INFECTION AND IMMUNITY, Mar. 1994, p. 1052-1057 Vol. 62, No. 30019-9567/94/$04.00+0Copyright © 1994, American Society for Microbiology

Binding Studies of a Monoclonal Antibody Specific for 3-Deoxy-D-manno-Octulosonic Acid with a Panel of Klebsiella

pneumoniae Lipopolysaccharides RepresentingAll of the 0 Serotypes

N. M. VAN DER MEER,' B. J. APPELMELK,I* A. M. J. J. VERWEIJ-VAN VUGHT,' W. NIMMICH,2P. KOSMA,3 L. G. THIJS,4 J. DE GRAAFF,' AND D. M. MACLAREN'

Department of Medical Microbiology' and Medical Intensive Care Unit,4 Vrije Universiteit, 1081 BTAmsterdam,The Netherlands; Institute for Medical Microbiology, University of Rostock, Rostock, Germany2; and

Institut fur Chemie der Universitat fur Bodenkultur, A-1235 Vienna, Austria3

Received 6 December 1993/Accepted 8 December 1993

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for ot-3-deoxy-D-manno-octulosonic acid (a-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiellapneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passivehemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representingall nine currently known 0 serotypes. MAb 20 showed reactivity with almost all 0 serotypes of K. pneumoniaeLPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of theseKlebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity betweenLPS of different strains belonging to the same 0 serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity ofMAb 20 was observed onlywith the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with thehigh-molecular-weight fraction that contained core material substituted with several units of 0-antigenbuilding blocks. The chemical basis for these differences in reactivity remains to be established. As far as weknow, this is the first report containing comprehensive immunochemical data on the LPS core of K.pneumoniae.

Gram-negative sepsis is associated with a high level ofmortality. Although the potent antibiotics currently availablekill the invading microorganisms, they do not neutralize endo-toxin. Endotoxins (lipopolysaccharides [LPS]) play a pivotalrole in the pathogenesis of the sepsis syndrome by inducinghigh levels of cytokines. Therefore, attempts have been madeto prepare endotoxin-neutralizing antisera, in particular asingle monoclonal antibody (MAb) reactive with LPS of Esch-erichia coli, Pseudomonas aeruginosa, and Klebsiella pneu-moniae, the organisms most often isolated in cases of gram-negative sepsis. Since the core and lipid A of LPS are highlyconserved among the different species of gram-negative bac-teria, MAbs to these regions have been raised (1). Indeed,some anti-lipid A and anticore MAbs have been shown toexhibit in vitro cross-reactivity with smooth LPS (28) andwhole bacteria (12). A clinical trial with such a MAb has shownprotection in patients suffering from gram-negative bacteremicsepsis and septic shock (32). A possible disadvantage of thisapproach is the generally low affinities of cross-genera, cross-reactive MAbs (1, 6).

This raises the question of whether it would not be better toprepare instead MAbs with high affinity for only a given speciesand then make a mixture of highly effective species-specificMAbs for clinical use. Much research has been done on thecore structure and immunochemistry of E. coli (15) and P.aeruginosa LPS, and high-affinity, species-specific MAbs have

* Corresponding author. Mailing address: Department of MedicalMicrobiology, van der Boechorststraat 7, 1081 BT Amsterdam, TheNetherlands. Phone: (31)(20)-5483952. Fax: (31)(20)-6447151.

already been described for these genera (10, 22). Klebsiellaeare the second most common microorganisms isolated, after E.coli, from gram-negative bacteremia patients (11, 17). Al-though the structures of the nine known 0 antigens (13, 16, 27)have been elucidated, very little research has been done on thecore and immunochemistry of Klebsiella LPS and very fewMAbs against its core and lipid A have been reported (20).

In this paper we describe the binding of a MAb (clone 20)specific for cx-3-deoxy-D-manno-octulosonic acid (a-Kdo) ofthe inner core with a large panel of LPS from K pneumoniaecomprising all nine currently known 0 serotypes (13, 27). Thegoal of our investigation was to demonstrate the presence of aconserved Kdo epitope in LPS ofK pneumoniae.

Clone 20 is already known to display complex bindingbehavior (1-5, 9, 29, 30). First of all, it binds to oa-Kdo but notto ,B-Kdo or 5-deoxy-Kdo; in other words, binding to Kdo isvery specific because reactivity is totally lost upon changing ofthe anomeric configuration or upon deletion of a singleoxygen. However, clone 20 is also capable of interactinghydrophobically with the fatty acyl chains of lipid A and ofbinding to nonrelated antigens. This promiscuous binding tononrelated antigens may be called a nonspecific interaction.This hydrophobic interaction was evident in enzyme-linkedimmunosorbent assay (ELISA) (1, 3) but not in passivehemolysis assay (PH) (9), in which the fatty acyl chains of thelipid A part of LPS are hidden in the membranes of sheeperythrocytes (SE). Thus, binding of clone 20 to an LPS inELISA is not necessarily indicative of the presence of a Kdoepitope in that LPS; it might simply be due to nonspecifichydrophobic interaction. Therefore, binding in both ELISAand PH was studied. Binding in the inhibition of passive

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hemolysis assay (PHI) was also studied because the syntheticKdo antigens we used for inhibition are not able to coat ELISAplates or SE. Finally, the binding to LPS separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was investigated by immunoblotting.

MATERIALS AND METHODS

LPS preparations and synthetic antigens. All LPS weredissolved in water, containing 0.01% merthiolate and 0.1%triethylamine, at a concentration of I mg of LPS per ml. Use oftriethylamine was needed because in its absence some LPS didnot coat well to SE (see below). The sources of the LPS, theirbacterial strains, and references concerning their purificationare given in Table 1. Smooth LPS had been extracted by thephenol-water procedure; for rough LPS the phenol-chloro-form-petroleum ether method had been used. All LPS ob-tained from a given scientist had been extracted by the sameextraction procedure from bacteria that had all been grownunder identical circumstances. LPS of K pneumoniae had been0 serotyped with monospecific antisera raised to 0-typestrains from the Klebsiella reference laboratory (Statens Serum-institut, Copenhagen, Denmark). Compound 506 representsthe synthetic counterpart of biphosphoryl hexaacyl lipid Afrom E. coli. The synthetic Kdo antigens were prepared bycopolymerization of allyl-Kdo haptens with acrylamide. Theresulting copolymers are polyvalent Kdo antigens with molec-ular weights of 60,000 to 100,000.

Antibodies. The preparation of clone 20 (murine immuno-globulin M) as well as its binding characteristics has beendescribed before (1-5, 9, 29, 30). Clone 20 (a gift from P.Trown, Berkeley, Calif.) had been purified to >95% purity.

ELISA. All assays were performed in 96-well flat-bottompolystyrene microtiter plates (Greiner, Alphen aan den Rijn,The Netherlands). The plates were incubated overnight atroom temperature with 100 ,u1 of the various LPS diluted inphosphate-buffered saline (PBS) to a concentration of 1 p.g/ml.Next, the plates were washed three times with PBS containing0.05% Tween 20 (PBST). Clone 20 was diluted with PBST toa concentration of 4 pLg/ml and titrated in twofold dilutions of100 pu1 per well. After the MAb was incubated overnight atroom temperature, the plates were washed. Goat anti-mouseimmunoglobulin M (American Qualex, La Miranda, Calif.)labeled with horseradish peroxidase was diluted to an endvolume of 100 .1I and supplemented with 0.5% normal goatserum. The plates were incubated for 3 h at 37°C and washed.A 10-min incubation with 100 p. of peroxidase substratesolution (citrate-phosphate buffer [pH 5.5], 1 mg of o-phe-nylenediamine per ml, 0.015% H2O,) at room temperaturewas performed and was terminated with 50 p.1 of 4 M H2S04.A492 was measured with a Bio-Rad (Richmond, Calif.) 2550enzyme immunoassay reader. The titer was defined as theMAb concentration (in nanograms per milliliter) at which theabsorbance was 0.2 higher than the absorbance for the negativecontrol (PBS coating, processed identically to LPS-coatedwells).

PH. Two- to three-week-old SE in Alsever buffer (Biotrad-ing, Mijdrecht, The Netherlands) were washed three times in asolution containing 15 mM NaH2PO4 and 150 mM NaCl, pH7.3, and were adjusted to a concentration of 5% in thesuspension. One milliliter of the 5% SE suspension wasincubated with 100 pg of the LPS for 30 min at 37°C. Thesensitized SE were washed and resuspended in barbiturate-buffered saline with Ca2' and Mg2' (pH 7.3) to a concentra-tion of 0.5% in the suspension. All assays were performed with96-well V-shaped polystyrene microtiter plates. Each well

TABLE 1. Information on LPS used

LPS origin Strain no." Source of LPS (reference)

K pneumoniaeOlK37

01K2201K46OK202K202K02K403K5803K3103K2504K1505K5705K7507K6708K6909K7201OK7301 2K800-K1803K1 '703- K1-Clinical isolate

8238

1996/495281505573805053Sf50636/5262582002/49Mich 614425/5 1645264 [2]88912053377081754/49Len 1Len 111

K. oxytoca clini-cal isolate

S. mitn.esota

E. coliRIR2R3R4K-1208K809K9019020

R595

F470F576F653F25 13W3100G3404-41Bi316-42

Lipid A com-pound 506

Poly-ot-Kdo(synthetic)

Poly-ot-(Kdo)2(synthetic)

Poly-P-Kdo(synthetic)

W. Nimmich, Rostock,Germany (23, 25)

W. Nimmich (23, 24)W. Nimmich (23)R. Zalisz, Osny, France (20)W. Nimmich (23)W. Nimmich (23)R. Zalisz (20)W. Nimmich (23, 25)W. Nimmich (23)R. Zalisz (20)W. Nimmich (23, 25)W. Nimmich (23, 25)W. NimmichW. Nimmich (23, 25)W. Nimmich (23, 25)W. Nimmich (23, 25)W. Nimmich (23, 25)W. Nimmich (23, 25)R. Zalisz (20)N. Kato, Nagoya, Japan (14)N. Kato (26)C. Easmon, London, UnitedKingdom

C. Easmon

H. Brade, Borstel, Germany

H. BradeH. BradeH. BradeH. BradeH. BradeW. NimmichW. NimmichK. Jann, Freiburg, GermanyK. Jann

Daiichi Fine Chemicals,Japan

P. Kosma, Vienna,Austria (9)

P. Kosma (9)

P. Kosma (9)

" Strains 8238 to 1754/49 are Orskov strains: the remainder are not Orskovstrains.

' K mutant of a K. pnelumonziae 03KI strain.0 antigen-deficient mutant of K ptneumoniace 03KI

contained 50 .1I of MAb solution titrated in twofold dilutionsteps, 50 p1 of sensitized SE, and 25 pL. of 3.3% guinea pigserum. After I h at 37°C and centrifugation, 100 p.l of thesupernatant was measured at an A4,4 with an enzyme immu-noassay reader. The titer was defined as the MAb concentra-tion which caused 50% specific lysis. Sensitized SE incubatedwith guinea pig serum but without MAb served as a negativecontrol.

PHI. PHI was essentially identical to the assay described

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above, with the exception that the SE were coated with 2 jig ofLPS per ml of 5% SE suspension for all LPS except that ofserotype O1K46, which required 10 jig/ml for acceptablecoating. A fixed concentration of clone 20 was used (twice theconcentration that caused 100% specific lysis) and was prein-cubated for 1 h at 37°C with the LPS, serially diluted (inbarbiturate-buffered saline with Ca2' and Mg2+) in twofoldsteps. The preincubated mixture (50 ,ul) was then introducedinto the PH system, and lysis was determined as describedabove. The coating concentration in PHI, which was muchlower than that in PH, was chosen to avoid the need to use

approximately 1-mg amounts of LPS as inhibitors in PHI.SDS-PAGE. LPS of 05K157 and 012K80 were subjected to

SDS-PAGE. For optimal separation of the higher-molecular-weight fractions containing the 0 antigen, the conventionalLaemmli system (18) was used with a 10% separating gel. Foroptimal separation of low-molecular-weight fractions, we usedthe Tricine system (19) with a 10% separating gel. LPS sampleswere diluted (3:1) in a sample buffer (0.25 M Tris-HCl [pH6.8], 8% SDS, 50% glycerol, 1 mg of bromophenol blue per ml,400 mM dithiothreitol, 20 mM EDTA) and heated for 10 minat 95 to 100°C. Electrophoresis was done for about 20 min at18 mA until the LPS entered the separating gel and then wasdone for 1 h at 30 mA. The amounts of LPS used for silverstaining and blotting are indicated in Results.

Silver stain. One half of the gel was silver stained, while theduplicate half was blotted (see below). The silver stain wasdone as described by Tsai and Frasch (31). This involvedovernight fixation in 40% ethanol-5% acetic acid; 10 min ofincubation with a solution containing 40% ethanol, 5% aceticacid, and 0.7% sodium metaperiodate; washing with H20(three times); 10 min of incubation with a 0.7% silver nitratesolution; washing (three times); and developing for 7 min in a0.24 mM citric acid-0.019% formaldehyde solution, with sub-sequent stopping of the reaction by washing with H20.Immunoblot. The blot buffer (pH 8.35) used contained 192

mM glycine, 25 mM Tris, and 20% methanol. A polyvinylidinedifluoride blot membrane (Millipore Co., Bedford, Mass.) wasused. Blotting was performed for 1 h at 350 mA. After beingblotted, the membranes were baked for 1 h at 70°C. All stepsof the immunostaining technique were performed at 37°C,except for the color-developing step, which was done at roomtemperature. During each procedure the membrane was gentlyrocked on an orbital shaker. First, the membrane was blockedwith a blocking reagent (Boehringer, Mannheim, Germany)for 45 min. After being washed three times with PBST, themembrane was incubated for 2 h with MAb 20 at 20 j,g/ml inPBST. After three washings with PBST, the membrane wasincubated for 1 h with horseradish peroxidase-conjugated goatanti-mouse immunoglobulin M (American Qualex) in PBSTwith 0.5% normal goat serum at a dilution of 1:1,000. Afterthree washes, color development was done with 25 ml ofcitrate-phosphate buffer containing 15 mg of 4-chloro-1-naph-thol (Bio-Rad) and 12.5 ,ul of 30% H202; staining was stoppedby rinsing the blot with water.

Chemicals. All chemicals not specified above were of thehighest obtainable purity.

Data. All data are representative results of at least threeexperiments.

RESULTS

Inhibition of passive hemolysis by synthetic Kdo and Sal-monella minnesota R595 LPS. The inhibition values (Table 2)obtained in the already-characterized (9) system (R595-coatedSE with R595 LPS and Kdo inhibitors) are in the same range

TABLE 2. Inhibition of MAb 20-induced passive hemolysis bysynthetic antigens and R595 LPS

Amt of inhibitor (ng/ml)aOrigin of LPScoating SE R595 LPS Poly-a- Poly-a- Po1y-p-

Kdo (Kdo)2 Kdo

S. minnesota R595 3 16 3 >500K pneumoniae01K37 0.5 0.5 0.75 >50001K46 16 8 8 >50002K2 0.75 2 1 >50003K58 3 4 3 >50004K15 16 24 16 >50008K69 0.5 0.75 1 >500a Data are amounts of inhibitor required to cause a 50% inhibition of

MAb-induced passive hemolysis of SE coated with various LPS.

as the values for a system with K pneumoniae LPS-coated SEwith the same inhibitors. These data indicate that MAb 20recognizes in K pneumoniae a monosaccharide ot-Kdo epitopevery similar to the one present in R595 LPS.

Reactivity ofMAb 20 with a panel of K. pneumoniae LPS. (i)ELISA. In ELISA, a solid-phase assay system in which the LPSis coated to the rigid wall of a microtiter plate, MAb 20 showedgenerally high levels of reactivity with the LPS of all serotypesofK pneumoniae (Table 3) except 05 and 09. Striking are theextreme differences in reactivity between LPS isolated fromdifferent strains of the same serotype, in particular the 01 and03 serotypes. K pneumoniae 01K37 and 01K46 showed veryhigh levels of reactivity, while K pneumoniae 011K22 showed avery low level of reactivity. These differences are not due todifferences in growth circumstances (21) of the bacteria or inLPS extraction methods. No reactivity was observed with the E.coli core types except R3. A drawback of ELISA was the use ofendpoint titers for expression of antibody reactivity. Whileclone 20 apparently reacted equally well with LPS of Kpneumoniae 01K46 and 02K4 (titers of 8 to 16 ng/ml),inspection of the full dose-response curves (not shown) revealsa different picture: the curve of binding to 01K46 was veryshallow, with no optical density higher than 1.5, not even at4,000 ,ug of MAb per ml; in contrast, the curve of binding to01K2 was very steep, with an optical density of >2 even in theconcentration range close to its endpoint titer. Thus, the use oftiter may hide large differences in reactivity; in PHI, however,this difference was apparent, and 01K46 reacted 50 times lessthan 01K2.

(ii) PH. In PH the coating of the SE leads to incorporationof hydrophobic domains of lipid A into the cell membrane.Thus, the reactivity for lipid A 506 in ELISA, which is absentin PH, is due to incorporation of the hydrophobic part of lipidA 506 into the cell membranes of the SE. As in the ELISA,MAb 20 shows broad reactivity in PH with LPS of all Kpneumoniae serotypes except 07 (Table 3); this means that aconserved Kdo epitope is indeed present. SE sensitized withKpneumoniae 05 and 010 LPS and with E. coli Rl and R2 LPSlysed completely in the presence of complement only, whichmakes it impossible to judge the reactivity of the MAb withthese LPS. We do not know whether this lysis is due to directcomplement activation by the LPS coated on the SE or,alternatively, whether lysis is mediated by natural anti-LPSantibodies present in the complement itself. As with theELISA measurements, within the K pneumoniae 01 groupthere is an extremely wide range of reactivities.

(iii) PHI. LPS is a heterologous mixture consisting ofmolecules that do not necessarily bind well to ELISA plates or

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TABLE 3. Reactivity in three assay systems of MAb 20with several LPS

Concn of MAb 2(0 (ng/ml) neededLPS origin for reactivity in:

ELISA" PH" PHI"

S. minnesotaR595 <8 8 <8

K oxytoca clinical sample <8 8 32

K pneumoniaeClinical sample <8 16 1601K37 <8 8 50001K22 >4,000 >4,000 >12,800O1K46 <8 <8 800011K2 > 4,000 > 4,000 > 64,00002K2 16 <8 1602K- 32 250 4,00002K4 < 8 64 < 803K1 32 <8 6403-Kl <8 8 1603K58 1,000 8 25003K31 2,000 <8 50003K25 1,000 32 25004K15 <8 16 1605K57 >4,000 -" 2,00005K75 >4,000 12,80007K67 125 > 4,000 6,00008K69 16 8 12509K72 >4,000 8 4,000101K73 500 >64,000

012K80 16 <8 500O-K18 >4,000 >4,000 24,000

E. coliR1 >4,000 >64,000R2 > 4,000 > 64,000R3 32 > 4,000 > 64,000R4 1,000 > 4,000 > 64,000K-12 > 4,000 > 4,000 > 64,000

Lipid A compound 506 32 >4,000 800

' Reactivity is defined as optical density 0.2 above that of the negative control.b Reactivity is defined as 50% lysis of SE.C R595-coated SE LPS are the inhibitors. Reactivity is defined as 50%

inhibition of passive hemolysis., 100% lysis without MAb.

incorporate efficiently into the membranes of SE. Thus, onecomponent of the mixture may coat well to plastic but veryinefficiently to SE, or vice versa. Clearly, selective coating maylead to discrepant outcomes between ELISA and PH. Tocircumvent these problems we also used PHI, in which LPS isin the fluid phase and coating is not a prerequisite forreactivity. Large differences in the abilities of the various LPSto inhibit the PH system of MAb 20 with S. minnesota R595LPS-sensitized SE were observed (Table 3), e.g., while lessthan 8 ng of 02K4 LPS per ml caused 50% inhibition of lysis,O1K2 caused no inhibition at 64,000 ng/ml.SDS-PAGE. (i) Silver stain. The periodate stain of the gels

shows separation of the LPS into a high- and a low-molecular-weight band (Fig. lA and B). A distinct ladder pattern is seenfor these LPS after separation by using the classical Laemmlisystem (18) (Fig. lA), while a better separation of the low-molecular-weight region took place in the Tricine system (19)(Fig. IB).

(ii) Immunoblot. Figure IC shows the immunoblot of LPS

H.-

1 2A

3 4B

5 6C

FIG. 1. SDS-PAGE silver stain (A and B) and immunoblot (C) ofLPS ofK pneumoniae 05K57 (lanes 1, 3, and 5) and 0 1 2K80 (lanes 2,4, and 6). For SDS-PAGE of the gels in panels A and B, glycine-Trisand Tricine-Tris were used, respectively. The blot in panel C was fromcompanion lanes of the same gel as that in panel B, and it wasdeveloped with MAb 20. The electrophoresis fronts of the gels and blotare aligned. The amounts of LPS used per lane for A, B, and C were1, 2, and 4 jug, respectively.

separated by using the Tricine system. MAb 20 reacted wellwith the fast-moving band (possibly nonsubstituted core mate-rial) of 012K80 LPS but not with those present in serotype05K57 (Fig. IC). The MAb did not react with the core of012K80 when linked to several units of 0-antigen buildingblock. A blot of LPS separated in the Laemmli system showedsimilar results (not shown).

DISCUSSION

We have previously observed the reactivity of one of ourMAbs raised against S. minnesota R595 with the LPS of oneKlebsiella strain (2). This raised our interest in researching thisgenus further. In this paper we report that clone 20, a MAbalready known to be specific for a-Kdo monosaccharide (9),reacted with many K pneumoniae LPS of almost all serotypes.On consideration of the inhibition of MAb 20-induced

passive hemolysis by synthetic antigens and R595 LPS (Table2), it is apparent that the inhibition patterns are similar for SEcoated with K pneumoniae LPS and SE coated with S.minnesota LPS. Consistent with the accepted theorem that theinner core and lipid A are the most conserved parts of LPS,these results suggest that the Kdo regions of K pneumoniaeand S. minnesota are closely similar. After establishing thatMAb 20 behaved as an c.-Kdo MAb, we tested its reactivitywith an extensive Klebsiella LPS panel in different assaysystems (Table 3). Comparatively, the tests showed that theLPS can be divided into three categories on the basis of theirreactivities with MAb 20. Members of the first category, whichincluded most of the serotypes of K pneumoniae LPS (e.g.,01K37, 02K2, and 04K15), showed consistently high levels ofreactivity in all three tests with MAb 20, i.e., in ELISA and PHreactivity levels ranged from <8 to 125 ng/ml, and in PHI

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inhibitory values of the LPS ranged from <8 to 500 ng/ml.Members of the second category showed very low levels ofreactivity in all assay systems (e.g., K pneumoniae O1K22,O1K2, and 0-K18). Members of the third category showedvariable results in the different assay systems, a phenomenonfor which several explanations can be offered. Some LPS in thethird category, e.g., K pneumoniae 07K67, E. coli R3, and lipidA 506, showed high levels of reactivity with MAb 20 in ELISAbut showed low levels of reactivity in PH. This may be causedby differences in the abilities of these LPS to coat SE in PH andplastic in ELISA. However, it is also possible that the LPS coatequally well in both systems but that differences in physico-chemical surroundings cause the difference in conformation(exposure) of the Kdo epitope (8). Lastly, it is known thatclone 20 may also interact with acyl residues (1, 3). Since in PHthe hydrophobic areas of the LPS are incorporated into themembranes of the SE, reactivities measured in PH are strongevidence of the presence of a Kdo epitope. The difference inreactivity in PH and ELISA may thus be caused by hydropho-bic interactions of MAb 20 with the LPS in ELISA which areimpossible in PH, as is certainly the case with lipid A 506 andpossibly with K pneumoniae 07K67 and E. coli R3. Other LPSin the third category, e.g., K pneumoniae 09K72, 03K58,03K31, and 03K25, showed low levels of affinity in ELISA butshowed high levels of reactivity in PH. Again, this difference inreactivity can be caused by differences in the coating abilities ofthese LPS in the different systems or differences in physico-chemical surroundings between the systems. It is interestingthat K pneumoniae 03K11- and 03- Kl - LPS react in allsystems but 03K58, 03K31, and 03K25 LPS react in PH only.The first two LPS were obtained from one source, and thelatter three were obtained from another. Thus, the differencesobserved between the two groups of 03 LPS may be due todifferences in growth medium or cultivation time (21) used forthe bacteria prior to LPS extraction, or they may be due tosmall variations in the phenol-water procedure. Other LPS inthe third category show differences in reactivity in ELISA, PH,and PHI, e.g., K pneumoniae 09K72 LPS, which did not reactin ELISA or PHI but reacted very well in PH. PHI has theadvantage that all LPS molecules of a given preparationremain present, in contrast to a possible selective coating inELISA and PH. Despite this, there can still be a much lowerreactivity level in PHI than in PH because the epitope recog-nized by MAb 20 might be hidden because of the formation ofmicelles of LPS in the fluid phase. The extreme differences inaffinity, even between isolates of one serotype, raise thequestion of which structural features are responsible for thisphenomenon; this is reminiscent of the occurrence of E. coliLPS of (immuno)chemically different core types (15, 27).SDS-PAGE, silver staining, and immunoblotting experimentswere done to further study the interactions of MAb 20 with theK pneumoniae LPS fractions separated according to molecularweight and degree of substitution of the core with 0 antigen(Fig. 1). MAb 20 is capable of recognizing only the fast-movingpart (likely representing the complete core without 0 antigen)of 012K80, a phenomenon that has been observed with otheranticore and anti-lipid A MAbs (7). The lack of reactivity ofMAb 20 with the higher-molecular-weight bands may haveseveral causes: (i) steric hindrance of the epitope of MAb 20 bythe 0 antigen, the epitope itself remaining the same butinaccessible; (ii) a change in conformation of the LPS when theO antigen is present, the epitope being changed because ofphysical forces; (iii) differences in covalent structures betweensubstituted and nonsubstituted core materials, e.g., in substi-tuted core material the Kdo might be substituted by phosphate.If MAb 20 recognizes nonsubstituted core material only, the

amount of nonsubstituted core material present in the variousLPS preparations tested, in addition to possible differences inchemical structure, may influence the reactivity with MAb 20.However, as judged by silver stain, similar amounts of nonsub-stituted core material are present in LPS of 05K57 and012K80. Consequently, the differences in reactivity with thesetwo LPS are due to differences in covalent structure. Thisfinding suggests that at least two distinct core structures of Kpneumoniae exist, of which only one contains the terminalxL-Kdo epitope that MAb 20 recognizes.Our results suggest that the use of more than one assay

system is to be recommended because variable reactivities maybe obtained in various assay systems. Furthermore, it isadvisable to use several LPS of relevant serotypes when testingMAbs for reactivity with LPS because large differences inreactivity may be obtained with the various LPS.

In conclusion, a MAb (MAb 20) specific for oL-Kdomonosaccharide reacts well with LPS of K pneumoniae, sug-gesting an epitope in the core of these LPS much like that inthe inner core of S. minnesota LPS. Some of the extremedifferences in reactivity with various LPS ofK pneumoniae arepossibly due to the existence of more than one core type in Kpneumoniae, as has been observed for E. coli.

ACKNOWLEDGMENTS

We thank N. Kato and R. Zalisz for the kind gift of many of the LPSused in this study.

REFERENCES1. Appelmelk, B. J., and J. Cohen. 1992. The protective role of

antibodies to the lipopolysaccharide core region, p. 375-410. InJ. L. Ryan and D. C. Morrison (ed.), Bacterial endotoxic lipopoly-saccharides, vol. 2. Immunopharmacology and pathophysiology.CRC Press, Boca Raton, Fla.

2. Appelmelk, B. J., J. Cohen, A. Silva, A. M. J. J. Verwei-van Vught,H. Brade, J. J. Maaskant, W. F. Schouten, 0. Mol, A. Honing,L. G. Thijs, and D. M. MacLaren. 1989. Further characterizationof monoclonal antibodies to lipopolysaccharide of Salmonellaminnesota strain R595, p. 319-330. In M. Nakano et al. (ed.),Endotoxin. Plenum Press, New York.

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