INFECTION AND IMMUNITY, Sept. 1989, p. 2829-28360019-9567/89/092829-08$02.00/0

Definition of Brucella A and M Epitopes by Monoclonal TypingReagents and Synthetic Oligosaccharidest

DAVID R. BUNDLE,'* JOHN W. CHERWONOGRODZKY,2t MARGARET ANNE J. GIDNEY,1PETER J. MEIKLE,1 MALCOLM B. PERRY,' AND THOMAS PETERS'

Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6,1 and Animal DiseaseResearch Institute, Agriculture Canada, Nepean, Ontario K2H 8P9,2 Canada

Received 6 March 1989/Accepted 5 June 1989

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within oneantigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclo-nal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysac-charides and 0 polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica0:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodiesavailable from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A-and M-antigen structures, an interpretation supported by competitive binding studies with 0 polysaccharidesand synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized.Antibodies specific for the A antigen required five contiguous al,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibitedby al,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a criticaldisaccharide element ft-D-Rha4NFo(l-*3)a-D-Rha4NFo bracketed by al,2-linked residues. The bindingprofiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A andM epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolatedal,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodiesare proposed as standard reagents for the detection and identification of Brucella A and M antigens.

The Brucella A and M antigens have been recognizedsince 1939 as important cell wall aminopolyhydroxy com-pounds (18, 19) that have been detected serologically byappropriately absorbed polyclonal rabbit sera (1, 33). Al-though these antigens can be identified serologically, theirstructural elucidation has only recently been accomplished(3, 9). Both antigens are now recognized as the 0 polysac-charides of the respective smooth lipopolysaccharide (LPS),and each is a homopolymer of 4,6-dideoxy-4-formamido-ct-D-mannopyranose residues. The A antigen extracted fromBrucella abortus 1119-3 is a linear, al,2-linked polymer (9),while the M antigen from Brucella melitensis is also a linearpolymer of pentasaccharide repeating units containing oneal,3-linked and four al,2-linked monosaccharide residues(Fig. 1) (3). The well-documented cross-reactivities of the Aand M antigens (10, 32) have their foundation in their closestructural similarities, and from these considerations alone itcould be well appreciated that antisera raised to eitherantigen would exhibit cross-reactions owing to the commonelements in each polysaccharide (3).Wilson and Miles (33) have concluded that Brucella A and

M antigens are simultaneously expressed on all smoothBrucella strains. Quantitative differences in the amounts ofcell surface antigen are thought to account for the threephenotypes A' M-, A- M+, and A' M+ identified byagglutination (1, 32). On the basis of the unique antigenstructures determined for prototype A-antigen (9) and M-

* Corresponding author.t Publication 30415 from the National Research Council of Can-

ada.t Present address: Department of National Defence, Defence

Research Establishment Suffield, Biomedical Defence Section, Ral-ston, Alberta TOV 2NO, Canada.

antigen (3) 0 polysaccharides, it has been suggested that theparadigm of Miles and Wilson (33) was a conceptual mistake(20). This was a reasonable conclusion based on our struc-tural studies of B. abortus 1119-3 (9) and B. melitensis 16M(3) 0 polysaccharides, which demonstrated extensive struc-tural homology. However, the original model proposed byWilson and Miles (33) has been reaffirmed based on thereproducibility of the binding profile of absorbed rabbitantibodies (31) and from considerations of DNA homology(30).

Elaboration of the fine structure of Brucella A and Mantigens in B. abortus, B. melitensis, and Brucella suisbiotypes established the existence of M epitopes in all Aantigens (17). This conclusion was based on the applicationof physical methods together with immunochemical tests inwhich monoclonal antibodies specific for A, M, and amixture of A and M antigens were used. Here we describethese new M-antigen-specific antibodies and provide a de-tailed comparison and analysis of the binding profiles of Aand M reagents produced both in this and earlier (5) studies.

MATERIALS AND METHODS

Antigens. Purified LPS and 0 polysaccharide from B.abortus (9), Yersinia enterocolitica 0:9 (8), and B. melitensis16M (3) were prepared as described previously.Immunization. Female BALB/c mice (age, 6 to 8 weeks;

Charles River Canada Inc., St. Constant, Quebec, Canada)were given two to four intraperitoneal injections over a

period of 4 to 15 weeks followed by one intravenous injec-tion 3 days prior to fusion. All injections consisted of 108 B.melitensis 16M formaldehyde-killed cells in 0.01 M phos-phate-buffered saline (PBS; pH 7.0).

Fusion, enzyme-linked immunosorbent assay screening, andcloning. Three separate fusion experiments were carried out.

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2830 BUNDLE ET AL.

CH3 CH3

HCOH olSo HCOH -

OH ~~~~~~~~~~~OH

SHOCH Ho A 0HCHHO

ot ~~H3 H3 t H3

NHOCH

H (< CH3HCOH -0

H

H'sX-NHOCH

So fH3 | OH

CH3i H O C~~~~~~~~~HOCCCH3° H3HCOH ~

H ~~~~~~~~~~~~~0

0~

A B

FIG. 1. Chemical structures of the Brucella prototype A (A) andM (B) antigens.

Spleen cells from two immunized mice were fused with thenonimmunoglobulin-producing Sp2/0 plasmacytoma cell line(26) (Institute for Medical Research, Camden, N.J.) asdescribed previously (5, 7). Putative hybridomas werescreened against B. melitensis and B. abortus LPSs byindirect enzyme-linked immunosorbent assay (ELISA) onculture supernatants 10 to 14 days postfusion.ELISA microdilution plates (Linbro; Flow Laboratories,

Mississauga, Ontario, Canada) were coated with LPS solu-tions (5), and ELISA testing was carried out as described byCarlin et al. (7). Bound antibody was detected by alkalinephosphatase-conjugated goat anti-mouse immunoglobulin M(IgM) and IgG antibody or alkaline phosphatase-conjugatedprotein A (Dimensions Laboratory Inc., Mississauga, On-tario, Canada). The A405 was read with a Titertek Multiscanapparatus (Flow Laboratories) after 60 min of incubation atroom temperature. Hybridomas with culture supernatantsgiving absorbance readings greater than 0.5 against thehomologous LPS with negligible background were cloned insemisolid agar or by serial dilution by using mouse spleencells as feeders. All hybrids were cloned twice to ensureclonality and stability before cell samples were frozen andascites fluid was raised.

Isotype analysis. Heavy-chain isotype analysis was per-formed by using an EIA mouse antibody screening kit(Hybri-cloned; Kirkegaard and Perry Laboratories Ltd.,Gaithersburg, Md.). Light-chain and IgG subclass identifi-cations were established by immunodiffusion with 10-foldconcentrations of culture supernatants and by precipitatingantisera specific for mouse K and X chains and IgG sub-classes (Cedarlane Labs, Hornby, Ontario, Canada).

Ascitic fluid. BALB/c mice primed by intraperitonealinjection with 0.5 ml of 2,6,10,14-tetramethylpentadecane(pristane) 1 to 4 weeks prior to injection with 106 hybridomacells were tapped for ascites fluid 10 days postinjection.

Immunodiffusion. Immunodiffusion tests were set up inplates of 1% agarose-Induboise A37 (Fisher Scientific Co.,Pittsburgh, Pa.) in PBS. Undiluted ascites fluids were testedagainst bacterial 0 polysaccharide and alkali-treated LPS at

concentrations of 1 mg/ml. Precipitin lines were recordedafter incubation at room temperature for 24 h.

Purification of monoclonal antibody. Ascites fluid wasapplied to a protein A-Sepharose CL-4B column system(Pharmacia Canada Inc., Dorval, Quebec, Canada) that wasmaintained at 4°C, and the IgG fraction was eluted. Mono-clonal antibody obtained in this way was immediately dia-lyzed against 50 mM Tris hydrochloride saline buffer (pH8.0).

Direct competitive binding. B. melitensis 16M 0-poly-saccharide-biotin conjugate was prepared as described pre-viously (P. J. Meikle and D. R. Bundle, submitted forpublication) and used in conjunction with streptavidin-horse-radish peroxidase conjugate (Sigma Chemical Co., St.Louis, Mo.) in a direct competitive enzyme immunoassay(EIA). The working concentrations of antibody and biotinconjugate required for the EIA were determined for eachantibody-conjugate pair by preliminary assays that variedthe concentrations of each (P. J. Meikle and D. R. Bundle,submitted for publication; E. Vorberg and D. R. Bundle,submitted for publication). Polysaccharide-biotin conjugateand inhibitor solutions were prepared in PBS containing 1%bovine serum albumin. In a typical assay, EIA microdilutionplates were coated with purified monoclonal antibody (5 to50 ,ug/ml) in PBS (100 RIi) for 3 h at 20°C. The plates werewashed three times with PBS, biotin-polysaccharide conju-gate solution (100 RI of a 1.5- to 100-ng/ml solution) with orwithout inhibitor (1 ng/ml to 1 mg/ml) was added to eachwell, and specific binding was allowed to reach equilibriumover 18 h of incubation at 20°C. The plates were againwashed three times with PBS, and streptavidin-horseradishperoxidase solution (100 ,ul, 25 ng/ml) was added to eachwell. After 1 h of incubation at 20°C, the peroxidase conju-gate was removed by washing, and substrate solution (100RI) was added to each assay well. The specific A414 was readafter incubation for 1 h at 20°C. The substrate used was2,2-azido-di-(3-ethyl benzthiazoline sulfonic acid) (0.55 mg/ml in 100 mM citrate-sodium hydroxide buffer [pH 4.0]containing 0.003% H202).

Oligosaccharide and polysaccharide inhibitors. Syntheticoligosaccharides (see Table 4) representing the possible A-and M-antigen determinants were available from previoussyntheses (4, 23, 24). The 0 polysaccharides used as inhib-itors in the direct competitive EIA were released from LPSby mild acid hydrolysis and purified by gel permeationchromatography on Sephadex G-50 (3, 8, 9).

RESULTS

Murine monoclonal antibodies to the 0 polysaccharide ofB. melitensis 16M LPS were prepared by the hybridomatechnique (14). Immunization of BALB/c mice with killed,whole cells of B. melitensis 16M induced a strong humoralresponse to the somatic antigen, and three separate fusionswere carried out. Indirect ELISA with the purified homolo-gous B. melitensis smooth LPS and heterologous B. abortussmooth LPS facilitated the selection of hybridomas thatsecreted antibody specific for the Brucella M antigen. Hy-bridomas producing antibody with strong titers against bothLPSs were also selected. The first fusion experiment pro-duced two stable hybridomas, the second fusion experimentproduced six, and the third fusion experiment produced ninecloned cell lines. From these 17 hybridomas, 9 were selectedfor further study on the basis of the specificity and quantityof secreted monoclonal antibody. The properties of six

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DEFINITION OF BRUCELLA A AND M EPITOPES 2831

TABLE 1. Precipitin and indirect ELISA profiles of B. melitensis and selected B. abortus monoclonal antibodies

Immunodiffusion of 0 Indirect ELISA titers of LPSs froma:Monoclonal Immunoglobulin and polysaccharide of:antibody light chain class

B. melitensis B. abortus B. melitensis B. abortus Y. enterocolitica 0:9

Bm2-10 IgG3(K) + + 3 x 104 8 x 103 1 X 102Bm2-15 IgG3(K) + - 3.4 x 103 1 X 102 1 x 101Bm2-28 IgG3(K) + - 1 x 104 4.4 x 104 1 x 10Bm3-2 IgG3(K) + - 1 x 105 2.5 x 102 0*Bm3-7 IgG2b(K) - - 1 x 106 1 x 102 ob

Bm3-8 IgG2a(K) + - 1 x 106 1 x 103 1 x 10lYsT9-1 IgG2b(K) - + 1 x 104 5 x 105 1 X 105YsT9-2 IgG3(K) + + 1 x 105 1 X 105 1 X 105a Titers were measured as the highest dilution reaching an optical density of 0.1 after 60 mim of substrate incubation.b No cross-reaction.

M-antigen-specific and two A-antigen-specific antibodieswere compared (Table 1).The binding characteristics of monoclonal antibodies were

assessed initially by indirect ELISA (Table 1). Endpointtitrations were conducted for each antibody with B. meliten-sis, B. abortus, and Y. enterocolitica 0:9 LPS antigen-coated ELISA plates. Three sets of binding patterns were

discerned. The first group was made up of antibodies thatexhibited comparable titers with all three LPS antigens;these were of no further interest. The second set of antibod-ies showed endpoint titers comparable to those of the A andM antigens but low titers compared with those of the Y.enterocolitica 0:9 antigen. Examples of this type wereBm2-10 and Bm2-28 (Fig. 2A). The third set, Bm2-15 (Fig.2B), Bm3-2, Bm3-7, and Bm3-8, showed the highest discrim-ination for the M antigen, as judged by endpoint titers (Table1). Indirect ELISA profiles for previously reported BrucellaA-antigen-specific antibodies (4) showed that the high-affini-ty A-antigen monoclonal antibody YsT9-1 readily discrimi-nated the A antigen from the M antigen (Fig. 2C). A valuableantibody, YsT9-2 (Fig. 2D), exhibited an equal affinity forthe A, M, and Y. enterocolitica 0:9 antigens.Immmunodiffusion experiments showed that seven of the

nine antibodies precipitated the B. melitensis LPS, and ofthese seven antibodies, six precipitated the M polysaccha-ride (Table 1).The most valuable comparison of antibody specificity and

relative affinity was obtained by direct competitive EIA(Meikle and Bundle, submitted; Vorberg and Bundle, sub-mitted). By using protein A-purified antibodies to coat EIAplates, the solid-phase antibodies were probed with biotin-labeled M polysaccharide (P. J. Meikle and D. R. Bundle,submitted for publication). The amount of antibody requiredto optimally coat plates, together with the concentration of

biotin conjugate required to produce an optical density of 1.0following separate incubations with streptavidin-horseradishperoxidase conjugate and horseradish peroxidase substrate,provided a measure of the intrinsic affinity of each antibody.For a given antibody coating concentration (assuming equiv-alent coating efficiencies), the amount of conjugate used toproduce an optical density of 1.0 was inversely proportionalto affinity. Thus, for the three antibodies that required 5 ,ugof antibody coating solution per ml, the order of affinity wasBm2-10 > Bm2-28 > Bm3-2 (Table 2). In other instances, itcan be seen that not only was it necessary to increase theconcentration of solid-phase antibody in order to reach theprescribed level of bound conjugate, but the concentration ofconjugate needed to drive the binding was also much higher.Thus, in contrast to the results of the indirect ELISA (Table1), antibody Bm3-7 had a relatively low affinity (Table 2).

Direct competitive EIA rather than indirect ELISA alsoprovided a more reliable test of specificity when the relativeinhibitory powers of the A, M, and Y. enterocolitica 0:9polysaccharides were measured for each antibody. Polysac-charides labeled or unlabeled with biotin competed for thesolid-phase antibody, and since the molecular weights ofeach polysaccharide were approximately the same, the con-centrations required for 50% inhibition of labeled polysac-charide binding were used to rank the specificities (Table 2).By this assay the selected antibodies were shown to exhibitvarying degrees of specificity for the M antigen, and in a lessmarked fashion, they bound the A antigen in preference tothe Y. enterocolitica 0:9 antigen. The most effective dis-crimination was achieved not by the highest-affinity antibod-ies Bm2-10 (Fig. 3A) or Bm2-28 but, rather, by the third-ranked antibody Bm3-2 (Table 2). This trend was alsoreflected in the higher discrimination exhibited by the anti-bodies with intermediate affinities (Bm2-15 and Bm3-8) (Fig.

TABLE 2. Direct competitive EIA profiles of B. melitensis and B. abortus monoclonal antibodies

Conditions for direct Competitive EIA Concn (ng/ml) of 0 polysaccharide for 50%7o inhibitionaMonoclonal antibody coating biotin conjugateantibody (pg/ml) (ng/ml) B. melitensis B. abortus Y. enterocolitica 0:9

Bm2-10 5 1.5 27 (1.0) 10,600 (392) 117,000 (4,744)Bm2-15 10 20 45 (1.0) 25,100 (558) 407,000 (9,053)Bm2-28 5 3 18 (1.0) 7,400 (403) 108,000 (6,294)Bm3-2 5 4 4.1 (1.0) 14,100 (3,445) 37,600 (9,167)Bm3-7 50 100 195 (1.0) 117,000 (603) 653,000 (3,349)Bm3-8 20 15 27 (1.0) 47,300 (1,752) 120,000 (4,453)YsT9-1 10 4 4,786 (1,167) 4.1 (1.0) 11.2 (273)Yst9-2 5 2 7.6 (1.0) 8.2 (1.0) 12.2 (1.5)a Values in parentheses indicate the ratios of nanogram quantities of 0 polysaccharides required for 50% inhibition.

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2832 BUNDLE ET AL. INFECT. IMMUN.

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FIG. 2. Indirect ELISA titration curves for ascites fluid containing hybridoma antibodies measured for B. abortus 1119-3 (0) and B.melitensis 16M (A) LPS-coated plates. (A) Bm2-28; high affinity; cross-reactive with A antigen. (B) Bm2-15; intermediate affinity;M-antigen-specific binding. (C) YST9-1; high affinity; A-antigen-specific binding. (D) YsT9-2; high affinity; A- and M-antigen-specific binding.

3B). Finally, as noted above, all nine antibodies bound the Aantigen 3- to 10-fold more strongly than the Y. enterocolitica0:9 antigen (Table 2).Comparison of the M-antigen-specific antibodies with two

antibodies obtained from a B. abortus fusion experiment (5)showed in one case (that for YsT9-1) a marked specificity forthe A antigen over the M antigen (Fig. 3C) and nearlyidentical binding specificities for the Y. enterocolitica 0:9and A antigens. The antibody YsT9-2, however, bound all

three antigens with an equal affinity (Fig. 3D). Both YsT9-1and YsT9-2 possessed relatively high affinities based on theamount of antibody required to effectively coat plates for thedirect EIA and the working concentration of biotin conjugate(Table 2).

High-affinity antibodies with balanced specificities for theA and M antigens were evaluated for use in agglutinationtests of B. abortus and B. melitensis biotypes (Table 3). TheA-antigen-specific antibody YsT9-1 accurately agglutinated

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DEFINITION OF BRUCELLA A AND M EPITOPES 2833

100

c

0

C

0Il

1.0 10 100 1000 10,000 100,000 1,000,000 1.0 10 100 1000 10,000 100,000 1,000,000

Inhibitor Concentration ng/mL Inhibitor Concentration ng/mL

D7

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~~~~~~~c600

1.0

cA40-

O~R40

20 20

1.010 100 1000 10,000 100,000 0.0 10 10 1000

Inhibitor Concentration ng/mL Inhibitor Concentration ng/mLFIG. 3. Measurement of relative inhibitory powers of the three O-polysaccharide antigens B. melitensis 16M (@), B. abortus 1119-3 (O),

and Y. enterocolitica 0:9 (A). Increasing amounts of polysaccharide inhibitors were added together with biotin-labeled 0 polysaccharide tothe wells of ELISA plates that were previously coated with the following monoclonal antibodies: Bm2-10, a high-affinity anti-M antigen (A);Bm3-8, a moderate-affinity anti-M antigen (B); YsT9-1, a high-affinity anti-A-antigen binding that is specific for the B. abortus A antigen aswell as the Y. enterocolitica 0:9 antigen (C); and YsT9-2, a high-affinity antibody with identical binding specificity for all three antigens (D).

the strains, in accordance with established serotypingschemes (32) (Table 3). Antibody YsT9-2, since it boundboth A and M antigens, agglutinated all smooth Brucellabacteria with a high titer. The high-affinity antibody Bm2-28showed the same agglutination profile as YsT9-2. Althoughthey exhibited different titers, Bm2-15 and Bm3-2 werespecific at high dilutions for the strains known to express M> A. However, those Brucella strains that were character-ized as serotype A also showed significant but lower titerswith these two antibodies.

Finally, a competitive EIA was used to characterize theantibody-combining sites in terms of the relative affinities fora series of synthetic oligomers ranging from di- to pentasac-charides. The synthetic structures studied are listed (Table4), and the inhibition data with M- or A-antigen-specificantibodies indicate that there were clear binding patterns(Table 5). The B. melitensis antibodies Bm2-10, Bm2-15, andBm2-28 were inhibited to some degree by oligosaccharidescontaining al-3, linked 4,6-dideoxy-4-formamido-D-man-

TABLE 3. Bacterial agglutination by Brucellamonoclonal antibodies

Bacterial strain, Reciprocal titers of monoclonal antibody:biotype, andserotype Bm2-15 Bm2-28 Bm3-2 YsT9-1a YsT9-2a

B. abortus1 A 40 320 160 640 2,5602 A 20 320 80 640 2,5603 A 40 320 80 640 2,5604 M 320 320 1,280 - 2,5605 M 160 320 640 - 2,5606 A 40 320 80 640 2,5609M 160 320 320 - 2,560

B. melitensis1 M 320 320 1,280 - 2,5602 A 10 320 80 640 2,5603 M 160 320 640 - 2,560

" Monoclonal antibodies generated as previously described (5).

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TABLE 4. Structures of synthetic M and A oligosaccharides

Oligosaccharide Structure"

1 a-D-Rha4NFo-(1--3)-at-D-Rha4NFo1---*OCH32 at-D-Rha4NFo-(1--*2)-Ca-D-Rha4NFo(1-*3)-a-D-Rha4NFo-1--*OCH33 CX-D-Rha4NFo-(1---2)-Ca-D-Rha4NFo(1l-2)-a-D-Rha4NFo-(1--*2)-a-D-Rha4NFo-(1--*3)-ct-D-Rha4NFo-

1-*OCH34 a-D-Rha4NFo-(1--*3)- X-D-Rha4NFo(1-*2)-(X-D-Rha4NFo-(1--*2)-a-D-Rha4NFo-(1--2)-aX-D-Rha4NFo-

1-*OCH35 cK-D-Rha4NFo-(1l-2)-cX-D-Rha4NFo(1--2)-cx-D-Rha4NFo-1-*OCH36 CX-D-Rha4NFo-(1-- 2)-a-D-Rha4NFo(1---2)-a(-D-Rha4NFo-(1--2)-a-D-Rha4NFo-1---OCH37 at-D-Rha4NFo-(1-*2)-Oa-D-Rha4NFo(1--*2)-(X-D-Rha4NFo-(1l-2)-a-D-Rha4NFo-(1--*2)-a-D-Rha4NFo-

1-*OCH3

a D-Rha4NFo, 4,6-Dideoxy-4-formamido-D-mannopyranosyl.

nose residues. The two Y. enterocolitica 0:9 antibodies wereinhibited by oligomers composed exclusively of al,2-linkedresidues. Whereas YsT9-1 required at least five, and possi-bly six, contiguous residues, YsT9-2 was effectively inhib-ited by a tetrasaccharide ligand. A pentasaccharide ligandwas no more active on a molar basis than the tetrasaccharideinhibitor (Table 5).

DISCUSSION

Unambiguous differentiation of homopolysaccharides asclosely related as the Brucella A and M antigens (3) byantibodies requires high binding specificities. The problem iscompounded by the controversial possibility that aspects offine structure are common to each antigen (20). In theprocess of attempting to generate monoclonal antibodies thateffectively discriminated between the two antigens, unre-solved details of antigen fine structure have been clarified(17). In conjunction with structural studies in which physicaltechniques were used, monoclonal antibody binding profilesof the A and M antigens suggest a model of antigen structurethat is consistent not only with observed serology andstructural epitopes but also with the early proposals ofWilson and Miles (33).

Selection of hybridoma cell lines by indirect ELISA ofculture supernatants provided a convenient assay format forthe rapid identification of potentially interesting antibodies.Despite the availability of highly purified A and M LPSantigens, the indirect ELISA titers masked, to some extent,antibody specificity. This was seen for such clones asBm2-10 and Bm2-28 (Table 1). These were the two highest-affinity M-antigen antibodies obtained from three separatefusion experiments, yet their endpoint titers with the M or Aantigens were virtually indistinguishable. This highlights a

deceptive feature of screening protocols based on indirectELISAs. Endpoint titrations alone do not provide adequatecriteria to select the most specific or high-affinity antibodies.Much discussion has surrounded this issue, and reports inthe literature generally indicate that indirect ELISA end-point titers are not a good indication of affinity (15, 16, 21,22, 28).

In order to carry out binding studies, a direct competitiveEIA was developed based on covalent attachment of enzymeto polysaccharides or oligosaccharides (Meikle and Bundle,submitted; Vorberg and Bundle, submitted). Competitivebinding assays which provide values for Ka, the associationconstant, are obtained for univalent inhibitors, while poly-valent inhibitors can also be used in this assay format. Avariant of the assay was applied in this study by using B.melitensis polysaccharide conjugated to biotin (Meikle andBundle, submitted). Comparison of both the amount ofantibody required to coat the EIA plates and the quantity ofenzyme conjugate used to provide a strong signal gave a

measure of the antibody avidity. Since the antigens hadcomparable molecular weights and the antibodies were ofthe bivalent IgG type, the high-affinity antibodies can beidentified as those that require low coating concentrations incombination with low conjugate concentrations. Not onlydoes a competitive assay of this type reflect affinity (Meikleand Bundle, submitted; Vorberg and Bundle, submitted) butit also permits a reliable assessment of specificity (29).Consistent with this, binding curves of M, A, and Y. entero-colitica 0:9 polysaccharide antigens with various antibodiesaccurately identified those antibodies that were capable ofdistinguishing M and A antigens (Fig. 3A to D). These datawere confirmed by functional assays such as agglutination

TABLE 5. Oligosaccharide inhibition by direct competitive EIA

Concn (p.mol/liter) of oligosaccharide required for 50% inhibition (oligosaccharide no.)Monoclonalantibody Disaccharide Trisaccharide Pentasaccharide Pentasaccharide Trisaccharide Tetrasaccharide Pentasaccharide

(1) (2) (3) (4) (5) (6) (7)

Bm2-10' >2,652 >1,815 >1,112 140 >573 >351Bm2-150 >2,652 >1,815 >1,112 222 >573 >351Bm2-28a 374 1.2 32 279 >573 >351Bm3-2a >2,652 >1,815 >1,112 >351 >573 >351Bm3-7a >2,652 >1,815 >1,112 >351 >573 >351Bm3-8a >2,652 >1,815 >1,112 >351 >573 >351YsT9-1b 1,210 174 62.4YsT9-2b 256 89.8 93.1

a Detected by B. melitensis 0 polysaccharide conjugated to biotin.b Detected by Y. enterocolitica 0:9 polysaccharide-biotin conjugate.

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DEFINITION OF BRUCELLA A AND M EPITOPES 2835

tests (Table 3) and precipitin tests, which also reflected theavidities and specificities of the antibodies (Table 1).The highest-affinity, or more accurately, highest-avidity,

antibodies Bm2-10, Bm2-28, and Bm3-2 showed good selec-tivities in the competitive binding assays (Table 2). Never-theless, Bm2-10 precipitated both the M and A polysaccha-rides and Bm2-28 did not differentiate A' M- from M+ A-strains in the agglutination tests (Table 3). This behavior maybe explained by two factors; high-affinity antibodies stillexhibit significant association constants for cross-reactiveantigens, whereas in the case of moderate-affinity antibod-ies, the effective binding to such antigens can drop belowthreshold levels. This reasoning would account for theprecipitin results of Bm2-10 (Table 1) and the agglutinationdata for Bm2-28 (Table 3), since it was demonstrated that allA antigens contained a small number of M epitopes. It wasalso seen that indirect ELISA (Table 1) failed to show thedramatic difference of binding between the M and A anti-gens, which was detected by the competitive binding assay

(Table 2). The third-ranked antibody, Bm3-2 (in avidityterms), showed a good compromise between high avidityand specificity. Its avidity for the A antigen was 3,000 timeslower than that for the M antigen, and it selectively precip-itated the M antigen. Furthermore, it showed higher agglu-tination titers with bacteria that express M > A antigens thanwith bacteria that express the A > M serotype. In thisrespect, it resembled the properties of those antibodies withintermediate avidities. Antibodies Bm2-15 and Bm3-8 boundthe M antigen 500 to 1,800 times more avidly than they didthe A antigen and were able to realize this discrimination inthe agglutination tests (Table 3).A consistent observation for all B. melitensis antibodies

was the 5- to 20-fold higher avidities for the Brucella Aantigen over the Y. enterocolitica 0:9 antigen (Table 2). Thediscovery of a small number of al,3 linkages in the A antigenaccounts for this behavior. This linkage defines the Mepitope (3), and its presence in A antigens accounts for thepreferential binding by B. melitensis antibodies. The pres-

ence in the A antigen of an average of one or two al,3linkages also explains the difficulties in deriving M-antigenantibodies that do not react (especially in agglutination tests)with A-antigen strains. Even the most selective M-antigenmonoclonal antibodies exhibited significant A-antigen titersin the agglutination test (Table 3).The combination of assay formats, most notably, the

competitive binding assay, with three distinct and chemi-cally defined bacterial polysaccharides provided a powerfultool to calibrate the binding characteristics of the monoclo-nal antibodies. However, a more precise definition of anti-genic determinants was possible through the availability of a

range of synthetic oligosaccharides. The structures of thesecompounds (Table 4) ranged from an al,3-linked disaccha-ride (oligosaccharide 1) through a trisaccharide (oligosaccha-ride 2) up to pentasaccharides in which the single al,3linkage occupied either the terminal reducing position (oli-gosaccharide 3) or the nonreducing position (oligosaccharide4). In order to characterize A-antigen determinants and theA-antigen monoclonal antibodies YsT9-1 and YsT9-2, an

a1,2-linked trisaccharide (oligosaccharide 5), a tetrasaccha-ride (oligosaccharide 6), and a pentasaccharide (oligosaccha-ride 7) were also investigated. The concentrations (in micro-molar) of these inhibitors which provided 50% inhibitionwere measured (Table 5). Antibodies Bm3-2, Bm3-7, andBm3-8 were not inhibited by any of the oligosaccharides.Antibodies Bm2-10 and Bm2-28 were inhibited by a range ofM-antigen-type oligosaccharides. The general picture that

emerged suggests that M-antigen-specific antibodies mostlikely bind to a determinant made up of an al,3-linkeddisaccharide flanked on either side by al,2-linked residues.Since this oligosaccharide fragment has yet to be synthe-sized, this hypothesis could only be inferred from the datapresented in Table 5. The most striking inhibition data,however, came from the studies of al,2 oligomers withYsT9-1 and YsT9-2. Optimum inhibition of YsT9-2 wasreached by a tetrasaccharide, and no improvement wasobserved when a pentasaccharide was used. A trend ofincreasing inhibitory power up to and presumably beyondthat of a pentasaccharide was observed when YsT9-1 wastested. These data nicely explain the polysaccharide-bindingprofiles of each antibody. A-antigen-specific YsT9-1achieved its specificity because the largest al,2 oligomericsequence available in an M antigen is a tetrasaccharide (3),and the inhibition data showed that the affinity of thisantibody for its ligand continued to rise at the pentasaccha-ride level, implying that this is a minimal size for theantibody-combining site. A tetrasaccharide, however, is theoptimal inhibitor size for the YsT9-2-combining site, andconsequently, this antibody should and does react equallywith A' or M+ antigens as well as Y. enterocolitica 0:9 0polysaccharide.The well-characterized binding profiles of the principal

Brucella M- and A-antigen antibodies developed in thislaboratory can be rationalized with the fine-structural detailsof the M and A antigens reported in the preceding paper (17).M antigens have a unique structure but cross-react with

those A-antigen-specific antibodies that have combiningsites capable of accepting al,2-linked tetrasaccharides witha sufficiently high affinity. Antibody YsT9-2 represents theextreme of this characteristic since it bound both A and Mantigens with an equal affinity. M-antigen-specific antibodiesBm2-10, Bm2-28, Bm2-15, and Bm3-2 showed 500- to 3,500-fold higher binding with the M antigen; but because the Aantigen contained a small number of otl,3 linkages, anyM-antigen-specific antibody must also bind the A antigen.The precise nature of the Brucella A and M epitopes can

be inferred from these data. The A epitope is a pentasaccha-ride or larger oligosaccharide composed of otl,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl (D-Rha4NFo) res-idues in which the formamido residue plays a crucial role (5)(unpublished data). The crucial element of the M epitope isa disaccharide, a-D-Rha4NFo(1---3)a-D-Rha4NFo, that re-quires adjacent al,2-linked residues for full activity. Thecommon epitope of A and M antigens is a linear al,2-linkedtri- or tetrasaccharide.

Finally, it is appropriate to comment on the paradigm ofWilson and Miles (33) from over 50 years ago, whichpostulated a qualitative similarity but quantitative variationin the amounts of A and M epitopes on the Brucella cellsurface. Since that time it has also been recognized that thetwo antigenic determinants might be carried on a single LPSmolecule. In the case of B. abortus biotypes, this is anaccurate model. However, the M antigen presents an Aepitope only in terms of a smaller common epitope. Poly-clonal antibodies must inevitably be composed of antibodieswith a spectrum of specificities, and it is most likely that thistri- or tetrasaccharide epitope accounts for the well-docu-mented reports in the literature of cross-reactive Brucellaantisera (1, 10, 32, 33).A number of monoclonal antibodies with varying specific-

ities for the Brucella A- and M-antigen determinants havebeen reported (2, 6, 11-13, 25, 27). For the most part, thesehave been tested by ELISA or agglutination protocols with

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2836 BUNDLE ET AL.

sera from infected cows or with whole cells. The results ofsuch studies and this study indicate that it is both practicaland desirable to consider replacing polyclonal sera for theidentification of Brucella biotypes. However, as we havealso demonstrated, the precise delineation of antigenic spec-ificity and the ability to discriminate such closely relatedhomopolysaccharide antigens demands the use of chemicallydefined polysaccharides and synthetic ligands. By thesecriteria, the three monoclonal antibodies YsT9-1, Bm3-2,and YsT9-2 proved to be well suited for use as standardBrucella typing or detection reagents.

ACKNOWLEDGMENTS

The assistance of B. Sinnott is gratefully acknowledged.T.P. was a National Research Council of Canada research asso-

ciate from 1986 to 1987. P.J.M. was a National Research Council ofCanada research associate from 1986 to 1988.

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3. Bundle, D. R., J. W. Cherwonogrodzky, and M. B. Perry. 1987.Structural elucidation of the Brucella melitensis M antigen byhigh-resolution NMR at 500 MHz. Biochemistry 26:8717-8726.

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