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Monoclonal antibodies specific for Shigella dysenteriae serotype 13 production, characterization, and diagnostic application
145
DIAGN MICROBIOLINFECT DIS 1994;18:145-149
Monoclonal Antibodies Specific for Shigella dysenteriae Serotype 13 Production, Characterization, and Diagnostic Application Firdausi Qadri, Tasnim Azim, Anwar Hossain, Ashrafuzzaman Chowdhury, and M. John Albert
Three mouse monoclonal antibodies (mAbs) (ICL3, ICL4, and ICL5) were produced that specifically recognized the lipopolysaccharide antigen of the newly recognized Shigella dysenteriae serotype-13 strain. All three mAbs reacted with all nine reference isolates of S. dysenteriae 13 in different tests. The mAbs also detected colonies of S. dysenteriae-13 isolates by direct slide agglutination test. The mAbs also reacted with the
reference Escherichia coli 0150 strain and showed its close antigenic relationship with S. dysenteriae 13. Use of these mAbs in our clinical laboratory during an 8-month period detected three S. dysenteriae-13 isolates that were also detected by a polyclonal rabbit antiserum. It should now be possible to define the epidemiologic importance of S. dysenteriae serotype 13 in diarrhea by using these mAbs.
INTRODUCTION
MATERIALS A N D METHODS
Shigella dysenteriae 13 is one of the recently recognized serotypes of Shigella in Bangladesh and other parts of the world from patients with bloody, mucoid diarrhea (Ansaruzzaman et al., 1993). Highly specific monoclonal antibodies (mAbs) have been useful for serotyping of isolates of S. dysenteriae type 1 and Shigella flexneri spp. (Carlin and Lindberg, 1983, 1986, and 1987; Islam and Stimson, 1987 and 1989). We report here the production and characterization of mAbs to the lipopolysaccharide (LPS) antigen of S. dysenteriae 13, which could be used to define the epidemiologic importance of this new serotype in the causation of diarrhea.
From the International Centre for Diarrhoeal Disease Research-Bangladesh, Dhaka, Bangladesh. Address reprint requests to Dr. F. Qadri, Laboratory Sciences Division, ICDDR,B, GPO Box 128, Dhaka 1000, Bangladesh. Received 13 September 1993; accepted 10 December 1993. © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/94/$7.00
Bacterial Strains, Media, and Antiserum Two strains of S. dysenteriae 13 (598-77 and 2489-78) were from the Centers for Disease Control (Atlanta, GA, USA), one strain (4/738/92) was from the National Bacteriological Laboratory (Stockholm, Sweden), and six strains (11357, 11389, 13561, 15357, 2845, and 4449) were recent isolates from Bangladeshi patients with diarrhea (Ansaruzzaman et al., 1993). An Escherichia coli serogroup 0150 strain that cross-reacts with S. dysenteriae 13 (WathenGrady et al., 1990; Ansaruzzaman et al., 1993) was obtained from the Central Public Health Laboratory (Colindale, England). Three strains each of S. dysenteriae serotypes 1-12 were from the National Bacteriological Laboratory (Stockholm), the Central Public Health Laboratory (Colindale), and the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), culture collection. Three strains each of S. flexneri (serotypes la, lb, 2a, 2b, 3a, 4a, and Y), and three strains each of Shigella boydii 1, S. sonnei (Form-I), Salmonella typhimurium, E. coli, Klebsiella sp., and Enterobacter sp., were also used as controls to check for cross-reactivity in the
146
study and were from our culture collection. A previously prepared rabbit polyclonal antiserum to S. dysenteriae 13 was used for comparative studies (Ansaruzzaman et al., 1993). For broth cultures, bacteria were grown in trypticase soy broth supplemented with 0.6% yeast extract (TSBY) (Difco, USA) at 37°C for 18 h in a shaker incubator (100 rpm/min).
Preparation of Lipopolysaccharide and Other Antigens of Bacteria LPS was purified by published methods (Westphal and Jann, 1965). Heat-killed cells were prepared by culturing bacteria in TSBY, washing in phosphatebuffered saline (PBS) (10 raM, pH 7.2), and heating in a water bath at 80°C for 1 h. After washing the cells in PBS, they were resuspended in PBS and used as antigen as described below. Acetone-treated whole cell extract was prepared from bacteria after culturing trypticase soy (TS) agar (Difco) as described previously (Edwards and Ewing, 1972).
Immunization of Mice and Production of Hybridoma Female BALB/c mice were immunized with 50 ~g of acetone extract of S. dysenteriae-13 strain 598-77 four times at weekly intervals as described previously (Qadri et al., 1993). Spleen cells from two immunized BALB/c mice were fused with SP2/0 myeloma cells (Carlin and Lindberg, 1986).
Screening of Hybridomas by ELISA and Production of Ascites Fluid S u p e r n a t a n t fluids harvested from hybridomas were tested in an enzyme-linked immunosorbent assay (ELISA) (Carlin and Lindberg, 1986) for reactivity against all nine S. dysenteriae-13 strains, the E. coli serogroup 0150 strain, and all control bacteria just described. Antigens used were acetone extract (50 ~g/ml), LPS (10 ~g/ml), or boiled cells (10s colony-forming units/ml) of bacteria. The hybridomas secreting antibody specific to LPS of S. dysenteriae 13 were cloned twice by limiting dilution. The ascites fluids produced in pristane-primed mice were diluted in PBS-Tween (0.05%) containing 0.1% bovine serum albumin and screened for reactivity to LPS of S. dysenteriae serotype-13 strains (598-77 and 4449) and other antigens described above in ELISA. A mAb, 4D3, of isotype IgG1 to Shiga toxin (Donohue-Rolfe et al., 1984) was used as a negative control in ELISAs. To determine the isotype of mAbs, supernatant obtained from hybridoma-serum-free medium was tested by ELISA (Carlin and Lindberg, 1986).
F. Qadri et al.
Lipopolysaccharide Patterns and Immunoblot LPS from S. dysenteriae serotype-13 strain (598-77 or 4449) was separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel (SDS--PAGE) (2.5 ~g/well) (Laemmli, 1970) and silver stained (Hitchcock and Brown, 1983). From duplicate unstained gels, the separated antigens were transferred to nitrocellulose membranes (Towbin et al., 1982) and treated with ascites fluid at 1:500 dilutions. The reaction was visualized as described previously (Carlin and Lindberg, 1986). mAb 4D3 was used as a control antibody to check for nonspecific reactions, while LPS obtained from an E. coli 08:K25 strain was used to check for nonspecific binding of S. dysenteriae-13specific mAbs.
Immunofluorescence Test Specificity of S. dysenteriae-13 mAbs was tested by an indirect immunofluorescence test (Weintraub et al., 1979). Heat-killed bacteria on glass slides mixed with mAbs (1:100 to 1:500 dilution in PBS) were treated with fluorscein-isothiocyanate-conjugated rabbit-anti-mouse immunoglobulin (Ig) (Silenus, Victoria, Australia) diluted 1:40 in PBS. The test bacteria included all isolates of S. dysenteriae 13. The negative controls included S. dysenteriae serotypes 1-12 and a mAb, MASF-1, specific for S. flexneri 1 (Carlin and Lindberg, 1986).
Slide Agglutination Test The ability of S. dysenteriae-13-specific mAbs to detect all nine S. dysenteriae-13 isolates and the single E. coli serogroup-0150 isolate was studied in a slide agglutination test with TSA agar (Difco)-grown bacteria at 37°C and diluted ascites fluid in PBS. Bacterial suspensions were made in 2 drops of physiologic saline placed on two different spots of a microscopic slide. To one suspension, 1 drop of ascites fluid was added and, to the other, saline was added. The slide was rocked gently, and clear agglutination within 2 min was considered positive. In addition, the ability of these mAbs to identify S. dysenteriae 13 was evaluated in our clinical laboratory over an 8-month period from November 1992 to July 1993. The earlier-listed control bacteria were also included. The test was carried out by at least two investigators independently.
RESULTS Production of Monoclonal Antibodies Specific for Lipopolysaccharide of Shigella dysenteriae 13 Three specific and stable clones were obtained: two mAbs, ICL3 and ICL4, were of IgM isotype and one mAb, ICL5, was of IgG1 isotype.
147
Use of Monoclonal Shigella Antibodies
The titers of ascites fluid corresponding to the five mAbs varied between 1.2 x 10~and 9.6 x 105 against LPS antigens from S. dysenteriae-13 strains 598-77 and 4449. All mAbs reacted in ELISA with heat-killed cells from the six Bangladeshi isolates and the three reference isolates of S. dysenteriae 13. They also reacted with LPS isolated from E. coli 0150. They did not react with any control bacteria.
A
B
",D
Lipopolysaccharide Pattern and Immunoblot The LPS separation pattern of S. dysenteriae-13 strain 598-77 on SDS-PAGE, which is presented in Figure 1 (lane A), showed the presence of the O-antigenic repeating units (ladderlike pattern in the upper portion of Figure 1, marked a) and the core polysaccharides (darkly staining portion in the bottom of Figure 1, marked b). In immunoblotting studies, all mAbs reacted with the O-antigenic polysaccharide (Figure 2, lane B; indicated by the arrow; data shown only for mAb ICL4). Nonspecific binding
A
a-tD
FIGURE 2 Immunoblot analysis of lipopolysaccharide (LPS) antigens separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis, blotted onto a nitrocellulose membrane, and probed with monoclonal antibody ICL4: lane A contains LPS from a negative control of Escherichia coli strain and lane B contains LPS from Shigella dysenteriae 13. The O-antigenic polysaccharide region of LPS is indicated by the arrow.
was not seen when the mAbs were tested against LPS from a negative control E. coli strain (Figure 2, lane A) or when mAb 4D3 was tested against LPS from S. dysenteriae 13 (data not shown).
Immunofluorescence Test The mAbs to S. dysenteriae 13 specifically recognized all isolates of S. dysenteriae 13 in this test. The optim u m dilution for mAbs ICL3 and ICL5 was 1:500, whereas it was 1:100 for mAb ICL4. None of the negative control bacteria reacted in this test. FIGURE 1 Pattern of separation of lipopolysaccharide (LPS) antigen from Shigella dysenteriae-13 strain 598-77. The antigen was separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis and visualized by silver staining. Lane A contains LPS from S. dysenteriae 13. Shown are O-antigenic polysaccharide (a) as well as core polysaccharide (b) indicated by arrows.
Slide Agglutination Test mAbs ICL3, ICL4, and ICL5 strongly agglutinated all S. dysenteriae-13 isolates and E. coli 0150. The optimum dilutions of these mAbs for agglutination tests were 1:200, 1:50, and 1:25, respectively. Non-
148
specific reaction was not detected with other bacteria tested. During the 8-month evaluation period of mAbs in the clinical laboratory, 35 Shigella-like colonies from diarrheal patients that did not agglutinate with polyclonal antisera to the conventional serotypes of shigellae were tested against S. dysenteriae-13 mAbs. Three of these isolates (nos. 18538, 19123, and 21333) turned out to be S. dysenteriae 13. The same three isolates only were identified as S. dysenteriae 13 by using the rabbit polyclonal antiserum w h e n tested independently. Identical results were obtained by the two different investigators.
DISCUSSION The results presented here show that ICL3-5 can be used for detection of isolates S. dysenteriae 13. They were highly specific, and the only other species of bacteria to react was E. coli 0150. Previous studies with rabbit polyclonal antiserum have suggested reciprocal cross-reactivities between S. dysenteriae 13 and E. coli 0150 (Wathen-Grady et al., 1990; Ansaruzzaman et al., 1993). This antigenic relatedness was demonstrated with all three mAbs of S. dysenteriae 13. Immunoblot studies suggested that these mAbs reacted with the epitopes on the polysaccharide side chain of the LPS antigen. Although these mAbs also recognize E. coli 0150, this will not limit their usefulness as diagnostic reagents, because we will be testing only pale colonies and not pink colonies from selective media used to isolate Shigella in the clinical laboratory (E. coli produce pink colonies due to fermentation of lactose, and Shigella produce pale colonies due to the absence of lactose fermentation in the selective media).
F. Qadri et al.
We have used these mAbs for 8 months (until this report was prepared) to screen for S. dysenteriae 13 in our clinical laboratory, and three isolates were identified. The same three isolates were also identified by the rabbit polyclonal antibody. Since the isolation rate of S. dysenteriae 13 was low during the study period, an extensive evaluation of the mAbs was not possible. The preliminary results obtained so far suggest, however, that these mAbs will be useful. Since S. dysenteriae 13 is a newly recognized serotype causing bloody, mucoid diarrhea, its worldwide importance has yet to be assessed. It should now be possible to define its epidemiologic importance with the help of these mAbs. The S. dysenteriae-13-specific mAbs now add to the list of other mAbs that have previously been proven to be useful for detection of other Shigella species (Carlin and Lindberg, 1983 and 1987; Islam and Stimson, 1987 and 1989).
This research was supported by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). The ICDDR,B is supported by countries and agencies that share its concern for the health problems of developing countries. Current donors include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Denmark, France, Japan, the Netherlands, Norway, Saudi Arabia, Sweden, Switzerland, the United Kingdom, and the United States; international organizations including the United Nations (UN) Children's Fund, the UN Development Program, the UN Population Fund, and the World Health Organization; and private foundations, including the Ford Foundation and the Sasakawa Foundation. We thank Mr. Manzurul Haque for secretarial assistance.
REFERENCES Ansaruzzaman M, Kibriya AKMG, Mitra AK, Sack RB, Albert MJ (1993) Isolation of Shigella dysenteriae serotypes 11, 12 and 13 from patients with diarrhea in Bangladesh. J Clin Microbiol 31:1392-1393. Carlin NIA, Lindberg AA (1983) Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri: clones binding to II, II:3, 4 and 7, 8 epitopes. J Clin Microbiol 18:1183-1189. Carlin NIA, Lindberg AA (1986) Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type 1 and type III, 6, 7, 8 antigens, group 6 antigens and a core epitope. Infect Immun 53:103-109. Carlin NIA, Lindberg AA (1987) Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV and VI antigen group 3, 4 antigen and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains. Infect Immun 55:14121420. Donohue-Rolfe A, Keusch GT, Edson C, Thorley-Lawson D, Jacewicz M (1984) Pathogenesis of Shigella diarrhea.
IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit specific monoclonal and polyclonal antibodies. J Exp Med 160:1767-1781. Edwards PR, Ewing WH (1972) In the genus Shigella. In The Identification of Enterobacteriaceae, 3rd ed. Minneapolis: Burgess, pp 108-142. Hitchcock PJ, Brown TM (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver stained polyacrylamide gels. J Bacteriol 154: 269-272. Islam MS, Stimson WH (1987) Production of monoclonal antibodies to Shigella: enzyme-linked immunosorbent assay for screening hybridoma antibodies with intact bacteria. Lett Appl Microbiol 4:85-89. Islam MS, Stimson WH (1989) Production and characterization of monoclonal antibodies with diagnostic potential against Shigella flexneri. J Clin Lab Immunol 29: 199-206. Laemmli UK (1970) Cleavage of the structural proteins
Use of Monoclonal Shigella Antibodies
during the assembly of the head of bacteriophage T4. Nature 227:680-685. Qadri F, Azim T, Hossain T, Islam D, Mondal G, Faruque SM, Albert MJ (1993) A monoclonal antibody to Shigella dysenteriae 13 cross-reacting with Shiga toxin. FEMS Microbiol Lett 107:343-348. Towbin H, Staehelim T, Gordon J (1982) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc Natl Acad Sci USA 76:4350--4354.
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Wathen-Grady HG, Britt LE, Strockbine NA, Wachsmuth IK (1990) Characterization of Shigella dysenteriae serotypes 11, 12 and 13. J Clin Microbiol 28:2580-2584. Weintraub A, Lindberg AA, Nord C-E (1979) Identification of Bacteroides fragilis by indirect immunofluorescence. Med Microbiol Immunol 167:223-230. Westphal O, Jann K (1965) Bacterial lipopolysaccharides: extraction with phenol-water and further application of the procedure. In Methods in Carbohydrate Chemistry, vol 5. Ed, RL Whistler. New York: Academic, pp 83-91.
DIAGN MICROBIOLINFECT DIS 1994;18:145-149
Monoclonal Antibodies Specific for Shigella dysenteriae Serotype 13 Production, Characterization, and Diagnostic Application Firdausi Qadri, Tasnim Azim, Anwar Hossain, Ashrafuzzaman Chowdhury, and M. John Albert
Three mouse monoclonal antibodies (mAbs) (ICL3, ICL4, and ICL5) were produced that specifically recognized the lipopolysaccharide antigen of the newly recognized Shigella dysenteriae serotype-13 strain. All three mAbs reacted with all nine reference isolates of S. dysenteriae 13 in different tests. The mAbs also detected colonies of S. dysenteriae-13 isolates by direct slide agglutination test. The mAbs also reacted with the
reference Escherichia coli 0150 strain and showed its close antigenic relationship with S. dysenteriae 13. Use of these mAbs in our clinical laboratory during an 8-month period detected three S. dysenteriae-13 isolates that were also detected by a polyclonal rabbit antiserum. It should now be possible to define the epidemiologic importance of S. dysenteriae serotype 13 in diarrhea by using these mAbs.
INTRODUCTION
MATERIALS A N D METHODS
Shigella dysenteriae 13 is one of the recently recognized serotypes of Shigella in Bangladesh and other parts of the world from patients with bloody, mucoid diarrhea (Ansaruzzaman et al., 1993). Highly specific monoclonal antibodies (mAbs) have been useful for serotyping of isolates of S. dysenteriae type 1 and Shigella flexneri spp. (Carlin and Lindberg, 1983, 1986, and 1987; Islam and Stimson, 1987 and 1989). We report here the production and characterization of mAbs to the lipopolysaccharide (LPS) antigen of S. dysenteriae 13, which could be used to define the epidemiologic importance of this new serotype in the causation of diarrhea.
From the International Centre for Diarrhoeal Disease Research-Bangladesh, Dhaka, Bangladesh. Address reprint requests to Dr. F. Qadri, Laboratory Sciences Division, ICDDR,B, GPO Box 128, Dhaka 1000, Bangladesh. Received 13 September 1993; accepted 10 December 1993. © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/94/$7.00
Bacterial Strains, Media, and Antiserum Two strains of S. dysenteriae 13 (598-77 and 2489-78) were from the Centers for Disease Control (Atlanta, GA, USA), one strain (4/738/92) was from the National Bacteriological Laboratory (Stockholm, Sweden), and six strains (11357, 11389, 13561, 15357, 2845, and 4449) were recent isolates from Bangladeshi patients with diarrhea (Ansaruzzaman et al., 1993). An Escherichia coli serogroup 0150 strain that cross-reacts with S. dysenteriae 13 (WathenGrady et al., 1990; Ansaruzzaman et al., 1993) was obtained from the Central Public Health Laboratory (Colindale, England). Three strains each of S. dysenteriae serotypes 1-12 were from the National Bacteriological Laboratory (Stockholm), the Central Public Health Laboratory (Colindale), and the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), culture collection. Three strains each of S. flexneri (serotypes la, lb, 2a, 2b, 3a, 4a, and Y), and three strains each of Shigella boydii 1, S. sonnei (Form-I), Salmonella typhimurium, E. coli, Klebsiella sp., and Enterobacter sp., were also used as controls to check for cross-reactivity in the
146
study and were from our culture collection. A previously prepared rabbit polyclonal antiserum to S. dysenteriae 13 was used for comparative studies (Ansaruzzaman et al., 1993). For broth cultures, bacteria were grown in trypticase soy broth supplemented with 0.6% yeast extract (TSBY) (Difco, USA) at 37°C for 18 h in a shaker incubator (100 rpm/min).
Preparation of Lipopolysaccharide and Other Antigens of Bacteria LPS was purified by published methods (Westphal and Jann, 1965). Heat-killed cells were prepared by culturing bacteria in TSBY, washing in phosphatebuffered saline (PBS) (10 raM, pH 7.2), and heating in a water bath at 80°C for 1 h. After washing the cells in PBS, they were resuspended in PBS and used as antigen as described below. Acetone-treated whole cell extract was prepared from bacteria after culturing trypticase soy (TS) agar (Difco) as described previously (Edwards and Ewing, 1972).
Immunization of Mice and Production of Hybridoma Female BALB/c mice were immunized with 50 ~g of acetone extract of S. dysenteriae-13 strain 598-77 four times at weekly intervals as described previously (Qadri et al., 1993). Spleen cells from two immunized BALB/c mice were fused with SP2/0 myeloma cells (Carlin and Lindberg, 1986).
Screening of Hybridomas by ELISA and Production of Ascites Fluid S u p e r n a t a n t fluids harvested from hybridomas were tested in an enzyme-linked immunosorbent assay (ELISA) (Carlin and Lindberg, 1986) for reactivity against all nine S. dysenteriae-13 strains, the E. coli serogroup 0150 strain, and all control bacteria just described. Antigens used were acetone extract (50 ~g/ml), LPS (10 ~g/ml), or boiled cells (10s colony-forming units/ml) of bacteria. The hybridomas secreting antibody specific to LPS of S. dysenteriae 13 were cloned twice by limiting dilution. The ascites fluids produced in pristane-primed mice were diluted in PBS-Tween (0.05%) containing 0.1% bovine serum albumin and screened for reactivity to LPS of S. dysenteriae serotype-13 strains (598-77 and 4449) and other antigens described above in ELISA. A mAb, 4D3, of isotype IgG1 to Shiga toxin (Donohue-Rolfe et al., 1984) was used as a negative control in ELISAs. To determine the isotype of mAbs, supernatant obtained from hybridoma-serum-free medium was tested by ELISA (Carlin and Lindberg, 1986).
F. Qadri et al.
Lipopolysaccharide Patterns and Immunoblot LPS from S. dysenteriae serotype-13 strain (598-77 or 4449) was separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel (SDS--PAGE) (2.5 ~g/well) (Laemmli, 1970) and silver stained (Hitchcock and Brown, 1983). From duplicate unstained gels, the separated antigens were transferred to nitrocellulose membranes (Towbin et al., 1982) and treated with ascites fluid at 1:500 dilutions. The reaction was visualized as described previously (Carlin and Lindberg, 1986). mAb 4D3 was used as a control antibody to check for nonspecific reactions, while LPS obtained from an E. coli 08:K25 strain was used to check for nonspecific binding of S. dysenteriae-13specific mAbs.
Immunofluorescence Test Specificity of S. dysenteriae-13 mAbs was tested by an indirect immunofluorescence test (Weintraub et al., 1979). Heat-killed bacteria on glass slides mixed with mAbs (1:100 to 1:500 dilution in PBS) were treated with fluorscein-isothiocyanate-conjugated rabbit-anti-mouse immunoglobulin (Ig) (Silenus, Victoria, Australia) diluted 1:40 in PBS. The test bacteria included all isolates of S. dysenteriae 13. The negative controls included S. dysenteriae serotypes 1-12 and a mAb, MASF-1, specific for S. flexneri 1 (Carlin and Lindberg, 1986).
Slide Agglutination Test The ability of S. dysenteriae-13-specific mAbs to detect all nine S. dysenteriae-13 isolates and the single E. coli serogroup-0150 isolate was studied in a slide agglutination test with TSA agar (Difco)-grown bacteria at 37°C and diluted ascites fluid in PBS. Bacterial suspensions were made in 2 drops of physiologic saline placed on two different spots of a microscopic slide. To one suspension, 1 drop of ascites fluid was added and, to the other, saline was added. The slide was rocked gently, and clear agglutination within 2 min was considered positive. In addition, the ability of these mAbs to identify S. dysenteriae 13 was evaluated in our clinical laboratory over an 8-month period from November 1992 to July 1993. The earlier-listed control bacteria were also included. The test was carried out by at least two investigators independently.
RESULTS Production of Monoclonal Antibodies Specific for Lipopolysaccharide of Shigella dysenteriae 13 Three specific and stable clones were obtained: two mAbs, ICL3 and ICL4, were of IgM isotype and one mAb, ICL5, was of IgG1 isotype.
147
Use of Monoclonal Shigella Antibodies
The titers of ascites fluid corresponding to the five mAbs varied between 1.2 x 10~and 9.6 x 105 against LPS antigens from S. dysenteriae-13 strains 598-77 and 4449. All mAbs reacted in ELISA with heat-killed cells from the six Bangladeshi isolates and the three reference isolates of S. dysenteriae 13. They also reacted with LPS isolated from E. coli 0150. They did not react with any control bacteria.
A
B
",D
Lipopolysaccharide Pattern and Immunoblot The LPS separation pattern of S. dysenteriae-13 strain 598-77 on SDS-PAGE, which is presented in Figure 1 (lane A), showed the presence of the O-antigenic repeating units (ladderlike pattern in the upper portion of Figure 1, marked a) and the core polysaccharides (darkly staining portion in the bottom of Figure 1, marked b). In immunoblotting studies, all mAbs reacted with the O-antigenic polysaccharide (Figure 2, lane B; indicated by the arrow; data shown only for mAb ICL4). Nonspecific binding
A
a-tD
FIGURE 2 Immunoblot analysis of lipopolysaccharide (LPS) antigens separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis, blotted onto a nitrocellulose membrane, and probed with monoclonal antibody ICL4: lane A contains LPS from a negative control of Escherichia coli strain and lane B contains LPS from Shigella dysenteriae 13. The O-antigenic polysaccharide region of LPS is indicated by the arrow.
was not seen when the mAbs were tested against LPS from a negative control E. coli strain (Figure 2, lane A) or when mAb 4D3 was tested against LPS from S. dysenteriae 13 (data not shown).
Immunofluorescence Test The mAbs to S. dysenteriae 13 specifically recognized all isolates of S. dysenteriae 13 in this test. The optim u m dilution for mAbs ICL3 and ICL5 was 1:500, whereas it was 1:100 for mAb ICL4. None of the negative control bacteria reacted in this test. FIGURE 1 Pattern of separation of lipopolysaccharide (LPS) antigen from Shigella dysenteriae-13 strain 598-77. The antigen was separated on 13.5% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis and visualized by silver staining. Lane A contains LPS from S. dysenteriae 13. Shown are O-antigenic polysaccharide (a) as well as core polysaccharide (b) indicated by arrows.
Slide Agglutination Test mAbs ICL3, ICL4, and ICL5 strongly agglutinated all S. dysenteriae-13 isolates and E. coli 0150. The optimum dilutions of these mAbs for agglutination tests were 1:200, 1:50, and 1:25, respectively. Non-
148
specific reaction was not detected with other bacteria tested. During the 8-month evaluation period of mAbs in the clinical laboratory, 35 Shigella-like colonies from diarrheal patients that did not agglutinate with polyclonal antisera to the conventional serotypes of shigellae were tested against S. dysenteriae-13 mAbs. Three of these isolates (nos. 18538, 19123, and 21333) turned out to be S. dysenteriae 13. The same three isolates only were identified as S. dysenteriae 13 by using the rabbit polyclonal antiserum w h e n tested independently. Identical results were obtained by the two different investigators.
DISCUSSION The results presented here show that ICL3-5 can be used for detection of isolates S. dysenteriae 13. They were highly specific, and the only other species of bacteria to react was E. coli 0150. Previous studies with rabbit polyclonal antiserum have suggested reciprocal cross-reactivities between S. dysenteriae 13 and E. coli 0150 (Wathen-Grady et al., 1990; Ansaruzzaman et al., 1993). This antigenic relatedness was demonstrated with all three mAbs of S. dysenteriae 13. Immunoblot studies suggested that these mAbs reacted with the epitopes on the polysaccharide side chain of the LPS antigen. Although these mAbs also recognize E. coli 0150, this will not limit their usefulness as diagnostic reagents, because we will be testing only pale colonies and not pink colonies from selective media used to isolate Shigella in the clinical laboratory (E. coli produce pink colonies due to fermentation of lactose, and Shigella produce pale colonies due to the absence of lactose fermentation in the selective media).
F. Qadri et al.
We have used these mAbs for 8 months (until this report was prepared) to screen for S. dysenteriae 13 in our clinical laboratory, and three isolates were identified. The same three isolates were also identified by the rabbit polyclonal antibody. Since the isolation rate of S. dysenteriae 13 was low during the study period, an extensive evaluation of the mAbs was not possible. The preliminary results obtained so far suggest, however, that these mAbs will be useful. Since S. dysenteriae 13 is a newly recognized serotype causing bloody, mucoid diarrhea, its worldwide importance has yet to be assessed. It should now be possible to define its epidemiologic importance with the help of these mAbs. The S. dysenteriae-13-specific mAbs now add to the list of other mAbs that have previously been proven to be useful for detection of other Shigella species (Carlin and Lindberg, 1983 and 1987; Islam and Stimson, 1987 and 1989).
This research was supported by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). The ICDDR,B is supported by countries and agencies that share its concern for the health problems of developing countries. Current donors include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Denmark, France, Japan, the Netherlands, Norway, Saudi Arabia, Sweden, Switzerland, the United Kingdom, and the United States; international organizations including the United Nations (UN) Children's Fund, the UN Development Program, the UN Population Fund, and the World Health Organization; and private foundations, including the Ford Foundation and the Sasakawa Foundation. We thank Mr. Manzurul Haque for secretarial assistance.
REFERENCES Ansaruzzaman M, Kibriya AKMG, Mitra AK, Sack RB, Albert MJ (1993) Isolation of Shigella dysenteriae serotypes 11, 12 and 13 from patients with diarrhea in Bangladesh. J Clin Microbiol 31:1392-1393. Carlin NIA, Lindberg AA (1983) Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri: clones binding to II, II:3, 4 and 7, 8 epitopes. J Clin Microbiol 18:1183-1189. Carlin NIA, Lindberg AA (1986) Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type 1 and type III, 6, 7, 8 antigens, group 6 antigens and a core epitope. Infect Immun 53:103-109. Carlin NIA, Lindberg AA (1987) Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV and VI antigen group 3, 4 antigen and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains. Infect Immun 55:14121420. Donohue-Rolfe A, Keusch GT, Edson C, Thorley-Lawson D, Jacewicz M (1984) Pathogenesis of Shigella diarrhea.
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