Production of Monoclonal Antibodies against Serotype Strains of ...

INFECTION AND IMMUNITY, Nov. 1987, p. 2854-2856

Vol. 55, No. 11

0019-9567/87/112854-03$02.00/0 Copyright © 1987, American Society for Microbiology

Production of Monoclonal Antibodies against Serotype Strains of Pseudomonas aeruginosa Department

JOSEPH S. LAM,* LESLIE A. MAcDONALD, AND MICHELE Y. C. LAM College of Biological Science, University of Guelph, Gielph, Ontario, Canada NJG 2W1

of Microbiology,

Received 14 May 1987/Accepted 16 July 1987

A panel of 22 monoclonal antibodies against 8 of the 17 International Antigenic Typing Scheme (IATS) serotypes of Pseudomonas aeruginosa was produced. The antibodies were characterized for cross-reactivities, isotypes, titers, and epitope specificities. The results complemented those of our previous study and marked the completion of a set of monoclonal antibodies for serotyping P. aeruginosa.

The most complete serotyping system of Pseudomonas aeruginosa, the International Antigenic Typing Scheme (IATS), consists of 17 standard 0 serotypes (8). A set of 0 antisera is commercially available from Difco Laboratories, Detroit, Mich. Although these polyclonal antibodies are effective in typing most clinical isolates, with the exception of cystic fibrosis isolates, they are known to vary in titer and specificity from batch to batch. Also, cross-reactions among certain serotypes are not uncommon (8). Hybridoma technology (6) has been well exploited in various areas of biological science, and monoclonal antibodies (MAbs) would be ideal to replace rabbit sera as serotyping reagents. Recently, we reported the production of MAbs against 13 serotypes of P. aeruginosa (7). This antibody panel contained strongly agglutinating MAbs suitable for serotyping 01, 03, 05, 06, 07, 08, 09, 011, 012, and 014 isolates. We now report the production and characterization of a panel of MAbs against P. aeruginosa serotypes 02, 04, 010, 013, 015, 016, and 017 and two additional MAbs against serotype 014. The P. aeruginosa serotype 01 to 017 strains used and their culture conditions were described in detail in our previous publication (7). Methods for the preparation and characterization of the MAbs using NS-1 myeloma cells to fuse with primed splenocytes were also exactly as described earlier (7). A short immunization schedule was used in which we injected BALB/c mice (Charles River Canada Inc., St. Constant, Quebec, Canada) intraperitoneally with 0.2 ml of a 1:1 mixture of 107 Formalin-fixed P. aeruginosa cells and Freund incomplete adjuvant (Difco) on day 1, followed by an intravenous booster of the same immunogen without adjuvant on day 12. The animals were sacrificed 4 days later, and the splenocytes were harvested. This was done to enhance the production of immunoglobulin M (IgM) antibodies, which should be better agglutinating antibodies than the IgG isotypes. Cross-reactions of each MAb with all 17 serotypes of P. aeruginosa were examined by both agglutination and Western immunoblotting (1, 12). Outer membranes of P. aeruginosa were isolated by the sucrose gradient method of Hancock and Carey (5), and the lipopolysaccharide (LPS) extraction method used was as described by Darveau and Hancock (2). Both types of antigens were used in immunoblotting, and the LPSs were also used in enzyme-linked immunosorbent assays (ELISAs) to screen for positive hybridomas. *

The short immunization schedule proved to be effective in that 17 of the 22 MAbs reported were IgM and 5 were IgG3 (Table 1). All of the IgM antibodies except MF60-2 and MF60-7 agglutinated strongly with homologous immunizing strains of P. aerui(ginosa. Both antibodies reacted with purified LPS of P. aeruginosa serotype 04 in the ELISA even though they did not agglutinate the bacterial cells. The specificity of these antibodies is possibly for a minor epitope of 04 LPS which is not readily accessible on the surface of live cells. Interestingly, MAbs MF57-1 and MF57-2 prepared against serotype 013 and MF58-2, MF58-3, and MF58-4 prepared against serotype 015 were of the IgG3 isotype. However, this was not surprising, because LPS is known to stimulate IgM and IgG3 responses in mice (3). When we examined the cross-reactivities of the antibodies with all 17 serotypes of P. aeruginosa, only 5 MAbs were found to cross-react with other serotype strains (Table 1). MAb MF71-1 prepared against the serotype 02 strain crossreacted with the 016 strain. In contrast, MAb MF47-4, which was specific for serotype 016, did not cross-react with the 02 strain. Thus, 02 and 016 LPSs might share a minor epitope, while their major epitopes differ. MAbs MF57-1 and MF57-2 prepared against serotype 013 agglutinated and immunoblotted ladderlike bands of repeating 0-antigen epitopes of both 013 and 014 LPSs. Similar results were obtained when MAbs MF55-1 and MF55-2 prepared against serotype 014 were characterized. Contrary to this, MAb MF45-4 from our previous studies (7) reacted only with serotype 014. Thus 013 and 014 strains might share a common 0-antigen epitope, while there still are significant differences in their LPS structures. This finding may explain why cross-reactions were often seen between these two serotypes in earlier reports (8). Apart from these cross-reacting antibodies, 17 of the 22 MAbs shown in Table 1 were serotype specific. Thus, we complemented our earlier study by successfully producing agglutinating MAbs against serotypes 02, 04, 010, 013, 015, 016, and 017 (Table 1). To better understand the epitope specificities of these MAbs, Western blotting with both purified LPS and outer membranes was performed. Most (14 of 22) of the strongly agglutinating and serotype-specific MAbs blotted with the ladderlike bands of LPS from about 10 repeating units to the top of the high-molecular-weight (HMW) 0-antigen bands (Fig. 1). This observation was in agreement with our earlier findings (7) as well as the findings of Gaston and co-workers (4) and Sadoff and co-workers (11). The two MAbs, MF71-1

Corresponding author. 2854

VOL. 55, 1987

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TABLE 1. Characterization of MAbs against P. aeriginosa serotype strains MAb

Specificitya

Isotype

agglutination

MF71-1 MF71-2e

02 02

IgM IgM

+ +

5 6

MF60-1 MF60-2 MF60-3 MF60-4 MF60-5e MF60-7

04 04 04 04 04 04

IgM 1gM IgM IgM 1gM IgM

+ + + + -

2 >8 6 6 >8 4

MF76-1 MF76-2e

010 010

lgM IgM

+ +

4 8

MF57-1 MF57-2e

013 013

IgG3 lgG3

+ +

2 3

014 014

O-Ag O-Ag

MF55-1 MF55-2

014 014

IgM IgM

+ +

6 5

013 013

O-Ag O-Ag

MF58-2e MF58-3 MF58-4

015 015 015

IgG3 IgG3 IgG3

+ + +

1 4 1

HMW LPS HMW LPS HMW LPS

MF47-4e

016

1gM

+

3

Core, OMPs

MF59-1 MF59-2e MF59-3

017 017 017 017

IgM IgM IgM IgM

+ + + +

2 6 4 2

O-Ag O-Ag O-Ag O-Ag

ELISA titerh

Cross-reaction'

Immunoblotd

016

HMW LPS HMW LPS

O-Ag OMPs

O-Ag O-Ag O-Ag OMPs

O-Ag O-Ag

MF59-4 "0 specificity represented positive reactivity with the LPS of the designated 0 group in an ELISA. Loglo ELISA titer was determined as the highest serial 10-fold dilution of the ascitic fluid that gave an optical density at 405 nm of .0.1 when reacted with 0.5 p.g of LPS. Alkaline phosphatase and p-nitrophenyl-phosphate was the enzyme-substrate system used. Cross-reactions of each MAb with other serotypes were tested by using both agglutination reactions and Western blots with purified LPS. d Immunoblots were done separately with both LPS and outer membranes of the homologous serotype strain. O-Ag, MAb interacted with ladderlike LPS bands; HMW LPS, MAb interacted with the HMW 0-antigen bands but not the lower ladderlike bands; OMPs, MAb interacted with major outer membrane bands; Core, MAb interacted with the core region of LPS. e Antibodies selected to represent a complete panel of MAbs for serotyping P. aeriuginosa 01 to 017. Other MAbs from our previous publication (7) included in this panel are MF25-1 (01), MF57-9 (03), MF15-2 (05), MF23-2 (06), MF29-2 (07), MF65-1 (08; not previously reported), MF43-3 (09), MF55-1 (011), MF35-4 (012), and MF45-4 (014) (the 0-antigen specificities of these MAbs are shown in parentheses).

and MF71-2, prepared against serotype 02 were found to recognize the more fully capped HMW 0-antigen bands but not the ladderlike 0-repeating units of LPS lower down on the gel. The 015-specific MAbs, MF58-2, MF58-3, and MF58-4, gave similar blotting results against the HMW 0-antigen bands of their homologous 015 LPS (Fig. 1). The latter was to be expected because such blotting profiles matched the silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of 015 LPS in which only two thick bands, a HMW 0-antigen band on top and a low-molecular-weight LPS band depicting the LPS core, were seen (9). The preferential reactivities of these MAbs with HMW LPS were likely due to the fact that the molecules in this region of the gel contained more repeating 0-antigen units which were more closely spaced. MAbs MF60-2 and MF60-7 prepared against serotype 04 did not blot with 0-antigen bands even though the antibodies gave ELISA titers of >8 and 4, respectively, when reacting to 0.5 ,ug of pure LPS. However, when outer membranes were used in the Western blots, these two antibodies were found to bind each of the major outer membrane protein bands; in particular, strong reactions with proteins F, G, and H2 were observed. This type of reaction is analogous to our earlier observations of nonagglutinating MAbs prepared against

other serotype strains of P. aeruginosa (7). Because of their reactivity with purified LPS and the blotting profile they

produced with outer membrane proteins, these antibodies likely recognize LPS structures that are closely associated with outer membrane protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as observed by Poxton et al. (10). The reactivities of 016-specific MAb MF47-4 in immunoblots were somewhat peculiar. This MAb blotted with the core region of the LPS but also interacted with each major outer membrane protein band. This is in contrast to most serotype-specific MAbs which recognize 0antigen bands. Thus, the epitope that this antibody recognized would be either at or near the core of the LPS and represented the LPS region that was also closely associated with outer membrane proteins. The fact that MAb MF47-4 agglutinated 016 cells illustrated the cell surface accessibility of such an epitope. We performed slide agglutination tests on 50 typable clinical isolates of P. aeruginosa using our complete panel of anti-Ol through anti-017 MAbs (see footnote e to Table 1) and found the results to conform directly with serotyping using the Difco antisera. The use of MAbs has also greatly reduced cross-reactions in serotyping P. aeruginosa which now occur only between 07 and 08 isolates and between 013 and 014 isolates, whereas in the earlier report by Liu et al. (8) cross-reactions were observed between serotypes 02 and 05, 07 and 08, 09 and 010, 013 and 014, 016 and 02, and 016 and 05. Further work is necessary to produce

2856

NOTES

INFECT. IMMUN.

A

B

FIG. 1. Western immunoblots of P. aeruginosa LPS. Lane A, 25 Fxg of LPS of serotype 04 interacting with MAb MF60-5. Note the reactivity of the ladderlike 0-antigen bands. Lane B, 25 ,ug of LPS of serotype 015 interacting with MAb MF58-3. Note the reactivity of the HMW band of the 0 antigen only. The blots were developed with an alkaline phosphatase-conjugated second antibody and the TR fast red substrate system as described previously (7).

antibodies specific for each of the 07, 08, and 013 serotypes. Although more tests with a larger number of clinical isolates of P. aeruginova from different medical centers need to be done in the near future, we believe that we have fulfilled our aim by completing the preparation of an entire set of well-characterized MAbs useful for P. aeruginosa serotyping. Since the publication of our previous paper on MAb production (7), some of our MAbs have been used by at least five other laboratories throughout North America for various research purposes. We thank Wendy Johnson for typing the manuscript and Spencer Lee for supplying the NS-1 myeloma cell line. This work was supported by grants to J.S.L. from both the Medical Research Council (MA9204) and the Natural Sciences and Engineering Research Council (NSERC) (A3020) of Canada. M.Y.C.L. is the recipient of an NSERC postgraduate studentship.

LITERATURE CITED

1. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radiodinated protein A. Anal. Biochem. 112:195-203. 2. Darveau, R. P., and R. E. W. Hancock. 1983. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseuidoinonas aeruginosa and Salmonella tvphimurium strains. J. Bacteriol. 155:831-838. 3. Davie, J. M. 1982. Antipolysaccharide immunity in man and animals, p. 129-134. In S. H. Sell and P. F. Wright (ed.), Haeemophiluis influenzae: epidemiology, immunology and prevention of disease. Elsevier Biomedical, New York. 4. Gaston, M. A., T. A. Vale, B. Wright, P. Cox, and T. L. Pitt. 1986. Monoclonal antibodies to the surface antigens of Pseudoinonas aeruiginosa. FEMS Microbiol. Lett. 37:357-361. 5. Hancock, R. E. W., and A. M. Carey. 1979. Outer membrane of Pseudomonas aerui,ginosa: heat- and 2-mercaptoethanol-modifiable proteins. J. Bacteriol. 140:902-910. 6. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 7. Lam, J. S., L. A. MacDonald, M. Y. C. Lam, L. G. M. Duchesne, and G. G. Southam. 1987. Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa. Infect. Immun. 55:1051-1057. 8. Liu, P. V., H. Matsumoto, H. Kusama, and T. Bergan. 1983. Survey of heat-stable, major somatic antigens of Pseudomonas aeruginosa. Int. J. Syst. Bacteriol. 33:256-264. 9. Maclntyre, S., T. McVeigh, and P. Owen. 1986. Immunochemical and biochemical analysis of the polyvalent Pseuidomonas aeruginosa vaccine PEV. Infect. Immun. 51:675-686. 10. Poxton, I. R., G. T. Bell, and G. R. Barclay. 1985. The association of SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseuidomonas aeruginosa as revealed by monoclonal antibodies and Western blotting. FEMS Microbiol. Lett. 27:247-251. 11. Sadoff, J. C., D. C. Wright, S. Futrovsky, H. Sidberry, H. Collins, and B. Kaufmann. 1985. Characterization of mouse monoclonal antibodies directed against Pseuidomonas aeraginosa lipopolysaccharides. Antibiot. Chemother. 36:134146. 12. Towbin, M., 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.