Vol. 39, No. 2INFECTION AND IMMUNITY, Feb. 1983, p. 623-6290019-9567/83/020623-07$02.00/0Copyright © 1983, American Society for Microbiology

Serological, Chemical, and Structural Analyses of theEscherichia coli Cross-Reactive Capsular Polysaccharides

K13, K20, and K23WILLIE F. VANN,'* TOMMY SODERSTROM,2 W. EGAN,1 F.-P. TSUI,1 RACHEL SCHNEERSON,1

IDA 0RSKOV,3 AND FRITS 0RSKOV3

Office ofBiologics, Food and Drug Administration, Bethesda, Maryland, 202051; Department of ClinicalImmunology, Institute of Medical Microbiology, University of Goteborg, Goteborg, Sweden2; andInternational Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, Denmark3

Received 10 August 1982/Accepted 22 October 1982

The Escherichia coli K13, K20, and K23 capsular polysaccharide antigens are

serologically related. All of these polysaccharides contain ribose and 2-keto-3-deoxyoctonate in equimolar quantities. The K13 and K20 polysaccharides are

partially 0-acetylated. A comparison of these polysaccharides after 0-deacetyla-tion, by nuclear magnetic resonance and permethylation analysis, showed thatthese polysaccharides contained the disaccharide repeat unit -+)-p-ribofuranosyl-(1-*7)-p-2-keto-3-deoxyoctonate. They differed in the presence and location of anacetyl moiety. The K13 polysaccharide was 0-acetylated at C4 of the 2-keto-3-deoxyoctonate. The K20 antigen was 0-acetylated at C-5 of the ribose moiety.The K23 polymer was nonacetylated. The cross-reactivity of these antigens was

demonstrated by tandem-crossed immunoelectrophoresis. Antibodies to K23could be completely absorbed from OK K23 serum by K13, K20, and K23antigenic extracts. The K13 and K20 antibodies could be completely absorbedfrom their respective antisera only by homologous antigenic extracts. Monoclonalantibodies were prepared against a protein conjugate of the K13 polysaccharide.Analyses of the reactions of these antibodies with the three polysaccharidessuggest that the K13 polysaccharide has at least three antigenic sites, one of whichis common to the K13, K20, and K23 polysaccharides.

Escherichia coli is the most common pathogenof the upper urinary tract, especially in children.Although there are approximately 100 K anti-gens of E. coli, only 5, namely, Kl, K2, K3,K12, and K13, are found in about 80%o of thedisease isolates (9, 20). This does not includeK5, which was recently found to be a common Kantigen (20; F. 0rskov and I. 0rskov, unpub-lished data). E. coli is often found in humanurinary tract infections (3, 21) and is especiallyprevalent in cystitis (9).The K13 antigen is an acidic polysaccharide

composed of a repeat unit of ribose and 0-acetyl-2-keto-3-deoxyoctonate (KDO) (23).Antisera raised in rabbits by injection of K13organisms react with the E. coli K20 and K23antigens (17). The availability of monoclonalantibodies (MCLA) which react with the K13,K20, and K23 polysaccharides provides an im-portant tool for the study of the serology of Kantigens and their cross-reactions. The chemicaland serological relationships among the K13,K20, and K23 capsular polysaccharides of E.coli are described in this report. A new classifi-cation of the corresponding antigens based upon

their structural characteristics is proposed, andthe relation between their structure and pathoge-nicity is discussed.

MATERIALS AND METHODSBacterial strains and K polysaccharides. The follow-

ing strains from the Staten Seruminstitut collection inCopenhagen were used: F2677 (= 06:K13), SU4344/41 (= O6:K13:H1) (World Health Organization teststrain for K13), H54 (= 025:K23:H1) (World HealthOrganization test strain for K23), E19a (=021:K20:H-) (World Health Organization test strainfor K20).The K polysaccharides from these three strains

were purified by the method of Gotschlich et al. (8,23). Briefly, bacteria together with acidic polysaccha-rides were precipitated from the culture medium bythe addition of cetyltrimethylammonium bromide (Ce-tavlon). The sedimented material was extracted withcalcium chloride. Purified polysaccharide was ob-tained by a sequence of steps involving alcohol pre-cipitation and extraction with cold buffered phenol.Polysaccharide was found to be homogeneous byDEAE-cellulose chromatography and immunoelectro-phoresis. Polysaccharides were 0-deacetylated with0.1 N sodium hydroxide at 37°C as reported previously(23).

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cm) in 0.5 ml of phosphate-buffered saline (pH 7.38)per plate. The suspension was heated at 60°C for 20min, sheared twice for 1 min in an Ultra Turraxdispersion apparatus at room temperature, and centri-fuged at 27,000 x g for 15 min. The resulting superna-tant was used undiluted as the "60°C extract". A"100°C extract" which gave identical results to the60°C extract in crossed immunoelectrophoresis wasprepared by heating the 60°C extract to 100°C for 1 h.

Analytical methods. Ribose was determined by gaschromatography as its alditol acetate (18). KDO wasdetermined by the thiobarbituric acid assay (24). Poly-saccharides were permethylated and analyzed by gas-liquid chromatography-mass spectrometry by pub-lished methods (13). Permethylated KDO wasanalyzed as an acetylated methyl glycoside (W. F.

100 90 80 70 60 Vann and L. R. Phillips, unpublished data). Gas chro-matography was performed on a Varian model 3700with a 3% ECNSS-M column at 180°C. Mass spectrawere recorded with an LKB 2091 gas chromatograph-mass spectrometer equipped with an LKB 2130 datasystem. Samples were introduced via a glass capillarycolumn wall (0.5 mm by 25 m) coated with SE-30.Proton and 13C spectra were recorded at pH -7.0 in aBruker WM-300 spectrometer, using quadrature phasedetection at spectrometer frequences of 300 MHz and75 MHz, respectively. Before Fourier transformation,

laX the free induction decay signal was zero-filled withu) 8,000 data points and exponentially multiplied so as to

result in additional line broadening in the frequency-domain spectrum (2 Hz for 13C and 0.5 Hz for 1H-_ l\i1Ow_nuclear magnetic resonance [NMR]). Sodium 2,2,3,3-tetradeutrio-4,4-dimethyl-4-silapentanoate (TSP) was

I I I added to polysaccharide solutions in D20 as an inter-150 100 50 0 nal chemical shift standard. Spectra of alkali-treated

d(p.p.m) polysaccharides were recorded by adjusting the pH ofthe polysaccharide solution in the NMR tube to 10

FIG. 1. "C-NMR spectrum of K23 polysaccha- with ammonium hydroxide and incubating at roomride. Chemical shifts are reported relative to the temperature until 0-deacetylation was complete.internal standard TSP.

Antisera. Antisera to E. coli 06:K13:H1,025:K23:H1, and 021:K20:H- (designated OK) wereprepared by the injection of rabbits with formalinizedbacteria as reported previously (14).MCLA from hybridoma cultures. Hybridomas were

prepared by fusion of the Sp20 cell line with spleencells from BALB/C NIH mice immunized with E. coliK13 polysaccharide conjugated to bovine serum albu-min via adipic acid dihydrazide and cyanogen bromideactivation of the polysaccharide, as reported previous-ly (10, 19). Antibody-producing cells were detected byenzyme-linked immuosorbent assay (6). The hybrido-ma antibodies were characterized by isoelectric focus-ing and analyzed for isotype by solid-phase radio-immunoassay, using 3H-labeled subclass-specificantisera (22; T. Soderstrom and K. E. Stein, unpub-lished data). The reactivity of these homogeneousantibodies with the K polysaccharides was studied byimmunodiffusion.

Tandem-crossed immunoelectrophoresis. Tandem-crossed immunoelectrophoresis was performed by theLaurell technique as modified by Weeke (25, 26) andAxelsen (1, 2).

Bacterial extracts for tandem-crossed immunoelectro-phoresis. Bacteria were collected from agar plates (7.5

RESULTSChemical analysis. The capsular polysaccha-

rides were isolated from strains F2677 (06:K13),Su4344/41 (06:K13:H1), E19a (021:K20:H-),and H54 (025:K23:H1). All polysaccharidepreparations, analyzed by gas chromatographyand the thiobarbituric assay, contained riboseand KDO in equimolar quantities (24). 0-Acetylgroups were assayed by NMR and detected inthe K13 and K20, but not in the K23, polysac-charides. Both the K13 and K20 polysaccharideswere 50 to 80% acetylated.The K13, K20, and K23 polysaccharides are

serologically related (17). The structural basisfor the serological cross-reactivity of these poly-mers was studied by "C-NMR. The 13C-NMRspectra recorded for K23 and 0-deacetylatedK13 and K20 polysaccharides were identical. Arepresentative spectrum (Fig. 1) displays 13 res-onances of approximately equal intensity and isin accord with the chemical composition pre-sented above. Most of the resonances in Table 1were assigned on the basis of comparison withmodel compounds. Previous results with chemi-

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E. COLI K13, K20, AND K23 CROSS-REACTIVITY 625

TABLE 1. 13C-NMR spectral data for the E. coli K13 and K20 polysaccharides before and after ammoniumhydroxide treatment and for the K23 polysaccharidesa

Carbon K13 K13 + NH4OH K23 K20 + NH4OH K20C1 175.77 176.33 176.26 176.14C2 104.1 104.52 104.44 104.45 104.42C3 34.6 37.27 37.24 37.27 37.1C4 73.53 70.13 70.16 70.13 70.2C5 63.6 67.93 67.97 68.0 68.03C6 74.85 74.92 74.93 74.89 75.11C7 77.72 77.8 77.8 77.8 77.57C8 62.33 61.75 62.07 62.17 63.2C-Acetylmethyl 23.4 23.16

C1' 106.91 106.13 106.43 106.55 108.08C2' 75.92 75.15 75.53 75.66 76.7C3' 76.9 76.89 76.83 76.76 77.2C4' 84.06 83.94 83.9 83.88 81.36C5, 65.5 65.31 65.34 65.34 68.16

a Chemical shifts are shown in parts per million relative to internal TSP at pH -7.0.

cal degradation and methylation analysis showthat the K13 polysaccharide contains a 3-glyco-sylated ribofuranose and a 7-glycosylated KDO(23). Since the 0-deacetylated polysaccharidesand K23 polymer have identical NMR spectra,they probably have identical substitution pat-terns. This was confirmed by methylation analy-sis. The K20 and K23 polymers were permethyl-ated (13) and converted into either partiallymethylated alditol acetates or methyl glyco-

100r

80F

C,)zw

zw

ui

60

40

20

I11L1

sides. These derivatives were analyzed by gas-liquid chromatography-mass spectrometry. Thespectrum obtained for partially methylated ribi-tol was similar to that reported for 1,3,4-0-acetyl-2,5-O-methylribitol. The ribosyl residuein these two polymers is substituted at C-3 asreported for the K13 polysaccharide. The spec-trum of the methyl ketoside methyl ester ofpartially acetylated permethylated KDO is pre-sented in Fig. 2. The primary fragment

aELL. 0,0

100. . I .

200m/e

FIG. 2. Mass spectrum of the partially acetylated permethylated KDO methyl ketoside obtained from K13,K23, and K20 polysaccharides.

11-- .

W-g"m w-1-9 -1 ,9 I I I I I I I I I I I I I t 1 1 . . I . .

--- 1. I.M AM.

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CH2OCH3 at m/z 45 is prominent and character-istic of an 0-methyl substitution at position 8.The primary fragments M-CH2OCH3 at m/z 305,M-CH3OCH2CH2OCH3 at m/z 261, andCH3OCH2CH2OCH3 at m/z 89 were not ob-served. These ions are prominent in spectra ofsimilar KDO derivatives having 0-methylgroups present at both position 7 and 8 (5). Thespectrum is consistent with KDO being 0-ace-tylated at position 7 and glycosylated in thepolysaccharide at this position. Thus, the K13,K20, and K23 polysaccharides are polymers of--3)-ribofuranosyl-(1--7)-2-keto-3-deoxyocton-ate. They differ in the presence and location ofan acetyl moiety. The K23 polysaccharide doesnot contain acetyl groups.The 13C- and 1H-NMR spectra were used to

determine the specific location of the acetylgroup on K13 and K20. After 0-deacetylation ofthe K13 polysaccharide, three resonances wereobserved to shift significantly (Table 1). Thesignal corresponding to the C-3 of KDO shifteddownfield 3 ppm, indicating that C-4 of thisresidue was 0-acetylated. The H-3 and H-3'proton resonances also shifted after 0-deacetyl-ation (data not shown). The signal at 63.6 ppmcorresponded to C-5 of the KDO since it shifteddownfield upon 0-deacetylation. The resonanceat 73.53 ppm corresponded to C-4 of the KDOsince it shifted upfield after 0-deacetylation.The site of 0-acetylation of the K20 polysac-

charide was established in a similar fashion. 0-

deacetylation resulted in a 3-ppm downfield shiftof the C-5 ribose resonance (65.34 in the deace-tylated polymer) and a 2.5-ppm upfield shift ofthe C-4 resonance (7). Thus, the K20 polysac-charide is 0-acetylated at C-5 of the ribose.The anomeric configurations of the ribosyl

and KDO residues were established by a combi-nation of 13C- and 'H-NMR spectroscopy. It hasbeen shown (8a) that the equatorial H-3 protonsignal of KDO is sensitive to anomeric stereo-chemistry. This signal occurs at ca. 2.1 ppm inthe a-anomer and 2.4 ppm in the ,-anomer. Theequatorial H-3 signal of the KDO residue in theK23 polysaccharide occurred at 2.47 ppm, indi-cating ,-stereochemistry. This assignment wassupported by 13C-NMR spectroscopy. The C-1resonance (at neutral pH) is sensitive to ano-meric configuration (4) (176.4 ppm in the ,3-anomeric form and 178.1 ppm in the a-anomericform). The C-1 resonance for the K23 polysac-charide occurred at -176.3 ppm. On the basis ofthese studies, the KDO is present as the -

anomer.A comparison of the 13C chemical shifts from

the ribofuranosyl residue of the K23 polysaccha-ride (see Table 1) with those from model com-pounds (2- and 3-0-alkyl, a- and -i-O-methylribofuranosides) did not provide much informa-

tion about the anomeric stereochemistry of theribofuranosyl ring. In the model compounds (3-0-methyl, 1-0-methyl ribofuranosides), the C-3shifts occurred at 81.7 ppm for the a- and 82.9ppm for the P-configuration. There was no cor-responding signal in this region in the K23polysaccharide as the 83.9 ppm signal was due toC-4.

Proton NMR was more informative. The sig-nal at 5.235 ppm due to the ribofuranosyl H-1was narrow, having no resolved couplings (cou-plings on the order of 2 Hz were resolved forother signals). It has been shown that 3J (H(1)-H(2)) is ca. 4 Hz for the a-anomer and 1.5 Hz forthe ,1-anomer (7). The lack of a resolvedJ(H(a)-H(2)) for the K23 polysaccharide indi-cated the 3-configuration. This was confirmedby the value of the H(1) chemical shift of theK23 polysaccharide. The ribofuranosyl residueis present as the f-anomer in these polysaccha-rides.

Serology. Cell extracts (60°C and 100°C) of thecorresponding test strains, Su4344/41(06:K13:H1), H54(025:K23:Hl), andE19a(021 :K20:H-), were examined againsttheir respective OK antisera in tandem-crossedimmunoelectrophoresis (11) to determine theserological relationships among these K anti-gens. These extracts or OK antisera were ap-plied in the intermediate gel for absorption insitu. The K13, K20, and K23 extracts in theintermediate gel completely absorbed K23 anti-bodies from OK K23 antiserum (Fig. 3B). Onlythe K13 antigen was able to completely absorbthe OK K13 antiserum (Fig. 3A). Only the K20extract was able to completely absorb the K20antibodies (Fig. 3C). The K20 and K23 antigensshowed reactions of identity against OK K13antiserum. The K13 antigen showed a reactionof partial identity with the K20 and K23 polysac-charide against OK K13 antiserum. Experimentsin which the antisera were added to the interme-diate gel (data not shown) confirmed the above-described relationships, thus justifying the fol-lowing new serotype formula for these antigens:K13 = Ki3ab; K20 = Ki3(a)c; and K23 = K13a.In a Danish collection of 457 strains of E. coliisolated from patients with septicemia, orga-nisms with the K13ab antigen were found withthe same frequency as K5 strains and less fre-quently than Ki strains. K13a (K23) was foundin only a few strains and K13(a)c not at all.K13ab occurred in different serotype combina-tions, 06:K13ab:H1 and 022:K13ab:H1 beingthe most common. In an American collection ofsepticemia isolates, K13ab strains were rare andK13a (K23) and K13(a)c (K20) were not detect-ed (I. 0rskov and F. 0rskov, personal commu-nication). Eight strains possessing the serotype06:H1 and the K13 complex were isolated from

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E. COLI K13, K20, AND K23 CROSS-REACTIVITY 627

A BOKK13 OKK23

0 00 00 0K13 K23 K20 K13 K23 K20

OKK13 OKK23

K13Ag K13Ag \ T

0 00 00 0K13 K23 K20 K13 K23 K20

OK K13 OK K23

K23 Ag K23Ag -\

0 0 0 0 00K13 K23 K20 K13 K23 K20

COK K20

I,,.-.AO O OK13 K23-K20

OKK20

K13Ag

K13 K23 K20

OK K20

K23 A

K13 K23 K20

FIG. 3. Tandem-crossed immunoelectrophoresis of K13 (A), K20 (B), and K23 (C) extracts and antisera. OKantisera are in the upper gels. Antigenic extracts (60°C) are in the intermediate gels and the wells of the lower gelsas indicated. The anode is to the left of the first dimension and to the top of the second dimension.

OK K20

-//////////ywK 20 Ag

0 0 0K13 K23 K20

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628 VANN ET AL.

urinary tract infections in Goteborg. All of thesestrains possessed the K13ab antigen.

Specificity of MCLA. The serological relation-ships of these E. coli K polysaccharides wereanalyzed in greater detail with MCLA. Of the 22MCLA prepared against the polysaccharide pro-tein conjugate, 17 precipitated with the partiallyacetylated K13 polysaccharide. DifferentMCLA showed partial and nonidentity reactionswhen reacted against the K13 polysaccharide.MCLA 150B2 gave a nonidentity reaction withthe partially related MCLA 156E8 and 147D11(Fig. 4). The K23 and K20 polysaccharides wererecognized by only two MCLA, 150B2 and148E4. Thus, although the K13 polysaccharideprecipitated with all of the MCLA, the antibod-ies are recognizing different antigenic sites onthe molecule. (The K13 polysaccharide is proba-bly a microheterogeneous population of chainspartially 0-acetylated at C-4 of the KDO moi-ety.) The antigenic sites recognized by MCLA150B2 were different from that recognized by156E8. The 156E8 and 147D11 sera showed linesof partial identity. These data show that the K13polysaccharide contains at least three antigenicsites, one of which is common to the K20 andK23 polysaccharides. The specificity of theseMCLA was investigated with derivatives of theK13 polysaccharide. O-Deacetylated K13 poly-saccharide gave a line of identity with K23polysaccharide when tested against 150B2 anti-body. Of 12 MCLA tested, 5 showed direct slideagglutination of fresh E. coli 06:K13 cultures.The bacterial agglutinating activity of 148E4 wasinhibited by ribofuranosyl (1->7) KDO whenMCLA were incubated with the dissaccharidebefore agglutination. This disaccharide containsonly one of the linkages in the repeating unit ofK13. None of the antisera tested showed cross-reaction with the meningococcal 29e capsularpolysaccharide, which also contains a 7-substi-tuted 4(5)-O-acetylated KDO (4). Unlike theK13 polysaccharide, the 29e polymer contains 3-substituted N-acetylgalactosamine. These data

0'c0

0(

are consistent with -,B-ribofuranosyl-(1--+7)-KDO being an important part of the 148E4MCLA specificity.

DISCUSSIONUsing the techniques of tandem-crossed immu-

noelectrophoresis and MCLA, we demonstratedthat there is a common component in the Kl 3,K20, and K23 polysaccharides. Characterizationby methylation analysis, gas chromatography, andNMR showed that the three polysaccharides con-tain the disaccharide -*3)-P-ribofuranosyl-(1-*7)-,B-2-keto-3-deoxyoctonate-(2--+ in its repeatingunit. The K13 polysaccharide is 0-acetylated onC-4 of the KDO. The majority of the MCLA werefound to react only with this 0-acetylated polysac-charide and not with the nonacetylated structure,represented by K23. This finding is consistentwith an earlier suggestion that 0-acetylated KDOis immunodominant (23). The MCLA 150B2formed precipitates with all of the polysaccha-rides. Thus, these antibodies are probably specificfor the ribosyl-KDO dissaccharide. The specific-ity of these antibodies must include the ribose,since no precipitates were formed with the menin-gococcal 29e capsular polysaccharide. This poly-saccharide, like the K13 antigen, contains a 7-substituted KDO residue which is 0-acetylated ateither position 4 or 5. However, unlike the K13complex, the KDO of the 29e polysaccharide issubstituted by N-acetylgalactosamine.The K13 and K23 polysaccharides did not

react strongly with the OK K20 antiserum, al-though the K20 extract could completely absorbthe K23 antiserum and was precipitated by theMCLA prepared against K13. The 0-acetylatedribose is probably serologically important whenanimals are immunized with the K20 antigen,and the 0-acetylated KDO is immunodominantwhen animals are immunized with the K13 anti-gen. On the basis of the results given above, wecan describe the relationship between the threepolysaccharides as:

(A

©W)

FIG. 4. Double diffusion of select MCLA against K13 (A), K20 (B), and K23 (C) polysaccharides. The MCLAdesignated 1 (156B8), 2 (15OE2), 3 (147D11), and 4 (148E4) were added in the outer wells as indicated.

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E. COLI K13, K20, AND K23 CROSS-REACTIVITY 629

K13 = K13ab =

3)-p-Ribofuranosyl-(1-+7)-P-KDO-(2--*4tAcetyl

K20 = K13(a)c =3)-p-Ribofuranosyl-(l13,7)-fi-KDO-(2--

5

Acetyl

K23 = K13a =

3)-p-Ribofuranosyl-(1-*7)-,B-KDO-(2--.We suggest that the nomenclature K13ab,K13(a)c, and K13a be used for E. coli strainscarrying these capsular antigens instead of usingK13, K20, and K23. It is not known whetherform variation exists in the K13 system. In theK2 system, the 0-acetylated antigen was morecommon among isolates than the nonacetylatedantigen. Although it is not known how 0-acetyl-ation relates to virulence, E. coli carrying the 0-acetylated form of the Kl antigen appeared to bemore virulent.Among urinary tract infection isolates, K13 is

common, although the K20 and K23 antigens arerarely detected. K13 often occurs with the 06antigen. Among eight E. coli isolates obtainedfrom Goteborg which had the serotype 06:H1,all possessed the K13ab antigen. Additional in-vestigation may show that the position of the 0-acetyl group and the occurrence of the 06antigen indicate that the K13 strains isolatedfrom disease are derived from a clone, as sug-gested previously (15).

ACKNOWLEDGMENTS

We are grateful to Lawrence R. Phillips for assistance withthe mass spectrometry.

T.S. received a Fogarty International Fellowship sponsoredby the Food and Drug Administration.

LITERATURE CITED1. Axelsen, N. H. 1973. Intermediate gel in crossed and fused

rocket immunoelectrophoresis. Scand. J. Immunol.2(Suppl.1):71-77.

2. Axeben, N. H., E. Bock, and J. Kroll. 1973. Comparisonof antigens: the reaction of identity. Scand. J. Immunol.2(Suppl. 1):91-94.

3.Bettiem, K. A., and J. Taylor. 1977. A study of Esche-richia coli isolated from chronic urinary infections. J.Med. Microbiol. 2:225-236.

4. Bhattacharjee, A. K., H. J. Jennings, and C. P. Kenny.1978. Structural elucidation of the 3-deoxy-D-manno-octulosonic acid containing meningococcal 29-e capsularpolysaccharide antigen using carbon-13 nuclear magneticresonance. Biochemistry 17:645-651.

5. Charon, D., and L. Szabo. 1980. Pyranoside derivatives of3-deoxyald-3-ulosonic acid. J. Chem. Soc. Perkins Trans.1:1971-1977.

6. Engval, E., and P. Perhmann. 1972. Enzyme linked im-munosorbent assay, ELISAIII. Quantitation of specificantibodies by enzyme labeled anti-immunoglobulin in

antigen coated tubes. J. Immunol. 109:129-135.7. Gorin, P. A. J., and M. Mazurek. 1976. Carbon-13 and

proton nuclear magnetic resonance studies on methylaldofuranosides and their 0-alkyl derivatives. Carbohydr.Res. 48:171-186.

8. GotshUch, E. C., M. Rey, C. Etlenne, W. R. Sanborn, R.Tradu, and E. Cvejtanovic. 1972. Immunological responseobserved in field studies in Africa with meningococcalvaccines. Prog. Immunobiol. Stand. 5:485-491.

8a.Jennings, H. J., K. G. Rosell, and K. G. Johnson. 1982.Structure of the 3-deoxy-D-manno-octulosonic acid-con-taining polysaccharide (K6 antigen) from Escherichia coliLP1092. Carbohydr. Res. 105:145-156.

9. Kaijser, B., L. A. Hanson, U. Jodal, G. Lindin-Janson,and J. B. Robbins. 1977. Frequency of E. coli K antigensin urinary tract infections in children. Lancet 1:663-664.

10. Kohler, G., and C. Milstein. 1975. Continuous cultures offused celis secreting antibody of predefined specificity.Nature (London) 256:495-497.

11. Kroll, J. 1973. Tandem-crossed immunoelectrophoresis.Scand. J. Immunol. 2:(Suppl. 1):57-59.

12. Larsen, J. C., F. 0rskov, I. 0rskov, M. A. Schmidt, E.Jann, and K. Jann. 1980. Crossed immunoelectrophoresisand chemical structural analysis used for characterizationof two varieties of Escherichia coli K2 polysaccharideantigen. Med. Microbiol. Immunol. 168:191-200.

13. Lindberg, B. 1972. Methylation analysis of polysaccha-rides. Methods Enzymol. 28:178-195.

14. 0rskov, F., and I.0rskov. 1978. Serotyping of Enterobac-teriaceae with special emphasis on K antigen determina-tion. Methods Microbiol. 11:1-77.

15. 0rskov, F., I.0rskov., D. J. Evams Jr., R. B. Sack, D. A.Sack, and T. Wadtromn. 1976. Special Escherichia coliserotypes among enterotoxigenic strains from diarrhea inadults and children. Med. Microbiol. Immunol. 162:73-80.

16. 0rskov, I., F. 0rskov, and A. Birch-Andersen. 1980.Comparison of Escherichia coli fimbrial antigen F7 withtype 1 fimbriae. Infect. Immun. 27:657466.

17. 0rskov, I., F. 0rskov, B. Jann, and K. Jann. 1977.Serology, chemistry, and genetics of 0 and K antigens ofEscherichia coli. Bacteriol. Rev. 41:667-710.

18. Sawardeker, J. S., J. B. Sloneker, and A. Jeanes. 1965.Quantitative determination of monosaccharides as theiralditol acetates by gas chromatography. Anal. Chem.37:1602-1604.

19. Schneerson, R., 0. Barrera, A. Sutton, and J. B. Robbins.1980. Preparation, characterization and immunogenicityof Haemophilus influenzae type b polysaccharide proteinconjugates. J. Exp. Med. 152:361-376.

20. Sjostedt, S. 1946. Pathogenicity of certain serologicaltypes of E. coli. Gleerupska Universitets Bokhandel,Lund, Sweden.

21. Spencer, A. G., D. Mulcahy, R. A. Shooter, F. W.O'Grady, K. A. Bettelheim, and J. Taylor. 1968. Esche-richia coli serotypes in urinary tract infections in amedical ward. Lancetii:839-842.

22. Stein, K. E., C. Bona, R. Lieberman, C. C. Chien, and W.Paul. 1980. Regulation of the anti-inulin antibody responseby a nonallotype-linked gene. J. Exp. Med. 151:1088-1102.

23.Vann,W. F., and K. Jann. 1979. Structure and serologicalspecificity of the K13-antigenic polysaccharide (K13 anti-gen) of urinary tract-infective Escherichia coli. Infect.Immun. 25:85-92.

24. Waravrekar,V. S., and L. D. Saslaw. 1959. A sensitivecolorimeteric method for the estimation of 2-deoxy sugarswith the use of the malonaldehyde-thiobarbituric acidreaction. J. Biol. Chem. 234:1945-1950.

25. Weeke, E. 1973. Crossed immunoelectrophoresis. Scand.J. Immunol. 2(Suppl. 1):47-56.

26. Weeke, E. 1973. General remarks on principles, equip-ment reagents and procedures. Scand. J. Immunol.2(Suppl. 1):15-35.

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