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0022-1767/86/1377-2361$02.00/0 THEJOURNAL OF IMMUNOLOGY Copyright Q 1986 by The American Asswlation of Immunologists

Vol. 137,2361 -2366.No. 7.October 1. 1986 Prlnted in U.S.A.

CHARACTERIZATION OF SHIGELLA FLEXNEN-SPECIFIC MURINE MONOCLONAL ANTIBODIES BY CHEMICALLY DEFINED GLYCOCONJUGATES' NILS I. A. CARLIN,* MARGARET A. J. CIDNEY,' A. A. LINDBERG,'

AND

DAVID R. BUNDLE'

From the *Departmentof Bacterlology, National Bacteriology Laboratory,S-10521 Stockholm, Sweden: the 'Division of Blofogicaf Sciences. National Research Council of Canada, Ottawa. Ontario, Canada K I A OR6; and 'Karolinska Institute, Department of Clinical Bacteriology, Huddlnge University Hospital. S-14689Huddinge. Sweden

established (4-6). The serotypeY antigen is defined by a Chemically defined glycoconjugates are demon(Fig. strated to have considerable potential for selectinglinear polymer with a tetrasaccharide repeating unit hybridoma antibodies directed toward 0-antigenic 1). and substitution by acetate and a-glucose residues determinants, especially when used in combination change and mask the antigenicspecificities of the serowith a panel of well-characterized LPS molecules. type and group determinants. The antigens of serotype X Monoclonal antibodiesspecific for the Shigellaflex- and 5 are related to type Y by a-glucosylation at two sites neri 0-antigens of serogroup 5b,variants X and Y, (Fig. 1).Because carbohydrates are conformationally well were generated after immunization of BALB/c mice defined, their interaction with antibody is one that may with killed bacterial cells, and active hybridswere be approached from a sound appreciation of the threeselected on the basis of ELISA performed with the dimensional shape of the antigen in solution. The oligopurified serotype-specific LPS antigen. Subsequent saccharides of S. flexneri serotype Y have been well screening witha variety of glycoconjugates, derivedstudied in this regard by nuclear magnetic resonance from synthetic oligosaccharides and larger structures obtained by phage SfGlendo-rhamnosidase hy- techniques, which haveprovided not only a three-dimensional model of the determinant insolution (7)but also of purified LPS established a detailed prodrolysis lead to identification of the biologic repeating unit (8). file of binding characteristics for Shigellaflexneri Monoclonal antibodies to these structurally interrelated variant Y-specific antibodies. Together with the results of precipitin analysis and heavychain isotyp- and well-characterized antigens have considerable poof antibodies tential both as standardized reagents for serodiagnosis ingexperiments,alimitednumber were selected as candidates for detailed studies of (9)and as potential candidatesfor detailed immunochemical analysis of the hitherto poorly characterized deterthe antibody combiningsite. The elucidation of the physical forces and atomic interactions betweena n antigen and the protein surface of the antibody combiningsite ultimately requiresthe combination of detailed ligand binding studies withthe precision of high resolution x-ray diffractionand amino acid sequencedata.The hybrid-myeloma technique (I), in conjunction with a discriminating antigen assay, is ideally suited to the production of large quantities of monoclonal antibody intendedfor this purpose. Bacterial polysaccharides not only offer awide selection of structurally interrelated antigens, well suited for such studies, but in addition possess practical significance a s serologic markers for diagnosis of infection (2). In differentiating bacterial serogroups and serotypes, antibodies exhibit exquisitespecificity for carbohydrate antigens, which may differ in stereochemistry or structure ata single carbon atom.The serologic classification of Shigellaflexneri is based on the 0-polysaccharide chains of the cell envelope lipopolysaccharide (LPS) (31, and the structural basis of these differences has been Received for publication April 14. 1986. Accepted for publication June 26. 1986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to \nd\cate this fact. ' This work was supported In part by the Swedish Medical Research Council (Grant 16X-656)and the Swedish Board for Technical DevelopThis is NRCC Publication No. 26336. ment (Grant 80-5589).

minants of S. flexneri 0-antigens. In this paper we describe the generation of monoclonal antibodies that bind the antigenic determinants of S. flexneri LPS of serogroups 5b,variants X and Y , and the characterization of Y variant specificities by use of synthetic glycoconjugates. Monoclonal antibodies suitable for detailed immunochemical studiesof the antibody site associated with various0-factors are also identified. MATERIALS AND METHODS

Bacterial strains. S. flexneri strains Y, X. 2b. and 5b were obtained from the AmericanType Culture Collection. Additional strains of serotypes la, Ib, 2a. 2b.3a. 4a, 5a, 5b, andX were from the strain collection, National Bacteriology Laboratory, Stockholm, Sweden and from Dr. E. Romanowska (4-6). Antigens. Lipopolysaccharides were extracted from S. JZexnerf strains grown in batch cultureby hot phenol-water (10).Purification was performed by repeated ultracentrifugation until each LPS was essentially protein and nucleic acid free (1 1). 0-Chain polysaccharides were liberated from lipid A by hydrolysis in 1%aqueous (v/v) acetic aclda t 100°C for 60 min and purified from low m.w. components bygel chromatography on a column of Sephadex LH20, 2.0 x 60 cm (4).Synthetic haptens linked to a nine carbon spacer were prepared according to published syntheses (12-17). deblocked and coupled to BSA by the modified Honzl and Ruddinger method (18). Oligosaccharides obtained by phage Sf6-mediated hydrolysis (8, 19) were also coupled to BSA via the 2-(4-aminophenyl)ethylamlne spacer as described (20). The structuresof the polysaccharides and oligosaccharides were confirmed by methylation analysis and nuclear magnetic resonance methods (4, 5, 7. 8).Alkali-treated LPS was prepared by incubation of LPS (1 mg) in 200 p l of 0.25 M sodium hydroxide solution a t 56°C for 1 hr.Theresulting solution was neutralized with 200 pl of 0.25 M hydrochloric acid and diluted to 1 rnl with phosphate-buffered saline(PBS). Immunization. Groups of six female BALB/c mice. 12 to 16 wk of

236 1

2362

GLYCOCONJUGATE-SELECTED

Glut

=‘I3

Glut

“13-

*

on

Figure I. 0-polysaccharide of Sflexnerf.

Shigella

structures

p

of the Y. X, 5a, and 5b antigens

age (Charles Rivers Canada Inc., St. Constant, Quebec), were immunized with phenol-killed cells of the appropriate S.flexnert species grown to late log phase in batch culture. Cells were washed twice with 10 mM PBS, pH 7.0. and suspended in PBS at about 10’ cells/ml. Three groups of six mice were immunized with S.flexneri strain X. Y. or 5b bacteria by i.p. injection (0.2 ml) on day 0, followed by iv. injections (0.2 ml) on days 7, 14, and 28 and splenectomy on day 31, protocol B (see Table I). Two other groups of mice were given an i.p. injection (0.2 ml) on day 0 with either S.flexnerf strain Y cells or S. flexnerf 5b cells. The mice receiving S. flexneri Y cells were immunized (0.2 ml) i.v. on day 7 followed by splenectomy on day 10. protocol A (see Table I). The Sflexnerl5b group received a second i.p. injection (0.2 ml) on day 7 followed by splenectomy on day 11. protocol A [Table I). In Tables II and IV, the fusion experiments and associated immunization protocols are referred to as YA, Y-B, 5b-A. and 5b-B. The immunization procedure used to generate the three antibodies MASFY-1 to -3 has been reported (9). Three sets of four mice were immunized i.p. with Y LPS 5. 10, and 20 &/injection on days 0 and 7. Splenectomy was performed on day 11 to measure LPS-specific plaque-forming cells. Cunningham plaque assay. The Cunningham direct plaque assay was performed on a sample of spleen cells (-lo6 cells/spleen) (21). Sheep red blood cells passively coated with alkali-treated variant Y LPS (22) were used together with guinea pig complement (Grande Island Biologics, Burlington, Ontario) twice absorbed against the LPS-coated sheep red blood cells. Plaques were read after incubation at 37OC for 60 min. Fusion and cloning. The fusion protocol was the modified procedure described by Kennett et al. (23). which combines the essential elements of orocedures described by Gafle et al. (24) and Gefter et al. (25). Spleen cells from two immunized mice werefused with the non-@-producing Sp2/0 plasmacytoma cell line [Institute for Medical Rese&h, Camden. NJ); as described (26). Putative hybrids were screened by an enzyme-linked immunosorbent assay (ELISA) on culture supernatants (100 al) 10 to 14 days postfusion. Hybrids exhibiting an absorbance reading greater than 0.3 against negligible background were cloned in “semi-solid agar’ by using mouse spleen cells as feeders (26). All hybrids were cloned twice to ensure stability before freezing cell samples and raising ascitic fluid. The three monoclonal antibodies, designated MASFY l-3 were produced against S.flexnert Y variant by a procedure identical to that reported for S. flexnert type II monoclonal antibodies (9). ELISA screentng. LPS-coated Linbro enzyme immunoassay (EIA)’ microtitration plates (Flow Laboratories, Mississauga. Ontario) were prepared by incubating with 100 pi/well of the appropriate LPS solution (10 p&ml) in 0.05 M sodium carbonate buffer, pH 9.8. for 3 2Abbreviations used enzyme immunoassay.

in this

paper:

AFC,

antibody-forming

cell:

EIA,

flexneri

MONOCLONAL

ANTIBODIES

hr at 37°C. Plates were sealed and stored at 4°C until needed. ELISA testing of cell supernatants and ascitic fluid was carried out as described (26). This employed an alkaline phosphatase-conjugated goat anti-mouse IgM and IgG antibody preparation in conjunction with p-nitrophenyl phosphate substrate (Sigma Chemical Co.. St. Louis, MO) [ 1 mg/ml p-nitrophenyl phosphate d&odium salt in 0.05 M sodium carbonate buffer, pH 9.8, containing 10e3 M magnesium chloride). Absorbance at 405 nm was read after 60 min at room temperature with a Titertek Multiscan (Flow Laboratories). Plates were coated as previously described (26) by employing glycoconjugates prepared from either synthetic or phage-derived oligosaccharides at 10 &/ml in PBS. ELISA end-point titrations were performed on ascites fluid diluted in 1 O-fold steps beginning at 10-z and ending at 1 Oe6. The end-point titer was defined as the reciprocal serum dilution giving an optical density of 0.1 at 405 nm after incubation at 37°C for 100 min. These values were calculated by linear regression on a Commodore CBM 8032 computer interfaced with the Titertek Multiscan (Flow Laboratories), utilizing ELISA-software (Meddata Digital AB. Solna. Sweden). lsotype analysis. Heavy chain isotype analysis was performed by using antigen-coated EIA plates, diluted ascites fluids, and a commercial class specific second antibody (Hybri-Clonal EIA mouse antibody screening kit: Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD). The IgG subclass of monoclonal antibodies was established by immunodiffusion. Subclass-specific antibodies (Cedarlane, Hornby. Ontario, Canada) were diluted (l/2) with PBS, according to the manufacturer’s directions, and run against monoclonal antibodies, partially purified by ammonium sulphate precipitation. MASFY l-3 antibodies were classed according to earlier work (9). Ascittcflutd. BALB/c mice were primed by Lp. injection of 0.5 ml 2,6,10,14-tetramethylpentadecane (pristane) 1 to 4 wk before injection with 1 O6 hybridoma cells. Ascitic fluid was tapped 7 to 10 days later and stored at -70°C. Immunodlffusfon. lmmunodiffusion plates were set up by using 1% agarose. Indublose A37 (Fisher Scientific Co., Pittsburgh, PA), in PBS. Ascitic fluids were added to the wells undiluted; antigen solutions [bacterial PS and alkali-treated LPS) were used at 1 mg/ml and 0.5 mg/ml concentrations. Precipitin lines were recorded after incubation at room temperature for 24 to 48 hr. RESULTS

When mice immunized with 5 pg per injection of purified S. flexneri LPS on days 0 and 7 were splenectomized at day 11, the average count of LPS-specific antibodyforming cells (AFC) was 1000 & 500/l 0’ spleen cells. Increasing the antigen dose to 10 pg resulted in the death of two out of four mice, and the AFC counts for the remaining mice were 3000 f 500/10’ spleen cells. None of the mice given 20 pg LPS doses survived. In contrast, when mice were given heat-inactivated whole S.flexneri bacteria, high AFC plaque counts, 25,000 & 500/108 spleen cells, were obtained. To ensure adequate numbers of LPS-specific AFC, all fusion experiments were performed with spleen cells of mice immunized with killed whole cell vaccines. Although groups of six mice were generally immunized, fusion experiments were conducted with spleen cells pooled from two mice, selected on the basis of the ELBA titers recorded for serum collected from the retroorbital plexus vein 7 days after the penultimate injection. Fusion experiments were screened by using the purified homologous LPS, and hybrid clones were selected on the basis of the magnitude of this signal. Only those wells recording a substantial response (OD > 0.3) were selected for recloning. The number of specific cell lines identified and successfully recloned together with their isotype distribution are recorded in Table I. Whereas the initial experiments conducted after only two injections of either serotype Y or 5b cells (protocol A, Table I) gave exclusively IgM clones, subsequent fusion after four injections (pro-

2363

GLYCOCONJUGATE-SELECTED Shigella flexneri MONOCLONAL ANTIBODIES TABLE I Sflexneri putatfue and speclffc LPS hybrid cell lfnes

Shearman and D. R. Bundle) with '251-labeled BSA glycoconjugates showed that independent of oligosaccharide hapten structure, themolar coating per well was similar.

Heavy Chaln lmmunlzlng S.Jexneri Strain

lmmunizat'on Procedure

Number of Clones Recorded as Posltlve by ELISA on Culture Supernatants"

Rat'0 Of Stab'e Recloned Lines to the Number Orlglnally Selected for

lsotype Of Clones

DISCUSSION

Chosen Further

The principal interest of the present study was to generate monoclonal antibodies toward the antigenic determinants of the s. flexneri variant Y, variant X, and CLoning ILM I& serogroup 5b 0-antigens and to use these for a detailed 5 A 69 14/16 Y 12/2 1 3 2 immunochemical analysis of their antibody combining Y B 21 20/20 a 3 95 B X sites. To approach this goal by using chemically synthe1 6 A 5b 1/2 sized oligosaccharides (12- 17) and semi-synthetic anti53 I 8/25 9 2 5b B gens (8). the combining site of the monoclonal antibodies Positive cell lines gave an ELISA OD. > 0.3 at 60 min. should ideally be complimentary to tetrasaccharide structures and not larger than penta- or hexasaccharides, in tocol B, Table I) gave a higher number of specific hybrids order to minimize synthetic obstacles. Further, because and several IgG clones, althoughIgM clones stillpredom- measurement of kinetic and thermodynamic data are most conveniently made with univalent molecules, moninated. Ascitic fluids were developed in mice for each of the oclonal antibodies were desired that would provide Fab recloned cell lines, and the antibodies produced were fragments in good yield. Such material would then also subjected todetailed ELISA testing withhomologous and have potential for x-ray diffraction studies.This made it heterologous LPS from each of the S . jlexneri serogroups. desirable that those antibodies withthe optimal binding The results of these assays are recorded in Tables I1 characteristic be of the IgG class and probably further through IV for eachof the strains Y, X, and 5b. In addition restricted to the IgGl or IgG2a/2b isotypes, for ease of to ELISA. the precipitation characteristics of each anti- Fab preparation (27). Nevertheless, IgM antibodies exbody with both alkali-treated and 0-polysaccharide were hibiting the requisite oligosaccharide binding profiles do also evaluated.The resultsof the latter experiments gave have direct potentialas agglutinating antibodies forbacsome indication of 0-chain specificity. Further evidence terial serogrouping (9).Therefore, in classifying binding for this type of binding was obtained by subjecting each characteristics via the use of chemically defined antigens antibody to a screening panel of S.flexneri smooth LPS and correlating these with isotype, improved 0-factor representing each serotype. The titersof the Y-, X-, and antibodies for either immunochemical studies or bacterial serogrouping could be identified. 5b-specific clones with these LPS preparations, which The use of whole cells to induce a vigorous humoral included onerough LPS to detectcore specific antibodies, are recorded in Tables I1 through IV. By employing gly- response to the LPS was indicated by the low number of coconjugates derived from syntheticoligosaccharides and LPS-specific AFC when purified LPS was used as antigen. larger oligosaccharides that originated from phage-me- This, together with the toxicity of the purified LPS, made diated degradation of LPS, the binding profiles of the it difficult to select an optimal immunizing dose of LPS apparently 0-chain-specific Y clones were evaluated by antigen. By comparison, the use of killed whole cells led ELISA (Table V). The glycoconjugates, bearing approxi- to significant numbers of LPS-specific AFC in plaque mately equal numbers of haptenic groups per mole of assays. Despite the useof whole cells and animmunizaBSA were used to coat ELISA plates, and the titerof the tion protocol intended to enhance a secondary response, ascitic fluid was takenas a direct measureof affinity for the largest number of antigen-specific hybrids belonged that determinant present as the haptenic group of the to the 1gM class (Table I). In contrast, a higher relative oligosaccharide (Table V). The coating efficiency of each proportion of IgG clones was observed in fusion experiglycoconjugate antigen was assumed tobe similar, since ments that produced clones MASFY-1 to -3 (Table 11), in unpublishedobservationsfrom this laboratory (P. J. which only two injections of whole cells were employed, TABLE I1 S.flexneri uarlant Y-speclflc monoclonal antibodies" Precipltatlon

ELSA Tlters

7-

Clone

Y-A1 Y-A2 Y-A3 Y-A4 Y-A5 Y-81 Y-82 Y-B3 Y-84 Y-€55 MASF-1 G2b MASF-2 MASF-3 a

Class

la

lb

M M M M M M G3 0.3 X lo4 M G2a 0.3 x IO4 >IOg 4 M 0.4 x 10" GI G1

-

-

-

Blanks indicate titers below lo2. ND = not determined.

2a

2b

3a

3b

-

-

-

-

-

-

-

-

x lo4

-

-

-

-

-

-

-

3 x lo5 1 x 103 1.5 x 103

0.2 x

4a

NDb 0.2 X 104 lo4 1.3 X 104

4b

5a

-

-

-

5b

"

X

- - _ - - - " - - _

treated Y 4bR

I x 104 1 x 104 5 x lo4 5 x 10" 1 x 103 I x 103

- " - 0.3 X 104 - " I x 104 - - _ 1 x 103 0.7 X 104 7 x 104 7 x lo5 4 x 10' 3 x io4 3 . 5 lo5 ~ - >106 1 X 105 0.4 X IO4 - 1 X lo3 5X IO3 - 4.0 x 104 - " 5 x 103 - " 5 x 103 - " 1 x 104 -

-

-

Alkall- 0 - p l y sacchaLPS rlde

-

+ + + + + + ND ND

-

++ + + -

ND ND

-

2364

GLYCOCONJUGATE-SELECTED Shigella flexneri

MONOCLONAL ANTIBODIES

TABLE 111 S.flexneri variant X-specific monoclonal antibodies" ELISA tlters Clone

Class 2b

Precipltatlon

0-plysaccharide

Alkali2al a

Ib

3a

3b

4a

4b

5a

2x104 1x103 >lo5 3x103 8X105 >lo5

-

-

-

-

-

-

5b

X

Y treated 4bR LPS

G2aX-1 X-4 X-5 X-9 X-13 X-15

G3

M M M G3

-

-

-

-

2X IO' -

-

-

-

-

-

-

-

2x 71 x0 13 03

0.7>lo5 X lo5 5x105 -

3 x 103

-

-

1 X 104 1x105 >lo5

+ + + + +

-

-

+

++ +

" Blanks indicate titers below 10'. TABLE IV S.flexnerf serogroup Bb-speciflc rltOiIOClOflQlanttbodfes" Preclpltatlon Clone

Class lb

la

2a

2b 4b

3a 4a

3b

5a

X

5b

AlkaliY 4bR rlde

5b-~1 5b-B1 5b-B2 5b-B6 5b-B8 5b-B10 G2a a

M M G3 M M

110"

-

2 x lo4 >lo5 2 x

-

-

-

-

-

-

-

lo3

4

-

x

-

io4 >io5

-

-

-

>io5 >lo5 >lo5

5 x io4 2 x io3 >io5

-

-

-

-

>lo5 >lo6

=-lo5

>lo5 >lo5

>lo5 45xx1100' '

>io5 >io5 >io5

-

-

-

-

-

-

-

treated LPS

O-pOlYsaccha-

+ + + + + +

++-

Blanks indicate titers below 10'. TABLE V ELISA screening of S.flexneri Y-specific monoclonalantibodies with g(yCOCOflJUgUteSa Ollgosaccharlde of Component MASFYClycoconjugate

ababccdaabcdbcdacdabdabccdabcdababcdabcdabY-LPS

Y-B3 Y -A2

Y-B2

Y-A3

1

-

-

-

0.9 x 104 5 x 10. 5 x 104

5 x lo4 5 X 104

-

-

0.9X 104

-

5 X 104 2 X 104 1 X 103 "Blanks indicate titers below 10'.

-

-

1x 1x 1x

lo4 lo4 lo4

0.4 X 103 1 x 105 1 X 105

-

0.6 X 104 5 x 104 7 X 104

0.4

x 103

-

1 x 103 0.7 x 103

-

0.3 x 105 >lo5 0.5 x lo4

MASFY-3 MASFY-2

-

>lo6

>lo6

0.5 x 104

0.6 x 105 >lob

0.7 x lo4 >lo6

5 x 105 1 X lo4

for screeningin combination with classical serogrouping thefirst of which was administeredtogetherwith Freund's complete adjuvant (9).In other experiments,the schemes developed with polyclonal rabbit sera(3). The basic structure of the S. flexneri variant Y 0incorporation of this adjuvant with whole cells has not chain repeating unit is shown (Fig. 1) as the biologic reliably induced a n IgM to IgG shift. Initial ELISA screening assays were based on homolo- repeating unit (8).the monosaccharide residuesof which gous LPS, and clones were selected primarily on the are designated a 4 . consistent with earlier publications magnitude of the ELISA signal withhomologous antigen. (7,8). The 0-polysaccharide of S . flexneri variant X Without further screening it was impossible to reliably carries a n a-D-glucopyranosylsubstituent at0-3of rhamidentify 0-antigen binding specificity. In S. flexneri a nose a, and theserogroup 5b polysaccharide has a second number of core stubs areleft uncapped by O-polysaccha- a-D-glucopyranosyl unit on rhamnose b. The first a-Dride chains (9), and consequently even smoothLPS prep- glucopyranosyl branch point on rhamnosea is associated arations canbind antibodies with core specificity as seen with 0-factors 7.8, andthe second glucose residue, of rhamnose b,is responsible for group for Y-B4 (Table 11) and 5b-A1 (Table IV). Inclusion of branchingat 0-3b The structural featuresof the variantY rough S. flexneri LPS in the screening panel of LPS V specificity (6). (Tables I1 through IV) permits this discrimination, be- antigen responsiblefor 0-factors 3.4 have notbeen identified but these may also be expressedby 0-chain struccause clones Y-B4 and 5B-A1, both of whichbound smooth LPS of all serogroups, also possessedhigh titers tures that possess a-glucosyl branch points at 0-3b. 0against the 4bR core LPS (Tables I1 and IV). Additional 4c, or 0-6d/0-4d,as occurs in types 2a, 3b. 4a, and 5a. problems arise since S. flexneri 0-chains of different Branching at 0-3a masks factors 3,4. S . flexneri Y-specific monoclonal antibodies (Table 11) serogroups share group determinants or type factors (e.g., factors 3,4 typical of Y LPS may also be expressed by resulted from three separate experiments, Y-A1 -A5, Yserotypes la, 2a, 3b, 4a, and 5a; factors 7.8 character- B1 -B5, and MASFY-1 -3,the lastof which were produced istic of X LPS may also be carried in groups 2b. 3a, and by procedures similar to those reported for S. flexneri 5b). Therefore, it is necessary to interpret screening datatype 2 monoclonal antibodies (9).The most suitable clone by considering the primary 0-chain structuresemployed for diagnostic work appeared to beMASFY- 1 witha high

GLYCOCONJUGATE-SELECTED Shigella flexneri MONOCLONAL ANTIBODIES

2365

Y titer and a lower but substantial 4atiter (4a 0-chains istics of each antibody could be precisely mapped (Table carry the3.4 0-factor as may types 2a, 3b, and 5a).Thus, VI. It is clearly seen that clones Y-A2 and MASFY-3 are on this basis, clones Y-A2,Y-A3,Y-B2, and Y-B3 in the only ones studied to possess significant binding to addition to MASFY-1 exhibit a Y reactivity profile that oligosaccharides smaller than a tetrasaccharide.MASFYthe oligosaccharide desconforms to conventionalserology. Immunoprecipitation 3 exhibits distinct preference for of alkali-treated LPS and 0-polysaccharide may be used ignated abcd to the extent that no binding is observed a containing to identify antibodies with high affinity for the antigen with the octasaccharide cdabcdab, structure of interest and also to distinguish those antibodies ex- essentially two chemical repeating units of the Y-polyhibiting possible chain-end specificity. Thus, antibodies saccharide (Fig. 2a). Binding to disaccharide ab, trisacthat precipitate the 0-polysaccharide (Y-A3, Y-A5, Y-B1, charide abc, tetrasaccharide abcd,and finally, the decaand Y-B2) have a high affinity for epitopes distributed saccharide derived from the LPS terminus (8)indicates along the 0-polysaccharide chain (7, 28). Those anti- that this antibody binds to the distal tip of the biologic bodies such as Y-B4 that bind LPS only in ELISA and fail repeating unit. Binding to the frame shifted tetrasacto precipitate either antigen possesscore or lipid-A spec- charides bcda, cdab. or its octasaccharide dimer is seen ificity. Precipitation of alkali-treated LPS but not O-pol- for clonesY-A3 and MASFY-1 and indicates a specificity for internal determinantsdisposed along the polysacchaysaccharide suggests a binding site complementary to univalent determinants located at the nonreducing end ride chain (Fig. 2b). Consistent with this interpretation, clone Y-A3 precipitates both the alkali-treated Y LPS and of the 0-antigen (7.28). Thus, although the obtained data with the serotype panel of LPS (Table 11) provide a gen- its 0-polysaccharide (Table 11). Antibodies Y-B2, Y-B3, MASFY-1, and MASFY-2 appear to possess combining eralized picture of the binding characteristics for each monoclonal antibody, a more detailed description of the sites complimentary to structureslarger than a tetrasacantigenic determinants is not possible without recourse charide since theyonly exhibit significant binding with to chemical structures representing more restricted ele- Y LPS, octasaccharide, and decasaccharide glycoconjugates. Slightly higher titers withthe latterglycoconjugate ments of the S-flexneri serotype polysaccharides. The availability of chemically defined glycoconjugates are seen for Y-B2, Y-B3, and MASFY-1 compared with addresses thisambiguity. Thus when synthetic glycocon- the octasaccharide conjugate, against which antibodyYjugates, containing oligosaccharidesfrom di- to tetrasac- B2 shows a very low titer. These four antibodies appear charide in length were used inELISA, together with larger to possess extended combining sites that require a seof four hexose units. glycoconjugates obtained by phage-associated endo- quence in excess Examination of the ELISA and precipitation data for rhamnosidase degradationof LPS, the binding character-

a

10s

-

Y-LPS

a b m

a a b b c c B S A

b d cd a b d a bc db ad C

BSABSABSA

c

C

d a b &A

Y-LPS

a b c d a b 8%

a b c aS

a bd c nd 8%-

b cd d a

c

c

a

b a o c b b d ~ s n ca d b a c b d BSA

a b BSA

Ffgure 2. Profile of bfnding to Y-LPS and synthetic glycoconjugates withthe following oligosaccharfde structurescoupled to BSA. a-L-Rha(l+Z)aL-Rha; a-b: a-L-Rha(l-r2)a-~-Rha( 1+3)a-~-Rha, a-b-c. a-~-Rha(1*2)a-~-Rha( 1+3)a-~-Rha( 14)BGlcNac. a-b-c-d; (a-L-Rha[ 1+3)@-o-GlcNAc( l-t2)a-~14)B-~-GlcNAc[l)~+2)a-~-Rha(l-+2)a-~-Rha. abcdabcdab. a. MASFY-3. an I@,, Rha(1+2)a-~-Rha]~. cdabcdab; [a-~-Rha(l+a)a-~-Rha( I--r3)a-~-Rha( antibody that exhibits chain- end [abcd) specificity. b, MASFY-1. an IgG2b. antibody that binds strongly with internal sequences bcda, cdab. or cdabcdab.

2366

GLYCOCONJUGATE-SELECTED Shigella flexnerf MONOCLONAL ANTIBODIES

the variant X (Table 111) and serogroup 5b (Table IV) antibodies showsthat with theexception of 5b-A1, a core or lipid A-specific antibody, all the X and 5bmonoclonal antibodies are directed toward the 0-antigen. ELISA titers indicate that clones X-l , X-4, and X-l 5 have high 0-factor titers and as IgG antibodies, theyare potentially candidates for combining site studies.Although X-1 appears to be directed toward the distal tipof the 0-antigen since thetwo do not forma precipitate, X- 15 precipitates the 0-polysaccharide and must recognize determinants along the polymer chain. The latter together withX-5 are a source of either IgG or IgM antibodies, suitable for classing S. flexneri of type X.Allof the 5b-B1 -B10 clones exhibittypical group V binding characteristicsand on the basis of titer, clone 5b-B10, a n IgG2a,would appear to bethe best candidateboth for immunochemical studies of the group 5b combining site and as a potential 5b typing reagent. Incorporation of chemically defined antigens in the screening protocols for0-antigen-specific monoclonal antibodies offers a precise assessment of binding specificity. In this way oligosaccharides attached to protein may be used to calibratethe size of the antibody combining site and difficulties encountered in assessment of specificity patterns on the basisof classical serology are avoided. Although purified LPS antigens of known structure offer an intermediate solution to specificity assessment, these methods are inevitably compromised by microheterogeneitywithin the 0-chain (29). In contrast, selection of S. flexneri variant Y-specific monoclonal antibodies by chemically defined antigens has identified a small number of antibodies of both the IgM or IgG classes suitable for extensive mapping of the combining site. With these well-defined antibodies, immunochemical studiesof specific 0-factor combining sites are greatly simplified, and by inference the structural features responsible for the type factors 3 and 4, characteristic of variant Y LPS, may be realized. Chemical synthesis of the S. flexneri X and 5b determinants (30)should permit further examination of the X and 5b monoclonal antibodies also described in this study. REFERENCES 1. Kohler. G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495. 2. Lindberg. A. A., R. Wollin, G. Bruse. E. Ekwall. andS. B. Svenson. 1983. Immunology and immunochemistry of synthetic andsemisynthetic Salmonella 0-antigen-specific glycocon]ugates. Am. Chem. SOC.Symp. Ser.231:83. 3. Edwards, P. R.. and W. H. Ewing. 1972. Identfffcatfonof Enterobacterfaceae. Burgess Publishing Co., Minneapolis. Pp. 126-131. 4. KeMe, L., B. Lindberg, K. Petersson, and E. Romanowska. 1977. Basic structure of the oligosaccharide repeating unit of the Shigella flexnerf 0-antigens. Carbohydr. Res.56:363. 5. KeMe, L..B. Lindberg, K. Peterson, E. Katzenellenbogen, andE. Romanowska. 1977. Structural studiesof the Shfgellaflexnerfvariant X, type 5a and type 5b 0-antigens.Eur. J. Blochem. 76:327. 6. Kenne, L., B. Lindberg. K. Peterson, E. Katzenellenbogen, andE. Romanowska. 1978. Structural studies of Shfgellaflexnerf0-antigens. Eur. J. Blochem. 91:279.

7. Bock,K., S. Josephson. and D.R. Bundle. 1982. Lipopolysaccharide solution conformation: antigen shape inferred from high resolution 'Hand "C nuclear magnetic resonance spectroscopy and hardsphere calculations. J. Chem. SOC. Perkln Trans. II P. 59. 8. Carlin, N. 1. A.. A. A. Lindberg, K. Bock, and D. R. Bundle. 1984. The Shfgellaflexnerf 0-antigenic polysaccharide chain: nature of the biological repeating unit. Eur. J. Bfochem. 139: 189. 9. Carlin, N. 1. A., and A. A. Lindberg. 1983. Monoclonal antibodies specific for the 0-antigenic polysaccharides of ShfgellQ flexnerf: clonesbinding to 11, k3.4 and 7.8 epitopes. J. Clln.Mlcrobfol. 18:1183. 10. Westphal. 0.. Luderitz. 0. and F. Bister. 1952. Uber die Extraktion von Bakterien mit Phenol/Wasser. 2. Naturforsch. (C)7:148. 11. Janda, J., and E. Work. 1971. A colourimetric estimation of lipopolysaccharides. FEBS Lett. 16:343. 12. Bundle, D. R., and S. Josephson. 1979. Artificial carbohydrate antigens: synthesis of rhamnose disaccharides common to Shigella flexnerf 0-antigen determinants. Can. J.Chem. 57:662. 13. Bundle, D. R.. and S. Josephson. 1979. Artificial carbohydrate antigens. Synthesis andconformation of a Shfgellaflexnerftrisaccharide hapten. J. Chern. SOC.Perkfn Trans.I P. 2736. 14. Josephson, S., and D. R. Bundle. 1980. Artificial carbohydrate antigens: synthesisof rhamnose trisaccharideanddisaccharide haptens common to Shfgellaflexnerf0-antigens. J. Chem. SOC. Perkfn Trans. I P. 297. 15. Josephson, S.,and D. R. Bundle. 1979. Artificial carbohydrate antigens: the synthesis of the tetrasacchariderepeating unit of Shfgellaflexnerf 0-antigen. Can. J. Chem. 57:3073. 16. Bundle, D. R..and S. Josephson. 1980. Artificial carbohydrate antigens: the synthesis of a tetrasaccharide hapten.a Shfgellaflexnerf 0-antigen repeating unit. Carbohydr. Res.80:75. 17. Wessel. H.-P.. and D. R. Bundle. 1983. Artificial carbohydrate antiof gens: a block synthesis of a linear, tetrasaccharide repeating unit the shfgella flexnerf variant Y polysaccharide. Carbohydr. Res. 124:301. 18. Pinto, B. M.. andD.R. Bundle. 1983. Preparation of glycoconjugates for use as artificial antigens: a simplified procedure. Carbohydr. Res. 1 2 4 r . 7 I.?. 19. Lindberg, A. A. R. , Wollin, P. Gemski, andJ. A. Wohlhieter. 1978. Interaction between bacteriophage Sf6 and Shfgella flexnerl. J. Virol. 27:38. 20. Svenson, S. B., and A. A. Lindberg. 1979. Coupling of actd labile Salmonella specific oligosaccharides to macromolecular carriers. J. Immunol. Methods 25:323. 21. Cunningham. A. J., and A. Szenberg. 1968. Further improvements in the plaque technique for detecting ~-single antibody-forming - cells. Immunology 14:599. 22. Neter, E., E. A. Gorzynski, R. M. Gino, 0.Westphal, and0.Luderitz. 1956. Theenterobacterial haemogglutination 'test and itsdiagnostic potential. Can. J. Mfcrobfol.2:232. 23. Kennett, R. H.. K. A. Denis, A. S. Tung, and N. R. Klinman. 1978. Hybrid plasmacytoma production fusions with adult spleen cells. monoclonal spleenfragment.neonatal spleencells andhuman spleen cells. Curr. Top. Mfcrobfol.Immunol. 81:77. 24. Gafle, G., S.C. Howe, C. Milstein, 0 . W. Butcher. andJ. C. Howard. 1977. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550. 25. Gefter, M. D., H. Margulies, and M. D. Scharff. 1977. A simple method for polyethylene-glycol promoted hybridization of mouse myeloma cells. Somatfc Cell Mol. Genet. 3:231. 26. Bundle. D. R., M. A. J. Gidney. N. Kassam, and A. F. R. Rahman. 1982. Hybridomas specific for carbohydrates;synthetic humanblood group antigens for the production, selection. and characterization of monoclonal typing reagents. J.Immunol. 129:678. 27. Parham, P. 1983. On the fragmentation of monoclonal IgGl, lgG2a. and IgG2b from BALB/c mice. J. Immunol. 131:2895. 28. Bundle, D. R., M. A. J. Gidney, M. B. Perry, J. R. and J. W. Cherwonogrodzky. 1984. Serological confirmation of Brucella abortus and Yersfnfa enterocolftfca 0:9 0-antigens by monoclonal antibodies. Infect. Immun. 46:389. 29. Elkins, K., and E. S. Metcalf. 1984. Monoclonal antibodies demonstrate multiple epitopes on the 0 antigens of Salmonella typhlrnurfum LPS. J. Immunol. 133:2255. 30. Wessel, H.-P., and D. R. Bundle. 1985. Strategies for the synthesis of branched oligosaccharides of the Shfgellaflexnerl 5a, 5b and variant X serogroups employing a multifunctional rhamnose precursor. J. Chem. SOC. Perkfn Trans. I P. 2251.

Duncan.