Vol. 29, No. 11

Anti-Salmonella Lipopolysaccharide Monoclonal Antibodies:Characterization of Salmonella BO-, CO-, DO-, and EO-Specific

Clones and Their Diagnostic UsefulnessJOHN M. C. LUK AND ALF A. LINDBERG*

Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital,S-141 86 Huddinge, Stockholm, Sweden

Received 20 May 1991/Accepted 2 August 1991

To facilitate the identification and serotyping of Salmonella species, we established a wide variety of murinemonoclonal antibodies (MAbs) that were reactive with the lipopolysaccharides (LPSs) of Salmonella serogroupsB to E. An effective approach for generating LPS-reactive hybridomas was used; this required immunizationof mice with LPS-coated bacteria. To screen for diagnostically useful MAbs, the MAbs were tested byenzyme-linked immunosorbent assay against a set of purified LPSs from smooth and rough Salmonella strains.At least four major groups of antibody specificities were identified: Salmonella (i) BO specific, (ii) CO specific,(iii) DO specific, and (iv) E0 specific. For a more detailed epitope analysis, a panel of eight differentserogroup-specific MAbs which were shown to bind the 0-antigenic polysaccharide chains, yielding charac-teristic ladder patterns in Western blots (immunoblots) against the LPS of Salmonella serogroups B to E, wereselected. The availability of various chemically defined LPS structures and Salmonella 0-antigen glycoconju-gates permitted the definition of 0-antigenic polysaccharide epitopes recognized by each MAb that serologicallycorresponded to factors 03, 04, 05, 06, 07, 08, 09, and 010 on the basis of the Kauffmann-White schemefor Salmonella classification. The diagnostic accuracy of these immunochemically defined 0-specific MAbs forSalmonella serotyping was demonstrated by correct identification of all 167 salmonellae (including 72 serotypesfrom serogroups B to E) among the 294 bacterial strains in a slide agglutination test. No false-positive reactionswere detected.

In Salmonella infections, the lipopolysaccharide (LPS) isof importance for both pathogenicity and virulence (20). TheLPS, which exists as a major surface component (105 to 106molecules per cell) of the Salmonella cell envelope, com-prises three structural domains: lipid A, core oligosaccha-ride, and 0-antigenic polysaccharide chain (14, 26). The0-antigenic polysaccharide chain is formed by polymeriza-tion of 0 repeating subunits which carry the 0-antigenicspecificity for a particular bacterium. Functionally, thepolysaccharide domain affects the virulence properties ofSalmonella species, mainly by modulating complement ac-tivation pathways and increasing resistance to phagocytosisby macrophages and, occasionally, by mimicking the hosttissue antigens so as to evade the immune defense mecha-nisms (20, 28). In addition to their prominent immunogenicproperties, the 3,6-dideoxyhexosyl residues of the Salmo-nella O-polysaccharide chains are also essential for thevirulence of these organisms, e.g., paratose in SalmonellaAO, abequose in Salmonella BO, and tyvelose in SalmonellaDO (Fig. 1).Although there are more than 2,000 serotypes of Salmo-

nella species, over 95% of the strains that cause infections inhumans and animals belong to serogroups A to E (21, 29).Definitive laboratory diagnosis of salmonellosis requires thedetection of the LPS 0 antigens that indicate the group towhich a given Salmonella organism belongs (6, 17). Conven-tional methods for rabbit 0 antiserum preparation are basedon cross-absorption by related bacteria (18), procedureswhich are cumbersome and time-consuming, and in addition,the antibody titers are often low and of variable specificity.

* Corresponding author.

This has drawn our interest to the production of trulymonospecific antibody preparations, i.e., monoclonal anti-bodies (MAbs), against the 0 antigens of Salmonella sero-groups A to E.By taking advantage of a recent development in our

laboratory that uses LPS-coated bacteria as the immunogen(24), a panel of specific MAbs against the LPS of Salmonellaserogroups A to E was successfully established. We previ-ously reported the identification of Salmonella serogroup Aby an 02-specific MAb (25). Here we describe the charac-terization of eight different 0-antigen-specific clones thatbind to the factors 03, 04, 05, 06, 07, 08, 09, and 010 ofSalmonella LPS. Our aim is to facilitate the routine identi-fication of Salmonella species by use of the present 0-spe-cific MAbs for serotyping.

MATERIALS AND METHODS

Buffer and media. We used the following media: Tris-buffered saline (TBS) containing 0.01 M Tris-HCl (pH 7.4)and 0.05 M NaCl, phosphate-buffered saline (PBS; pH 7.4),PBS supplemented with 0.05% (vol/vol) Tween 20(PBS-Tween), and PBS containing 1% (wt/vol) bovine serumalbumin (BSA). Complete and incomplete Freund's adju-vants were obtained from Difco Laboratories (Detroit,Mich.). Buffers were made by using Milli Q water, and pHmeasurements were made at 23°C. RPMI 1640 (GIBCO,Paisley, Scotland) supplemented with 10% heat-inactivatedfetal calf serum (Flow Laboratories, Irvine, Scotland), 1 mNsodium pyruvate, 4 mM L-glutamine, and 100 U of penicillin-streptomycin (GIBCO) was the complete medium for in vitrocell cultures. The complete medium plus hypoxanthine-aminopterin-thymidine was used to select hybridomas in

2424

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2424-24330095-1137/91/112424-10$02.00/0Copyright C) 1991, American Society for Microbiology

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SALMONELLA O-ANTIGEN-SPECIFIC MAbs 2425

OH

HO0

HO

CH,

OH\OH

HO O

HO] OM

HO aH

OH

HOa

OMeHO 0

OHCH3

1 3 1 1 3 1 1 3 1

Parp---*o-.Manp----.&OM9 Abep-j-wco-Manp-a-.oMq TYvp--..o-Manp----*.OM9

Serogroup A Serogroup B Serogroup D

FIG. 1. Conformations of the synthetic 3,6-dideoxy-a-D-hexosyl-a-D-mannopyranoside disaccharides corresponding to Salmonellaserogroups A (02), B (04), and D (09).

fusion experiments. Hank's buffered salt saline from FlowLaboratories was used for cell washings.

Bacterial strains and culture conditions. Table 1 lists thereference laboratory strains used in this study and their0-antigen formulas. All other strains were recent clinicalisolates (mainly from fecal samples) obtained from theroutine diagnostic service of the Clinical Microbiology Cen-ter of Huddinge Hospital, Huddinge, Sweden. Bacterialisolates were grown in nutrient L broth or on blood agar

(Oxoid Ltd., Hampshire, England) at 37°C overnight. Cul-tures were inactivated with 1% formaldehyde, washed twicewith cold, sterile PBS, and then harvested by centrifugationat 5,000 x g for 15 min.

LPSs, phage-hydrolyzed oligosaccharides, and syntheticsaccharides. LPS was extracted from smooth bacterialstrains by the hot phenol-water method (38) and from roughstrains by the phenol-chloroform petroleum method (11).Further treatments with proteinase K and RNase were doneto ensure less than 5% protein and less than 10% nucleic acidcontaminations of the LPS preparations.Salmonella 0-antigen glycoconjugates were used to study

the epitope specificities of LPS-reactive hybridomas. LPSsfrom S. typhimurium and S. enteritidis were treated with 1%acetic acid (100°C, 60 min). The lipid A-free polysaccharideswere then subjected to specific degradation with bacterio-phage P22 endo-ot-rhamnosidase (33). Preparation of 0-spe-cific oligosaccharides from S. thompson was done similarly

by using the endo-at-mannosidase activity of bacteriophage14 (9). The resulting tetrasaccharides, octasaccharides, de-casaccharides, and dodecasaccharides (representing one tothree repeating units of the 0-polysaccharide chain) were

separated by Bio-Gel P2 and P4 chromatography (Bio-RadLaboratories, Richmond, Calif.). Artificial Salmonella 0

antigens representing the disaccharides of serogroups A:02,ox-Par(1--+3)Man; B:04, oa-Abe(1->3)Man; C:08, ox-Abe(1-*3)Rha; and D:09, a-Tyv(1->3)Man, as well as the trisac-charide of E:03, ,B-Man(1--*4)-a-Rha-(1--)3)-a-Gal, were

available from earlier studies (22). Saccharide products were

covalently linked to BSA as a protein carrier, and theresulting conjugates had an average ratio of 15 to 25 mol ofsaccharide per mol of BSA, as determined by sugar andprotein analysis (31).

Immunization, hybridoma fusion, and screening. MAbsdirected against the Salmonella LPS antigens were raised byusing two types of immunogens: heat-killed bacteria (80°C, 1h) and LPS-coated bacteria (10 mg of bacteria additionallycoated with 0.2 mg of homologous, free LPS) (24). Forimmunization, male BALB/c mice were inoculated with a

total of 0.2 ml of antigen solutions (containing 108 cells or 0.1mg of protein) emulsified with an equal volume of Freund'sadjuvants or sterile PBS. All immunizations were made byintraperitoneal injection, except for the final intravenousbooster. The timetables of administration are given in Table2. Mice were bled 3 days after the last injection, and the

TABLE 1. Salmonella reference strains used for LPS extraction and mice immunization

Salmonella strain Serogroup 0-antigenic formulaa Sourceb

S. paratyphi A IS2 A 0:2,12 L. Le MinorS. typhimurium SH4809 B 0:4,5,12 P. H. MakelaS. thompson IS40 C1 0:6,7 L. Le MinorS. newport IS50 C2 0:6,8 L. Le MinorS. kentucky IS98 C3 0:8,20 L. Le MinorS. typhi 253Ty D 0:9,12 B. A. D. StockerS. typhi T2 D 0:9,12 B. A. D. StockerS. enteritidis SH1262 D 0:9,12 P. H. MakelaS. anatum IS78 E1 0:3,10 L. Le MinorS. illinois IS133 E3 0:3,15 L. Le MinorS. senftenberg SH1707 E4 0:1,3,19 P. H. MakelaS. typhimurium TV119 Rough (Ra chemotype) 0-antigen deficiency B. A. D. Stocker

a Descriptions of the 0-antigen formulas can be found in reference 21.b L. Le Minor, World Health Organization Salmonella Reference Center, Institute Pasteur, 199 Institut National de la Santd et de la Recherche Mddicale, Paris,

France; P. H. Makela, National Public Health Institute, Mannerheimintie 166 Helsinki, Finland; B. A. D. Stocker, Department of Microbiology and Immunology,Stanford University School of Medicine, Stanford, Calif.

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2426 LUK AND LINDBERG

TABLE 2. Immunization, hybridoma generation, and characterization of MAbs against Salmonella LPS

Administration No. of stable, No. of clones with the following No. of MAbs bindingFusion Immunogen schedule LPS-reactive antibody isotype distribution: to LPS antigensbno. (days)_hybrid_cloneS(days) hybrid clonesa IgM IgGl IgG2a IgG2b IgG3 IgA 0 chains Ra core

1 S. typhimurium, 0,10, 24, 38 1 1 0 0 0 0 0 1 0heated bacteria

2 S. typhimurium, LPS- 0, 28, 42 9 1 0 2 5 1 0 9 0coated bacteria

3 S. newportHeated bacteria 0, 28, 42LPS-coated bacteria 56, 70 20 10 2 2 3 3 0 20 0

4 S. typhi, heated 0, 28, 42 1 1 0 0 0 0 0 1 0bacteria

5 5. typhimurium, LPS- 0, 28, 42coated bacteria

S. typhi, LPS-coated 56, 70 23 17 2 1 2 1 0 22 1bacteria

6 S. anatum, LPS- 0, 28, 42 10 0 8 0 2 0 0 7 3coated bacteria

a A total of 64 (30 IgM, 34 IgG) LPS-reactive hybrid clones were generated in six fusion experiments.b Tested by ELISA against the LPS of homologous immunizing strains and the rough S. typhimurium TV119 (Ra) mutant.

subject that gave the highest antibody response to theimmunizing strain was splenectomized, and subsequently itsspleen was used for fusion.Hybridoma fusions were accomplished by standard pro-

cedures (12). Briefly, spleen cells from an immune BALB/cmouse and murine plasmacytoma cells (Sp2/O-Agl4) werefused at a ratio of 2.5:1 in serum-free medium by usingpolyethylene glycol (molecular weight, 4,000; Merck, Darm-stadt, Federal Republic ofGermany) as the fusion agent. Thefused cells were grown in hypoxanthine-aminopterin-thymi-dine medium, with mouse peritoneal cells used as feeders.After 10 to 14 days, hybridoma culture supernatants werescreened by enzyme-linked immunosorbent assay (ELISA)for antibodies against the homologous LPS antigens. Thosereactive (A405, >0.5) and stable (over two passages) hybridswere selected and cloned at least three to four times bylimiting dilution (1 and 0.2 cells per well).

Production and characterization of MAbs. Large-scale pro-duction of MAbs was achieved by harvesting spent culturesupernatants or by ascites production. For the latter,BALB/c mice were primed with pristane (2,6,10,14-tetra-methylpentadecane; Merck) from 10 to 14 days prior tointraperitoneal injection of pure hybridoma cells (106 to 107cells). Antibodies from culture supernatants and asciticfluids were partially purified by 50% ammonium sulfateprecipitation, and isotypes were determined by the doubleimmunodiffusion method by using mouse MAb typing kitMMT 02K (Serotec, Bicester, England) (12). The specificityof each MAb was determined in an ELISA by testing eachMAb against a set of purified LPSs from S. paratyphi A IS2,S. typhimurium SH4809, S. thompson IS40, S. newportIS50, S. typhi 253Ty, S. anatum IS78, S. typhimuriumTV119 (Ra chemotype), Shigella flexneri type Y, and Esch-erichia coli 0111.Enzyme immunoassay. Microtiter enzyme immunoassay

plates (Costar, Cambridge, Mass.) were coated with 0.1 ml(10 ,ug/ml) of purified LPS or glycoconjugates in 0.05 Mcarbonate-bicarbonate buffer (pH 9.6) at room temperature

overnight. Unsaturated binding sites in the wells wereblocked with PBS-1% BSA, and then the plates wereincubated at 37°C for 90 min following the addition ofantibodies in culture supernatants to antigen-coated wells.After three washes with PBS-Tween, a 1:1,000 dilution ofalkaline phosphatase-conjugated goat antimouse immuno-globulins was added (Sigma Co., St. Louis, Mo.), and theplate was again incubated at 37°C for 90 min. After threemore washes with PBS-Tween, the enzyme reaction wasallowed to proceed at 37°C with the Sigma 104 phosphatasesubstrate disodium p-nitrophenylphosphate (10 mg/ml) in10% diethanolamine-HCI buffer (pH 9.8). The A405 wasmeasured after 30 min of incubation by using an ELISAprogram (Medata Digital, Solna, Sweden) on a PET Com-modore business computer interfaced with a Flow photom-eter.

Electrophoresis of LPS and immunoblotting. LPS prepara-tions from Salmonella serogroups A to E were analyzed bydiscontinuous sodium dodecyl sulfate-polyacrylamide gelelectrophoresis in a 13.5% resolving gel. A sample prepara-tion of smooth LPS (10 jig) or rough LPS (5 ,ug) was treatedwith an equal volume of sample buffer (2% [wt/vol] sodiumdodecyl sulfate, 4% [vol/voll 2-mercaptoethanol, 10% [wt/vol] sucrose, and 0.01% Congo red in 0.0625 M Tris-HCI [pH6.8]) in a boiling water bath for 10 min before it was loadedinto the sample wells. Neither the stacking nor the resolvinggel (1.5 mm, 14 by 16 cm) contained sodium dodecyl sulfate.After electrophoresis at a constant 35 mA, the resolved LPScomponents were visualized by the silver stain method ofHitchcock and Brown (13).The Western blot (immunoblot) technique was applied to

study the binding characteristics of different MAbs andpolyvalent antisera against the LPS species. The resolvedLPS components were transferred onto a nitrocellulosemembrane (pore size, 0.45 ,um; Schleicher & Schuell, Das-sel, Federal Republic of Germany) at a constant voltage of 30V overnight in the Bio-Rad Transblot system. After electro-transfer, the nitrocellulose membrane was blocked with

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TABLE 3. ELISA reactivities of LPS-reactive MAbs against purified LPSs from various Salmonella species, E. coli, and S. flexneriELISA reactivity (A405)a

Group Clone Isotype Specificity Salmonella LPSb Other bacterial LPS

AO BO CO DO EO Ra S. flexneri E. coli 0111

1 MABO-8 IgG2b Group B O chain - 3.2MABO-10 IgG3 - 2.9 - - - - - -

2 MACO-18 IgM Group C O chain - - 3.2MACO-26 IgGl - - 2.0 - - - - -

3 MADO-9 IgM Group D O chain - - - 2.0 -

4 MAEO-7 IgGl Group E O chain - - - - 3.0MAEO-10 IgGl - - - - 2.3 - - -

5 MADR-5 IgM Ra core LPS 1.6 2.0 0.9 1.5 1.7 2.0a MAb culture supernatants diluted 1:10 were tested, and optical densities of less than 0.1 determined by ELISA were not recorded and are indicated by a minus

sign.b Salmonella LPSs were from S. paratyphi A (AO), S. typhimurium (BO), S. newport (CO), S. typhi (DO), S. anatum (EO), and S. typhimurium TV119 (Ra).

PBS-2% casein and was then incubated with antibody solu-tion (1:500 rabbit polyvalent serum or 1:10 hybridoma cul-ture supernatant diluted in sterile PBS) at room temperature(20 to 24°C) for 2 to 3 h. After three washings in PBS-Tween,the antibodies bound to the LPS antigens on the nitrocellu-lose membrane were detected by using an indicator systemwith peroxidase-conjugated swine anti-rabbit or rabbit anti-mouse immunoglobulins (Dakopatts AS, Copenhagen, Den-mark), and the color bands were developed with 4-chloro-1-naphthol (Sigma).

RESULTS

High yield of LPS-reactive hybridomas by immunizationswith LPS-coated bacteria. Two immunization strategies wereused to raise antibody responses in mice for hybridomageneration against the LPSs of S. typhimurium SH4809, S.newport IS50, S. typhi 253Ty, and S. anatum IS78: the use ofheat-killed bacteria and LPS-coated bacteria, i.e., wholebacterial cells additionally coated with homologous pureLPS. Immunizations with heated-killed bacteria, either S.typhimurium or S. typhi, elicited only one LPS-reactive,immunoglobulin M (IgM)-secreting hybridoma. By contrast,use of LPS-coated bacteria gave rise to 62 hybrid clones (infour fusions) reactive against the LPS antigens. Moreover, awide distribution of antibody isotypes (IgM [28 clones], IgGl[12 clones], IgG2a [5 clones], IgG2b [12 clones], IgG3 [5clones]) was isolated. Fifty-eight of these clones were shownto secrete antibodies that bound the 0-antigenic polysaccha-ride chains, while four clones secreted antibodies that werereactive with the complete core rough mutants (Ra chemo-type) (Table 2).

Binding specificities of MAbs to the SalmoneUa LPS anti-gens. The resulting hybridomas were screened by ELISA forantibody specificity against a battery of LPSs from Salmo-nella serogroups A to E as well as the S. typhimurium roughmutant TV119 (Ra chemotype). Two major categories ofanti-LPS specificities were found (Table 3). (i) 0-chaingroup-specific antibodies bound selectively to a particularSalmonella 0 serogroup (or subgroup), e.g., MABO-8 andMABO-10 were BO-specific MAbs; MACO-3, MACO-18,and MACO-26 were CO-specific MAbs; MADO-9 was aDO-specific MAb; and MAEO-7 and MAEO-10 were EO-specific MAbs. (ii) Four LPS core-specific MAbs (e.g.,

MADR-5) were found to react not only with the LPS of S.typhimurium TV119 (Ra chemotype) but also with the LPSsof serogroup A to E salmonellae. The presence of nakedcores in these LPS preparations was demonstrated in silver-stained gels (Fig. 2). Eight different 0-chain-specific MAbs(MABO-8, MABO-10, MACO-3, MACO-18, MACO-26,MADO-9, MAEO-7, and MAEO-10) were selected for amore detailed immunochemical characterization. In screen-ing tests, all eight clones gave interesting specificity pat-terns, with a high affinity for the 0-antigenic polysaccharidechains (Table 3).Immunoblotting analysis of 0-antigen-specific clones. Silver

staining of the electrophoresis-separated LPSs from Salmo-

. 4* 4

'.4

FIG. 2. LPS patterns of Salmonella species. Electrophoresis in13.5% polyacrylamide gel (35 mA, 3.5 h) of purified LPS prepara-tions (10 ,ug per lane) from S. typhimurium (lane 1), S. thompson(lane 2), S. newport (lane 3), S. typhi (lane 4), S. anatum (lane 5),and S. typhimurium TV119 Ra LPS (5 ,ug per lane) (lane 6).Migration profiles of each LPS species were visualized by silverstaining.

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2428 LUK AND LINDBERG

I23 4 5 6 1 2 3 4 5 6

B

I 2 3 4 5 t

Usno

A C

FIG. 3. Immunoblotting analysis of Salmonella 0-specific antibody binding to the high-molecular-weight LPS patterns. After sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, the separated LPS patterns were electroblotted onto a nitrocellulose membrane and thenprobed with MACO-3 (1:10 culture supematant) (A); a pool of MABO-10, MACO-3, MACO-18, MADO-9, and MAEO-7 antibodies (1:5supernatant) (B); and a 1:500 dilution of rabbit polyvalent anti-Salmonella poly 0 antisera (C). Lanes are as described in the legend to Fig.2.

nella serogroups B to E revealed the characteristic ladderpattern (with each band differing from the next one by an0-repeating unit), while the TV119 (Ra) LPS lacked all of thehigh-molecular-weight bands and gave core bands only at thebottom region on polyacrylamide gels (Fig. 2) (13, 15).Essentially, immunoblotting of these resolved LPS antigenswith the selected 0-chain-specific MAbs gave ladder pat-terns similar to those obtained in the silver-stained gels. Forexample, the MAC0-3 antibody was strongly reactiveagainst nearly all of the different-molecular-weight bands ofS. thompson LPS (Fig. 2, lane 2), but no specific binding tothe LPSs of other serogroups or the core bands was ob-served (Fig. 3A). Likewise, MABO-10, MACO-18, MADO-9, and MAEO-7 MAbs also bound selectively to the high-molecular-weight ladders of the LPSs of S. typhimurium(Fig. 2, lane 1), S. newport (Fig. 2, lane 3), S. typhi (Fig. 2,lane 4), and S. anatum (Fig. 2, lane 5), respectively.The 0-antigen-specific MAbs MABO-10, MACO-3, MACO-

18, MADO-9, and MAEO-7 were combined and tested fortheir binding characteristics with the same LPS prepara-tions. The MAb pool reacted specifically with all the fiveSalmonella serogroup B to E LPSs at the high-molecular-weight region, but it was unreactive with all of the LPS cores(Fig. 3B). For comparison, similar staining patterns wereobtained by probing with a commercial rabbit polyvalentanti-Salmonella poly-O antiserum (Fig. 3C). The immuno-blot reactions between the antibodies (both monoclonal andpolyclonal) and the S. typhi LPS in lane 4 of Fig. 3Cappeared to be relatively weak, and this might be attributedto the preparation and treatment of this particular LPS typeitself. The use of LPS from another S. typhi strain gavestrong reactions (data not shown).

Epitope analysis of 0-specific MAbs by LPS, oligosaccha-rides, and synthetic saccharide glycoconjugates. To furthercharacterize the epitope specificities of the 0-antigen-spe-cific MAbs, we tested the clones against a series of structur-ally related Salmonella LPSs and defined O-antigen-BSAglycoconjugates by ELISA titration.

The reactivities of the BO-, DO-, and EO-specific MAbswith the LPSs and glycoconjugates of Salmonella sero-groups A, B, D, and E are given in Table 4. First, MABO-8and MABO-10 (elicited by LPS-coated S. typhimurium bac-teria) were found to bind selectively to the BO (0:4,5) LPS.While MABO-8 seemed to recognize the 05 epitope,MABO-10 was 04 specific because it bound to the alkaline-treated S. typhimurium (04) LPS and the 04-containingocta- and dodecasaccharide conjugates. Second, MADO-9(induced by LPS-coated S. typhi bacteria) reacted specifi-cally with the DO (0:9) LPSs of S. typhi (0:9,12) and S.enteritidis (0:9,12). In testing the 09-containing glycoconju-gates, MADO-9 was shown to bind the strain SH1262dodecasaccharide-BSA conjugates. For the EO-specificclones, MAEO-7 and MAEO-10 (raised against LPS-coatedS. anatum bacteria) reacted strongly with the E1 (0:3,10)LPS, but only MAEO-7 showed cross-reactivity with theLPSs of serogroups E1 to E4. Notably, MAEO-7 was alsoable to recognize the synthetic 03 epitope in the EO-trisaccharide conjugate. None of the murine MAbs showedany detectable binding to the synthetic AO-, BO-, or DO-disaccharide conjugates.The CO-specific MAbs were tested against a panel of

Salmonella C1, C2, and C3 LPSs as well as the 07 and 08glycoconjugates (Table 5). MACO-3 was raised against LPS-coated S. thompson bacteria in our previous study (24), andit bound specifically to the LPS of Salmonella serogroup C1(0:6,7) and reacted equally well with the 07-containingdecasaccharide-BSA. On the other hand, MACO-18 andMACO-26, both of which were elicited by LPS-coated S.newport bacteria, bound to the LPS of serogroup C2 (0:6,8).However, only MACO-18 was reactive against serogroup C3(0:8,20) LPS. We surmise that MACO-26 identified the 06epitope which is specific for C2 only, whereas MACO-18recognized the 08 epitope, which is shared among serogroupC2 and C3 salmonellae.

Identification and serotyping of salmonellae by 0-specificMAbs. To study the potential diagnostic usefulness of these

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TABLE 4. Reactivities of Salmonella BO-, DO-, and EO-specific MAbs against chemically definedSalmonella LPSs and glycoconjugates

Structure of Reciprocal endpoint titer of MAb supernatantbAntigen 0 factor oligosaccharide units' MABO-8 MABO-10 MADO-9 MAEO-7 MAEO-10 Other MAbsc

Purified LPSParot 4 1,3

S. paratyphi A IS2 0:2,12 [a-Man(1---*4)os-Rha(l--*3)a-Gal(l--*2)]nAbe-(OAc)a 4 1,3

S typhimurium 0:4,5,12 [a-Man(1--4)a-Rha(1---3)a-Gal(--->2)]nSH4809

Abea 4 1,3

S. typhimurium 0:4,12 [a-Man(1-*4)a-Rha(1--*3)a-Gal(1-- 2)]nSH4809 (NaOHtreated)

Tyvao 1,3

S. enteritidis SH1262 0:9,12 [a-Man(1--4)a-Rha(1l-3)a-Gal(1-*2)]n

Tyva 4 1,3

0:9,12 [a-Man(1--*4)a-Rha(1--3)a-Gal(1--*2)],,

OAc

S. anatum IS78 0:3,10 [,-Man(1--*4)a-Rha(1--*3)a-Gal(1-*6)]nS. illinois IS133 0:3,15 [P-Man(1-*4)a-Rha(1-*3)P-Gal(1--*6)],,

Glca 4 1,6

S. senftenberg 0:1,3,19 [p-Man(1--*4)a-Rha(1--*3)a-Gal(1-+6)1,SH1707

GlycoconjugateSH4809

Abea4 1,3

Tetrasaccharide- 0:4 [a-Gal(1-*2)a-Man(l->4)a-Rha(1-*3)]nBSA (n = 1)

Abea 4 1,3

Octasaccharide- 0:4 [a-Gal(1-*2)a-Man(l-->4)a-Rha(1---*3)]nBSA (n = 2)

Abea 4 1,3

Dodecasaccharide- 0:4 [a-Gal(1---2)a-Man(1--*4)a-Rha(1--*3)]nBSA (n = 3)

SH1262Tyva4 1,3

Octasaccharide- 0:9 [a-Gal(l-*2)a-Man(l---4)a-Rha(l--3)]nBSA (n = 2)

Tyvao 1,3

Dodecasaccharide- 0:9 [a-Gal(1---2)a-Man(l--*4)a-Rha(1-*3)tnBSA (n = 3)

<1 <1 <1 <1 <1 <1

2,200 1,800 <1 <1 <1 <1

<1 1,500 <1 <1 <1 <1

<1 <1 160 <1 <1 <1

<1 <1 280 <1 <1 <1

<1 <1 <1 3,500 3,500 <1

<1 <1 <1 80 <1 <1

<1 <1 <1 620 <1 <1

<1 <1 <1 <1 <1 <1

<1 80 <1 <1 <1 <1

<1 1,024 <1 <1 <1 <1

<1 <1 <1 <1 <1 <1

<1 <1 240 <1 <1 <1

AO disaccharideBO disaccharideCO disaccharideDO disaccharaideEO trisaccharide

0:20:40:80:90:3

a-Par(1--*3)a-Man(1--*BSA)a-Abe(1--+3)a-Man(1l-BSA)a-Abe(1--*3)P-Rha(1-*BSA)a-Tyv-(1-->3)a-Man(1--+BSA)P-Man(1--4)a-Rha(1--*3)a-Gal(1---*BSA)

<1 <1 <1 <1<1 <1 <1 <1<1 <1 <1 <1<1 <1 <1 <1<1 <1 <1 2,048

S. typhi T2

a Abe, abequose; Gal, galactose; Glc, glucose; Man, mannose; Par, paratose; Tyv, tyvelose; OAc, O-acetyl. Cleavage sites of the BO and DO repeating units

by bacteriophage P22 endo-rhamnosidase are indicated by 11*. LPS structures are from reference 21.b Endpoint titer represents the highest dilution of antibody supernatant to give an A405 of 0.2 by ELISA.- MACO-3, MACO-18, and MACO-26 MAbs.

<1 <1<1 <1<1 <1<1 <1<1 <1

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TABLE 5. Reactivities of Salmonella CO-specific MAbs against LPSs from serogroups C1 to C3 and the 0:7 glycoconjugate

Reciprocal endpoint titer of MAbsupernatantb

Antigen 0 factor Structure of oligosaccharide unitsaMACO-3 MACO-18 MACO-26 MAbsc

Glcaj 1,3

S. thompson IS40 LPS 0:6,7 [P-Man(l >2)a-Man(l2)o-Man(1 >2),-Man(1 >3)P-GIcNAc(l2)]" 1,024 <1 <1 <111*

IS40 decasaccharide-BSA 0:7 [,-Man(1 >3),-GIcNAc(l s2),-Man(12)a-Man(l s2)a-Man(l >2)]2 2,048 <1 <1 <1glycoconjugate

Abe Glc-OAcatc1,3 at 1,3

S. newport IS50 LPS 0:6,8 [,-Rha(1-*2)a-Man(1-- 2)a-Man(1-- 3)I3-Gal(1-*4)]n <1 2,048 1,024 <1

Abe Glc-OAcaj 1,3 ax 1,3

S. kentucky IS98 LPS 0:8,20 [,-Rha(1 >2)a-Man(l2)a-Man(l >3)P-Gal(l4)]n <1 1,024 <1 <1

a Abe, abequose; Gal, galactose; Glc, glucose; Man, mannose; NAc, N-acetyl; OAc, 0-acetyl. Cleavage site of the CO repeating units by bacteriophageendo-mannosidase is indicated by ft*. LPS structures are from reference 21.

b Endpoint titer represents the highest dilution of antibody supernatant to give an A405 of 0.2 by ELISA.c MABO-8, MABO-10, MADO-9, MAEO-7, and MAEO-10 MAbs.

antibodies, mouse ascitic fluids containing MABO-10 (IgG3),MACO-3 (IgM), MACO-18 (IgM), MADO-9 (IgM), andMAEO-7 (IgGl) were pooled and then used as poly (sero-groups B to E) 0 reagents in slide agglutination tests for theidentification of salmonellae among 294 bacterial strains(Table 6). The MAb "cocktail" agglutinated strongly andrapidly all of the 167 clinical isolates of commonly encoun-tered salmonellae from serogroups B to E (including 72serotypes), whereas none of the 127 strains from otherbacterial species gave a positive reaction. In addition, theseSalmonella strains were unequivocally serotyped intogroups B to E by the individual BO-, CO-, DO-, andEO-specific antibodies. The routinely used factor-specificrabbit anti-Salmonella 0 antisera yielded similar results, butcross-reactivities against 10 other strains of related membersof the family Enterobacteriaceae were detected with thecommercial serotyping antisera.

DISCUSSION

Specific antibodies directed against the LPS 0 antigensare of interest because the bacterial LPS is a virulence factorwhich resists the host defense mechanisms (28) and theO-polysaccharide chain carries distinct diagnostic markersuseful for serotyping (21). Moreover, there is cumulativeevidence pointing toward the immunoprophylactic and pro-tective capacities of anti-O-polysaccharide antibodiesmainly against homologous gram-negative bacterial infec-tions (2, 4, 22, 32). Therefore, antibodies specific for the 0antigens of bacterial LPSs are useful not only for mappingpotential virulence epitopes but also for serological identifi-cation of etiologic agents (5, 27). In attempts to improve thediagnostic accuracy, we have considered two possible ap-

proaches, one by immunizing animals with synthetic 0-an-

tigen-specific glycoconjugates (7-9, 22, 34) and the other byraising MAbs specific for the 0-antigen epitopes (1). In thisstudy, we combined the hybridoma technique for continuousproduction of LPS-specific antibody reagents and the use ofchemically defined glycoconjugates for mapping the epitopespecificity of MAbs reactive against the Salmonella 0-anti-genic polysaccharides.For the optimum yield of 0-antigen-specific hybridomas,

we preferably centered on the use of LPS-coated bacteria asthe immunogen, which has previously been shown to besuperior to the use of heat-killed bacteria and glycoconjugateas the immunogen (24). Apparently, this favorable immuniz-ing effect is attributed to the increased amount of LPSantigens on the bacterial cell envelope and is perhaps in abetter form of presentation to the host for immune recogni-tion. On the basis of this effective immunization strategy, awide variety of LPS-specific MAbs with different reactivitypatterns and antibody isotypes were thus generated (Table2). In our initial selection procedures with a battery ofpurified LPSs from Salmonella serogroups A to E, we beganto distinguish those useful MAbs with interesting epitopespecificities which could be applied prospectively as diag-nostic tools.MAbs MABO-8, MABO-10, MACO-3, MACO-18, MACO-

26, MADO-9, MAEO-7, and MAEO-10 are directed againstthe 0-antigenic polysaccharide chain of Salmonella LPSssince (i) they bound directly to the cell surface antigens ofSalmonella bacteria but did not bind any of the otherbacterial species tested; (ii) they reacted with the smoothLPSs extracted from Salmonella BO, CO, DO, and EO, butdid not react with that purified from the TV119 (Ra) roughmutant, which is genetically defective in the biosynthesis ofthe 0-antigenic polysaccharide chain; and (iii) these MAbsshowed the strongest reactivity with the high-molecular-weight LPS components which contain a relative abundanceof 0-antigen repeating units (Fig. 3). In a further study, wedetermined the epitope specificities of the 0-antigen-specificMAbs by using a set of chemically defined Salmonella0-antigen glycoconjugates and related Salmonella LPSstructures which differed from their counterparts by a sugarsubstitution or their anomeric linkages. The specificities thatwe found can be summarized as follows.

Epitopes 04, 05, and 09. The structures of Salmonella0-antigenic polysaccharide chains in serogroups A, B, D,and E are very similar except for the nature of their3,6-dideoxyhexosyls, e.g., paratose in group A, abequose ingroup B, and tyvelose in group D (Fig. 1), which are linkedto the D-mannose residue in the tetrasaccharide-repeatingunits. The dideoxyhexose acts as an immunodominant sugarmarker in determining the serogroup specificity of Salmo-

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TABLE 6. Agglutination tests of salmonellae and other bacteria with mouse ascitic fluids containing 0-specific MAbs incomparison with rabbit polyvalent serotyping antisera

No. of strains No. of positive with MAbsb No. positive with rabbit polyvalent antiseracBacterial strain testedaese ~BO CO DO EO Poly O BO CO DO EO Poly O

Salmonella serogroup BS. typhimuriumS. agonaS. bredeneyS. chesterS. saintpaulS. stanleyOther 7 serotypes

Salmonella serogroup CS. thompsonS. braenderupS. infantisS. virchowS. choleraesuisS. newportS. litchfieldS. blockleyS. muenchenS. kentuckyOther 27 serotypes

Salmonella serogroup DS. enteritidisS. dublinS. typhiS. javianaOther 5 serotypes

Salmonella serogroup ES. anatumS. londonS. senftenbergS. giveS. lexingtonS. krefeldS. ugandaOther 6 serotypes

Other EnterobacteriaceaeEscherichia coliShigella spp.Citrobacter spp.Yersinia enterocoliticaMorganella spp.Enterobacter spp.Proteus spp.Klebsiella pneumoniae

Other gram-negative bacteriaHaemophilus influenzaePseudomonas aeruginosaAcinetobacter spp.Campylobacterjejuni

8 8 0 0 0 8 85 5 0 0 0 5 53 3 0 0 0 3 32 2 0 0 0 2 22 2 0 0 0 2 22 2 0 0 0 2 29 9 0 0 0 9 9

4 0 4 0 0 43 0 3 0 0 33 0 3 0 0 33 0 3 0 0 32 0 2 0 0 24 0 4 0 0 43 0 3 0 0 32 0 2 0 0 22 0 2 0 0 22 0 2 0 0 2

36 0 36 0 0 36

23 0 0 23 0 237 0 0 7 0 75 0 0 5 0 52 0 0 2 0 25 0 0 5 0 5

5 0 0 0 5 54 0 0 0 4 44 0 0 0 4 43 0 0 0 3 32 0 0 0 2 22 0 0 0 2 22 0 0 0 2 28 0 0 0 8 8

3520151510652

00000000

10531

0000

a A total of 294 bacterial strains were tested (167 salmonellae, 108 Enterobacteriaceae, 19 other gram-negative bacteria).b A 1:10 dilution of mouse ascitic fluid was used for agglutination. Poly 0 is a cocktail of ascitic fluids containing BO-, CO-, DO-,c Antisera were from the National Bacteriology Laboratory, Stockholm, Sweden.

nella AO, BO, and DO (21, 26). The 04 epitope, which is

represented by the a-Abe(1-*3)Man disaccharide, is themajor determinant that identifies all serogroup B salmonel-lae; and 0 acetylation of the abequose forms anotherepitope, 05, which is easily removed by alkaline treatment.The present data indicate that the epitope recognized by

and EO-specific MAbs.

MABO-10 is 04 and the epitope recognized by MABO-8 is05 (Table 4). When we tested MABO-10 with different sizesof 04-containing glycoconjugates, a lower limit of octasac-charide determinant (two BO-repeating units) was neededfor MABO-10 antibody recognition and a higher enzymeimmunoassay titer (10-fold enhancement) was detected with

8532229

4333243222

36

4333243222

36

237

25

237S25

5 54 44 43 32 22 22 28 8

26011000

0000

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2432 LUK AND LINDBERG

the dodecasaccharide conjugate (i.e., three repeating units).This finding is consistent with our previous suggestion,based on rabbit anti-04 antibodies, that the sizes of Salmo-nella BO determinants for antibody binding are larger than a

tetrasaccharide but smaller or equal in size to an octasac-charide (16).The MADO-9 antibody showed selective reactivity only

with the LPS of serogroup D. Therefore, we suggest that thea-Tyv(1->3)Man disaccharide of factor 09 is important forMADO-9 recognition. Further proof of its 09 specificity isbased on the reactivity of MADO-9 MAbs with the strainSH1262 09-BSA conjugate. Here, a dodecasaccharide (three0 repeating units) was required for antibody binding.

Epitopes 03 and 010. The difference between the 0-polysaccharide structure of Salmonella serogroup E andthose of serogroups A, B, and D is the lack of a 3,6-dideoxyhexose and the fact that the D-mannose residue isP-1,4-linked to L-rhamnose. This imparts distinct serologicalspecificities to EO. In a previous study, Uchida et al. (36)suggested that the P-Man(1-4)-oa-Rha disaccharide of the 0chain was the 03 determinant of serogroup E salmonellae.The reactivity of MAEO-7 with the synthetic ,-Man(1-+4)-a-Rha(1->3)-aL-Gal trisaccharide-BSA glycoconjugate pro-vides strong evidence that the MAEO-7 combining siterecognizes the 03 determinant. Compared with the E1 LPS,the LPSs from E3 and E4 salmonellae had lower reactivitieswith MAEO-7, which made us surmise that the x-galactose,and perhaps the O-acetyl group, is partially involved in therecognition. On the other hand, failure of the MAEO-10antibody to bind the E3 and E4 LPSs clearly shows that thea-GalOAc residue in the serogroup E1 LPS is recognized bythis antibody, i.e., epitope 010.

Epitopes 06, 07, and 08. The structures of the 0-polysac-charide chains of serogroup C salmonellae differ markedlyfrom those of groups A, B, D, and E (Tables 4 and 5). Asexpected, none of the Salmonella CO-specific MAbs showedcross-reactivity with serogroups A, B, D, or E or vice versa.On the basis of reactivity against the strain IS40 decasac-charide, which was previously shown to contain the 07determinant (9), the epitope recognized by MACO-3 istentatively identified as factor 07, which is unique forSalmonella serogroup C1. However, little is known aboutthe structural element representing the 07 determinant andawaits a detailed immunochemical characterization withMACO-3 and other related 07-specific MAbs which showexquisite specificity for Salmonella serogroup C1.The specific 0 antigen in Salmonella serogroups C2 and C3

is factor 08, and the immunodominant sugar is abequose.Unlike the 04 epitope in serogroup B, here, the abequose isao(l-3) linked to L-rhamnose, thereby introducing 08 spec-ificity to the C2 and C3 serogroups. Since MACO-18 wascross-reactive with serogroups C2 and C3 but did not reactwith serogroup B, we therefore concluded that the epitopespecificity of MACO-18 is attributed to factor 08, which ischaracterized by the ac-Abe(1--3)Rha determinant. In con-

trast, the failure of MACO-26 to identify serogroup C3 iSlargely due to the structural difference between these twoserogroups. Therefore, we suggest that the ot-GlcOAc(1->3)Man disaccharide is essential for MACO-26 recogni-tion, which in turn corresponds to the 06 epitope specific forSalmonella serogroup C2. This 06 epitope differs from thatseen in serogroup C1, since MACO-26 did not bind to any C1salmonellae (Table 5).Our interest is to develop new and more efficient immu-

noassays on the basis of 0-antigen-specific MAbs (1). Re-cently, we established an MAb-based immunomagnetic tech-

nique to open a new possibility for the rapid detection ofsalmonellae in clinical and food specimens (23). Further-more, there have been several reports on the use of MAbprobes to detect salmonellae in clinical specimens, and theirpotential diagnostic value was also demonstrated (3, 19, 30,35), even though either the epitope specificity of the de-scribed MAbs were not immunochemically defined or thevariety of salmonellae identified by these agents was oftenlimited. With a satisfactory performance in slide agglutina-tion tests, the present 0-antigen-specific MAbs correctlyidentified all 167 strains of salmonellae from serogroups B toE (Table 6). Altogether, a complete panel of the Salmonella02-specific (25) and 03-, 04-, 07-, 08-, and 09-specific (thisstudy) MAbs are now available, covering more than 95% ofthe Salmonella isolates in Europe and the United States.One prospective clinical implication is that the MAbs withthe described 0 specificities are useful as diagnostic reagentsto replace the conventional rabbit polyvalent factor-specificantisera for identification and serotyping of Salmonellaspecies. At present, we are investigating the possibility ofusing the MAb-based immunomagnetic system to isolatesalmonellae in clinical specimens, with a subsequent DNAamplification and rapid detection by the powerful polymer-ase chain reaction technique (10, 37).

In summary, we demonstrated that the Salmonella 0-spe-cific MAbs that we studied recognize 0-antigen epitopescorresponding to 03, 04, 05, 06, 07, 08, 09, and 010, asdescribed by the Kauffmann-White scheme (17), and that thesize of the 0-specific antigenic determinant for antibodyrecognition ranged from a trisaccharide (epitope 03), to anoctasaccharide (04), a decasaccharide (07), and finally, adodecasaccharide (09) of the Salmonella 0-antigenic poly-saccharide chains.

ACKNOWLEDGMENTS

This work was supported by grant 16X-656 from the SwedishMedical Research Council.We thank Sten Karsbrink of the Clinical Research Center of

Huddinge Hospital for photographic work.

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