Vol. 59, No. 2
INFECTION AND IMMUNITY, Feb. 1991, p. 712-715
0019-9567/91/020712-04$02.00/0 Copyright © 1991, American Society for Microbiology
Four Epitopes of Pseudomonas aeruginosa Elastase Defined by Monoclonal Antibodies JACQUELINE LAGACE* AND MANON FRECHETTE Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, P.O. 6128, Station A, Montreal, Quebec, Canada H3C 3J7 Received 12 July 1990/Accepted 7 November 1990
Monoclonal antibodies against the elastase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified elastase of P. aeruginosa and P3-X63-Ag8-U1 myeloma cells. The six clones established generated antibodies which reacted with a 33,000-Da peptide and recognized four different elastase epitopes by a competitive binding enzyme-linked immunosorbent assay. The monoclonal antibodies designated as ELA-17 and ELA-42 that recognize two different epitopes reacted by dot-enzyme immunoassay and by Western immunoblotting with all clinical and International Antigen Typing Scheme strains of P. aeruginosa positive for elastase.
Pseudomonas aeruginosa is a common opportunistic pathogen which can cause fatal infections in vulnerable hosts. It is the primary pulmonary pathogen in patients with cystic fibrosis (CF) and elicits significant morbidity in patients with thermal burns, cancer, and an immunosuppressed status (15). The pathogenesis of P. aeruginosa is multifactorial and involves a large number of extracellular products which may act as bacterial virulence factors (5, 11, 18). Among them, elastase, a Zn-metalloproteinase produced by most clinical strains of P. aeruginosa, has been largely characterized (12, 13). The elastase gene has been cloned and sequenced, and its mature protein product has been identified as a 32,926- to 33,000-Da polypeptide with a fully defined amino acid sequence (1, 2). Despite recent documentation of its complete primary structure (2, 7), little is known about the immunochemistry of elastase, and no specific epitopes have been identified or characterized in this protein molecule. To the best of our knowledge, no monoclonal antibodies (MAbs) that react with P. aeruginosa elastase have yet been identified. In the present study, we isolated and characterized six MAbs which react specifically with P. aeruginosa elastase. Using a competition assay, these MAbs were shown to react to four different epitopes on elastase. Antigenic conservation studied with the MAbs was demonstrated for two of these epitopes among all clinically testqd and representative strains of the International Antigen Typing Scheme (IATS) positive for P. aeruginosa elastase activity. P. aeruginosa PA103 was chosen for its absence of detectable elastase and P. aeruginosa 388 leu::Tnl was chosen because of its known capacity to secrete elastase (16). These strains were kindly provided by B. Iglewski and T. I. Nicas. A set of 17 serotype-specific representative strains of the IATS was obtained from E. Toma, Hotel-Dieu de Montreal (P. aeruginosa types 1 to 8, 10 to 12, and 14 to 16 as defined by a IATS kit commercially marketed by Difco Laboratories, Detroit, Mich.) and from the American Type Culture Collection: type 9 (ATCC 33356), type 13 (ATCC 33360), and type 17 (ATCC 33364). Clinical strains of P. aeruginosa were isolated from the sputum of CF patients of la Clinique de fibrose kystique de l'Hopital Sainte-Justine de *
Montrdal. Identification of the clinical P. aeruginosa strains was confirmed by culture on a selective medium for P. aeruginosa with phenanthroline (Sigma Chemical Co., St. Louis, Mo.); and 9-chloro-9-[4-(diethylamino)phenyl]-9,10dihydro-10-phenylacrinehydrochloride (C-390; kindly provided by Norwich Eaton Pharmaceuticals, Norwich, N.Y.) as described by Campbell et al. (4). P. aeruginosa strains were grown for 24 h at 37°C with constant agitation in medium containing- 0. 8% (wt/vol) tryptone, 0.5% (wt/vol) yeast extract, and 0.5% (wt/vol) NaCl. Following incubation, the cultures were centrifuged at 20,000 x g for 20 min at 4°C. Ammonium sulfate was added slowly to the culture supernatant to 60% saturation. After overnight incubation at 4°C, the material was centrifuged at 15,000 x g for 30 min to precipitate proteins. The pellet was dissolved in 1:20 or 1:80 of the initial volhme in 0.005 M Tris hydrochloride buffer (pH 8.0)-0.01 M NaCl and dialyzed against six changes of phosphate-buffered saline (PBS) (pH 7.2) over a 48-h period: All samples were stored at -70°C. BALB/c mice were immunized once a week for 4 weeks. For the first immunization, thXy were inoculated intraperitoneally with 10 ,ug of purified elastase (Nagase Biochemicals, Osaka, Japan) emulsified in an equal amount of complete Freund adjuvant (GIBCO Laboratories, Grand Island, N.Y.). For immunizations 2 through 4, the mice were given the immunogen intraperitoneally in incomplete Freund adjuvant. Serum titers of antibodies to elastase were determined by enzyme-linked immunosorbent assay (ELISA) (as outlined below) and were 1: 10,000 in the mice selected for use in the MAb isolation. For MAb production, the method described by l3rodeur et al. (3) was used with modifications. The spleens from selected mice were removed 3 days after the last injection for fusion with the mouse myeloma cell line P3-X63-Ag8-U1 (American Type Culture Collection) at a 10:1 ratib, employing 50% polyethylene glycol 1500 (Kodak) as a fusogen. RPMI 1649 medium (GIBCO) used for growth of the fused cells was supplemented with 20% fetal calf serum (GIBCO), 2 mM glutamine, 100 FM hypoxanthine, 1 ,M aminopterin, 16 ,uM thymidine, 50 ,ug of gentamicin per ml, and 104 normal m,use peritoneal cells per 0.2 ml. MAb titration was performed by ELISA. The hybridoma was subcloned three times by limiting dilution in 96-well plates (Nunc, GIBCO) in the same medium without aminopterin. MAbs were pro-
Corresponding author. 712
VOL. 59, 1991
duced in large quantities in 75-cm2 tissue culture flasks and in mouse ascitic fluid. MAbs in ascitic fluid were partially purified by precipitation with 50% ammonium sulfate. Hybridoma culture supernatants were obtained from cells transferred at a high concentration (4 x 106 per ml) in culture medium without fetal calf serum for 3 days and partially purified by precipitation with polyethylene glycol 6000 (Fisher, Montreal, Quebec, Canada) for 2 h at 4°C at a final concentration of 10% (wt/vol) (6). Purified MAbs were conjugated to biotin as described elsewhere (19). Briefly, purified MAbs (10 mg/ml) in 100 mM carbonate buffer (pH 8.5) were mixed with 0.1 volume of a fresh N,N-dimethylformamide solution (Fisher) in 6 mg of biotinyl-N-hydroxysuccinimide (Sigma) per ml. The mixture was allowed to react for 1 h at room temperature, dialyzed against PBS until free of biotin, and frozen at -70°C in aliquots. ELISA was performed as described previously (10), using a commercial preparation of purified elastase (Nagase Biochemicals) as a coating antigen at a final concentration of 2 ,ug/ml in 0.05 M sodium carbonate buffer (pH 9.6). The 96-well plates (Nunc, GIBCO) were incubated overnight at room temperature. Controls included unrelated MAb and preimmune and immune mouse sera. Preliminary studies have shown that the elastase enzyme used as a coating antigen did not manifest immunoglobulin G (IgG) proteolytic activity in our ELISA protocol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SI)S-PAGE) was performed under reducing conditions by the Laemmli method (9) in a minigel system (Bio-Rad Laboratories, Richmond, Calif.). Electrophoresis was conducted in 12% slab gels at 200 V for 50 min, using appropriate molecular weight markers (Bio-Rad Laboratories). Subs'equently, the gels were either stained with silver nitrate (Canlab, Toronto, Ontario, Canada) as described previously (14) or electrophoretically transferred to nitrocellulose by the method of Towbin et al. (20). Electrophoretic transfers onto nitrocellulose (Bio-Rad Laboratories) were performed at 100 V for 60 min. Immunospecific reactions were detected by autoradiography (8) or with avidin-peroxidase conjugates used in concert with biotinylated secondary antibodies according to the manufacturer's recommendations (Vector Laboratories, Burlingame, Calif.). To assess whether MAbs recognize the same epitopes, we performed a competitive binding assay using ELISA essentially as described above unless otherwise specified. Wells of polystyrene microdilution plates were coated with 100 RI of purified elastase at a concentration of 0.5 ,ug/ml as determined by preliminary assays with biotinylated MAb dilutions to optimize assay conditions. After the blocking step, 50 ,ul of MAb undiluted culture supernatant was added in triplicate to each well. The MAbs were allowed to bind to elastase for 30 min at room temperature. Then, 50 pul of a biotinylated MAb dilution per well was added. Plates were incubated for 1 h at 37°C. After washing, the amount of biotinylated MAb bound was measured by reaction with avidin-conjugated peroxidase as recommended by the manufacturer (Vector Laboratories). The A490 was measured with a Titertek Multiskan MC spectrometer. A dot-enzyme immunoassay was used for screening MAbs against 10 P. aeruginosa clinical strains isolated from the sputum of CF patients. Elastase (2 pI, produced as above) from each of the P. aeruginosa strains was applied to nitrocellulose paper and allowed to dry at room temperature. Immune specificity was revealed with biotinylated aintimouse IgG as previously described. Protein concentration was measured with a commercial
NOTES
713
100
no
60 60
40
20 FIG. 1. Competitive inhibition of the binding of biotin-conjugated MAb to elastase by various unlabeled MAbs (undiluted culture supernatants). Percent binding was calculated relative to the degree of binding of biotin-conjugated MAb in the presence of an unrelated MAb as a control. Symbols: 0, ELA-17; X, ELA-42; *, ELA-207; El, ELA-73; , ELA-45; U, ELA-56.
protein assay reagent (Bio-Rad Laboratories). The subisotype of MAbs was determined with a mouse subisotyping kit (Serotec, Oxford, United Kingdom) used as recommended by the manufacturer. Elastolytic activity was assayed by dye release from elastin-Congo red (17). Six hybridomas producing antibodies specific to elastase were isolated by a single fusion of P3-X63-Ag8-U1 cells and spleen cells from mice immunized with purified elastase. Positive clones were detected by ELISA with purified elastase as the antigen. These MAbs were designated ELA42, ELA-17, ELA-56, ELA-45, ELA-73, and ELA-207. The first three MAbs were of the IgGl subclass, and the latter three were of the IgG3 subclass. To test whether ELA-56, ELA-42, ELA-17, ELA-207, ELA-45, and ELA-73 recognize different epitopes, we performed a competitive binding assay using the ELISA. The results of these experiments are summarized in Fig. 1. Data are expressed as percent binding of biotin-conjugated MAb (b-cMAb) in the presence of an unlabeled and undiluted MAb culture supernatant; binding in the presence of the negative control (an unrelated MAb) was taken as 100%. Binding of the b-cMAb ELA-42 was inhibited only by its unlabeled homologous MAb; b-cMAb ELA-45 binding was inhibited by its unlabeled homologous MAb and by ELA-73; and b-cMAb ELA-17 binding was inhibited by its unlabeled homologous MAb and ELA-56 (Fig. 1). b-cMAb ELA-42, ELA-45, and ELA-17 binding in the presence of heterologous MAbs was in the range of 67 to 100% compared with 30 to 10% in the presence of homologous MAbs (Fig. 1). We concluded that the six MAbs recognized four different epitopes on elastase: one epitope was recognized by ELA42, a second by ELA-207, a third by ELA-45 and ELA-73, and a fourth by ELA-17 and ELA-56. The ability of our six different MAbs to recognize purified elastase and elastase from cell-free culture supernatant of P.
714
NOTES
INFECT. IMMUN. 2
a
66KD
a b
I
d
gh
t
a
aeruginosa 388 leu: :Tnl and of the negative control strain P. iaeruginosa PA103 was examined by reacting them with Western immunoblots. In Fig. 2a, purified elastase presents a single protein band of 33 kDa as stained by the Bio-Rad biotin-blot total protein assay. By immunodetection (Fig. 2a), the six MAbs reacted to the same purified elastase band of 33 kDa with different intensities and patterns not related
4
3 c
f,i
--
45KD --
36KD 29KD -_
mEa
a
to their concentration. The reactions to ELA-42 and ELA-17 antibodies were darker and larger than those to ELA-45 and ELA-207. This was confirmed by Western immunoblotting
3
24KD -
20KD -_
of the culture supernatant of strain 388 leu::Tnl, for which the same larger reactions to ELA 42 and ELA-17 were observed compared with ELA-45 and ELA-207 antibodies (Fig. 2b). No reaction was seen when the six MAbs were tested with Western immunoblots of culture supernatant of P. aeruginosa PA103 negative for elastase secretion (data
14KD _
66kd
3 ow
2
b
1
a
b
c
d
r-,-%
4
i>
g ;h
not
-
45kd 36kd 29 kd -_
,--.
m
a
24kd _
2Okd
shown).
The ability of the four different MAbs to react to concentrated cell-free culture supernatants of clinical and IATS P. aeruginosa strains was examined by dot-enzyme immunoassays or by reacting Western immunoblots of SDS-polyacrylamide gels of supernatants with these antibodies. ELA-42 and ELA-17 reacted by dot-enzyme assay with supernatants of the 10 clinical strains isolated from CF patients; ELA-207 and ELA-45 reacted, respectively, with five and four of them (Table 1). Eight supernatants of IATS strains (numbers 3 to 8, 14, and 16) reacted with ELA-45; ELA-42 and ELA-17 reacted with supernatants of 12 of 17 IATS strains examined by Western immunoblotting (Table 1). The five negative IATS strains by Western immunoblotting with the latter MAbs were 1, 9, 10, 15, and 17. The elastolytic activity of concentrated supernatants of negative strains by Western immunoblotting was tested on elastin-Congo red; a total lack of elastolytic activity was recorded with all these strains. Positive control strains showed an elastolytic activity corresponding to more than 1 mg of purified elastase. ELA-207
-e
14kd _
FIG. 2. All these MAbs (ELA-56 [lane b], ELA-42 [lane c], ELA-17 [lane d], ELA-207 [lane fi, ELA-45 [lan(e h], ELA-73 [lane i]) and an unrelated MAb (lanes a, e, and g) werre reacted with the 33-kDa purified elastase (a) and a 33-kDa proteiln band in supernatant from strain 388 leu::Tnl (b). KD, kd, Kilod,altons.
TABLE 1. Reactivity of MAbs specific for P. aeruginosa elastase, determined by dot-enzyme immunoassay and Western immunoblotting aginst concentrated cell-free culture supernatant from clinical and IATS P. aeruginosa strains
ELA42
PA103 (negative control) 388 leu::Tnl 1 2 3 4 5
6 7 8 9 10 11 12
IATS strain examined by
CF strain examined by dot-enzyme immunoassay'
P. aeruginosa straina
+ + +
+ + + + + + + +
immunoblotting'
ELA-17 + + + + + +
ELA-45 + +
ELA-207 + +
-
-
+ + + + +
+ _
+ -
+ + + -
ELA42 + _ + + + + + + +
BLA-17 + _ + + + + + + +
+ +
+ +
+
+ +
+
+
+
+
A3+
14 15 16 17 a The 10 clinical strains were isolated from sputum of CF patients. b Positive reactions were identified colorimetrically. Each strain was tested more than two times. c ELA-207 was not tested.
Wester ELA45 _ _ + + + + + +
NOTES
VOL. 59, 1991
was not tested against IATS strains because of the instability of this MAb with time. In this report, we described the first isolation and characterization of six MAbs directed against P. aeruginosa elastase. These MAbs can be divided in two groups based on their IgG subclasses, their pattern of immunoblotting reactions, their degree of affinity, and the predominance of their antigenic sites among P. aeruginosa strains. Thus, MAbs ELA-17, ELA-42, and ELA-56, all of the IgGl subclass, reacted more strongly in immunologic tests and presented a wider band in Western immunoblots and their antigenic sites were more common among P. aeruginosa strains compared with MAbs of the IgG3 subclass, ELA-45, ELA-73, and ELA-207. Because very strong reactions with MAbs ELA17, ELA-42, and ELA-56 were observed on Western immunoblots (denaturing conditions), epitopes bound by these MAbs are apparently sequence determined, whereas MAbs ELA-45, ELA-73, and ELA-207 possibly react with an epitope partially dependent on conformational arrangement since reactivity in Western immunoblots was decreased compared with that in ELISA. Moreover, these latter MAbs show a lower affinity to their epitope since whatever their concentration was, their measured reactivity was never as high as that for the three other MAbs (data not shown). An interesting finding in this study is that all clinical and IATS P. aeruginosa strains presenting elastase as demonstrated by SDS-PAGE and/or elastolytic activity shared the two same separate epitopes as revealed by their interaction with the MAbs ELA-17 and ELA-42 (Table 1). The pattern of conservation of the two other epitopes is less common since ELA-207 and ELA-45 reacted, respectively, with only 50 and 40% of the clinical strains tested and the latter reacted with 66% of the IATS strains. We must consider that this difference in epitope specificities of elastase among P. aeruginosa strains may also influence the structure and/or activity of the elastase molecule secreted by the different groups of P. aeruginosa strains. We thank Barbara Iglewski and Thalia I. Nicas for providing bacterial strains. This work was supported by a grant from the Canadian Cystic Fibrosis Foundation. J.L. is a research scholar from Fonds de la Recherche en Santd du Quebec, and M.F. was a research scholar from the Canadian Cystic Fibrosis Foundation.
REFERENCES 1. Bakala, H., J. Waliach, and M. Hanss. 1978. Determination of elastolytic activity using a conductimetric method. Biochimie 60:1205-1207. 2. Bever, R. A., and B. H. Iglewski. 1988. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J. Bacteriol. 170:4309-4314. 3. Brodeur, B. R., J. Lagace, and Y. Larose. 1987. Mouse-human myeloma partners for the production of heterohybridomas, p. 51-63. In L. B. Schook (ed.), Monoclonal antibody production techniques and applications, vol. 33. Marcel Dekker, Inc., New
715
York. 4. Campbell, M. E., S. W. Farmer, and D. P. Speert. 1988. New selective medium for Pseudomonas aeruginosa with phenanthroline and 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro10-phenylacridine hydrochloride (C-390). J. Clin. Microbiol.
26:1910-1912. 5. Cash, H. A., D. C. Straus, and J. A. Bass. 1983. Pseudomonas aeruginosa exoproducts as pulmonary virulence factors. Can. J. Microbiol. 29:448-456. 6. Digeon, M., M. Laver, J. Riza, and J. F. Bach. 1977. Detection of circulating immune complexes in human sera by simplified assays with polyethylene glycol. J. Immunol. Methods 16:165183. 7. Fukushima, J., S. Yamamoto, K. Morihara, Y. Atsumi, H. Takeuchi, S. Kawamoto, and K. Okuda. 1989. Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase. J. Bacteriol. 171:1698-1704. 8. Hamel, J., B. R. Brodeur, Y. Larose, P. S. Tsang, A. Belmaaza, and S. Montplaisir. 1987. A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b. J. Med. Microbiol. 23:163-170. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 10. Lagace, J., J. Mercier, M. Frechette, D. Fournier, M. Dubreuil, A. Lamarre, J. R. Lapointe, and S. Montplaisir. 1989. Circulating immune complexes, antibodies to Pseudomonas aeruginosa, and pulmonary status in cystic fibrosis. J. Clin. Lab. Immunol. 30:7-11. 11. Liu, P. V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130(suppl.):S94-S99. 12. Morihara, K., and H. Tsuzuki. 1975. Pseudomonas aeruginosa elastase: affinity chromatography and some properties as a metallo-neutral proteinase. Agric. Biol. Chem. 39:1123-1128. 13. Morihara, K., H. Tsuzuki, T. Oka, H. Inoue, and M. Ebata. 1965. Pseudomonas aeruginosa elastase isolation, crystallization, and preliminary characterization. J. Biol. Chem. 240:32953304. 14. Morrissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. 15. Neu, H. C. 1983. The role of Pseudomonas aeruginosa in infections. J. Antimicrob. Chemother. 11(suppl. B):1-13. 16. Nicas, T. I., and B. H. Iglewski. 1985. The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can. J. Microbiol. 31:387-392. 17. Ohman, D. E., S. J. Cryz, and B. H. Iglewski. 1980. Isolation and characterization of a Pseudomonas aeruginosa PAO mutant that produces altered elastase. J. Bacteriol. 142:836-842. 18. Stiver, G. H., K. Zachidniak, and D. P. Speert. 1987. Inhibition of polymorphonuclear leukocyte chemotaxis by the mucoid exopolysaccharide of Pseudomonas aeruginosa. Clin. Invest. Med. 11:247-252. 19. Tijssen, P. 1985. Practice and theory of enzyme immunoassay, p. 29. In Laboratory techniques in biochemistry and molecular biology, vol. 15. Elsevier Science Publishers, Amsterdam. 20. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.
INFECTION AND IMMUNITY, Feb. 1991, p. 712-715
0019-9567/91/020712-04$02.00/0 Copyright © 1991, American Society for Microbiology
Four Epitopes of Pseudomonas aeruginosa Elastase Defined by Monoclonal Antibodies JACQUELINE LAGACE* AND MANON FRECHETTE Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, P.O. 6128, Station A, Montreal, Quebec, Canada H3C 3J7 Received 12 July 1990/Accepted 7 November 1990
Monoclonal antibodies against the elastase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified elastase of P. aeruginosa and P3-X63-Ag8-U1 myeloma cells. The six clones established generated antibodies which reacted with a 33,000-Da peptide and recognized four different elastase epitopes by a competitive binding enzyme-linked immunosorbent assay. The monoclonal antibodies designated as ELA-17 and ELA-42 that recognize two different epitopes reacted by dot-enzyme immunoassay and by Western immunoblotting with all clinical and International Antigen Typing Scheme strains of P. aeruginosa positive for elastase.
Pseudomonas aeruginosa is a common opportunistic pathogen which can cause fatal infections in vulnerable hosts. It is the primary pulmonary pathogen in patients with cystic fibrosis (CF) and elicits significant morbidity in patients with thermal burns, cancer, and an immunosuppressed status (15). The pathogenesis of P. aeruginosa is multifactorial and involves a large number of extracellular products which may act as bacterial virulence factors (5, 11, 18). Among them, elastase, a Zn-metalloproteinase produced by most clinical strains of P. aeruginosa, has been largely characterized (12, 13). The elastase gene has been cloned and sequenced, and its mature protein product has been identified as a 32,926- to 33,000-Da polypeptide with a fully defined amino acid sequence (1, 2). Despite recent documentation of its complete primary structure (2, 7), little is known about the immunochemistry of elastase, and no specific epitopes have been identified or characterized in this protein molecule. To the best of our knowledge, no monoclonal antibodies (MAbs) that react with P. aeruginosa elastase have yet been identified. In the present study, we isolated and characterized six MAbs which react specifically with P. aeruginosa elastase. Using a competition assay, these MAbs were shown to react to four different epitopes on elastase. Antigenic conservation studied with the MAbs was demonstrated for two of these epitopes among all clinically testqd and representative strains of the International Antigen Typing Scheme (IATS) positive for P. aeruginosa elastase activity. P. aeruginosa PA103 was chosen for its absence of detectable elastase and P. aeruginosa 388 leu::Tnl was chosen because of its known capacity to secrete elastase (16). These strains were kindly provided by B. Iglewski and T. I. Nicas. A set of 17 serotype-specific representative strains of the IATS was obtained from E. Toma, Hotel-Dieu de Montreal (P. aeruginosa types 1 to 8, 10 to 12, and 14 to 16 as defined by a IATS kit commercially marketed by Difco Laboratories, Detroit, Mich.) and from the American Type Culture Collection: type 9 (ATCC 33356), type 13 (ATCC 33360), and type 17 (ATCC 33364). Clinical strains of P. aeruginosa were isolated from the sputum of CF patients of la Clinique de fibrose kystique de l'Hopital Sainte-Justine de *
Montrdal. Identification of the clinical P. aeruginosa strains was confirmed by culture on a selective medium for P. aeruginosa with phenanthroline (Sigma Chemical Co., St. Louis, Mo.); and 9-chloro-9-[4-(diethylamino)phenyl]-9,10dihydro-10-phenylacrinehydrochloride (C-390; kindly provided by Norwich Eaton Pharmaceuticals, Norwich, N.Y.) as described by Campbell et al. (4). P. aeruginosa strains were grown for 24 h at 37°C with constant agitation in medium containing- 0. 8% (wt/vol) tryptone, 0.5% (wt/vol) yeast extract, and 0.5% (wt/vol) NaCl. Following incubation, the cultures were centrifuged at 20,000 x g for 20 min at 4°C. Ammonium sulfate was added slowly to the culture supernatant to 60% saturation. After overnight incubation at 4°C, the material was centrifuged at 15,000 x g for 30 min to precipitate proteins. The pellet was dissolved in 1:20 or 1:80 of the initial volhme in 0.005 M Tris hydrochloride buffer (pH 8.0)-0.01 M NaCl and dialyzed against six changes of phosphate-buffered saline (PBS) (pH 7.2) over a 48-h period: All samples were stored at -70°C. BALB/c mice were immunized once a week for 4 weeks. For the first immunization, thXy were inoculated intraperitoneally with 10 ,ug of purified elastase (Nagase Biochemicals, Osaka, Japan) emulsified in an equal amount of complete Freund adjuvant (GIBCO Laboratories, Grand Island, N.Y.). For immunizations 2 through 4, the mice were given the immunogen intraperitoneally in incomplete Freund adjuvant. Serum titers of antibodies to elastase were determined by enzyme-linked immunosorbent assay (ELISA) (as outlined below) and were 1: 10,000 in the mice selected for use in the MAb isolation. For MAb production, the method described by l3rodeur et al. (3) was used with modifications. The spleens from selected mice were removed 3 days after the last injection for fusion with the mouse myeloma cell line P3-X63-Ag8-U1 (American Type Culture Collection) at a 10:1 ratib, employing 50% polyethylene glycol 1500 (Kodak) as a fusogen. RPMI 1649 medium (GIBCO) used for growth of the fused cells was supplemented with 20% fetal calf serum (GIBCO), 2 mM glutamine, 100 FM hypoxanthine, 1 ,M aminopterin, 16 ,uM thymidine, 50 ,ug of gentamicin per ml, and 104 normal m,use peritoneal cells per 0.2 ml. MAb titration was performed by ELISA. The hybridoma was subcloned three times by limiting dilution in 96-well plates (Nunc, GIBCO) in the same medium without aminopterin. MAbs were pro-
Corresponding author. 712
VOL. 59, 1991
duced in large quantities in 75-cm2 tissue culture flasks and in mouse ascitic fluid. MAbs in ascitic fluid were partially purified by precipitation with 50% ammonium sulfate. Hybridoma culture supernatants were obtained from cells transferred at a high concentration (4 x 106 per ml) in culture medium without fetal calf serum for 3 days and partially purified by precipitation with polyethylene glycol 6000 (Fisher, Montreal, Quebec, Canada) for 2 h at 4°C at a final concentration of 10% (wt/vol) (6). Purified MAbs were conjugated to biotin as described elsewhere (19). Briefly, purified MAbs (10 mg/ml) in 100 mM carbonate buffer (pH 8.5) were mixed with 0.1 volume of a fresh N,N-dimethylformamide solution (Fisher) in 6 mg of biotinyl-N-hydroxysuccinimide (Sigma) per ml. The mixture was allowed to react for 1 h at room temperature, dialyzed against PBS until free of biotin, and frozen at -70°C in aliquots. ELISA was performed as described previously (10), using a commercial preparation of purified elastase (Nagase Biochemicals) as a coating antigen at a final concentration of 2 ,ug/ml in 0.05 M sodium carbonate buffer (pH 9.6). The 96-well plates (Nunc, GIBCO) were incubated overnight at room temperature. Controls included unrelated MAb and preimmune and immune mouse sera. Preliminary studies have shown that the elastase enzyme used as a coating antigen did not manifest immunoglobulin G (IgG) proteolytic activity in our ELISA protocol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SI)S-PAGE) was performed under reducing conditions by the Laemmli method (9) in a minigel system (Bio-Rad Laboratories, Richmond, Calif.). Electrophoresis was conducted in 12% slab gels at 200 V for 50 min, using appropriate molecular weight markers (Bio-Rad Laboratories). Subs'equently, the gels were either stained with silver nitrate (Canlab, Toronto, Ontario, Canada) as described previously (14) or electrophoretically transferred to nitrocellulose by the method of Towbin et al. (20). Electrophoretic transfers onto nitrocellulose (Bio-Rad Laboratories) were performed at 100 V for 60 min. Immunospecific reactions were detected by autoradiography (8) or with avidin-peroxidase conjugates used in concert with biotinylated secondary antibodies according to the manufacturer's recommendations (Vector Laboratories, Burlingame, Calif.). To assess whether MAbs recognize the same epitopes, we performed a competitive binding assay using ELISA essentially as described above unless otherwise specified. Wells of polystyrene microdilution plates were coated with 100 RI of purified elastase at a concentration of 0.5 ,ug/ml as determined by preliminary assays with biotinylated MAb dilutions to optimize assay conditions. After the blocking step, 50 ,ul of MAb undiluted culture supernatant was added in triplicate to each well. The MAbs were allowed to bind to elastase for 30 min at room temperature. Then, 50 pul of a biotinylated MAb dilution per well was added. Plates were incubated for 1 h at 37°C. After washing, the amount of biotinylated MAb bound was measured by reaction with avidin-conjugated peroxidase as recommended by the manufacturer (Vector Laboratories). The A490 was measured with a Titertek Multiskan MC spectrometer. A dot-enzyme immunoassay was used for screening MAbs against 10 P. aeruginosa clinical strains isolated from the sputum of CF patients. Elastase (2 pI, produced as above) from each of the P. aeruginosa strains was applied to nitrocellulose paper and allowed to dry at room temperature. Immune specificity was revealed with biotinylated aintimouse IgG as previously described. Protein concentration was measured with a commercial
NOTES
713
100
no
60 60
40
20 FIG. 1. Competitive inhibition of the binding of biotin-conjugated MAb to elastase by various unlabeled MAbs (undiluted culture supernatants). Percent binding was calculated relative to the degree of binding of biotin-conjugated MAb in the presence of an unrelated MAb as a control. Symbols: 0, ELA-17; X, ELA-42; *, ELA-207; El, ELA-73; , ELA-45; U, ELA-56.
protein assay reagent (Bio-Rad Laboratories). The subisotype of MAbs was determined with a mouse subisotyping kit (Serotec, Oxford, United Kingdom) used as recommended by the manufacturer. Elastolytic activity was assayed by dye release from elastin-Congo red (17). Six hybridomas producing antibodies specific to elastase were isolated by a single fusion of P3-X63-Ag8-U1 cells and spleen cells from mice immunized with purified elastase. Positive clones were detected by ELISA with purified elastase as the antigen. These MAbs were designated ELA42, ELA-17, ELA-56, ELA-45, ELA-73, and ELA-207. The first three MAbs were of the IgGl subclass, and the latter three were of the IgG3 subclass. To test whether ELA-56, ELA-42, ELA-17, ELA-207, ELA-45, and ELA-73 recognize different epitopes, we performed a competitive binding assay using the ELISA. The results of these experiments are summarized in Fig. 1. Data are expressed as percent binding of biotin-conjugated MAb (b-cMAb) in the presence of an unlabeled and undiluted MAb culture supernatant; binding in the presence of the negative control (an unrelated MAb) was taken as 100%. Binding of the b-cMAb ELA-42 was inhibited only by its unlabeled homologous MAb; b-cMAb ELA-45 binding was inhibited by its unlabeled homologous MAb and by ELA-73; and b-cMAb ELA-17 binding was inhibited by its unlabeled homologous MAb and ELA-56 (Fig. 1). b-cMAb ELA-42, ELA-45, and ELA-17 binding in the presence of heterologous MAbs was in the range of 67 to 100% compared with 30 to 10% in the presence of homologous MAbs (Fig. 1). We concluded that the six MAbs recognized four different epitopes on elastase: one epitope was recognized by ELA42, a second by ELA-207, a third by ELA-45 and ELA-73, and a fourth by ELA-17 and ELA-56. The ability of our six different MAbs to recognize purified elastase and elastase from cell-free culture supernatant of P.
714
NOTES
INFECT. IMMUN. 2
a
66KD
a b
I
d
gh
t
a
aeruginosa 388 leu: :Tnl and of the negative control strain P. iaeruginosa PA103 was examined by reacting them with Western immunoblots. In Fig. 2a, purified elastase presents a single protein band of 33 kDa as stained by the Bio-Rad biotin-blot total protein assay. By immunodetection (Fig. 2a), the six MAbs reacted to the same purified elastase band of 33 kDa with different intensities and patterns not related
4
3 c
f,i
--
45KD --
36KD 29KD -_
mEa
a
to their concentration. The reactions to ELA-42 and ELA-17 antibodies were darker and larger than those to ELA-45 and ELA-207. This was confirmed by Western immunoblotting
3
24KD -
20KD -_
of the culture supernatant of strain 388 leu::Tnl, for which the same larger reactions to ELA 42 and ELA-17 were observed compared with ELA-45 and ELA-207 antibodies (Fig. 2b). No reaction was seen when the six MAbs were tested with Western immunoblots of culture supernatant of P. aeruginosa PA103 negative for elastase secretion (data
14KD _
66kd
3 ow
2
b
1
a
b
c
d
r-,-%
4
i>
g ;h
not
-
45kd 36kd 29 kd -_
,--.
m
a
24kd _
2Okd
shown).
The ability of the four different MAbs to react to concentrated cell-free culture supernatants of clinical and IATS P. aeruginosa strains was examined by dot-enzyme immunoassays or by reacting Western immunoblots of SDS-polyacrylamide gels of supernatants with these antibodies. ELA-42 and ELA-17 reacted by dot-enzyme assay with supernatants of the 10 clinical strains isolated from CF patients; ELA-207 and ELA-45 reacted, respectively, with five and four of them (Table 1). Eight supernatants of IATS strains (numbers 3 to 8, 14, and 16) reacted with ELA-45; ELA-42 and ELA-17 reacted with supernatants of 12 of 17 IATS strains examined by Western immunoblotting (Table 1). The five negative IATS strains by Western immunoblotting with the latter MAbs were 1, 9, 10, 15, and 17. The elastolytic activity of concentrated supernatants of negative strains by Western immunoblotting was tested on elastin-Congo red; a total lack of elastolytic activity was recorded with all these strains. Positive control strains showed an elastolytic activity corresponding to more than 1 mg of purified elastase. ELA-207
-e
14kd _
FIG. 2. All these MAbs (ELA-56 [lane b], ELA-42 [lane c], ELA-17 [lane d], ELA-207 [lane fi, ELA-45 [lan(e h], ELA-73 [lane i]) and an unrelated MAb (lanes a, e, and g) werre reacted with the 33-kDa purified elastase (a) and a 33-kDa proteiln band in supernatant from strain 388 leu::Tnl (b). KD, kd, Kilod,altons.
TABLE 1. Reactivity of MAbs specific for P. aeruginosa elastase, determined by dot-enzyme immunoassay and Western immunoblotting aginst concentrated cell-free culture supernatant from clinical and IATS P. aeruginosa strains
ELA42
PA103 (negative control) 388 leu::Tnl 1 2 3 4 5
6 7 8 9 10 11 12
IATS strain examined by
CF strain examined by dot-enzyme immunoassay'
P. aeruginosa straina
+ + +
+ + + + + + + +
immunoblotting'
ELA-17 + + + + + +
ELA-45 + +
ELA-207 + +
-
-
+ + + + +
+ _
+ -
+ + + -
ELA42 + _ + + + + + + +
BLA-17 + _ + + + + + + +
+ +
+ +
+
+ +
+
+
+
+
A3+
14 15 16 17 a The 10 clinical strains were isolated from sputum of CF patients. b Positive reactions were identified colorimetrically. Each strain was tested more than two times. c ELA-207 was not tested.
Wester ELA45 _ _ + + + + + +
NOTES
VOL. 59, 1991
was not tested against IATS strains because of the instability of this MAb with time. In this report, we described the first isolation and characterization of six MAbs directed against P. aeruginosa elastase. These MAbs can be divided in two groups based on their IgG subclasses, their pattern of immunoblotting reactions, their degree of affinity, and the predominance of their antigenic sites among P. aeruginosa strains. Thus, MAbs ELA-17, ELA-42, and ELA-56, all of the IgGl subclass, reacted more strongly in immunologic tests and presented a wider band in Western immunoblots and their antigenic sites were more common among P. aeruginosa strains compared with MAbs of the IgG3 subclass, ELA-45, ELA-73, and ELA-207. Because very strong reactions with MAbs ELA17, ELA-42, and ELA-56 were observed on Western immunoblots (denaturing conditions), epitopes bound by these MAbs are apparently sequence determined, whereas MAbs ELA-45, ELA-73, and ELA-207 possibly react with an epitope partially dependent on conformational arrangement since reactivity in Western immunoblots was decreased compared with that in ELISA. Moreover, these latter MAbs show a lower affinity to their epitope since whatever their concentration was, their measured reactivity was never as high as that for the three other MAbs (data not shown). An interesting finding in this study is that all clinical and IATS P. aeruginosa strains presenting elastase as demonstrated by SDS-PAGE and/or elastolytic activity shared the two same separate epitopes as revealed by their interaction with the MAbs ELA-17 and ELA-42 (Table 1). The pattern of conservation of the two other epitopes is less common since ELA-207 and ELA-45 reacted, respectively, with only 50 and 40% of the clinical strains tested and the latter reacted with 66% of the IATS strains. We must consider that this difference in epitope specificities of elastase among P. aeruginosa strains may also influence the structure and/or activity of the elastase molecule secreted by the different groups of P. aeruginosa strains. We thank Barbara Iglewski and Thalia I. Nicas for providing bacterial strains. This work was supported by a grant from the Canadian Cystic Fibrosis Foundation. J.L. is a research scholar from Fonds de la Recherche en Santd du Quebec, and M.F. was a research scholar from the Canadian Cystic Fibrosis Foundation.
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