FEMS MicrobiologyLetters 97 ( 19921173-178 ~3 1992Federationof European MicrobiologicalSocieties11378-1(XJ7/92/$I)5.(X) Publishedby Elsevier
FEMSLE 1151184
Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, 09) of Salmonella typhi John M.C. Luk, C.-R. Zhao, Kerstin M. Karlsson and AIf A. Lindberg Department of ClinicalBacteriology, Karolin,~kahzstimte. Ihlddingc tto.~piml,lhuhlinge, Sweden Received 15June 1002 Revisionreceived 13Jul.v1992 Accepted 16July 1992 Key words: Salmonella ~phi; Vi polysacc~,aride; Lipopolysaccharidc; Mont~clonal antibodies; Serotyping 1. SUMMARY A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Sabnonella typhi. Four clones (lgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (lgM) and MATy-O9 (lgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific antiSalmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-'O9 were significantly inhibited by the anti-Vi and anti~O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D Correspondence to: A.A. Lindberg, Department of Clinical Bacteriology, KarolinskaInstitute,HuddingeHospital. S-141 86 Huddinge.Sweden.
salmonellae among a total of 1411 bacterial isolates representing eight different Enterobacteriaceae genera tested.
2. INTRODUCTION Typhoid fever, a frequent and severe enteric disease in many developing countries [!], is due to a systemic infection with Sabnonella typhi in susceptible patients. Serologically, $. typhi belongs to serogroup D and is characterized by the capsular Vi and somatic 0 9 antigens and also its flagellar H antigens [2]. The Vi and 0 9 antigens are both envelope constituents of S. t)'phi and important virulence factors. Chemically, the Sabnonella Vi capsular polysaccharide is a linear homopolymer of ct(l---) 4)-O-acetyI-N-acetylgalactosaminuronic acid (Fig. 1) [3], and the O antigens of the lipopolysaccharide (LPS) are formed by polymerization of a tetrasaccharide repeating unit which is composed of [tyveiose-a(1 -~ 3)]-D-mannose-a(l -~ 3)-i.-rhamnose.a( ! -~ 3)Da(l ~ 2) [4]. Here the dideoxyhexosyl tyvelose is the immunodomii~ant sugar of the Sabnonella 0 9 epitope.
3.2 Preparation of bacterial lipopolysacdlaride and Vi polysaccharide O30H
Ae Fig. I. SahntmellaVi capsularpolysaccharide. The Vi antigen is besides present in almost all clinical isolates of S. typhi, also found in S. parat)'phi C, and rarely in S. dublin. Strains of Citrobacter freundii [5] and Escherichia coli [6] have also been reported to contain the Vi polysaccharide. The virulence of Vi-bearing salmonellae is attributed to its ability to make it possible for the bacteria to evade both specific and non-specific host defence mechanisms [7]. The Vi capsule shields the bacteria against phagocytosis by its surface negative charge properties and by covering cell-envelope components sensitive to serum opsonins (e.g. lgG or C3b ligands). The damage to the host is largely an effect of the endotoxin (or LPS) [8]. The lipid A moiety of LPS elicits a wide range of pathophysiological and immunological effects, and thc O-polysaccharide modulates the host immune defense mechanisms by interference with the normal serum bactericidal activity and/or the process of phagocytosis.
3. MATERIALS AND METHODS
3.1. Bacterials strains Laboratory reference strains of salmonellae and other Enterobacteriaceae (Table 2) were obtained from the National Bacteriological Laboratory (SBL), Stockholm, Sweden. Other clinical isolates were collected from the routine diagnostic service of the Clinical Microbiology Laboratory at Huddinge Hospital, Huddinge, Sweden. Bacterial strains were grown on nutrient, blood and Cled agar (Difco Lab., Detroit, Ml) at 37°C overnight, and identified by standard biochemical and serological tests [2].
Bacteria were cultivated in LKB fermentor at 37°C overnight as previously described [9] and LPS was extracted with hot phenol-water [10] or, for rough mutants, with phenol-chloroform-petroleum ether [ll]. LPS from rough strains were also electrodialysed and neutralized with triethylamine. Highly purified Vi capsular polysaccharide was prepared from S. typhi strain 10086 by precipitation with ethanoi-Cetavlon as described by Wong and Feeley [12]. Purity of the Vi-polysaccharide was assessed in enzymc immunoassay by using rabbit anti-Vi and anti-O9 LPS antibodies in order to monitor the contamination with trace amount ( ~ 1% or less) of S. typhi LPS.
3.3. Production of monoclonal antibodies Three BALB/c mice were inoculated intraperitoneally (i.p.) with ![) 7 heat-killed Sahnonella typhi 253 Ty (with rich expression of O, H, Vi antigens) cells suspensed in sterile, cold phosphate-buffered saline (PBS) pH 7.5. A final injection of 2 p.g pure Vi capsular material was given i.p. on day 10. One week later, spleen cells from a mouse that had the highest serum antibody titer against the S. typhi Vi antigen were fused with mouse plasmacytoma cell Sp2/0 line. mAbs were produced by the method of K6hler and Milstein [13], with polyethylene glycol as the fusion agent. Culture supernatant from growing clones were screened for antibodies against homologous LPS and Vi antigens by an enzyme immunoassay (EIA), and antibody-secreting h~bridomas were cloned two to three times by limiting dilution. Cloned hybridomas were later injected into BALB/c mice primed with Pristane (2,6,10,14-tetramethyl pentadecane) for the production of ascitic fluid.
3.4. Purification and characterization of monoclonal antibodies Antibodies from culture supernatant and ascitic fluid were partially purified by 50% ammonium sulphate precipitation. Further purification was done by affinity chromatography using protein A-Sepharose or by exclusion gel chromato-
175 graphy on Sephacryl S-300, following standard procedures [14]. Antibody class and subclass of each monoclonal cell lines were determined by the line blot immunoassay by using the Inno-LIA mouse mAb isotyping kit (innogenetics S.A., Antwerp, Belgium). The specificity of each mAb was determined in an EIA by testing against a set of purified LPS from Salmonella serovars Paratyphi A IS2, Typhimurium SH4809, Thompson IS40. Newport IS50, Typhi T2, Enteritidis SH1262, Anatum IS78, Typhimurium T V I I 9 (Ra chemotype), Escherichia coli O111 and ShigeUa flexneri type Y.
3.5. Enzyme immunoassays The E I A for the detection of anti-LPS antibodies was performed as described [15]. Briefly, microtiter EIA plates (Costar, Cambridge, MA) were coated with 0.1 ml (10 p,g/ml) of purified Vi and LPS or (5/.tg/ml) glycoconjugate antigens in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at room temperature overnight. Binding of antibodies was determined with an alkaline phosphatase-eonjugated rabbit anti-mouse immunoglobulin antibodies (Dakopatts AS) in I:I(XI0 dilution with p-nitrophenyl phosphate (Sigma) as substrate.
In the competition EIA, 5(1 #1 of a 1/10 diluted mAb supernatant was mixed with an equal volume of competing antibody (rabbit monospecific Sabnonella agglutinating sera obtained from the SBL, Stockholm) to microtiter wells precoated with a limiting concentration of the purified S. typhi T2 LPS (11.5/.tg/ml) or Sabnmzella Vi (2 /.tg/ml) antigens. The rest of the procedures were the same as those described above.
4. R E S U L T S A N D DISCUSSION The major objective of this paper was to generate monoclonals against the 0 9 and Vi polysaccharide antigens of S. typhi, and to characterize their speeificities. Conventional immunization procedures, i.e. repeated injections of heat-killed S. typhi failed to generate any desired Vi-specific hybridomas (our unpublished data, and ref. 16). Bacterial capsular polysaccharides like the Vi antigen have T-independent properties, and repeated immunizations with the highly polymerized capsular antigens may paralyse the immune response in animals or human subjects [1,12,17]. We therefore tried a short immunization scheme combined with dual immunogen preparations:
=:
it
,
FEMSLE 1151184
Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, 09) of Salmonella typhi John M.C. Luk, C.-R. Zhao, Kerstin M. Karlsson and AIf A. Lindberg Department of ClinicalBacteriology, Karolin,~kahzstimte. Ihlddingc tto.~piml,lhuhlinge, Sweden Received 15June 1002 Revisionreceived 13Jul.v1992 Accepted 16July 1992 Key words: Salmonella ~phi; Vi polysacc~,aride; Lipopolysaccharidc; Mont~clonal antibodies; Serotyping 1. SUMMARY A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Sabnonella typhi. Four clones (lgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (lgM) and MATy-O9 (lgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific antiSalmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-'O9 were significantly inhibited by the anti-Vi and anti~O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D Correspondence to: A.A. Lindberg, Department of Clinical Bacteriology, KarolinskaInstitute,HuddingeHospital. S-141 86 Huddinge.Sweden.
salmonellae among a total of 1411 bacterial isolates representing eight different Enterobacteriaceae genera tested.
2. INTRODUCTION Typhoid fever, a frequent and severe enteric disease in many developing countries [!], is due to a systemic infection with Sabnonella typhi in susceptible patients. Serologically, $. typhi belongs to serogroup D and is characterized by the capsular Vi and somatic 0 9 antigens and also its flagellar H antigens [2]. The Vi and 0 9 antigens are both envelope constituents of S. t)'phi and important virulence factors. Chemically, the Sabnonella Vi capsular polysaccharide is a linear homopolymer of ct(l---) 4)-O-acetyI-N-acetylgalactosaminuronic acid (Fig. 1) [3], and the O antigens of the lipopolysaccharide (LPS) are formed by polymerization of a tetrasaccharide repeating unit which is composed of [tyveiose-a(1 -~ 3)]-D-mannose-a(l -~ 3)-i.-rhamnose.a( ! -~ 3)Da(l ~ 2) [4]. Here the dideoxyhexosyl tyvelose is the immunodomii~ant sugar of the Sabnonella 0 9 epitope.
3.2 Preparation of bacterial lipopolysacdlaride and Vi polysaccharide O30H
Ae Fig. I. SahntmellaVi capsularpolysaccharide. The Vi antigen is besides present in almost all clinical isolates of S. typhi, also found in S. parat)'phi C, and rarely in S. dublin. Strains of Citrobacter freundii [5] and Escherichia coli [6] have also been reported to contain the Vi polysaccharide. The virulence of Vi-bearing salmonellae is attributed to its ability to make it possible for the bacteria to evade both specific and non-specific host defence mechanisms [7]. The Vi capsule shields the bacteria against phagocytosis by its surface negative charge properties and by covering cell-envelope components sensitive to serum opsonins (e.g. lgG or C3b ligands). The damage to the host is largely an effect of the endotoxin (or LPS) [8]. The lipid A moiety of LPS elicits a wide range of pathophysiological and immunological effects, and thc O-polysaccharide modulates the host immune defense mechanisms by interference with the normal serum bactericidal activity and/or the process of phagocytosis.
3. MATERIALS AND METHODS
3.1. Bacterials strains Laboratory reference strains of salmonellae and other Enterobacteriaceae (Table 2) were obtained from the National Bacteriological Laboratory (SBL), Stockholm, Sweden. Other clinical isolates were collected from the routine diagnostic service of the Clinical Microbiology Laboratory at Huddinge Hospital, Huddinge, Sweden. Bacterial strains were grown on nutrient, blood and Cled agar (Difco Lab., Detroit, Ml) at 37°C overnight, and identified by standard biochemical and serological tests [2].
Bacteria were cultivated in LKB fermentor at 37°C overnight as previously described [9] and LPS was extracted with hot phenol-water [10] or, for rough mutants, with phenol-chloroform-petroleum ether [ll]. LPS from rough strains were also electrodialysed and neutralized with triethylamine. Highly purified Vi capsular polysaccharide was prepared from S. typhi strain 10086 by precipitation with ethanoi-Cetavlon as described by Wong and Feeley [12]. Purity of the Vi-polysaccharide was assessed in enzymc immunoassay by using rabbit anti-Vi and anti-O9 LPS antibodies in order to monitor the contamination with trace amount ( ~ 1% or less) of S. typhi LPS.
3.3. Production of monoclonal antibodies Three BALB/c mice were inoculated intraperitoneally (i.p.) with ![) 7 heat-killed Sahnonella typhi 253 Ty (with rich expression of O, H, Vi antigens) cells suspensed in sterile, cold phosphate-buffered saline (PBS) pH 7.5. A final injection of 2 p.g pure Vi capsular material was given i.p. on day 10. One week later, spleen cells from a mouse that had the highest serum antibody titer against the S. typhi Vi antigen were fused with mouse plasmacytoma cell Sp2/0 line. mAbs were produced by the method of K6hler and Milstein [13], with polyethylene glycol as the fusion agent. Culture supernatant from growing clones were screened for antibodies against homologous LPS and Vi antigens by an enzyme immunoassay (EIA), and antibody-secreting h~bridomas were cloned two to three times by limiting dilution. Cloned hybridomas were later injected into BALB/c mice primed with Pristane (2,6,10,14-tetramethyl pentadecane) for the production of ascitic fluid.
3.4. Purification and characterization of monoclonal antibodies Antibodies from culture supernatant and ascitic fluid were partially purified by 50% ammonium sulphate precipitation. Further purification was done by affinity chromatography using protein A-Sepharose or by exclusion gel chromato-
175 graphy on Sephacryl S-300, following standard procedures [14]. Antibody class and subclass of each monoclonal cell lines were determined by the line blot immunoassay by using the Inno-LIA mouse mAb isotyping kit (innogenetics S.A., Antwerp, Belgium). The specificity of each mAb was determined in an EIA by testing against a set of purified LPS from Salmonella serovars Paratyphi A IS2, Typhimurium SH4809, Thompson IS40. Newport IS50, Typhi T2, Enteritidis SH1262, Anatum IS78, Typhimurium T V I I 9 (Ra chemotype), Escherichia coli O111 and ShigeUa flexneri type Y.
3.5. Enzyme immunoassays The E I A for the detection of anti-LPS antibodies was performed as described [15]. Briefly, microtiter EIA plates (Costar, Cambridge, MA) were coated with 0.1 ml (10 p,g/ml) of purified Vi and LPS or (5/.tg/ml) glycoconjugate antigens in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at room temperature overnight. Binding of antibodies was determined with an alkaline phosphatase-eonjugated rabbit anti-mouse immunoglobulin antibodies (Dakopatts AS) in I:I(XI0 dilution with p-nitrophenyl phosphate (Sigma) as substrate.
In the competition EIA, 5(1 #1 of a 1/10 diluted mAb supernatant was mixed with an equal volume of competing antibody (rabbit monospecific Sabnonella agglutinating sera obtained from the SBL, Stockholm) to microtiter wells precoated with a limiting concentration of the purified S. typhi T2 LPS (11.5/.tg/ml) or Sabnmzella Vi (2 /.tg/ml) antigens. The rest of the procedures were the same as those described above.
4. R E S U L T S A N D DISCUSSION The major objective of this paper was to generate monoclonals against the 0 9 and Vi polysaccharide antigens of S. typhi, and to characterize their speeificities. Conventional immunization procedures, i.e. repeated injections of heat-killed S. typhi failed to generate any desired Vi-specific hybridomas (our unpublished data, and ref. 16). Bacterial capsular polysaccharides like the Vi antigen have T-independent properties, and repeated immunizations with the highly polymerized capsular antigens may paralyse the immune response in animals or human subjects [1,12,17]. We therefore tried a short immunization scheme combined with dual immunogen preparations:
=:
it
,
