Vol. 29, No. 7
JOURNAL OF CLINICAL MICROBIOLOGY, July 1991, p. 1407-1412
0095-1137/91/071407-06$02.00/0 Copyright © 1991, American Society for Microbiology
Production and Characterization of Monoclonal Antibodies Directed against the Lipopolysaccharide of Francisella tularensis MARK J. FULOP,* TIMOTHY WEBBER, RICHARD J. MANCHEE, AND DAVID C. KELLY Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 OJQ, United Kingdom Received 22 October 1990/Accepted 10 April 1991
Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 103 organisms, whereas FT2F11 was able to detect only 105 organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified 0 side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the 0 side chain and that FT2F11 is directed against the core.
Francisella tularensis is a small gram-negative coccobacillus which causes the disease tularemia in humans and many mammals. Its host range is considerable, but for reasons which are not fully understood, the disease is endemic only in northern and temperate regions of the Northern Hemisphere. The species is divided into two subspecies, namely, F. tularensis subsp. tularensis (type A) and ?; tularensis subsp. palaearctica (type B). Type A strains, confined to Nearctic regions, cause a more serious disease with a higher mortality than that caused by type B strains, which are of Holarctic distribution. The subspecies are indistinguishable by serological methods (25), although recent work has shown that it may be possible to distinguish them by differences in their 16S rRNAs in a few hours (9). The agent is extremely infectious (10), and it is thought that as few as 10 organisms can cause infection in humans (1). Current methods of identification of F. tularensis from environmental samples and pathological material from apparently infected animals and humans are both time-consuming to perform and slow in giving results. Existing serological tests based on polyclonal serum tend to produce crossreactions with other microorganisms, thus preventing conclusive identification. Monoclonal antibodies against the 0 side chain of the lipopolysaccharides (LPSs) of other gram-negative bacteria such as Bordetella pertussis (11) and Vibrio cholerae (12) have been shown to be species specific, whereas those against core (23) and lipid A show extensive cross-reaction (2). The carbohydrates of the 0 side chain have been shown to be important surface antigens in a number of studies on F. tularensis (20) and are probably the molecules which confer specificity (8). Although the bacterial cell of F. tularensis is said to be surrounded by a capsule (19), this does not appear to mask the 0 antigen of the LPS and has allowed the development of an enzyme-linked immunosorbent assay (ELISA) based on this antigen (3). *
Here we report the production and characterization of two monoclonal antibodies directed against F. tularensis LPS and assess the suitability for their eventual use in a kit for the specific, sensitive, and rapid identification of this infectious agent.
MATERIALS AND METHODS Bacterial strains and cultivation. F. tularensis LVS, Sverige 121, A308/67, HN63, 3202, A362/67, and Jap Down; Yersinia pestis TS; and Brucella suis were all obtained from the U.S. Army Medical Research Institute of Infectious Diseases. The F. tularensis LVS cap mutant was obtained from the Swedish Defence Research Establishment, Umea, Sweden. Y. pestis EV76 was obtained from the Institut Pasteur, Paris, France. Salmonella typhimurium ATCC 13311 and Legionella pneumophila were obtained from The Centre for Applied Microbiology and Research, Porton Down, Salisbury, United Kingdom. Escherichia coli 018 Kwas obtained from Edinburgh University, Edinburgh, United Kingdom. F. tularensis strains were grown on blood cysteine glucose agar (BCGA) plates (5) and MCPH broth (14). Y. pestis strains were grown on blood agar base (Oxoid, Basingstoke, United Kingdom). S. typhimurium and E. coli strains were grown on nutrient agar (Oxoid). L. pneumophila was grown on charcoal yeast extract agar (Oxoid). B. suis was grown on Iso-Sensitest agar with the addition of 5% horse serum in a 5% CO2 atmosphere. LPS preparation. Twenty liters of F. tularensis LVS was grown in 250-ml shake flasks (100 ml per flask) at 100 rpm at 37°C for 48 h in MCPH broth. The flasks were checked for purity by Gram staining and culture on BCGA plates, and the bulked cultures were centrifuged at 10,000 x g for 30 min. The pellets were washed twice in 5 mmol phosphatebuffered saline (PBS; pH 7.2) and freeze-dried, and LPS was extracted by the hot phenol method of Westphal and Jann (26). The crude LPS was purified by treatment with pronase and RNase and by high-speed centrifugation as described by Smith et al. (22).
Corresponding author. 1407
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Protein content. The protein content of the final product was determined by use of the Farbstott-Kongentrat reagent (Bio-Rad, Watford, United Kingdom). 0 side chain preparation. A total of 10 mg of purified LPS was subjected to mild acid hydrolysis in 1% acetic acid for 90 min at 100°C under nitrogen, and lipid A was removed with hexane. The resulting crude 0 side chain was rotary evaporated and resuspended in 1 ml of pyridinium acetate (pH 5.3; 4 ml of pyridine and 10 ml of acetic acid made to 1 liter with distilled water), and the solution was applied to a column (25 by 1.6 cm) of Sephadex G50 (Sigma, Poole, United Kingdom). Sixty 1-ml fractions were collected and assayed for carbohydrate by the phenol-sulfuric acid method described by Dubois et al. (6); and the peak fractions were pooled, dried by rotary evaporation, and resuspended in PBS. Production of monoclonal antibodies. Female BALB/c mice (age, 6 to 9 weeks) were immunized with 50 p1l of 105 heat-killed F. tularensis LVS organisms per ml at weekly intervals for 28 days, followed by 50 ,ul of 1 mg of F. tularensis LVS LPS per ml at weekly intervals for 5 weeks. Fusion and cloning were carried out as described elsewhere for V. cholerae LPS (12). Briefly, B lymphocytes from immunized mice were fused with cells from the mouse myeloma cell line SP2/0-Agl4 (21) by using polyethylene glycol 1500 as the fusion agent. Primary selection of hybrids was performed by growing the cells in 96-well microtiter plates on hypoxanthine-aminopterin-thymidine medium (12) at 37°C with 80% humidity and 5% C02; antibody-producing hybrid cells of interest were recloned by limiting dilution. Antibodies were produced in pristane (Sigma)-primed mice (17) by injecting 5 x 106 hybridoma cells intraperitoneally; the resulting ascites antibodies were stored at -20°C. Indirect ELISA. A total of 100 pAl of 10 ,g of LPS in antigen-binding buffer (50 mmol carbonate buffer; pH 9.6) per ml or 100 RI of F. tularensis at various concentrations in PBS was added to each well of a 96-well microtiter plate (M 129 A; Dynatech Irvine, United Kingdom) and incubated at 4°C overnight. After washing with PBS, the wells were filled with BLOTTO (1% milk powder in PBS) and incubated for 1 h at 37°C to block nonspecific binding sites. After three rinses with PBS containing 0.05% Tween 20 (PBS-Tween), 100 1±1 of antibody diluted in BLOTTO was added to each well and incubated for 1 h at 37°C. The plate was rinsed again three times in PBS-Tween, and 100 ,ul of horseradish peroxidase goat anti-mouse immunoglobulin (Sigma) diluted in BLOTTO (1/1,000) was added to each well and incubated for 1 h at 37°C. The plate was washed five times in PBS-Tween, and 100 RI of enzyme substrate [55 mg of 2,2'-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) in 56 ml of 0.1 M citric acid-44 ml of 0.2 M Na2HPO4-10 pI of H202; pH 5.51 was added to each well. The optical density after 1 h at 20°C was read in a Dynatech microplate reader at 414 nm. A reading of 0
0
0
x 106
x 106 x 106 x 106 x 106 x 106 x 106 x 106 x 106 x 106
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a 0
U)
-,00
°
°O,O-C
0.4-
cn n
010-0
0.3 +
0.2 0.4
10
1 0
Side chain
~
~
100
~
1 000
(/ig/ml)
FIG. 1. An inhibition ELISA was completed as described in the
a An optical density at 414 nm of >0.1 was used.
text. Before the primary antibody was added to the microtiter plate,
it was incubated with various concentrations of 0 side chain from F. tularensis LPS. 0, FT14; 0, FT2F11.
After treatment with chemicals, the antigens were evaluated for their immunological activities. RESULTS Purity of extracted LPS. LPS extracted from F. tularensis LVS was of high purity. Spectrophotometric analysis showed that there was no detectable nucleic acid and only 0.75% protein contamination. The 0 side chain was confined to the peak eluting with the void volume of the Sephadex column, with each 10 mg of purified LPS producing a yield of 1.3 mg of the 0 side chain. Characteristics of monoclonal antibodies against LPS. From the initial screen, two lines of hybridoma cells producing antibody to F. tularensis LVS LPS were isolated and cloned. The antibodies were designated FT14 and FT2F11, and in an indirect ELISA, both were able to detect approximately 3 pg of F. tularensis LPS. Both FT14 and FT2F11 were shown to be IgM antibodies by ELISA. To test the sensitivity of the antibodies in detecting the whole bacterial cells used as antigens in the indirect ELISA, 10 different strains of F. TABLE 2. ELISA inhibition of clones FT2F11 and FT14 with LPS from different strains used as inhibitors Inhibitor (LPS)U
Escherichia coli 055:B5 Escherichia coli 0111:B4 Escherichia coli 0127:B8 Escherichia coli 0128:B12 Escherichia coli K235 Klebsiella pneumoniae Pseudomonas aeruginosa S. abortus equi Salmonella enteritidis Salmonella minnesota Salmonella typhimurium Salmonella typhosa Serratia marcescens
Shigella flexneri Vibrio cholerae Inaba Francisella tularensis LVS
50%o Inhibitory value (~Lg/ml)b FT2F11
FT14
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 20
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 100 15
All LPSs except the F. tularensis LVS LPS were commercial preparations. b Concentration of LPS required to reduce the optical density by 50%o. a
tularensis were examined (Table 1). FT2F11 was less sensitive, with 105 organisms being required for a positive identification, whereas FT14 was able to detect 103 organisms. To indicate specificity, FT2F11 and FT14 were reacted with 16 commercially prepared LPS samples from 5 strains of E. coli and 10 species of other gram-negative bacteria, as well as with our LPS from F. tularensis LVS; residual antibody was measured by ELISA. None of the commercial preparations was able to produce a 50%o inhibition value with FT14, whereas this could be achieved with 20 ,ug of F. tularensis LVS LPS per ml. FT2F11 was inhibited by V. cholerae Inaba LPS, but a high concentration, 125 ,ug/ml, was required for 50% inhibition, whereas only 15 ,ug/ml was required to produce the same level of inhibition for F. tularensis LPS (Table 2). When FT14 was incubated with the isolated 0 side chain and residual antibody activity was measured by ELISA, there was a sharp fall in antibody titer compared with that of antibody incubated with PBS, indicating that it is directed against 0 side chain components. By contrast, the titer of FT2F11 was only slightly reduced even after incubation with a large amount of 0 side chain, showing that this antibody is directed against other epitopes of the LPS (Fig. 1). When the antibodies were incubated with whole organisms of a number of other gram negative species, there were no falls in titer (Table 3). Comparison of antibodies by competitive ELISA. To deter-
TABLE 3. Cross-reaction studies with gram-negative bacteria by an inhibition ELISA Bacteria
Yersinia pestis TS Yersinia pestis EV76 Brucella suis Escherichia coli 018 K-
Salmonella typhimurium ATCC 13311 Legionella pneumophila Vibrio cholerae Inaba Francisella tularensis Schu 4
No. of organisms/ml required to achieve a 50% inhibition of color
FT2F11
FT14
>107
>107
i04
102
>107 >107 >107 >107 >107 >107
>107 >107 >107 >107 >107 >107
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J. CLIN. MICROBIOL.
FULOP ET AL. 1.5 T
A B C O
E
1.0
o I/ .--\._ X
I
a)
C:0
E.0
0
.0
0.5 t
a ~~~~~~~ 0.:
v
n~~~~~"--
., 5
10
1000 100 Reciprocal Antibody Titre x 101
10000
FIG. 2. Competition ELISA. Peroxidase-labeled FT2F11 was used as the primary antibody, and either unlabeled FT14 (0) or FT2F11 (0) was used as the second antibody. The ELISA was then developed as described in the text.
mine whether monoclonal antibodies FT14 and FT2F11 competed for the same epitope, a competitive ELISA was devised. Unlabeled FT14 (ascites) antibody was mixed with purified peroxidase-labeled FT2F11, and the mixture was titrated in plates coated with F. tularensis LPS; there was no reduction in titer compared with that of FT2F11 alone. As a positive control, labeled and unlabeled FT2F11 was titrated in the same way; inhibition of color development occurred at titers of 1:10,000 and below (Fig. 2). Comparison of antibodies by gel electrophoresis and immunoblotting. Silver staining of F. tularensis LPS showed the typical banding pattern of the repeating 0 side chain of the structure as seen with other gram-negative bacteria such as E. coli and S. typhimurium (Fig. 3). No bands were visible following staining with Coomassie blue. Western immunoblotting gave a characteristic banding pattern when FT14 was used as the primary antibody (Fig. 4), but no banding
FIG. 4. Western immunoblot of F. tularensis LVS LPS probed with monoclonal antibodies FT2F11 (lanes A and B) and FT14 (lanes C and D).
was seen when FT2F11 was used as the primary antibody. To determine the reason for the failure of FT2F11 to immunoblot, a chemical inhibition ELISA was devised. LPS from F. tularensis was first boiled in the sample buffer used for the preparation of specimens for SDS-PAGE and was then reacted with the two antibodies. FT2F11 failed to react with the treated LPS, but FT14 was reacted normally. If LPS was treated with the separate components of the sample buffer, it was found that SDS was the chemical which destroyed its ability to react with FT2F11. Triton X-100 produced a similar lack of reaction with FT2F11. Treatment of LPS with 5% (vol/vol) formaldehyde and glutaraldehyde had no effect on the ability of either FT2F11 or FT14 to recognize F. tularensis LPS (Table 4).
DISCUSSION
Most studies on the antigenic determinants of F. tularensis have used ill-defined antigens (13), although one used purified LPS as the antigen in an ELISA to diagnose tularemia (3). If diagnosis of the disease is the sole requirement, however, crude mixtures of antigens are easier to prepare and are effective (24), with reservations about a tendency of
TABLE 4. Effect of chemical modification of F. tularensis LPS on antibody recognition measured in an indirect ELISA Effecta Chemical FT14
FT2F11
SDS (2% [wt/vol]) Triton X-100 (2% [wt/vol])
-
+ +
Formaldehyde (5% [vol/vol]) Glutaraldehyde (5% [vol/vol])
-
2-p-Mercaptoethanol (5% [vol/vol])
FIG. 3. SDS-PAGE of LPS isolated from F. tularensis LVS and visualized by silver staining.
-
a -, chemical has no effect on the ability of antibody to recognize the LPS; +, chemical modified LPS so that antibody failed to recognize the LPS.
VOL. 29, 1991
MONOCLONAL ANTIBODIES AGAINST F. TULARENSIS LPS
the antigens to cross-react with antibodies invoked by infection with some other gram-negative bacteria. The cross-reaction studies with either whole killed cells or commercially prepared LPS showed that the epitope that FT14 recognized was unique to F. tularensis and that FT2F11 cross-reacted with V. cholerae LPS at a low avidity. We conclude that the epitopes that the monoclonal antibodies bind to are specific to F. tularensis but are conserved throughout the genus Francisella. However, all the strains were grown under the same cultural conditions. It is possible that alteration of growth conditions could modify the structure of the LPS, which has been shown to occur with some strains of E. coli (4). Diagnosis of tularemia has been based on immunoserological methods, because direct culture of specimens from infected sites has proved to be difficult and is a potential hazard to laboratory personnel (25). The ability of FT14 to detect low numbers of bacteria could be used to detect F. tularensis directly in infected sites by labeling the antibody with a fluorescent tag. The specificity of the antibodies could also be important in preventing incorrect diagnosis, because it has been reported that antibodies against L. pneumophila gave a false diagnosis of Legionnaires disease when the patient was suffering from tularemia (18). Inhibition studies showed that FT14 reacted strongly with isolated 0 side chain but that FT2F11 did not, indicating that the latter was directed against components of LPS other than the 0 side chain, and the results of the competition assay between the two monoclonal antibodies showed clearly that they do not compete for the same epitope. Studies with an acapsular rough mutant of F. tularensis (Cap-) indicated that the presence of a capsule aided survival (19). However, the study did not investigate the possibility that the rough colonial appearance was caused by structural changes in the LPS, although rough LPS strains have not been reported. FT14 reacted as strongly with the Cap- strain as it did with the other strains, implying that this mutant has a functional 0 side chain and therefore is not a rough LPS strain. Other rough colonial mutants have been isolated (7), but their LPSs have not been analyzed. FT14 could prove to be valuable in isolating and identifying rough LPS mutants as an aid to evaluating the role of the 0 side chain in the disease process. The results of the chemical inhibition ELISA show clearly that detergents affected LPS to such an extent that FT2F11 was prevented from recognizing it either by ELISA or Western blotting (immunoblotting). This destruction of biological activity was not only confined to SDS, which carries a strong negative charge, but was also manifest with Triton X-100, which is nonionic. Formaldehyde and glutaraldehyde, which cross-link protein, did not affect the binding of FT2F11, showing that this antibody is not directed against the small contaminating protein part of the LPS preparation. The most likely scenario is that the detergents caused structural changes in lipid A which induced a conformational change in the core but not in the 0 side chain. Because the core region is less exposed on the bacterial surface, it is to be expected that antibodies directed against this region would be less sensitive in detecting whole organisms. Before FT14 and FT2F11 are incorporated into a kit to rapidly identify F. tularensis, we intend to test them against wild-type strains isolated from patients and the environment. REFERENCES 1. Bell, J. F., C. R. Owen, and C. C. Larson. 1955. Virulence of Bacterium tularense: a study of Bacterium tularense in mice, guinea pigs, and rabbits. J. Infect. Dis. 97:162.
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2. Bogard, W., D. L. Dunn, K. Abernethy, C. Kilgarriff, and P. C. Kung. 1987. Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity. Infect. Immun. 55:899-908. 3. Carlsson, H. E., A. A. Lindberg, G. Lindberg, B. Hederstedt, K.-A. Karisson, and B. 0. Agell. 1979. Enzyme-linked immunosorbent assay for immunological diagnosis of human tularemia. J. Clin. Microbiol. 10:615-621. 4. Dodds, K. L., M. B. Perry, and I. J. McDonald. 1987. Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli 0157:H7 as a function of growth rate and growth-limiting nutrient. Can. J. Microbiol. 33:452-458. 5. Downs, C. M., L. L. Coriell, S. S. Chapman, and A. Klauber. 1947. The cultivation of Bacterium tularense in embryonated eggs. J. Bacteriol. 53:89-100. 6. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related compounds. Anal. Chem. 28:350-356. 7. Eigelsbach, H. T., W. Braun, and R. D. Herring. 1951. Studies on the variation of Bacterium tularense. J. Bacteriol. 61:557569. 8. Eliwood, D. C. 1970. The distribution of 2-keto-3-deoxy-octonic acid in bacterial walls. J. Gen. Microbiol. 60:373-380. 9. Forsman, M., G. Sandstrom, and B. Jaurin. 1990. Identification of Francisella species and discrimination of type A and type B strains of F. tularensis by 16S rRNA analysis. Appl. Environ. Microbiol. 56:949-955. 10. Greisman, S., R. B. Hornick, F. A. Carozza, and T. E. Woodward. 1963. The role of endotoxin during typhoid fever and tularemia in man. I. Acquisition of tolerance to endotoxin. J. Clin. Invest. 42:1064-1075. 11. Gustafsson, B., U. Lindquist, and M. Andersson. 1988. Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide. J. Clin. Microbiol. 26:188-193. 12. Gustafsson, B., A. Rosen, and T. Holme. 1982. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide. Infect. Immun. 38:449-454. 13. Holm, S. E., A. Tarnvik, and G. Sandstrom. 1980. Antigenic composition of a vaccine strain of Francisella tularensis. Int. Arch. Allergy Appl. Immun. 61:136-144. 14. Hood, A. M. 1977. Virulence factors of Francisella tularensis. J. Hyg. 79:47-60. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London) 227: 680-685. 16. Nakane, P. 1979. In R. M. Nakamura, W. R. Dito, and E. S. Tucker (ed.), Immunoassays in the clinical laboratory, p. 81. Alan R. Liss, New York. 17. Potter, M., J. G. Pumphrey, and J. L. Walters. 1972. Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J. Natl. Cancer Inst. 49:305-308. 18. Roy, T., D. Fleming, and W. H. Anderson. 1989. Tularemic pneumonia mimicking Legionnaires' disease with false positive direct fluorescent antibody stains for legionella. South. Med. J. 82:1429-1431. 19. Sandstrom, G., S. Lofgren, and A. Tarnvik. 1988. A capsuledeficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by polymophonuclear leukocytes. Infect. Immun. 56:1194-1202. 20. Sandstrom, G., A. Tarnvik, H. Wolf-Watz, and S. Lofgren. 1984. Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect. Immun. 45:101-106. 21. Schulman, M., C. D. Wilde, and G. Kohler. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276:269-270. 22. Smith, A. R. W., S. Zamze, and R. C. Hignett. 1985. Composition of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum and its digestion by bacteriophage A7. J. Gen. Microbiol. 131:963-974. 23. Tsang, R. S. W., K. H. Chan, P. Y. Chan, K. C. Wan, M. H. Ng,
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and S. Schlecht. 1987. A murine monoclonal antibody specific for the outer core oligosaccharide of Salmonella lipopolysaccharide. Infect. Immun. 55:211-216. 24. Viljanen, M. K., T. Nurmi, and A. Salminen. 1983. Enzymelinked immunosorbent assay (ELISA) with bacterial sonicate antigen for IgM, IgA, and IgG antibodies to Francisella tularensis: comparison with bacterial agglutination test and ELISA with lipopolysaccharide antigen. J. Infect. Dis. 148:715-720.
J. CLIN. MICROBIOL. 25. Weaver, R. E., D. G. Holis, and E. J. Bottone. 1985. Gramnegative fermentation bacteria and Francisella tularensis, p. 316-319. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 26. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
JOURNAL OF CLINICAL MICROBIOLOGY, July 1991, p. 1407-1412
0095-1137/91/071407-06$02.00/0 Copyright © 1991, American Society for Microbiology
Production and Characterization of Monoclonal Antibodies Directed against the Lipopolysaccharide of Francisella tularensis MARK J. FULOP,* TIMOTHY WEBBER, RICHARD J. MANCHEE, AND DAVID C. KELLY Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 OJQ, United Kingdom Received 22 October 1990/Accepted 10 April 1991
Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 103 organisms, whereas FT2F11 was able to detect only 105 organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified 0 side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the 0 side chain and that FT2F11 is directed against the core.
Francisella tularensis is a small gram-negative coccobacillus which causes the disease tularemia in humans and many mammals. Its host range is considerable, but for reasons which are not fully understood, the disease is endemic only in northern and temperate regions of the Northern Hemisphere. The species is divided into two subspecies, namely, F. tularensis subsp. tularensis (type A) and ?; tularensis subsp. palaearctica (type B). Type A strains, confined to Nearctic regions, cause a more serious disease with a higher mortality than that caused by type B strains, which are of Holarctic distribution. The subspecies are indistinguishable by serological methods (25), although recent work has shown that it may be possible to distinguish them by differences in their 16S rRNAs in a few hours (9). The agent is extremely infectious (10), and it is thought that as few as 10 organisms can cause infection in humans (1). Current methods of identification of F. tularensis from environmental samples and pathological material from apparently infected animals and humans are both time-consuming to perform and slow in giving results. Existing serological tests based on polyclonal serum tend to produce crossreactions with other microorganisms, thus preventing conclusive identification. Monoclonal antibodies against the 0 side chain of the lipopolysaccharides (LPSs) of other gram-negative bacteria such as Bordetella pertussis (11) and Vibrio cholerae (12) have been shown to be species specific, whereas those against core (23) and lipid A show extensive cross-reaction (2). The carbohydrates of the 0 side chain have been shown to be important surface antigens in a number of studies on F. tularensis (20) and are probably the molecules which confer specificity (8). Although the bacterial cell of F. tularensis is said to be surrounded by a capsule (19), this does not appear to mask the 0 antigen of the LPS and has allowed the development of an enzyme-linked immunosorbent assay (ELISA) based on this antigen (3). *
Here we report the production and characterization of two monoclonal antibodies directed against F. tularensis LPS and assess the suitability for their eventual use in a kit for the specific, sensitive, and rapid identification of this infectious agent.
MATERIALS AND METHODS Bacterial strains and cultivation. F. tularensis LVS, Sverige 121, A308/67, HN63, 3202, A362/67, and Jap Down; Yersinia pestis TS; and Brucella suis were all obtained from the U.S. Army Medical Research Institute of Infectious Diseases. The F. tularensis LVS cap mutant was obtained from the Swedish Defence Research Establishment, Umea, Sweden. Y. pestis EV76 was obtained from the Institut Pasteur, Paris, France. Salmonella typhimurium ATCC 13311 and Legionella pneumophila were obtained from The Centre for Applied Microbiology and Research, Porton Down, Salisbury, United Kingdom. Escherichia coli 018 Kwas obtained from Edinburgh University, Edinburgh, United Kingdom. F. tularensis strains were grown on blood cysteine glucose agar (BCGA) plates (5) and MCPH broth (14). Y. pestis strains were grown on blood agar base (Oxoid, Basingstoke, United Kingdom). S. typhimurium and E. coli strains were grown on nutrient agar (Oxoid). L. pneumophila was grown on charcoal yeast extract agar (Oxoid). B. suis was grown on Iso-Sensitest agar with the addition of 5% horse serum in a 5% CO2 atmosphere. LPS preparation. Twenty liters of F. tularensis LVS was grown in 250-ml shake flasks (100 ml per flask) at 100 rpm at 37°C for 48 h in MCPH broth. The flasks were checked for purity by Gram staining and culture on BCGA plates, and the bulked cultures were centrifuged at 10,000 x g for 30 min. The pellets were washed twice in 5 mmol phosphatebuffered saline (PBS; pH 7.2) and freeze-dried, and LPS was extracted by the hot phenol method of Westphal and Jann (26). The crude LPS was purified by treatment with pronase and RNase and by high-speed centrifugation as described by Smith et al. (22).
Corresponding author. 1407
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FULOP ET AL.
Protein content. The protein content of the final product was determined by use of the Farbstott-Kongentrat reagent (Bio-Rad, Watford, United Kingdom). 0 side chain preparation. A total of 10 mg of purified LPS was subjected to mild acid hydrolysis in 1% acetic acid for 90 min at 100°C under nitrogen, and lipid A was removed with hexane. The resulting crude 0 side chain was rotary evaporated and resuspended in 1 ml of pyridinium acetate (pH 5.3; 4 ml of pyridine and 10 ml of acetic acid made to 1 liter with distilled water), and the solution was applied to a column (25 by 1.6 cm) of Sephadex G50 (Sigma, Poole, United Kingdom). Sixty 1-ml fractions were collected and assayed for carbohydrate by the phenol-sulfuric acid method described by Dubois et al. (6); and the peak fractions were pooled, dried by rotary evaporation, and resuspended in PBS. Production of monoclonal antibodies. Female BALB/c mice (age, 6 to 9 weeks) were immunized with 50 p1l of 105 heat-killed F. tularensis LVS organisms per ml at weekly intervals for 28 days, followed by 50 ,ul of 1 mg of F. tularensis LVS LPS per ml at weekly intervals for 5 weeks. Fusion and cloning were carried out as described elsewhere for V. cholerae LPS (12). Briefly, B lymphocytes from immunized mice were fused with cells from the mouse myeloma cell line SP2/0-Agl4 (21) by using polyethylene glycol 1500 as the fusion agent. Primary selection of hybrids was performed by growing the cells in 96-well microtiter plates on hypoxanthine-aminopterin-thymidine medium (12) at 37°C with 80% humidity and 5% C02; antibody-producing hybrid cells of interest were recloned by limiting dilution. Antibodies were produced in pristane (Sigma)-primed mice (17) by injecting 5 x 106 hybridoma cells intraperitoneally; the resulting ascites antibodies were stored at -20°C. Indirect ELISA. A total of 100 pAl of 10 ,g of LPS in antigen-binding buffer (50 mmol carbonate buffer; pH 9.6) per ml or 100 RI of F. tularensis at various concentrations in PBS was added to each well of a 96-well microtiter plate (M 129 A; Dynatech Irvine, United Kingdom) and incubated at 4°C overnight. After washing with PBS, the wells were filled with BLOTTO (1% milk powder in PBS) and incubated for 1 h at 37°C to block nonspecific binding sites. After three rinses with PBS containing 0.05% Tween 20 (PBS-Tween), 100 1±1 of antibody diluted in BLOTTO was added to each well and incubated for 1 h at 37°C. The plate was rinsed again three times in PBS-Tween, and 100 ,ul of horseradish peroxidase goat anti-mouse immunoglobulin (Sigma) diluted in BLOTTO (1/1,000) was added to each well and incubated for 1 h at 37°C. The plate was washed five times in PBS-Tween, and 100 RI of enzyme substrate [55 mg of 2,2'-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) in 56 ml of 0.1 M citric acid-44 ml of 0.2 M Na2HPO4-10 pI of H202; pH 5.51 was added to each well. The optical density after 1 h at 20°C was read in a Dynatech microplate reader at 414 nm. A reading of 0
0
0
x 106
x 106 x 106 x 106 x 106 x 106 x 106 x 106 x 106 x 106
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a 0
U)
-,00
°
°O,O-C
0.4-
cn n
010-0
0.3 +
0.2 0.4
10
1 0
Side chain
~
~
100
~
1 000
(/ig/ml)
FIG. 1. An inhibition ELISA was completed as described in the
a An optical density at 414 nm of >0.1 was used.
text. Before the primary antibody was added to the microtiter plate,
it was incubated with various concentrations of 0 side chain from F. tularensis LPS. 0, FT14; 0, FT2F11.
After treatment with chemicals, the antigens were evaluated for their immunological activities. RESULTS Purity of extracted LPS. LPS extracted from F. tularensis LVS was of high purity. Spectrophotometric analysis showed that there was no detectable nucleic acid and only 0.75% protein contamination. The 0 side chain was confined to the peak eluting with the void volume of the Sephadex column, with each 10 mg of purified LPS producing a yield of 1.3 mg of the 0 side chain. Characteristics of monoclonal antibodies against LPS. From the initial screen, two lines of hybridoma cells producing antibody to F. tularensis LVS LPS were isolated and cloned. The antibodies were designated FT14 and FT2F11, and in an indirect ELISA, both were able to detect approximately 3 pg of F. tularensis LPS. Both FT14 and FT2F11 were shown to be IgM antibodies by ELISA. To test the sensitivity of the antibodies in detecting the whole bacterial cells used as antigens in the indirect ELISA, 10 different strains of F. TABLE 2. ELISA inhibition of clones FT2F11 and FT14 with LPS from different strains used as inhibitors Inhibitor (LPS)U
Escherichia coli 055:B5 Escherichia coli 0111:B4 Escherichia coli 0127:B8 Escherichia coli 0128:B12 Escherichia coli K235 Klebsiella pneumoniae Pseudomonas aeruginosa S. abortus equi Salmonella enteritidis Salmonella minnesota Salmonella typhimurium Salmonella typhosa Serratia marcescens
Shigella flexneri Vibrio cholerae Inaba Francisella tularensis LVS
50%o Inhibitory value (~Lg/ml)b FT2F11
FT14
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 20
>1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 100 15
All LPSs except the F. tularensis LVS LPS were commercial preparations. b Concentration of LPS required to reduce the optical density by 50%o. a
tularensis were examined (Table 1). FT2F11 was less sensitive, with 105 organisms being required for a positive identification, whereas FT14 was able to detect 103 organisms. To indicate specificity, FT2F11 and FT14 were reacted with 16 commercially prepared LPS samples from 5 strains of E. coli and 10 species of other gram-negative bacteria, as well as with our LPS from F. tularensis LVS; residual antibody was measured by ELISA. None of the commercial preparations was able to produce a 50%o inhibition value with FT14, whereas this could be achieved with 20 ,ug of F. tularensis LVS LPS per ml. FT2F11 was inhibited by V. cholerae Inaba LPS, but a high concentration, 125 ,ug/ml, was required for 50% inhibition, whereas only 15 ,ug/ml was required to produce the same level of inhibition for F. tularensis LPS (Table 2). When FT14 was incubated with the isolated 0 side chain and residual antibody activity was measured by ELISA, there was a sharp fall in antibody titer compared with that of antibody incubated with PBS, indicating that it is directed against 0 side chain components. By contrast, the titer of FT2F11 was only slightly reduced even after incubation with a large amount of 0 side chain, showing that this antibody is directed against other epitopes of the LPS (Fig. 1). When the antibodies were incubated with whole organisms of a number of other gram negative species, there were no falls in titer (Table 3). Comparison of antibodies by competitive ELISA. To deter-
TABLE 3. Cross-reaction studies with gram-negative bacteria by an inhibition ELISA Bacteria
Yersinia pestis TS Yersinia pestis EV76 Brucella suis Escherichia coli 018 K-
Salmonella typhimurium ATCC 13311 Legionella pneumophila Vibrio cholerae Inaba Francisella tularensis Schu 4
No. of organisms/ml required to achieve a 50% inhibition of color
FT2F11
FT14
>107
>107
i04
102
>107 >107 >107 >107 >107 >107
>107 >107 >107 >107 >107 >107
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J. CLIN. MICROBIOL.
FULOP ET AL. 1.5 T
A B C O
E
1.0
o I/ .--\._ X
I
a)
C:0
E.0
0
.0
0.5 t
a ~~~~~~~ 0.:
v
n~~~~~"--
., 5
10
1000 100 Reciprocal Antibody Titre x 101
10000
FIG. 2. Competition ELISA. Peroxidase-labeled FT2F11 was used as the primary antibody, and either unlabeled FT14 (0) or FT2F11 (0) was used as the second antibody. The ELISA was then developed as described in the text.
mine whether monoclonal antibodies FT14 and FT2F11 competed for the same epitope, a competitive ELISA was devised. Unlabeled FT14 (ascites) antibody was mixed with purified peroxidase-labeled FT2F11, and the mixture was titrated in plates coated with F. tularensis LPS; there was no reduction in titer compared with that of FT2F11 alone. As a positive control, labeled and unlabeled FT2F11 was titrated in the same way; inhibition of color development occurred at titers of 1:10,000 and below (Fig. 2). Comparison of antibodies by gel electrophoresis and immunoblotting. Silver staining of F. tularensis LPS showed the typical banding pattern of the repeating 0 side chain of the structure as seen with other gram-negative bacteria such as E. coli and S. typhimurium (Fig. 3). No bands were visible following staining with Coomassie blue. Western immunoblotting gave a characteristic banding pattern when FT14 was used as the primary antibody (Fig. 4), but no banding
FIG. 4. Western immunoblot of F. tularensis LVS LPS probed with monoclonal antibodies FT2F11 (lanes A and B) and FT14 (lanes C and D).
was seen when FT2F11 was used as the primary antibody. To determine the reason for the failure of FT2F11 to immunoblot, a chemical inhibition ELISA was devised. LPS from F. tularensis was first boiled in the sample buffer used for the preparation of specimens for SDS-PAGE and was then reacted with the two antibodies. FT2F11 failed to react with the treated LPS, but FT14 was reacted normally. If LPS was treated with the separate components of the sample buffer, it was found that SDS was the chemical which destroyed its ability to react with FT2F11. Triton X-100 produced a similar lack of reaction with FT2F11. Treatment of LPS with 5% (vol/vol) formaldehyde and glutaraldehyde had no effect on the ability of either FT2F11 or FT14 to recognize F. tularensis LPS (Table 4).
DISCUSSION
Most studies on the antigenic determinants of F. tularensis have used ill-defined antigens (13), although one used purified LPS as the antigen in an ELISA to diagnose tularemia (3). If diagnosis of the disease is the sole requirement, however, crude mixtures of antigens are easier to prepare and are effective (24), with reservations about a tendency of
TABLE 4. Effect of chemical modification of F. tularensis LPS on antibody recognition measured in an indirect ELISA Effecta Chemical FT14
FT2F11
SDS (2% [wt/vol]) Triton X-100 (2% [wt/vol])
-
+ +
Formaldehyde (5% [vol/vol]) Glutaraldehyde (5% [vol/vol])
-
2-p-Mercaptoethanol (5% [vol/vol])
FIG. 3. SDS-PAGE of LPS isolated from F. tularensis LVS and visualized by silver staining.
-
a -, chemical has no effect on the ability of antibody to recognize the LPS; +, chemical modified LPS so that antibody failed to recognize the LPS.
VOL. 29, 1991
MONOCLONAL ANTIBODIES AGAINST F. TULARENSIS LPS
the antigens to cross-react with antibodies invoked by infection with some other gram-negative bacteria. The cross-reaction studies with either whole killed cells or commercially prepared LPS showed that the epitope that FT14 recognized was unique to F. tularensis and that FT2F11 cross-reacted with V. cholerae LPS at a low avidity. We conclude that the epitopes that the monoclonal antibodies bind to are specific to F. tularensis but are conserved throughout the genus Francisella. However, all the strains were grown under the same cultural conditions. It is possible that alteration of growth conditions could modify the structure of the LPS, which has been shown to occur with some strains of E. coli (4). Diagnosis of tularemia has been based on immunoserological methods, because direct culture of specimens from infected sites has proved to be difficult and is a potential hazard to laboratory personnel (25). The ability of FT14 to detect low numbers of bacteria could be used to detect F. tularensis directly in infected sites by labeling the antibody with a fluorescent tag. The specificity of the antibodies could also be important in preventing incorrect diagnosis, because it has been reported that antibodies against L. pneumophila gave a false diagnosis of Legionnaires disease when the patient was suffering from tularemia (18). Inhibition studies showed that FT14 reacted strongly with isolated 0 side chain but that FT2F11 did not, indicating that the latter was directed against components of LPS other than the 0 side chain, and the results of the competition assay between the two monoclonal antibodies showed clearly that they do not compete for the same epitope. Studies with an acapsular rough mutant of F. tularensis (Cap-) indicated that the presence of a capsule aided survival (19). However, the study did not investigate the possibility that the rough colonial appearance was caused by structural changes in the LPS, although rough LPS strains have not been reported. FT14 reacted as strongly with the Cap- strain as it did with the other strains, implying that this mutant has a functional 0 side chain and therefore is not a rough LPS strain. Other rough colonial mutants have been isolated (7), but their LPSs have not been analyzed. FT14 could prove to be valuable in isolating and identifying rough LPS mutants as an aid to evaluating the role of the 0 side chain in the disease process. The results of the chemical inhibition ELISA show clearly that detergents affected LPS to such an extent that FT2F11 was prevented from recognizing it either by ELISA or Western blotting (immunoblotting). This destruction of biological activity was not only confined to SDS, which carries a strong negative charge, but was also manifest with Triton X-100, which is nonionic. Formaldehyde and glutaraldehyde, which cross-link protein, did not affect the binding of FT2F11, showing that this antibody is not directed against the small contaminating protein part of the LPS preparation. The most likely scenario is that the detergents caused structural changes in lipid A which induced a conformational change in the core but not in the 0 side chain. Because the core region is less exposed on the bacterial surface, it is to be expected that antibodies directed against this region would be less sensitive in detecting whole organisms. Before FT14 and FT2F11 are incorporated into a kit to rapidly identify F. tularensis, we intend to test them against wild-type strains isolated from patients and the environment. REFERENCES 1. Bell, J. F., C. R. Owen, and C. C. Larson. 1955. Virulence of Bacterium tularense: a study of Bacterium tularense in mice, guinea pigs, and rabbits. J. Infect. Dis. 97:162.
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2. Bogard, W., D. L. Dunn, K. Abernethy, C. Kilgarriff, and P. C. Kung. 1987. Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity. Infect. Immun. 55:899-908. 3. Carlsson, H. E., A. A. Lindberg, G. Lindberg, B. Hederstedt, K.-A. Karisson, and B. 0. Agell. 1979. Enzyme-linked immunosorbent assay for immunological diagnosis of human tularemia. J. Clin. Microbiol. 10:615-621. 4. Dodds, K. L., M. B. Perry, and I. J. McDonald. 1987. Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli 0157:H7 as a function of growth rate and growth-limiting nutrient. Can. J. Microbiol. 33:452-458. 5. Downs, C. M., L. L. Coriell, S. S. Chapman, and A. Klauber. 1947. The cultivation of Bacterium tularense in embryonated eggs. J. Bacteriol. 53:89-100. 6. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related compounds. Anal. Chem. 28:350-356. 7. Eigelsbach, H. T., W. Braun, and R. D. Herring. 1951. Studies on the variation of Bacterium tularense. J. Bacteriol. 61:557569. 8. Eliwood, D. C. 1970. The distribution of 2-keto-3-deoxy-octonic acid in bacterial walls. J. Gen. Microbiol. 60:373-380. 9. Forsman, M., G. Sandstrom, and B. Jaurin. 1990. Identification of Francisella species and discrimination of type A and type B strains of F. tularensis by 16S rRNA analysis. Appl. Environ. Microbiol. 56:949-955. 10. Greisman, S., R. B. Hornick, F. A. Carozza, and T. E. Woodward. 1963. The role of endotoxin during typhoid fever and tularemia in man. I. Acquisition of tolerance to endotoxin. J. Clin. Invest. 42:1064-1075. 11. Gustafsson, B., U. Lindquist, and M. Andersson. 1988. Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide. J. Clin. Microbiol. 26:188-193. 12. Gustafsson, B., A. Rosen, and T. Holme. 1982. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide. Infect. Immun. 38:449-454. 13. Holm, S. E., A. Tarnvik, and G. Sandstrom. 1980. Antigenic composition of a vaccine strain of Francisella tularensis. Int. Arch. Allergy Appl. Immun. 61:136-144. 14. Hood, A. M. 1977. Virulence factors of Francisella tularensis. J. Hyg. 79:47-60. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London) 227: 680-685. 16. Nakane, P. 1979. In R. M. Nakamura, W. R. Dito, and E. S. Tucker (ed.), Immunoassays in the clinical laboratory, p. 81. Alan R. Liss, New York. 17. Potter, M., J. G. Pumphrey, and J. L. Walters. 1972. Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J. Natl. Cancer Inst. 49:305-308. 18. Roy, T., D. Fleming, and W. H. Anderson. 1989. Tularemic pneumonia mimicking Legionnaires' disease with false positive direct fluorescent antibody stains for legionella. South. Med. J. 82:1429-1431. 19. Sandstrom, G., S. Lofgren, and A. Tarnvik. 1988. A capsuledeficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by polymophonuclear leukocytes. Infect. Immun. 56:1194-1202. 20. Sandstrom, G., A. Tarnvik, H. Wolf-Watz, and S. Lofgren. 1984. Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect. Immun. 45:101-106. 21. Schulman, M., C. D. Wilde, and G. Kohler. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276:269-270. 22. Smith, A. R. W., S. Zamze, and R. C. Hignett. 1985. Composition of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum and its digestion by bacteriophage A7. J. Gen. Microbiol. 131:963-974. 23. Tsang, R. S. W., K. H. Chan, P. Y. Chan, K. C. Wan, M. H. Ng,
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J. CLIN. MICROBIOL. 25. Weaver, R. E., D. G. Holis, and E. J. Bottone. 1985. Gramnegative fermentation bacteria and Francisella tularensis, p. 316-319. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 26. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
