Microbial Pathogenesis 1998; 24: 299–308

PATHOGENESISMICROBIAL

Monoclonal antibodies to the epitopea-Gal-(1-4)-b-Gal-(1- of Moraxella catarrhalisLPS react with a similar epitope in type IVpili of Neisseria meningitidisMotiur Rahman∗, Ann-Beth Jonsson & Tord Holme

Microbiology and Tumorbiology Center, Karolinska Institute, S-171 77 Stockholm, Sweden

(Received October 14, 1997; accepted December 22, 1997)

Murine monoclonal antibodies (MAbs) against the A, B and C LPS serotypes of M. catarrhalis weregenerated and their binding specificity was examined in an enzyme-linked immunosorbent assay(ELISA). Two broadly cross-reactive monoclonal antibodies (MCA1 and MCC2) against the outercore region of LPS were further characterized. A panel of synthetic glycoproteins and glycolipidswas used to determine the binding specificity of the MAbs. MCA1 and MCC2 bound specificallyto a-Gal-(1-4)-b-Gal of galabiose and globotriose glycoconjugates. The reactivity of the MAbs withgalabiose was higher than that with globotriose. The MAbs could recognize the a-Gal-(1-4)-b-Galepitope only when it was in a terminal position. MCA1 was further shown to react with a similarepitope in the glycosylated type IV pili of N. meningitidis, which has been shown to contain a 1-4linked digalactose at the terminal part of the saccharide present in the pili. MCA1 could efficientlyrecognize this epitope indicating that it was exposed on the surface of the pili.

1998 Academic Press Limited

Key words: Moraxella catarrhalis, LPS, monoclonal antibody, epitope, Neisseria meningitidis, pili,glycosylation.

side chains [1]. Each of these components hasIntroductionits specific role in pathogenesis and es-tablishment of infection [2]. Moraxella catarrhalis

Lipopolysaccharides (LPS) are critical structural is a gram-negative bacterium causing childhoodand antigenic components of the outer mem- otitis media and maxillary sinusitis [3]. It alsobrane of gram-negative bacteria. LPS consists of causes respiratory disease in patients withlipid A, core oligosaccharide and O-antigenic chronic bronchitis and in immunocompromised

hosts [4]. The lipopolysaccharide of M. catarrhalisconsists of lipid A linked to the core oli-gosaccharide but lacks the extended O-antigenic∗Author for correspondence: Laboratory Science Division,

ICDDR, B, G.P.O. Box 128, Dhaka, Bangladesh. side chains that are characteristic of gram-

0882–4010/98/050299+10 $25.00/0/ 1998 Academic Press Limitedmi970191

M. Rahman et al.300

negative enteric pathogens, thus being similar selected for further investigation: two againstserotype A LPS (MCA1, IgG2a and MCA2, IgM),in general structure to the LPS of Neisseria men-

ingitidis, Neisseria gonorrhoeae, Haemophilus in- two against serotype B LPS (MCB1, IgM andMCB2, IgG2b) and two hybridomas against ser-fluenzae and Bordetella pertussis [5]. Serological

typing of M. catarrhalis LPS using hyperimmune otype C (MCC1, IgM and MCC2, IgG2b). Theculture supernatants from hybridomas were ex-rabbit sera demonstrated the presence of three

major serotypes accounting for 95% of the isol- amined against a panel of LPS from differentM. catarrhalis strains, Salmonella typhimurium (Ra-ates. Among the clinical isolates serotype A

accounts for 60%, serotype B for 30%, serotype Re type), Aeromonas caviae, Vibrio cholerae andpurified lipid A from M. catarrhalis. MAbs MCA1C for 5% and 5% of the isolates were nontypable

[6]. Cross-reactions between serotypes indicate and MCC2 reacted with all different LPS sero-types of M. catarrhalis but did not react with thethe presence of common immunogenic epitopes.

Although the pathogenesis of M. catarrhalis is purified lipid A. The MAbs did not react withany of the other LPSs tested (Table 1).not fully understood, recent studies indicate that

LPS of M. catarrhalis contributes to its pathogenic MAbs MCB1 and MCB2 were specific forserotype B LPS, and did not react with any otherpotential [7].

The chemical structure of the polysaccharide serotype indicating that these MAbs recognizean epitope present only in serotype B LPS. MAbsportion of LPS from all three serotypes has been

reported [5, 8, 9]. The determinant for serotype MCA2 and MCC1 had variable reactivity withdifferent LPS serotypes. MAb MCA2 was lessA is a terminal a-GlcNAc-(1-2)-b-Glc epitope,

for serotype B is b-Gal-(1-4)-a-Glc and for ser- reactive to all LPS serotypes than MCA1. MAbMCC1 was reactive against homologous LPS butotype C is a terminal b-Gal-(1-4)-a-GlcNAc. A

terminal a-Gal-(1-4)-b-Gal-(1-4)-Glc epitope was weakly reactive to serotype A, B and anotherserotype C LPS (RS 10).found to be common to the three serotypes. This

common epitope a-Gal-(1-4)-b-Gal-(1-4)-Glc is In order to investigate the expression of epi-topes recognized by MCA1 and MCC2 we ex-also found in the LPS of other gram-negative

non-enteric pathogens such as H. influenzae, N. amined 28 defined strains of M. catarrhalisbelonging to serotypes A, B and C in whole cellgonorrhoeae and N. meningitidis [10]. In-

terestingly, the terminal disaccharide of this tri- ELISA and 19 strains of gram-negative entericbacteria. Twenty-six out of twenty-eight strainssaccharide has been shown to be present in the

glycosylated pili of N. meningitidis but not in the of M. catarrhalis reacted with MCA1 and MCC2(Table 2). None of these MAbs reacted withglycosylated pili of N. gonorrhoeae [11, 12]. The

presence of this epitope in a number of gram- any of the gram-negative enteric bacteria. Thesefindings indicate that the epitopes recognizednegative bacteria indicates the presence of com-

mon mechanisms for pathogenesis. by MCA1 and MCC2 are accessible to the MAbson the surface of intact bacteria. These two broadIn this paper we describe monoclonal anti-

bodies against different serotypes of M. ca- reacting MAbs were selected for further charac-terization.tarrhalis LPS and characterization of an a-Gal-

(1-4)-Gal specific monoclonal antibody and itsspecific recognition of a similar epitope in N.meningitidis type IV pili.

Characterization of the binding epitopefor MCA1 and MCC2

On the basis of the known chemical structure ofResultsthe A, B and C serotypes of M. catarrhalis LPSand considering the saccharide parts in commonProduction and selection of MAbs

specific for LPS to all serotypes, we examined the reactivity ofMCA1 and MCC2 against a panel of syntheticglycolipid or glycoproteins. Both MAbs showedBALB/c mice were immunized with LPS pre-

pared from strains belonging to serotype A, B high titres with galabiose and globotriose gly-coconjugates [Fig. 1(a) and (b)]. The MAbs didand C. After fusion, hybridomas were screened

with homologous LPS in ELISA and recloned not react with any of the other glycoconjugatesor glycolipids examined and not with gly-by limiting dilution. After subsequent recloning

and screening a total of six hybridomas was coconjugates with an internal a-Gal-(1-4)-Gal.

Monoclonal antibodies against M. catarrhalis LPS 301

Table 1. Reactivity of MAbs to LPS from M. catarrhalis and other bacteria

Lipopolysaccharide (LPS) Monoclonal antibodies (isotype)

MCA1 MCA2 MCB1 MCB2 MCC1 MCC2(Ig2a) (IgM) (IgM) (IgG2b) (IgM) (IgG2b)

M. catarrhalis LPS(Serotype A)

ATCC 25238 +++ ++ − − + +++RS272 +++ +++ − − + +++CCUG 18283 ++ ++ − − + +++CCUG 18284 +++ + − − + ++

(Serotype B)CCUG 3292 +++ ++ +++ +++ + +++RS 13 ++ + +++ +++ + ++

(Serotype C)RS 26 ++ ++ − − ++ ++RS 10 +++ ++ − − + +++

M. catarrhalis lipid AATCC 25238 − − − − − −

S. typhimuiumSL 1102 (Re) − − − − − −SL 3769 (Rd1) − − − − − −SL 1181 (Rd2) − − − − − −SL 805 (Rc) − − − − − −TV 119 (Ra) − − − − − −TV 161 (Rb2) − − − − − −SL 733 (Rb1) − − − − − −TV 148 (Rb3) − − − − − −

A. caviae 11212 − − − − − −

V. cholerae 420264 − − − − − −

Microtitre plates were coated with 10 lg/ml of LPS. Culture supernatants were added at 1/16 dilutionin duplicate wells. Results are expressed as (+++) strong reactivity (absorbance >1.0 at 450 nm),(++) moderate reactivity (absorbance <1.0 to >0.7 at 450 nm), (+) mild reactivity (absorbance <0.7to >0.4 at 450 nm) and (−) no reactivity (absorbance <0.4 at 450 nm). The results indicated here arethe mean of three experiments.

The reactivity with galabiose was stronger than and 6.5 lg/ml of globotriose. No other gly-coconjugate or glycolipid was found to be in-with globotriose.

In order to determine the specificity of reaction hibitory in the concentrations tested.between MAbs and glycoconjugates ELISA in-hibition experiments were performed. Gly-coconjugates were used as inhibitors of reactions Cross-reaction between type IV pili

and MAbsbetween homologous LPS and MAbs. Table 3shows the reactions between ATCC 25238 LPS(serotype A) and MCA1 and MCC2 MAb. In the It has recently been shown that type IV pili

N. gonorrhoeae, N. meningitidis and Pseudomonasreaction between ATCC 25238 LPS and MCA1MAb a 50% inhibition was obtained at a con- aeruginosa are glycosylated. The pili of N. men-

ingitidis are O-glycosylated with a terminal 1-4-centration of 0.5 lg/ml of galabiose and 4.5 lg/ml of globotriose. A similar inhibition in the linked digalactose moiety covalently linked to

a 2,4-diacetamido-2,4,6-trideoxyhexose within areaction between ATCC 25238 LPS and MCC2MAb was obtained wth 0.9 lg/ml of galabiose peptide spanning amino acid residues 45 to 73

M. Rahman et al.302

Table 2. Binding of MCA1 and MCC2 MAbs to M. catarrhalis andother gram-negative non-enteric bacteria in whole cell ELISA

Strains Number of strains reacting with MAbs

MCA1 MCC2

M. catarrhalisSerotype A (20) 18 18Serotype B (6) 6 6Serotype C (2) 2 2

S. typhimurium (4) — —A. caviae (3) — —E. coli (6) — —K. pneumoniae (2) — —Y. enterocolitica (2) — —P. vulgaris (2) — —

Microtitre plates were coated with whole bacterial cells (OD 600 nm=0.01). MAb culture supernatants at 1/16 dilution in PBS-T were addedin duplicates. An absorbance value >0.5 at 450 nm was considered aspositive and <0.5 was considered as negative. The results indicated hereare the mean of three different experiments.

of PilE [11]. The gonococcal pili carry an O- from N. meningitidis FAM20 and 002 contain thea-Gal-(1-4)-Gal epitope and that this epitope islinked disaccharide a-GlcNAc-(1-3)-Gal at serine

63 [12]. Highly purified pili preparations from exposed on the surface of the pili (Fig. 3). Thespecificity of MAbs for a-Gal-(1-4)-Gal epitopeN. gonorrhoeae MS11 and N. meningitidis FAM20

were examined for glycosylation. The saccharide of the saccharide moiety of pili was examinedby inhibition ELISA where synthetic gly-moiety of PilE was detected by treating SDS-

PAGE separated pili preparations with sodium coconjugates were used as inhibitor of the bind-ing of MAbs to pili. The reaction between piliperiodate under conditions that preferentially

oxidises the sugar moieties followed by biotin- and MAbs could be efficiently inhibited by thegalabiose glycoconjugates but not by any otherhydrazine treatment. Figure 2(a) shows that PilE

of N. gonorrhoeae MS11 and N. meningitidis glycoconjugates or glycolipids tested (data notshown). These results indicate that an a-Gal-(1-FAM20 are glycosylated. Chemical de-

glycosylation of the pili and fetuin was done by 4)-Gal epitope is exposed on the meningococcalpili and accessible to the MAb MCA1.trifluoro-methanesulphonic acid. The de-

glycosylated pili and fetuin along with pili andfetuin were separated on SDS-PAGE and trans- Discussionferred to nitrocellulose filter and the extent ofdeglycosylation was examined. Complete de-glycosylation of pili and fetuin was observed as In this paper we describe the generation and

characterization of monoclonal antibodiesshown in Fig. 2(b).N. meningitidis C311 pili have been shown to against different LPS serotypes of M. catarrhalis.

MAbs MCB1 and MCB2 were generated againstcontain a terminal 1-4 diagalactose moiety. Weexamined if pili prepared from N. meningitidis serotype B LPS and reacted only with serotype

B LPS. No reactivity could be observed with anyFAM20 and 002 contain a similar diagalactosemoiety. We used the a-Gal-(1-4)-Gal-specific other LPS serotypes indicating that it specifically

recognized the serotype B determinant. The ser-MAbs to recognize this disaccharide. In solidphase ELISA MAb MCA1 recognized the di- otype determinant for type B LPS is a di-

saccharide moiety b-Gal-(1-4)-a-Glc-(1 at thesaccharide moiety only in pili preparations ofN. meningitidis FAM20 and 002, but not in N. nonreducing end and it is probable that these

monoclonal antibodies specifically recognizegonorrhoeae MS11 pili or deglycosylated pili.These results indicate that the pili preparations this epitope.

Monoclonal antibodies against M. catarrhalis LPS 303

We have previously reported the chemicalstructure of serotype A, B and C LPS of M.catarrhalis [5, 8, 9]. A terminal a-Gal-(1-4)-b-Gal-(1 epitope was found to be present in allserotypes. In direct binding assays MAbs MCA1and MCC2 reacted with galabiose and glo-botriose glycoconjugates indicating that theminimum recognition marker is a-Gal-(1-4)-b-Gal-(1. The MAbs recognized this epitope onlywhen it was in a terminal position. No reactivitywas obtained with globotetrosylceramide whichcontains the same epitope in an internal position.In inhibition experiments, galabiose was a moreefficient inhibitor than globotriose and in directbinding assays the binding of the MAbs wasstronger with galabiose glycoconjugates thanglobotriose glycoconjugates. These results sup-port the assumption that MCA1 and MCC2 MAbspecifically recognize the a-Gal-(1-4)-b-Gal-(1epitope.

Twenty-eight defined isolates of M. catarrhaliswere examined for expression of the a-Gal-(1-4)-b-Gal-(1 epitope on their surface. Twenty-sixout of twenty-eight reacted with the monoclonalantibodies indicating that this epitope is ex-pressed in most of the isolates and exposed onthe surface of the bacteria.

Pili of N. gonorrhoeae MS11 carry a covalentlybound O-linked N-acetyl-glucosamine-a1-3-galactose (a-GlcNAc-(1-3)-Gal) at serine 63 [12].Pili of N. meningitidis C311 contain an O-linkeddiagalactosyl 2,4-diacetamido-2,4,6-trideoxy-hexose within a peptide spanning amino acidresidues 45 to 73 [11]. We have examinedwhether the terminal 1-4 linked digalactose moi-ety of pili is accessible to the MAbs. In ourexperiments MAb MCA1 reacts with purifiedtype IV pili of N. meningitidis FAM20 and 002,but not with pili of N. gonorrhoeae MS11. Theseresults are in agreement with the known struc-ture of the saccharide moiety of the pili of N.

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meningitidis. Crystallographic studies of type IVpili of N. gonorrhoeae MS11 have suggested thatFigure 1. Reactivity of MAb MCA1 (a) and MCC2the saccharide moiety could be exposed on the(b) with different synthetic glycoproteins and glyco-

lipids. Microtitre plates were coated with 5 lg/ml of surface of the pilus fibre [12]. Our results showglycoconjugates and incubated with a two-fold serial that the a-Gal-(1-4)-b-Gal-(1 moiety is accessibledilution of culture supernatants in duplicate wells. to the MAbs and is therefore exposed on theThe results are the average of two experiments and surface of the pilus fibre.are represented as optical density reading re- In recent years it has been shown that acorded at 450 nm. Symbols used for coating antigen number of bacterial surface components displayare ATCC 25238 LPS (Ε), lactose (Ο), maltose (Χ), molecular mimicry to human glycoprotein andglobotriose (Β), cellobiose (+), galabiose (Φ), asialo

glycolipids and are called mimotopes. Mi-GM1 (Μ) and 3′sialylactose (Α).motope expression is a way to evade hostimmune responses and could possibly havesome other yet unidentified function. The

M. Rahman et al.304

Table 3. Concentration of inhibitors (lg/ml) required to obtain a 50% inhibition of binding of monoclonalantibodies MCC1 and MCC2 to M. catarrhalis LPS in inhibition ELISA

Inhibitor Chemical structure Monoclonal antibodies

MCC1 MCC2

Cellobiose Glcb1-4Glcb1-O-PAP-HSA >40 >40Maltose Gala1-4Glca1-O-PAP-HSA >40 >40Lactose Galb1-4Glcb1-O-PAP-HSA >40 >40Galabiose Gala1-4Galb1-O-PAP-HSA 0.5 0.9Globotriose Gala1-4Galb1-2Glc-O-PAP-HSA 4.5 6.53′-Sialyllactose Neu5Aca2-3Galb1-4(Glc)-APD-HSA >40 >40Lacto-N-fucopentaoseII Galb1-3GlcNAcb1-3Galb1-4(Glc)-APD-HSA >40 >40

4Fuca1

Lewis Y-tetrasaccharide Fuca1-2Galb1-4GlcNAcb1-O-APE-HSA >40 >403

FUca1LewisB-HSA Fuca1-2Galb1-3GlcNAcb1-3Galb1-4(Glc)-APD-HSA >40 >40

4Fuca1

Globotetrosylceramide GalNAcb1-3Gala1-4Galb1-Glcb1-1-Cer >40 >40AsialoGM1-gaglioside Galb1-3GalNAcb1-4Galb1-4Glcb-1-Cer >40 >40

capsular polysaccharide of Group B men- in human glycosphingolipids in the urinary tractand binds the Pap pili of uropathogenic Es-ingococci is a polymer of a-(2-8) polysialic acid

which is present on N-CAM of mammalian cherichia coli [18]. It is also expressed on gastro-intestinal epithelium and binds the B subunit ofcells and is essential for neuronal development

and kidney organogenesis [13]. Lacto-N-neo- Shiga toxin and members of verotoxin family[19]. It is also associated with Pk antigen of redtetraose (LNnT) oligosaccharide is present at

the nonreducing end of oligosaccharides in blood cells which is the receptor for parvovirusB19 [20, 21].immunotype 2, 3, 4, 5, 7 and 9 lipopoly-

saccharide of N. meningitidis. This oligo- Spontaneous phenotypic variants of H. in-fluenzae that produce LPS with a lower apparentsaccharide is also present in human milk.

The glycolipid LNnT-ceramide and its sialyated molecular mass and do not express the Gal-(1-4)-b-Gal-(1 digalactose were found to be lessform have been found in human erythrocytes,

lymphocytes and granulocytes [14]. Helicobacter virulent than the parental strain indicating thatthis disaccharide is important for virulence [22].pylori contains LPS epitopes that mimic blood

group antigens Lewis X, Y and H I and it The role of Gal-(1-4)-b-Gal-(1 epitope in thepathogenesis of M. catarrhalis is unknown buthas recently been demonstrated that H. pylori

infection give rise to autoantibodies that play expression of a similar epitope in a number ofgram-negative bacteria indicates the presence ofan important role in pathogenesis [15]. This

Campylobacter jejuni contains LPS epitopes that a common mechanism involved in the patho-genesis of these bacteria.mimic gangliosides and human antibodies

against the gangliosides and is believed toplay a central role in the pathogenesis ofGuillain-Barre syndrome [16]. Materials and methodsLipopolysaccharides of some species of gram-negative non-enteric bacteria, for example, N. Bacterial strainsgonorrhoeae, N. meningitidis, H. influenzae and M.catarrhalis express a disaccharide a-Gal-(1-4)-b- Twenty-eight strains of Moraxella catarrhalis were

used in this study. Four of them (ATCC 25238,Gal-(1 which was found to be expressed by anumber of host cells [17]. This epitope is present CCUG 18283, CCUG 18284 and CCUG 3292),

Monoclonal antibodies against M. catarrhalis LPS 305

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Figure 3. The binding of monoclonal antibody (MAb)MCA1 to purified pili from N. gonorrhoeae MS11(Β), N. meningitidis FAM20 (Χ), 002 (Φ) and N.meningitidis FAM20 deglycosylated pili (Ε) wasmeasured by ELISA. Microtitre plates were coatedwith 5 lg/ml of pili preparations in 0.05 M sodiumcarbonate coating buffer (pH 9.4), and incubated with10-fold serial dilution of MAb for 1 h at 37°C. Theresults are represented here as the optical density

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Figure 2. Demonstration of glycosylation of pili. (a)Purified pili from N. meningitidis FAM20 and N.gonorrhoeae MS11 were separated on SDS-PAGE and

from January 1988 to May 1991 and have beentransferred to nitrocellulose filter. The saccharide moi-described [23]. Four strains from culture col-ety was detected by using periodate oxidation fol-

lowed by biotin hydrazine treatment. Pili preparations lections (ATCC 25238, CCUG 18283, CCUGfrom FAM20 (lane 1) and MS11 (lane 2). Molecular 18284 and CCUG 3292) and four clinical isolatesweight markers are on the left. (b) Glycosylated pili (RS 10, 13, 26 and 272) used for LPS preparationfrom FAM20 and fetuin were deglycosylated by tri- were described earlier [24]. Salmonella typhi-fluoromethanosulphonic acid. Deglycosylated pili murium, A. caviae, E. coli, Klebsiella pneumoniae,and fetuin along with native pili and fetuin were sep- Yersinia enterocolitica and Proteus vulgaris strainsarated on SDS-PAGE and transferred to nitro-cellulose were obtained from the culture collection offilter and the saccharide moiety was detected as de-

MTC, Karolinska Institute. Neisseria gonorrhoeaescribed in Materials and methods. Pili preparationsMS11, N. meningitidis FAM20 and 002 were de-from FAM20 (lane 1), deglycosylated pili (lane 2), fet-scribed earlier [25]. LPS preparations from threeuin (lane 3) and deglycosylated fetuin (lane 4).strains of M. catarrhalis ATCC 25238 (serotypeA), CCUG 3292 (serotype B) and RS26 (serotypeC) were used as antigens for generation of hy-bridomas and monoclonal antibodies.were obtained from American Type Culture Col-

lection and the Culture Collection of University Moraxella catarrhalis strains were grown over-night at 37°C on horse blood agar plates. Ne-of Gothenburg. Twenty-four strains were isol-

ated from patients at the Roslagstull Hospital issoria gonorrhoeae and N. meningitidis strains

M. Rahman et al.306

were grown at 37°C in a 5% CO2 atmosphere on immunoglobulins [27]. Positive clones were fur-ther tested against a panel of LPS from M.Difco GCB agar containing Kellogg’s sup-

plement [26]. Escherichia coli, S. typhimurium, A. catarrhalis, S. typhimurium (core type Ra-Re), A.caviae and V. cholerae in ELISA.caviae, Y. enterocolitica and P. vulgaris were grown

on LB agar and K. pneumoniae was grown onblood agar plates.

The synthetic glycoconjugates cellobiose- ELISAHSA, maltose-HSA, melibiose-HSA, galabiose-HSA and globotriose-HSA were purchased from Flat bottom microtitre plates (Immunolon II,

Dynatech Laboratories, Virginia, U.S.A.) wereAccurate Chemical Inc. (Westbury, NY, U.S.A.).3′-Sialyllactose-HSA, lacto-N-fucopentaose- coated with LPS (10 lg/ml), or 5 lg/ml of gly-

coconjugates or 5 lg/ml purified pili pre-HSA, Lewis Y-tetrasaccharide-HSA, Lewis B-HSA and globotetraosylceramid were purchased paration or bacterial suspension (OD 0.01 at

600 nm) in sodium carbonate coating bufferfrom Iso-sep AB (Sweden). Asialo-GM1 gan-glioside was purchased from Sigma. The (pH 9.4) and kept overnight at room tem-

perature. The plates were washed four timesglycoconjugates contained 10–20 mol oligo-saccharide/mol HSA, and the carbohydrates are with phosphate buffered saline containing 0.05%

Tween 20 (PBS-T) and all the other washingsattached to the protein via a spacer. The spacerused were p-aminophenyl (PAP), amino- were done in the same way. Plates were then

incubated with 100 ll of suitable concentrationphenylethyl (APE) and acetylphenylenediamine(APD). PAP and APE are attached to the car- of antibodies for 2 h at 37°C. After washing,

plates were incubated with 100 ll of rabbit anti-bohydrate glycosidically and APD by reductiveamination as thus the terminal monosaccharide mouse horseradish peroxidase conjugated anti-

body at 1/1000 for 1 h at 37°C. The substrateis reduced and is present as aminoadetol. Sugarunits modified in this way are given in par- used was 3,5,3′,5′-tetra-methyl-benzidin,

0.1 mg/ml in 0.1 M sodium acetate (pH 6.0) andenthesis.0.002% H2O2. The reaction was stopped after10 min by adding 2 M H2SO4 and the plates wereread at 450 nm.Immunization and generation

of hybridomas

A group of four male 6–8 week BALB/c mice Inhibition ELISAwere inoculated with 0.2 ml of each LPS antigen(50 lg/ml) solution emulsified in an equal vol- For inhibition ELISA two-fold serial dilutions

of inhibitors were preincubated with MAbs inume of Freund’s complete adjuvant for 1 weekand then the same amount with Freund’s in- culture supernatant in appropriate dilution for

1 h at 37°C. It was then added to the microtitrecomplete adjuvant for 1 week. Mice were in-oculated with 0.2 ml of antigen solution over a plates and incubated for 2 h at 37°C. The rest

of the procedures are as described above. Theperiod of 16 weeks. Serum samples were col-lected and tested for antibody response against antibody dilutions for inhibition experiments

were calculated from the linear portion of theLPS. Finally the mice were boosted 3 days beforefusion. Hybridomas were generated by fusing titration curve against homologous LPS.

The inhibitory value was calculated as themouse plasmocytoma cell lines Sp2/0 with thespleen cells of an immunized mouse using stand- percentage reduction in absorbance compared

to a control without inhibitor. The concentrationard protocol as described elsewhere [27].Three weeks after fusion, culture supernatants required for 50% inhibition was determined for

each inhibitor.were tested for their reactivity to the ho-mologous LPS as antigen. Positive clones(OD>1.0 at A450) were recloned twice by limitingdilution and further tested against LPS of M. Preparation of LPS and type IV pilicatarrhalis. Culture supernatants from positiveclones were concentrated by 50% ammonium Cultivation of M. catarrhalis and extraction of

LPS was carried out as described earlier [5].sulphate precipitation and the antibody classand subclass of the MAbs were determined by Type IV pili were prepared from N. gonorrhoeae

and N. meningitidis as described earlier [28].immunodiffusion using rabbit anti-mouse

Monoclonal antibodies against M. catarrhalis LPS 307

2 Roantree RJ. Salmonella O-antigens and virulence. AnnLPS contamination of pili preparations wereRev Microbiol 1967; 21: 443–66.examined by a KDO specific monoclonal anti-

3 Catlin BW. Branhamella catarrhalis: an organism gainingbody and no such contamination was detected. respect as a pathogen. Clin Mirobiol Rev 1990; 3: 293–320.4 Murphy TF. Branhamella catarrhalis: epidemiology, sur-

face antigenic structure and immune response. MicrobiolDetection of saccharide moiety of pili Rev 1996; 60: 267–79.

5 Edebrink P, Jansson P-E, Rahman MM, et al. Structuralstudies of the O-polysaccharide from the lipo-Pili preparations from N. gonorrhoeae strain MS11polysaccharide of Moraxella (Branhamella) catarrhalis ser-and N. meningitidis FAM20 were separated on otype A (strain ATCC 25238). Carbohyd Res 1994; 257:

SDS-PAGE and glycosylation was detected by 269–84.Glycotract Kit (Oxford Glycosystems). Purified 6 Vaneechoutte M, Verschraegen G, Claeys G, van den

Abeele A-M. Serological typing of Branhamella ca-pili preparations were separated on a 12% SDS-tarrhalis strains on the basis of lipopolysaccharide an-PAGE and then transferred to nitro-cellulosetigens. J Clin Microbiol 1990; 28: 182–7.membrane. Sugar moieties on the protein were 7 Storm Fomsgaard J, Fomsgaard A, Hoiby N, Bruun B,

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