel chloride, and 0.1 ml of 30% H202 (Sigma). The color reaction was stopped by washing the membranes in water. PAGE and immunoblotting. Lyophilized CCSP samples from Listeria spp. were analyzed by SDS-PAGE (12% polyacrylamide) by the procedure described by Smith (33). A 2-mg amount of CCSP from each culture was dissolved in 1 ml of sample solvent (5) and loaded onto each of several wells for SDS-12% PAGE. At the end of electrophoresis (20 mA, 3.5 h), the gel was either stained with Coomassie blue
R250 or transblotted to Immobilon P membrane with the Bio-Rad transblot apparatus. The blotted membranes were immunoprobed with either MAb EM-7G1 or MAb EM-6E11 as described for dot blotting with the modification that an alkaline phosphatase-conjugated IgG (heavy and light chain) was used as the secondary antibody. The substrate mixture contained 0.33 mg of nitroblue tetrazolium per ml and 0.165 mg of 5-bromo-4-chloro-3-indolylphosphate per ml of alkaline phosphatase buffer, pH 9.6 (100 mM Tris, 100 mM NaCl, 5 mM MgCl,). IEF and immunoblotting. The CCSP from L. monocytogenes cells were focused in an 8% acrylamide gel containing ampholyte (pH 3 to 10) according to the Pharmacia isoelectric focusing (IEF) manual (Pharmacia Fine Chemical, Uppsala, Sweden) for 3 h at 2 W (constant power) (22). The proteins from IEF gel were transblotted to Immobilon P membrane and immunoprobed with either MAb EM-7G1 or MAb EM-6E11. Surface proteolysis. Surface proteolysis of L. monocytogenes V7 and Scott A cells was performed as described by Olson et al. (28). Trypsin or protease (pronase E) at a concentration of 200 pg/ml (both from Sigma) was added to 100 ml of PBS-washed cells and incubated in shaker incubators at 37°C for 1 h. Trypsin and protease reactions were terminated by the addition of either trypsin inhibitor (200 ,ug/ml) or EDTA (10 mM/ml) (Sigma), and the cells were sedimented at 15,300 x g at 10°C, washed once with PBS, and resuspended with 100 ml of PBS. Aliquots (0.5 ml each) of each of the two enzyme treatments along with untreated control cells were tested by dot blotting. The remaining cell suspensions were subjected to protein extraction by the method described above, and the extracts were analyzed by SDS-12% PAGE and by immunoblotting with MAb EM-7G1 and compared with untreated protein extracts. The enzymetreated cells were also checked by phase-contrast microscopy for motility and cell lysis and also streaked on TSA-YE agar plates to assess viability. RESULTS ChymoPeptide map analysis and antigen preparation. from L. monocytotrypsin treatment of the 66-kDa proteins genes and L. innoclua generated several protein fragments and L. ranging from 20 to 66 kDa for both L. monocytogenes not blue Coomassie shown). innocua upon staining (data Immunoblotting with MAb C11E9 showed that most of the
APPL. ENVIRON. MICROBIOL.
BHUNIA AND JOHNSON
1926
seeligeri, L. welshimeri, L. grayi, L. murrayi, and L. ivanovii. MAbs EM-7G1 and EM-2C1 reacted only with L. monocytogenes, whereas MAbs EM-7H10 and EM-6E11 reacted with all the species of Listeria. Therefore, EM-7G1 and EM-6E11 were selected and cloned by limiting dilution, and the MAbs from the two clones described above were used for further studies. Partially purified MAbs EM-7G1 and EM-6E11 from ascites fluid contained about 12 and 15 mg of protein per ml and belonged to immunoglobulin subclasses Gl and G2b, respectively, with light chains (Table 1). ELISA and dot and colony blots. ELISA and dot and colony blotting tests of MAbs EM-7G1 and EM-6E11 against several Listeria spp. along with several other gram-positive and gram-negative organisms indicated that EM-7G1 reacted with all the strains of L. monocytogenes tested, of which 11 strains were human isolates (20), 11 were animal or food isolates (20, 31), 10 were transposon mutants (7), and 1 was a pediocin-resistant isolate (6). EM-7G1 did not react with six strains of L. innocua, with two strains of each L. welshimeri, or with L. seeligeri, L. ivanovii, L. murrayi, L. grayi (31, 32). Similarly, EM-7G1 did not react with any other gram-positive test organisms (namely, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Leuconostoc oenos) or with gram-negative bacteria (including Pseudomonas putrefacens, Pseudomonas fluorescens, Escherichia coli and Salmonella typhimurium). In contrast, EM-6E11 reacted with all the species of Listeria and did not show reaction with any gram-positive or gram-negative organisms mentioned above. However, both MAbs gave a weak nonspecific cross-reaction with S. aureus because of the presence of surface protein A, whereas a protein A-negative S. aureus strain (SANSORBIN) did not show any reaction. PAGE and immunoblotting. SDS-PAGE (12% acrylamide under reducing conditions) analysis of CCSP from L. monocytogenes V7, Scott A, F4262, F1057, or Tn543 and from L. innocua LA-1 cells indicated the presence of several protein bands ranging from 10 to 105 kDa upon Coomassie blue staining. Immunoblot results indicated that MAb EM-7G1 reacted with proteins of 66 kDa from all the tested strains of L. monocytogenes (Fig. 2B, lanes 1 to 5) but not with CCSP from L. innocua (Fig. 2B, lane 6) or other Listeria spp. (data not presented). In contrast, SDS-PAGE (under both reducing and nonreducing conditions) and immunoblotting with MAb EM-6E11 of CCSP from L. monocytogenes V7, L. innocua LA-1, L. welshimeri ATCC 35897, L. ivanovii KC1714, L. seeligeri LA-15, L. grayi ATCC 19120, and L. K
FIG. 1. Western blot of the 66-kDa protein from L. monocyto(lanes 1 and 2) and L. innocua (lanes 3 and 4) after limited proteolysis with chymotrypsin. The enzyme-digested protein fragments (lanes 2 and 4) along with untreated protein bands (lanes 1 and 3) were then probed with MAb C11E9. Several of the protein fragments from L. monocytogenes (lane 2) gave a strong reaction with C11E9. In contrast, the undigested 66-kDa protein from L. innocua (lane 4) gave a weak reaction. Lane M contained fast-greenstained molecular mass standards (MW-SDS-7; Sigma).
genes
protein fragments from the 66-kDa protein of L. monocytothe antibody (Fig. 1, lane 2), whereas fragments from the same protein of L. innocua reacted with MAb C11E9 and only the undigested 66-kDa protein band did react (Fig. 1, lane 4). This result indicated that there were differences in the spatial arrangement of the C11E9-reactive epitopes in the 66-kDa proteins of L. monocytogenes versus L. innocua. According to this model, it was possible to mask the epitopes which the 66-kDa proteins of both L. monocytogenes and L. innocua organisms have in common while leaving some epitopes, unique to L. monocytogenes, unreacted and therefore available to serve as immunogens. We tested this hypothesis by reacting MAb C11E9 with heat-killed L. monocytogenes cells and then injecting these treated cells into mice. MAbs. Several hybridomas were generated after two fusions. Five hybridomas were selected on the basis of their reaction with heat-killed cells of L. monocytogenes in ELISA. The MAbs from EM-7G1, 2C1, 7H10, 6E11, and 1A5 were further tested in ELISA against live cells of several strains of L. monocytogenes, L. innocua, L.
genes reacted with none of the protein
TABLE 1. Reactivities of MAbs from selected hybridoma clones with different Listeria spp. in ELISAa
Reactivity" with clone (immunoglobulin subclass')
Organism
L. L. L. L. L. L. L.
monocytogenes innocua
seeligeri welshimen grayi murrayi ivanovii a
b
c
EM-7G1 (IgGl)
EM-2C1 (IgG2a)
EM-7H10 (IgGl)
EM-6E11 (IgG2b)
EM-lA5 (IgGl)
9/9 0/6 0/2 0/2 0/2 0/1 0/1
9/9 0/6 0/2 0/2 0/2 0/1 0/1
9/9 6/6 2/2 2/2 1/1 1/1 1/1
9/9 6/6 2/2 2/2 1/1 1/1 1/1
9/9 4/6 0/2 2/2 0/1 0/1 1/1
ELISA values of .0.2 were considered positive, and values of
