Vol. 20, No. 6JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1984, p. 1180-11850095-1137/84/121180-06$02.00/0Copyright © 1984, American Society for Microbiology

Monoclonal Antibody-Based Enzyme-Linked ImmunosorbentAssays for Identification and Serotyping of Vibrio cholerae 01

BJORN GUSTAFSSONDepartment of Bacteriology, Karolinska Institute, Stockholm, Sweden

Received 21 May 1984/Accepted 13 August 1984

Monoclonal antibodies directed against 0-specific antigens of Vibrio cholerae 01 lipopolysaccharide were

used in two different enzyme-linked immunosorbent assays (ELISAs), designed for identification andserotyping of V. cholerae 01. In the sandwich ELISA, a monoclonal antibody against the group-specific antigenwas used as capture antibody, whereas peroxidase-conjugated monoclonal antibodies directed against group-

and type-specific antigens were used as the second antibodies. Monoclonal antibodies were also used in ELISAinhibition tests with whole bacteria as inhibitors in microtiter trays coated with V. cholerae 01 lipopolysaccha-ride. In addition, the monoclonal antibodies were shown to be useful in slide agglutination tests. The enzyme

immunoassays were equally sensitive, showing positive reactions with all V. cholerae 01 strains tested, whereasall V. cholerae non-O1 as well as strains of Escherichia coli, Shigella sonnei, Salmonella spp., Citrobacterfreundii, and Brucella abortus were negative. The microtiter application makes the immunoassays suitable withlow consumption of reagents for screening of samples from suspected cases as well as from the environment.

Vibrio cholerae is divided into several serogroups, on thebasis of their 0 antigens. The disease Asiatic cholera iscaused only by strains belonging to serogroup 01, character-ized by the group-specific antigen A. This serogroup issubdivided into two main serotypes, Ogawa and Inaba,owing to the presence of type-specific antigens B and C,respectively. In addition, a third serotype, Hikojima, con-taining all three antigens has been described (2, 3, 11, 16, 25).These antigens are thermostable polysaccharides and partsof the cell wall lipopolysaccharide (LPS) (14, 20, 27). V.cholerae strains other than serogroup 01 are designatedNAG strains or V. cholerae non-O1 (20).The identification of V. cholerae 01 and its serotypes is

based on the use of group-specific (anti-A) and type-specific(anti-B, anti-C) antisera. These are produced by rabbitsimmunized with heat-inactivated whole bacteria. Type-spe-cific antisera are prepared by cross-absorption of the polyva-lent antiserum with bacteria of the Ogawa or Inaba type toremove the antibodies directed against the A antigen. How-ever, since there is often a concomitant loss of type-specificantibodies with this procedure, such antisera are difficult toproduce (20). Cross-reactions have been reported to occurbetween type-specific V. cholerae antiserum and some sero-types of Salmonella spp., Citrobacter spp., Escherichia coli,and Shigella spp. (32). Furthermore, laboratories havewrongly diagnosed V. cholerae non-O1 as V. cholerae 01when commercially available antisera was used (6).The clinical spectrum of cholera is broad, ranging from

severe cholera (10 to 25% of all cases) to inapparent infec-tions (60 to 75%) (19). It is important from both a diagnosticas well as an epidemiological point of view that accurate andreproducible methods are available for the certain identifica-tion of V. cholerae 01. The aim of this study was to developa rapid and highly specific immunoassay based on monoclo-nal antibodies directed against the somatic group- and type-specific antigens A, B, and C for eventual use in thescreening of clinical specimens and for epidemiologicalpurposes.

MATERIALS AND METHODSBacterial strains and cultivation. The strains used in this

study are listed in Table 1. Strains used in the sandwich

enzyme-linked immunosorbent assay (ELISA) and ELISAinhibition were cultivated overnight in 5 ml of nutrient broth(Oxoid Ltd., London, England) in test tubes at 37°C. Forslide agglutination, strains were cultivated overnight onnutrient agar at 37°C. LPS was prepared from V. cholerae34, 35, and NCTC 4711, cultivated in a tryptone-yeastextract (TY-2) (15) medium in an aerated, stirred, 12-literfermentor at 37°C at a constant pH of 7.2.LPS preparation. LPS was extracted from the bacteria by

the phenol-water method (31) and purified by high-speedcentrifugation as described earlier (13).Hybridomas. Production and characterization of the hybri-

domas H8 and C6 have been described elsewhere (12).Establishment of the hybridomas 110-1-SB and 112-3-2G wasas follows.

(i) Antigen preparation. V. cholerae 569B was cultivatedovernight in tryptic soy broth (Difco Laboratories, Detroit,Mich.) in shake flasks at 37°C. The bacteria were killed byadding Formalin to a final concentration of 1.5% (vol/vol),washed twice in 10 mM phosphate-buffered saline (PBS), pH7.2, and incubated at 65°C for 1 h (5).

(ii) Immunization. Female BALB/c mice, 6 to 10 weeks ofage, were immunized intraperitoneally twice with 107 cells ata 2-week interval, followed by immunization with 108 and1010 cells at a 3-week interval. Fusion was performed on day3 after the last immunization.

(iii) Fusion and cloning. Hybridomas were established bythe method of Nowinski et al. (21) as described earlier (13)with modifications (12). Briefly, B lymphocytes from immu-nized mice were fused with mouse myeloma cells from thecell line SP2/0-Ag 14 (26) by using polyethylene glycol (PEG1500; E. Merck AG, Darmstadt, Federal Republic of Germa-ny) as the fusion agent. Primary selection of hybrids wasperformed by growing the cells in 96-well microtiter plates(3042; Falcon Plastics, Oxnard, Calif.) on HAT medium (18)in a tissue culture incubator at 37°C, 80% humidity, and 5%CO2. Hybrid cells of interest were recloned by limitingdilution.

(iv) Production of antibodies. Monoclonal antibodies wereproduced by growing the cells in 250-ml tissue culture flasks(3075; Costar Data Packaging, Cambridge, Mass.) in RPMI1640 medium (GIBCO Laboratories, Glasgow, Scotland)

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TABLE 1. Bacterial strainsaStrain Serogroup Sourceb

Vibrio choleraeOgawa

34145113141824VRL 3VRL 19VRL 59VRL 84VRL 88

Inaba35VRL 10VRL 48VRL 58VRL 86VRL 87569BV63P

Hikojima1602160343311689

010101010101010101

0101010101010101

01010101

Not groupedNCTC 4711NCTC 4716B4202-647007-628394-6210317-62112-68218-6810843-62211-7211416-62B8645-64103-68316-71110-68B5257-64139-6810332-62109-68317-7114438-6214821-62344-7210438-6212630-62161-6812795-625473-62171-68151-68152-681311-691321-691322-69215-68225-68

E. coli611617Bi 7455/41

02040506070809010011012013014015016017018019020021023024025026027028029030031032033034035036037038039

06:K15:H1608:K40:H9043:H2

AAAABBBBB

ABBBBBAA

GGGG

TABLE 1-ContinuedStrain Serogroup Sourceb

055:B5 C614 078:H11 C

085:H1 D0104 D0105:H8 D0111:K58 D

1935 0150:K93 E

Salmonella spp.S. bergen IS 701 47 FS. urbana IS 122 30 FS. enteritidis IS 64 1, 9, 12 FS. paratyphi A IS 1 1, 2, 12 FS. godesberg S:2996/80 30 FS. arizonae 23:33:25 47 FS. kaolack IS 786 47 FS. kentucky IS 98 8 FS. typhimurium 1, 4, 12 C

Shigella sonnei C

Citrobacter freundii FATCC 8090a The V. cholerae strains are designated as described in Shimada and

Sakazaki (28).bStrains were provided by: A, J. Holmgren, University of Gothenburg,

Sweden; B, H. Smith, Jefferson Medical College, Philadelphia; C, our straincollection; D, B. Kaijser, University of Gothenburg, Sweden; E, F. Orskov,C! +A+A_c fis+_A)AbbsT-e+ IC ' nto ;

A Statens Seruminstutut, Copennagen, DenmarK; r, The National Dacteriulogi-A cal Laboratory, Stockholm, Sweden; and G, T. J. Donovan, Preston Hall

A Hospital, England.A

AA supplemented with 10%o fetal calf serum, L-glutamine (1A mM), penicillin (100 U/ml), and streptomycin (100 p.g/ml), orA the antibodies were produced in pristane (Sigma ChemicalA Co., St. Louis, Mo.)-primed mice (22) by injecting 5 x 106A cells intraperitoneally into the mice. The antibodies wereA stored at -20°C until they were used.A Immunoglobulin class, subclass, and light chain. Immuno-A globulin class, subclass, K and X light chains were deter-A mined by immunodiffusion by the method of OuchterlonyA with 1% agarose (Marine Colloids, Rockland, Maine) in PBSA and specific rabbit antiserum to mouse immunoglobulin MA (IgM), IgG (7S), IgGl, IgG2a, IgG2b, IgG3, and light chainA (Bionetics Laboratory Products, Charleston, S.C.).A ELISA. (i) Direct ELISA. Antibody production was mea-A sured by ELISA (9), performed in 96-well microtiter traysA (Dynatech M 129 A; Flow Laboratories, Irwine, Scotland)A (30) as described earlier (13). Briefly, 50 ,ul of cell cultureA supernatant or ascitic fluid was added to wells previouslyA coated with LPS from V. cholerae 34 (Ogawa) and 35A (Inaba), respectively. The immune reaction was performedA by adding 50 ,u of rabbit anti-mouse immunoglobulin (Dako,A Copenhagen, Denmark), followed by incubation with 50 ,ulA of horseradish peroxidase-conjugated sheep anti-rabbitA immunoglobulin. A 100-,u amount of 3 mM 1,2-phenylene-A diaminedihydrochloride (Fluka, Buchs, Switzerland) in 40A mM Tris-hydrochloride buffer (pH 7.6) was used as theA substrate. The optical density at 450 nm was measured in a

A Titertek Multiscan (Flow Laboratories) spectrophotometer.An optical density of 0.2 above background was consideredto be a positive result. As a negative control, wells were

C coated with RPMI 1640 medium or ascitic fluid from miceC injected with cells of the myeloma SP2/0-Ag 14.D (ii) ELISA inhibition. Inhibition of the monoclonal anti-

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1.0

c

L_

E

Cna.

0.6

0.2

0.5 50 5000 0.5 50 5000

Inhibitor (pg LPS/ml)FIG. 1. ELISA inhibition of monoclonal antibodies 112-3-2G (A) and 110-1-5B (B) with LPS from V. cholerae 34 (A), 35 (0), and NCTC

4711 (0). The microtiter trays were coated with LPS from strain 34. The antibodies were prepared from cell culture supernatants.

bodies with LPS preparations or whole bacteria was per-formed in glass tubes as described earlier (13). Briefly,monoclonal antibodies were incubated with the inhibitors,serially diluted in PBS, for 30 min at 22°C. The remainingantibodies, not neutralized by the inhibitors, were measuredin ELISA as described above. The percent inhibition wascalculated from the absorbance values obtained in the directELISA with antibodies preincubated with bacteria or PBS.Inhibition of the antibodies by more than 20% was consid-ered a positive result.

(iii) Sandwich ELISA. The sandwich ELISA (29) was

performed in 96-well microtiter trays (Titertek PVC EIA173; Flow Laboratories). Each well was coated overnight at37°C with ascites antibodies from the hybridoma 110-1-SBwhich was diluted 1/1,000 in 50 mM carbonate buffer (pH9.6). The remaining binding sites were blocked by theaddition of 100 p.l of 5% bovine serum albumin in 10 mMPBS-0.05% Tween 20. After a 15-min incubation at 22°C, thewells were rinsed three times with 100 p.l of PBS-Tween 20.Samples (100 pl1 each) of bacteria, suspended in PBS, wereadded in duplicate to each well, and the trays were incubatedfor 1 h at 37°C. After three rinses with PBS-Tween 20, 100 pL.of peroxidase-conjugated monoclonal antibodies (112-3-2G,H8, or C6) was added to each well. The trays were incubatedfor 45 min at 37°C before being rinsed with PBS-Tween 20.The enzyme reaction was performed as described above. Anoptical density value of 0.2 above background was consid-ered to be a positive result.

Enzyme-antibody conjugate. Monoclonal antibodies (asci-tes) were purified on a 5-ml protein A-Sepharose CL-4B(Pharmacia Fine Chemicals, Uppsala, Sweden) column (34)and coupled to peroxidase (Sigma Chemical Co.) by theperiodate method (1).

Agglutination. Slide agglutination was performed by sus-

pending a single colony of bacteria in 20 p.l of monoclonalantibodies (ascites), serially diluted in PBS, on a test slide.Visible agglutination within 1 min was considered to be a

positive result.

RESULTS

ELISA inhibition with LPS. Monoclonal antibodies 112-3-2G and 110-1-SB were tested in ELISA inhibition with LPSfrom V. cholerae 34 (Ogawa), 35 (Inaba), and NCTC 4711(02). Inhibition of monoclonal antibody 112-3-2G with LPSfrom strains 34 (Ogawa) and 35 (Inaba) showed almost

identical results (Fig. 1A). Total inhibition of monoclonalantibody 110-1-SB was also obtained with these antigens.The amount of antigen needed to obtain a 50% decrease ofthe absorbance in ELISA, however, was somewhat higherwith 110-1-SB antibodies as compared with 112-3-2G anti-bodies (Fig. 1B). No inhibition of any of the two antibodieswas obtained with LPS from strain NCTC 4711 (Fig. 1). Thisindicates that the antibodies are directed against antigen A,common to both Ogawa and Inaba.

Characterization of monoclonal antibodies H8 and C6 hasbeen described elsewhere (12).

Characteristics of the monoclonal antibodies used in thisstudy are summarized in Table 2.Sandwich ELISA. All V. cholerae 01 strains tested were

positive when tested with the monoclonal antibody 112-3-2G-peroxidase conjugate (Fig. 2A). All Ogawa strains testedwere positive when tested with monoclonal antibody H8,whereas the Inaba strains were negative (Fig. 2B). Whenmonoclonal antibody C6 was used as the second antibody,all Inaba strains were positive, whereas all Ogawa strainswere negative (Fig. 2C). Results obtained with the Hikojimastrains are described below. All other strains listed in Table 1were negative when tested in the sandwich ELISA.The amount of bacteria usually obtained after overnight

cultivation was 2 x 108 to 1 x 101( CFU/ml. Most 01 vibriostested showed positive reactions in the sandwich ELISA inconcentrations of 3 x 106 to 4 x 107 CFU/ml.ELISA inhibition with whole bacteria. All V. cholerae 01

strains tested showed significant inhibition when tested withmonoclonal antibody 110-1-SB (Fig. 3A). All but two of theOgawa strains and all but one of the Inaba strains were

TABLE 2. Monoclonal antibody characteristics and titers inELISA

Monoclonal Class or Light Titer'antibody subclass chain Specificity In vitro In vivo

110-1-SB 1gM K A 64 32,000112-3-2G IgG3 K A 64 16.000H8 IgG3 X B 512 16,000C6 IgG3 K C 128 8 000

' Endpoint titers of cell culture or ascitic fluid in ELISA. Endpoint titerswere defined as the highest dilution in a twofold serial dilution still giving anoptical density of >0.2 at 450 nm.

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1.0 [

I 0.2

A80

- a~ 8A °00

B0

CP0

0

0

- - - - - -

C00

80o0

Ogawa Inaba Ogawa Inaba Ogawa InabaFIG. 2. Sandwich ELISA absorbance values of V. cholerae 01 cultures. The assays were performed in microtiter wells coated with

antibody 110-1-SB by using the peroxidase-conjugated antibodies 112-3-2G (A), H8 (B), and C6 (C) as second antibodies.

positive when tested with monoclonal antibody 112-3-2G(Fig. 3B). Significant inhibition of antibody H8 was obtainedwith the Ogawa strains, whereas inhibition obtained with theInaba strains was less than 6% (Fig. 3C). All Inaba strainstested showed positive inhibition of monoclonal antibodyC6, whereas all Ogawa strains tested were negative in theELISA inhibition test (Fig. 3D). Results obtained with theHikojima strains are described below. None of the otherstrains listed in Table 1 inhibited any of the monoclonalantibodies by more than 6%.To obtain a positive reaction in the ELISA inhibition test

with antibodies 110-1-SB, H8, and C6, a minimum concen-tration of 5 x 106 to 5 x 107 CFU/ml was usually required,whereas concentrations of 2 108 CFU/ml were required withantibody 112-3-2G.

Slide agglutination. Monoclonal antibodies 110-1-5B and112-3-2G, both directed against antigen A, reacted somewhatdifferently when mixed with 01 vibrios. 110-1-5B antibodies

showed positive agglutinations in dilutions ranging from 1/40to 1/160 with the Ogawa strains, whereas the Inaba strainsonly agglutinated with 110-1-SB antibodies in dilutions rang-ing from 1/20 to 1/80. Such differences in reactivity were notobserved when 112-3-2G antibodies were used; however, theconcentrations needed to obtain a positive reaction weregenerally higher (Table 3).H8 antibodies agglutinated only Ogawa strains, whereas

Inaba strains were unaffected. All Inaba strains agglutinatedwhen tested with C6 antibodies which were diluted 1/80 to 1/160. Two of the Ogawa strains agglutinated in the lowestdilution tested, whereas the remaining Ogawa strains werenegative. The Hikojima strains agglutinated with both H8and C6 antibodies diluted 1/10 to 1/160 (Table 3). Noagglutination was obtained with V. cholerae non-O1 strainsor with any of the strains of E. coli, Salmonella spp.,Shigella sonnei, and Citrobacter freundii listed in Table 1.Two different preparations of Brucella abortus, heat-killed

and suspended in phenol-saline (kindly provided by A.TABLE 3. Slide agglutination of different strains of V. cholerae

with monoclonal antibodies (ascites)Agglutination with monoclonal antibodya:

Strain110-1-5B 112-3-2G H8 C6

Ogawa34 160 20 40 <101451 80 10 40 <101314 80 20 80 101824 160 10 20 <10VRL 19 80 10 40 <10VRL 59 160 10 80 <10VRL 84 160 10 80 <10VRL 88 40 10 40 <10VRL3 80 10 20 10

Inaba35 40 10 <10 160569B 20 10 <10 160VRL 10 20 20 <10 160VRL 48 40 20 <10 160VRL 58 40 20 <10 160VRL 86 80 20 <10 160VRL 87 20 10 <10 160V63P 40 20 <10 80

Hikojima433 160 10 10 1601602 40 10 40 801603 40 10 80 16011689 80 10 20 80

a Reciprocal of twofold serial dilutions of antibodies still giving a visibleagglutination within 1 min.

100

60

20

S

0-

z1001

60 F

20

Ogc0wa Inaba

C

CP0

Ogawa Inaba

B

*0%0 1%0 C

80

8

Og0wa Inaba

D00

Go

00

0- ---- ---9

Ogawa InabaFIG. 3. Inhibition of the monoclonal antibodies 110-1-SB (A),

112-3-2G (B), H8 (C), and C6 (D) by V. cholerae 01 bacteria.Percent inhibition was calculated from the absorbance values ob-tained in ELISAs with antibodies preincubated with bacteria orPBS.

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TABLE 4. Sandwich ELISA and ELISA inhibition of monoclonalantibodies with Hikojima strains of V. cholerae

Optical density in sand-Straintwichal ofnt iantody- % ELISA inhibition of antibodyb:

112-3-2G H8 C6 110-1-5B 112-3-2G H8 C6

433 0.33 0.02 0.30 100 30 12 91602 0.61 0.21 0.64 100 38 11 891603 0.57 0.29 0.65 99 53 19 9211689 0.55 0.04 0.54 99 33 3 92

a Optical density was obtained at 450 nm.Percent inhibition wa$ calculated as described in the legend to Fig. 3.

Lindberg, National Bacteriological Laboratory, Stockholm,Sweden and M. J. Corbel, Central Veterinary Laboratory,Surrey, England, respectively), were found negative in slideagglutination, ELISA inhibition, and sandwich ELISA.Also, a crude cell wall extract (M. J. Corbel) containing LPSshowed no inhibitory effect in the ELISA inhibition test.However, a weak agglutination of both Ogawa and Inabastrains was obtained with a polyclonal antiserum against B.abortus.Typing of Hikojima strains. Four V. cholerae 01 strains,

originally characterized as Hikojima strains (T. Donovan,Ph.D. thesis, University of Surrey, Guildford, United King-dom, 1982), were tested in the various assays described inthis study. All four strains agglutinated when they weresuspended in ascitic fluid containing antibodies directedagainst group- and type-specific antigens of V. cholerae 01(Table 3). No agglutination was obtained with any of thestrains when they were suspended in ascitic fluid obtainedfrom a mouse previously injected with cells from the mousemyeloma SP2/0. All strains showed optical density values of>0.2 above background in the sandwich ELISA when theywere incubated with the peroxidase-conjugated second anti-bodies 112-3-2G and C6. However, only strains 1602 and1603 were positive, whereas strains 433 and 11689 werenegative when incubated with Ogawa-specific antibody H8(Table 4). Monoclonal antibody 110-1-5B was completelyinhibited by the Hikojima strains, whereas inhibition ofmonoclonal antibody 112-3-2G ranged from 30 to 53%. Poorinhibition of H8 antibodies was obtained with the strains 433,1602, and 1603, whereas the inhibition obtained with strain11689 did not exceed background values. The strains 1602,1603, and 11689 clearly inhibited antibody C6, whereas theinhibition obtained with strain 433 was close to backgroundvalues (Table 4).

DISCUSSIONTo date, serological identification of V. cholerae 01 has

mainly been performed by slide agglutination of bacteriasuspended in polyclonal antisera. Group-specific antiseraare fairly easy to produce, whereas preparation of Ogawa-and Inaba-specific antisera is more laborious, owing to thecross-absorption procedure needed to remove antibodiesagainst the group antigen (20). Each preparation varies inquality, so comparisons of results obtained with differentpreparations are difficult to interpret. The use of monoclonalantibodies circumvents these problems and offers an almostunlimited- supply of defined antibody preparations. Theenzyme immunoassay approach makes the screening proce-dure more convenient and also increases the sensitivity ofthe test as compared with conventional slide agglutination.One obstacle in the development of a sandwich ELISA is

to find a suitable antibody that fulfills the requirements of a

good coating antibody. In this case, such an antibody should(i) be specific for the group-antigen A and thus react with all01 vibrios, irrespective of serotype, (ii) show good adhesiveproperties to the plastic of the microtiter tray, and (iii)preferentially be of the IgM class to enhance immobilizationof the bacteria. Monoclonal antibody 110-1-SB fulfilled theserequirements.The second antibodies used in the sandwich ELISA were

conjugated to peroxidase. This procedure minimizes thenumber of incubations needed to complete the assay butincreases costs, owing to the need for different enzyme-conjugated antibodies. The introduction of an anti-mouseIgG conjugate as a third antibody, together with unconju-gated second antibodies to antigens A, B, and C, wouldincrease the number of incubations but cut costs, as only oneconjugate would be needed. This enzyme-antibody conju-gate could also be used not only in assays of cholera antigensbut also in other tests.ELISA inhibition of monoclonal antibodies with whole

bacteria was almost equally sensitive as the sandwichELISA when used for identification of V. cholerae 01. Bothassays measured not only the presence of whole V. cholerae01 bacteria but also free LPS released from the bacteriaduring cultivation, and this characteristic probably contrib-utes to the sensitivity of the assays.

Conflicting results were obtained with the Hikojimastrains. By definition, Hikojima strains contain both antigensB and C in addition to the group antigen A (20, 24). Althoughall four strains agglutinated with antibodies against all threeantigens, results from the sandwich ELISA indicated thatonly V. cholerae 1602 and 1603 were true Hikojima strains,whereas strains 433 and 11689 were of the Inaba type.However, the very weak inhibition of both H8 and C6antibodies obtained in ELISA inhibition with strain 433indicates that this strain is better classified as semirough.

Earlier reports have indicated cross-reactivity between V.cholerae and B. abortus (7, 8, 10, 33). It has been proposedthat B. abortus should share a heat-stable antigenic compo-nent with both serotypes of V. cholerae and that the cross-reacting antigenic component should be more dominant instrains of serotype Inaba (10). In a study presented by Caroffet al. (4), the structure of the 0-specific side chain ofYersinia enterocolitica 09 LPS was shown to be a homopol-ymer of 4,6-dideoxy-4-formamido-D-mannose. In this re-port, it is also stated that the 0 chain of LPS from B. abortusis identical to the 0 chain of Y. enterocolitica 09 LPS. V.cholerae 01 LPS, on the other hand, contains a homopoly-saccharide of 4-amino-4,6-dideoxy-D-mannopyranosyl resi-dues with the amino groups acylated by 3-deoxy-glycero-tetronic acid (17, 23). It is possible that these sugars areresponsible for the cross-reactions observed with polyclonalantisera. However, the fact that the monoclonal antibodiesused in this study did not react with B. abortus does notexclude a possible similarity between the antigens, as thisfinding merely could be an expression of the extreme speci-ficity of monoclonal antibodies.

Preliminary experiments have shown that the monoclonalantibodies are able to inhibit motility of the bacteria. Al-though motility is promoted by the flagella, inhibition ofmotility probably is caused by steric hindrance or is due justto an overload of antibodies on the bacterial surface.The immunoassays presented in this study offer an oppor-

tunity to measure the relative antigen content of each antigenwithin a given strain. This will not only improve serotypingof V. cholerae 01, but will also be valuable for the develop-ment of improved vaccines against cholera.

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ELISA FOR IDENTIFICATION OF V. CHOLERAE 01 1185

ACKNOWLEDGMENTS

I thank U. Bjork for skillful technical assistance.This study was supported by the Karolinska Institute, the Swed-

ish Medical Research Council (project no. 656), and the Emil andVera Cornell Foundation.

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