Vol. 174, No. 7
JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2178-2184 0021-9193/92/072178-07$02.00/0 Copyright ©) 1992, American Society for Microbiology
Monoclonal Antibodies as Probes To Examine Serotype-Specific and Cross-Reactive Epitopes of Lipopolysaccharides from Serotypes 02, 05, and 016 of Pseudomonas aeruginosa JOSEPH S. LAM,1,2* MICHELE Y. C. HANDELSMAN,'t TERESA R. CHIVERS,"12 AND LESLIE A. MACDONALD"12 Department of Microbiology' and Canadian Bacterial Diseases Networkl2 College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 11 November 1991/Accepted 21 January 1992
Serotypes 02, 05, and 016 of Pseudomonas aeruginosa are chemically related, and the 0 antigens of their
lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the 02, 05, and 016 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum 0-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks 0 antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the 0 repeat that is common to all three serotypes of the 02-05-016 group, while the 0-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more 0-antigen repeat units are present on the LPS molecule. Thus, the 0-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the 0 side chains.
the 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid residue. Serotype differences among strains in this group were believed to be caused by configurational variations in the diacetamidohexuronic acid and the glycosidic bond of N-acetyl fucosamine (15). In order to improve the efficiency of serotyping P. aeruginosa, monoclonal antibodies (MAbs) would be a logical alternative to the polyclonal antisera. Gaston et al. (7) and our laboratory (21, 22) have separately reported the production of MAbs against the IATS serotypes. Results from our laboratory have shown that the use of MAbs greatly reduced the number of crossreactions observed in clinical isolates of P. aeruginosa (20, 28). Interestingly, while 0-specific MAbs were successfully produced against each of the IATS serotypes, we were also able to isolate MAbs that cross-reacted only with strains of a chemically related group and not with other serotype strains. In this study, we report the use of several 0-specific and cross-reactive MAbs to examine common and serotypespecific LPS epitopes among the 02-05-016 serotype group. The use of these highly specific immunological probes will enable us to shed some light on what constitutes an LPS epitope.
Differences in the chemistry of the 0 antigens of lipopolysaccharide (LPS) among various strains form the basis of serotyping in Pseudomonas aeruginosa. In the International Antigenic Typing Scheme (IATS) proposed by Liu et al. (26), 17 standard serotypes were described, and in a recent paper by Liu and Wang (27), another 3 new serotypes were added to the scheme. Until recently, serotyping of P. aeruginosa was largely done by using rabbit antisera raised against each of the serotypes. The use of these typing sera has certain disadvantages, including variability from batch to batch and a high incidence of cross-reactivity. Even after extensive cross absorptions, cross-reactions among closely related serotypes could be observed (10, 32). Closely related groups in which cross-reactions could be observed include serotypes 02, 05, and 016; serotypes 07, 08, and 09; and serotypes 013 and 014. From the data reported by Knirel et al. (14) on the structural determination of 0 antigens of P. aeruginosa, it became apparent that these cross-reactions could be attributed to the similarities in the chemical structure of the 0-antigen repeats common to all strains of the same group. Among 02, 05, and 016 serotypes, the 0 antigens share a trisaccharide repeating unit which consists of two uronic acid derivatives and one N-acetyl fucosamine residue (Fig. 1). These three serotypes were designated by Lanyi and Bergan (25) as 02a,c, 02a,d, and 02a,b, respectively, where the group 0 factor, 02a, was represented by
MATERIALS AND METHODS Bacterial strains. P. aeruginosa serotype strains 02
(ATCC 33349), 05 (PA01 H103), and 016 (ATCC 33363) of the IATS have been described previously in Lam et al. (21, 22). Strains 170006 and Fisher 7 were two serotype 05 strains kindly provided by P. V. Liu, University of Louisville, Louisville, Ky. Strain AK1380, also described as strain PAO(D3) by Kuzio and Kropinski (19), was a PA01 strain
Corresponding author. as Michele Y. C. Lam. Present address: Department of Microbiology and Immunology, Health Science Centre, University of Western Ontario, London, Ontario, Canada N6A 3K7. *
t Formerly known
2178
MAb PROBES OF P. AERUGINOSA LPS
VOL. 174, 1992 PAO1 (serotype 05)
-4)-D-ManNAcAmA-(p1-4)-D-Man(NAc)2A-(Pl-3)-D-FucNAc-(al170006
-4)-DManNAcAmA-(Pl-4)-L-Gul(NAc)2A-(al 3-D-FucNAc-(alFisher 7
-4)-L-GulNAcAmA-(al-4)-D-Mn(NAc)2A-(Pl 3-DFucNAc-(alsertype 02
-4)-D-MaNAcAmA-(Pl1-4)-L-Gul(NAc)2A-(al-3)-D-FucNAc-(Pl1serotype 016
-4)-D-ManNAcAmA-(P1-4)-D-Man(NAc)2A-(p1-3)-D-FucNAc-(P1 AK1380
-4)-D-ManNAcAmA-(Pl-4)-D-Man(NAc)2A-(Pl-3)-FucNAc-(Pl
41OAc
FIG. 1. Chemical structures of the 0-antigen repeats of P. aeruginosa strains used in this study. Some of the unusual monosaccharides are as follows: D-ManNAcAmA, 3-acetamidino2-acetamido-2,3-deoxy-D-mannuronic acid; L-GulNAcAmA, 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid; D-Man(NAc)2A, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid; L-Gul(NAc)2A, 2,3-diacetamido-2,3-dideoxy-L-guluronic acid; D-FucNAc, 2-acetamido-2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine). (Adapted from Knirel et al. [14]).
that had undergone phage conversion and now possessed an altered 0 antigen which typed 016. Strain AK1401 contained core plus one 0 side chain (1), and strain AK44 contained complete core but was 0 antigen deficient (17). All P. aeruginosa strains were maintained on tryptic soy agar (Difco Laboratories, Detroit, Mich.). MAb production. The production of LPS-specific MAbs was done precisely as described by Lam et al. (21). MAb MF15-4 and MAb MF47-4, raised against 05 and 016 LPS, respectively, were previously reported (21, 22). We now report two new MAbs, MAb MF71-3 (02 specific) and MAb 18-19. MAb 18-19 was raised against 05 LPS and appeared to be cross-reactive with all strains belonging to the 02-05016 serogroup. LPS and 0-antigen preparation. LPS from all strains was isolated by the phenol-hot water method of Westphal and Jann (33). This LPS was subsequently used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. 0 antigen was prepared by hydrolysis in 1% (vol/vol) acetic acid for 1.5 h at 100°C by the method of Kropinski et al. (18). 0 antigen was further purified by column chromatography with a Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, N.J.) column (50 cm by 30 mm) and a pyridinium acetate buffer (pH 5.3). Column chromatography. The separation of LPS by gel filtration chromatography was done essentially as described by Lam et al. (24). Briefly, 25 to 30 mg of LPS was applied to a Sephadex G-200 superfine (Pharmacia) column (65 cm
2179
by 25 mm) and run at room temperature with a deoxycholate buffer system. Fractions (2.5 ml each) were collected at a flow rate of 3 ml/h and were assayed for the presence of amino sugars by the method of Gatt and Berman (8) and for 2-keto-3-deoxyoctulosonic acid by the method described by Osborn (29). LPS fractions were stored at room temperature until use. SDS-PAGE. LPS samples were analyzed in a discontinuous SDS-PAGE system using a 15% running gel essentially as described by Hancock et al. (9). LPS bands were visualized by the silver staining method of Dubray and Bezard (3). Western immunoblotting and immunodiffusion. The reactivity of the MAbs with purified LPS and chemically modified LPS was examined by Western immunoblotting by the method of Burnette (2) but with slight modifications. Nitrocellulose blots were blocked with 1.5% gelatin (Sigma Chemical Co., St. Louis, Mo.) and then incubated with hybridoma culture supernatant containing either an 0-specific MAb or the cross-reactive MAb. The blots were developed at room temperature with a goat anti-mouse F(ab')2 fragment antibody conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) and a substrate consisting of 30 mg of Nitro Blue Tetrazolium (Sigma) and 15 mg of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 100 ml of 0.1 M bicarbonate buffer (pH 9.8). Ouchterlony immunodiffusion was performed according to standard protocols using 1% SeaKem agarose (grade ME; FMC Bioproducts, Rockland, Maine) in the following barbital buffer (pH 8.2): 4.48 g of barbituric acid C IV (0.024 M) (Fisher Scientific Co., Ottawa, Canada), 8.86 g of Tris base (0.073 M) (Sigma), and 0.108 g of calcium lactate (0.00035 M) (Fisher). The pH was adjusted to 8.2 with 1 N NaOH. GelBond film (FMC Bioproducts) was used as the support surface for the agarose in an agarose/surface area ratio of 0.18 ml/cm2. Sample wells were filled with LPS or 0 antigen (8 to 10 ,ug) and the 0-specific MAbs (8 to 10 ,ul of undiluted or twofold-diluted ascitic fluid). Chemical modification of LPS. (i) Mild alkali hydrolysis. To examine the effect of deacetylation of lipid A fatty acids on antibody reactivity, 2 mg of LPS was hydrolyzed in 200 ,ul of 0.25 M NaOH for 1 h at 56°C and then neutralized with 200 ,ul of 0.25 M HCl (method described by M. Perry [29a]). Distilled water (600 ,ul) was added to yield a final solution containing LPS suspended in saline (0.1 M NaCI). (ii) Carboxyl reduction of uronic acids. Carboxyl groups on the uronic acid residues of the 0-antigen molecule were converted to the corresponding alcohol groups by derivatization with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma) followed by reduction with sodium borohydride according to the method of Taylor and Conrad (31). Briefly, 2 to 3 mg of each LPS sample was suspended in 3 ml of distilled water and reacted with 0.5 g of AG 5OW-X8 hydrogen-form ion-exchange resin (Bio-Rad Laboratories, Richmond, Calif.) to convert all carboxyl groups to the free acid. The samples were centrifuged at 10,000 x g for 2 min in an Eppendorf microfuge (Brinkmann Instruments Inc., Westbury, N.Y.), and the supematant was retained. Derivatization of the carboxyl group was performed by the addition of 2 mg of EDC. The samples were stirred at room temperature for 2 h while maintaining the pH at 4.8 with 0.1 N NaOH. Then, 1 ml of 2 M sodium borohydride was added, and the mixture was incubated overnight at 37°C. The reaction was stopped by adjusting the pH to 5.0 with 10% acetic acid. The samples were dialyzed against distilled water and lyophilized. HPLC analysis of LPS. The degree of LPS 0 acetylation in
2180
LAM ET AL.
J. BACTERIOL.
co 0 0
0
r
a
_
b
c
o
,-
.(
CD
0
08-o)
FIG. 2. Silver-stained SDS-PAGE gel of LPS from strains of the 02-05-016 group. Approximately 20 p.g of LPS was loaded per lane. Note that although LPS from these three groups are structurally similar, there are still subtle differences in the banding profiles among the standard 02, 05, and 016 serotype strains in terms of the locations of the HMW bands and the interband distances.
strains of P. aeruginosa was determined by measuring the amount of acetic acid released by mild base hydrolysis (0.02 M NaOH, 25°C, 16 h) and detected by high-pressure liquid chromatography (HPLC) analysis. Acetate quantitation by HPLC was performed on a Beckman system containing two model 110B pumps, a model 167 dual-channel rapid-scanning UV-visible-light detector, a model 406 analog interface, and an IBM-XT computer controller with Beckman Gold chromatography software and Bio-Rad HPLC column heater. Appropriate controls included incubation of LPS with phosphate-buffered saline (pH 6.7) instead of 0.02 M NaOH. No significant release of 0-acetyl groups in the controls was detected. Number of nanomoles of acetic acid released was determined by using the following formula: response factor (Ri) = nanomoles/area, where a 2 mM acetic acid standard was used to determine Rf.
RESULTS Numerous plasmacytoma fusion experiments were performed in order to isolate 0-specific and cross-reactive antibodies against LPS of each of the 02, 05, and 016 serotypes. Using an enzyme-linked immunosorbent assay with either formalin-fixed bacterial cells or purified LPS, 186, 298, and 151 clones of positive hybridomas for serotypes 02, 05, and 016, respectively, were identified. MAbs secreted from all of these hybridoma clones were further characterized by Western immunoblotting and were designated 0-specific antibodies when reactions occurred only between the MAb and its homologous LPS or were designated cross-reactive antibodies when reactions between the MAb and heterologous antigens were seen. The MAbs reported in this study are all of the immunoglobulin M isotype and are representative of the 0-specific or crossreactive reactions among the antibodies tested against the 02, 05, and 016 serotypes. Analysis of LPS from the 02, 05, and 016 serotypes by SDS-PAGE and silver staining revealed that there are differences in the banding patterns (Fig. 2). It appears, therefore,
0
co
9
t-
CD
0C) oo le v
o
o
JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2178-2184 0021-9193/92/072178-07$02.00/0 Copyright ©) 1992, American Society for Microbiology
Monoclonal Antibodies as Probes To Examine Serotype-Specific and Cross-Reactive Epitopes of Lipopolysaccharides from Serotypes 02, 05, and 016 of Pseudomonas aeruginosa JOSEPH S. LAM,1,2* MICHELE Y. C. HANDELSMAN,'t TERESA R. CHIVERS,"12 AND LESLIE A. MACDONALD"12 Department of Microbiology' and Canadian Bacterial Diseases Networkl2 College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 11 November 1991/Accepted 21 January 1992
Serotypes 02, 05, and 016 of Pseudomonas aeruginosa are chemically related, and the 0 antigens of their
lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the 02, 05, and 016 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum 0-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks 0 antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the 0 repeat that is common to all three serotypes of the 02-05-016 group, while the 0-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more 0-antigen repeat units are present on the LPS molecule. Thus, the 0-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the 0 side chains.
the 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid residue. Serotype differences among strains in this group were believed to be caused by configurational variations in the diacetamidohexuronic acid and the glycosidic bond of N-acetyl fucosamine (15). In order to improve the efficiency of serotyping P. aeruginosa, monoclonal antibodies (MAbs) would be a logical alternative to the polyclonal antisera. Gaston et al. (7) and our laboratory (21, 22) have separately reported the production of MAbs against the IATS serotypes. Results from our laboratory have shown that the use of MAbs greatly reduced the number of crossreactions observed in clinical isolates of P. aeruginosa (20, 28). Interestingly, while 0-specific MAbs were successfully produced against each of the IATS serotypes, we were also able to isolate MAbs that cross-reacted only with strains of a chemically related group and not with other serotype strains. In this study, we report the use of several 0-specific and cross-reactive MAbs to examine common and serotypespecific LPS epitopes among the 02-05-016 serotype group. The use of these highly specific immunological probes will enable us to shed some light on what constitutes an LPS epitope.
Differences in the chemistry of the 0 antigens of lipopolysaccharide (LPS) among various strains form the basis of serotyping in Pseudomonas aeruginosa. In the International Antigenic Typing Scheme (IATS) proposed by Liu et al. (26), 17 standard serotypes were described, and in a recent paper by Liu and Wang (27), another 3 new serotypes were added to the scheme. Until recently, serotyping of P. aeruginosa was largely done by using rabbit antisera raised against each of the serotypes. The use of these typing sera has certain disadvantages, including variability from batch to batch and a high incidence of cross-reactivity. Even after extensive cross absorptions, cross-reactions among closely related serotypes could be observed (10, 32). Closely related groups in which cross-reactions could be observed include serotypes 02, 05, and 016; serotypes 07, 08, and 09; and serotypes 013 and 014. From the data reported by Knirel et al. (14) on the structural determination of 0 antigens of P. aeruginosa, it became apparent that these cross-reactions could be attributed to the similarities in the chemical structure of the 0-antigen repeats common to all strains of the same group. Among 02, 05, and 016 serotypes, the 0 antigens share a trisaccharide repeating unit which consists of two uronic acid derivatives and one N-acetyl fucosamine residue (Fig. 1). These three serotypes were designated by Lanyi and Bergan (25) as 02a,c, 02a,d, and 02a,b, respectively, where the group 0 factor, 02a, was represented by
MATERIALS AND METHODS Bacterial strains. P. aeruginosa serotype strains 02
(ATCC 33349), 05 (PA01 H103), and 016 (ATCC 33363) of the IATS have been described previously in Lam et al. (21, 22). Strains 170006 and Fisher 7 were two serotype 05 strains kindly provided by P. V. Liu, University of Louisville, Louisville, Ky. Strain AK1380, also described as strain PAO(D3) by Kuzio and Kropinski (19), was a PA01 strain
Corresponding author. as Michele Y. C. Lam. Present address: Department of Microbiology and Immunology, Health Science Centre, University of Western Ontario, London, Ontario, Canada N6A 3K7. *
t Formerly known
2178
MAb PROBES OF P. AERUGINOSA LPS
VOL. 174, 1992 PAO1 (serotype 05)
-4)-D-ManNAcAmA-(p1-4)-D-Man(NAc)2A-(Pl-3)-D-FucNAc-(al170006
-4)-DManNAcAmA-(Pl-4)-L-Gul(NAc)2A-(al 3-D-FucNAc-(alFisher 7
-4)-L-GulNAcAmA-(al-4)-D-Mn(NAc)2A-(Pl 3-DFucNAc-(alsertype 02
-4)-D-MaNAcAmA-(Pl1-4)-L-Gul(NAc)2A-(al-3)-D-FucNAc-(Pl1serotype 016
-4)-D-ManNAcAmA-(P1-4)-D-Man(NAc)2A-(p1-3)-D-FucNAc-(P1 AK1380
-4)-D-ManNAcAmA-(Pl-4)-D-Man(NAc)2A-(Pl-3)-FucNAc-(Pl
41OAc
FIG. 1. Chemical structures of the 0-antigen repeats of P. aeruginosa strains used in this study. Some of the unusual monosaccharides are as follows: D-ManNAcAmA, 3-acetamidino2-acetamido-2,3-deoxy-D-mannuronic acid; L-GulNAcAmA, 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid; D-Man(NAc)2A, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid; L-Gul(NAc)2A, 2,3-diacetamido-2,3-dideoxy-L-guluronic acid; D-FucNAc, 2-acetamido-2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine). (Adapted from Knirel et al. [14]).
that had undergone phage conversion and now possessed an altered 0 antigen which typed 016. Strain AK1401 contained core plus one 0 side chain (1), and strain AK44 contained complete core but was 0 antigen deficient (17). All P. aeruginosa strains were maintained on tryptic soy agar (Difco Laboratories, Detroit, Mich.). MAb production. The production of LPS-specific MAbs was done precisely as described by Lam et al. (21). MAb MF15-4 and MAb MF47-4, raised against 05 and 016 LPS, respectively, were previously reported (21, 22). We now report two new MAbs, MAb MF71-3 (02 specific) and MAb 18-19. MAb 18-19 was raised against 05 LPS and appeared to be cross-reactive with all strains belonging to the 02-05016 serogroup. LPS and 0-antigen preparation. LPS from all strains was isolated by the phenol-hot water method of Westphal and Jann (33). This LPS was subsequently used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. 0 antigen was prepared by hydrolysis in 1% (vol/vol) acetic acid for 1.5 h at 100°C by the method of Kropinski et al. (18). 0 antigen was further purified by column chromatography with a Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, N.J.) column (50 cm by 30 mm) and a pyridinium acetate buffer (pH 5.3). Column chromatography. The separation of LPS by gel filtration chromatography was done essentially as described by Lam et al. (24). Briefly, 25 to 30 mg of LPS was applied to a Sephadex G-200 superfine (Pharmacia) column (65 cm
2179
by 25 mm) and run at room temperature with a deoxycholate buffer system. Fractions (2.5 ml each) were collected at a flow rate of 3 ml/h and were assayed for the presence of amino sugars by the method of Gatt and Berman (8) and for 2-keto-3-deoxyoctulosonic acid by the method described by Osborn (29). LPS fractions were stored at room temperature until use. SDS-PAGE. LPS samples were analyzed in a discontinuous SDS-PAGE system using a 15% running gel essentially as described by Hancock et al. (9). LPS bands were visualized by the silver staining method of Dubray and Bezard (3). Western immunoblotting and immunodiffusion. The reactivity of the MAbs with purified LPS and chemically modified LPS was examined by Western immunoblotting by the method of Burnette (2) but with slight modifications. Nitrocellulose blots were blocked with 1.5% gelatin (Sigma Chemical Co., St. Louis, Mo.) and then incubated with hybridoma culture supernatant containing either an 0-specific MAb or the cross-reactive MAb. The blots were developed at room temperature with a goat anti-mouse F(ab')2 fragment antibody conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) and a substrate consisting of 30 mg of Nitro Blue Tetrazolium (Sigma) and 15 mg of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 100 ml of 0.1 M bicarbonate buffer (pH 9.8). Ouchterlony immunodiffusion was performed according to standard protocols using 1% SeaKem agarose (grade ME; FMC Bioproducts, Rockland, Maine) in the following barbital buffer (pH 8.2): 4.48 g of barbituric acid C IV (0.024 M) (Fisher Scientific Co., Ottawa, Canada), 8.86 g of Tris base (0.073 M) (Sigma), and 0.108 g of calcium lactate (0.00035 M) (Fisher). The pH was adjusted to 8.2 with 1 N NaOH. GelBond film (FMC Bioproducts) was used as the support surface for the agarose in an agarose/surface area ratio of 0.18 ml/cm2. Sample wells were filled with LPS or 0 antigen (8 to 10 ,ug) and the 0-specific MAbs (8 to 10 ,ul of undiluted or twofold-diluted ascitic fluid). Chemical modification of LPS. (i) Mild alkali hydrolysis. To examine the effect of deacetylation of lipid A fatty acids on antibody reactivity, 2 mg of LPS was hydrolyzed in 200 ,ul of 0.25 M NaOH for 1 h at 56°C and then neutralized with 200 ,ul of 0.25 M HCl (method described by M. Perry [29a]). Distilled water (600 ,ul) was added to yield a final solution containing LPS suspended in saline (0.1 M NaCI). (ii) Carboxyl reduction of uronic acids. Carboxyl groups on the uronic acid residues of the 0-antigen molecule were converted to the corresponding alcohol groups by derivatization with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma) followed by reduction with sodium borohydride according to the method of Taylor and Conrad (31). Briefly, 2 to 3 mg of each LPS sample was suspended in 3 ml of distilled water and reacted with 0.5 g of AG 5OW-X8 hydrogen-form ion-exchange resin (Bio-Rad Laboratories, Richmond, Calif.) to convert all carboxyl groups to the free acid. The samples were centrifuged at 10,000 x g for 2 min in an Eppendorf microfuge (Brinkmann Instruments Inc., Westbury, N.Y.), and the supematant was retained. Derivatization of the carboxyl group was performed by the addition of 2 mg of EDC. The samples were stirred at room temperature for 2 h while maintaining the pH at 4.8 with 0.1 N NaOH. Then, 1 ml of 2 M sodium borohydride was added, and the mixture was incubated overnight at 37°C. The reaction was stopped by adjusting the pH to 5.0 with 10% acetic acid. The samples were dialyzed against distilled water and lyophilized. HPLC analysis of LPS. The degree of LPS 0 acetylation in
2180
LAM ET AL.
J. BACTERIOL.
co 0 0
0
r
a
_
b
c
o
,-
.(
CD
0
08-o)
FIG. 2. Silver-stained SDS-PAGE gel of LPS from strains of the 02-05-016 group. Approximately 20 p.g of LPS was loaded per lane. Note that although LPS from these three groups are structurally similar, there are still subtle differences in the banding profiles among the standard 02, 05, and 016 serotype strains in terms of the locations of the HMW bands and the interband distances.
strains of P. aeruginosa was determined by measuring the amount of acetic acid released by mild base hydrolysis (0.02 M NaOH, 25°C, 16 h) and detected by high-pressure liquid chromatography (HPLC) analysis. Acetate quantitation by HPLC was performed on a Beckman system containing two model 110B pumps, a model 167 dual-channel rapid-scanning UV-visible-light detector, a model 406 analog interface, and an IBM-XT computer controller with Beckman Gold chromatography software and Bio-Rad HPLC column heater. Appropriate controls included incubation of LPS with phosphate-buffered saline (pH 6.7) instead of 0.02 M NaOH. No significant release of 0-acetyl groups in the controls was detected. Number of nanomoles of acetic acid released was determined by using the following formula: response factor (Ri) = nanomoles/area, where a 2 mM acetic acid standard was used to determine Rf.
RESULTS Numerous plasmacytoma fusion experiments were performed in order to isolate 0-specific and cross-reactive antibodies against LPS of each of the 02, 05, and 016 serotypes. Using an enzyme-linked immunosorbent assay with either formalin-fixed bacterial cells or purified LPS, 186, 298, and 151 clones of positive hybridomas for serotypes 02, 05, and 016, respectively, were identified. MAbs secreted from all of these hybridoma clones were further characterized by Western immunoblotting and were designated 0-specific antibodies when reactions occurred only between the MAb and its homologous LPS or were designated cross-reactive antibodies when reactions between the MAb and heterologous antigens were seen. The MAbs reported in this study are all of the immunoglobulin M isotype and are representative of the 0-specific or crossreactive reactions among the antibodies tested against the 02, 05, and 016 serotypes. Analysis of LPS from the 02, 05, and 016 serotypes by SDS-PAGE and silver staining revealed that there are differences in the banding patterns (Fig. 2). It appears, therefore,
0
co
9
t-
CD
0C) oo le v
o
o
