Báo cáo Y học: Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd–/b+ of Haemophilus influenzae docx

Chemical structure and immunoreactivity of the lipopolysaccharide
of the deep rough mutant I-69 Rd
–
/b
+
of
Haemophilus influenzae
Sven Mu¨ ller-Loennies, Lore Brade and Helmut Brade
Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany
From the lipopolysaccharide of the deep rough mutant I-69
Rd
–
/b
+
of Haemophilus influenzae two oligosaccharides
were obtained after de-O-acylation and separation by
high-performance anion exchange chromatography.
Their chemical structures w ere determined by one- a nd
two-dimensional
1
H-,
13
C- and
31
P-NMR spectroscopy
as aKdo-4P -(2 fi 6)-bGlcN-4P-(1 fi 6)- aGlcN-1P and
aKdo-5P-(2 fi 6)- bGlcN-4P-(1 fi 6)-aGlcN-1P. The spe-
cificity of mAbs S42-21 and S42-16 specific for Kdo-4P or
Kdo-5P, r e spectively [Rozalski, A., Brade L., Kosma P.,
Moxon R., Kusumoto S., & B rade H. (1997). Mol. Micro-
biol. 23, 569–577] was confirmed with neoglycoconjugates

obtained by conjugation of the isolated oligosaccharides to
BSA. In addition, a mAb S42-10-8 with unknown epitope
specificity could be assigned using the neoglycoconjugates
described herein. This mAb binds to an epitope composed of
the b isphosphorylated glucosamine b ackbone of lipid A a nd
Kdo-4P, w hereby the latter determines t he specificity strictly
by the position of the phosphate group.
Keywords: carbohydrate antibody; Kdo-phosphate;
neoglycoconjugate; serology; sugar phosphate.
Haemophilus influenzae normally colonizes the human
nasopharynx but may cause severe infections, in particular
meningitis, in children. A m ajor virulence factor of this
human p athogen is the type b capsule, an acidic polysac-
charide composed of ribose, ribitol a nd phosphate and
which i s the basis of an e ffec tive conjugate vaccine [1].
Among other viru lence factors i s the lipopolysacchari de
(LPS) in which we are interested for various reasons: (a)
LPS is an essential component of the outer membrane in all
Gram-negative bacteria; (b) LPS is the endotoxin of Gram-
negative bacteria; (c) LPS is a major surface antigen leading
to the induction of protective a ntibodies; and (d) the
understanding of the biosynthesis of LPS may allow
the distinct blockage of e ssential steps as a new strategy
for the development of antibiotics [2,3].
The smallest LPS structure which still allows the bacter-
ium t o survive was f ound in the mutant strain I-69 Rd
–
/b
+
of H. influenzae (referredtohereasI-69)wherea

single phosphorylated 3-deoxy-
D
-manno-oct-2-ulopyrano-
sonic acid (Kdo) residue is linked to the lipid A moiety.
Helander et al. h ave shown t hat t he I-69 LPS was composed
of two molecular s pecies with Kdo phosphorylated at either
position 4 or 5 [4].
The Kdo transferase of I-69 has been cloned and
characterized and the phosphokinase adding the phospho-
ryl group to position 4 of the Kdo residue has also been
cloned [5,6]. Coexpression of both e nzymes in an Escheri-
chia coli strain lacking its own Kdo transferase led to the
synthesis o f an LPS which contained exclusively Kdo-4P [7].
For this study mAbs were useful to identify the secondary
gene products. We have reported earlier on mAb recogni-
zing either t h e 4- or 5-phosphorylated Kdo which was
chemically synthesized and c onjugated to BSA [8]. In
addition, we found mAb S 42-10-8 w hich was specific f or the
I-69 LPS but did not react with Kdo-4P or Kdo-5P alone.
Therefore, this antibody was assumed to recognize an
epitope requiring, i n addition to a phosphorylated Kdo
residue, the phosphorylated lipid A backbone. As the LPS
species containing the Kdo-4P or Kdo-5 P could not be
separated at t hat time and were not yet chemically
synthesized, the s pecificity of this m Ab has not yet been
elucidated. Here, we report on: (a) t he successful separation
of the deacylated carboh ydrate backbone of I-69 LPS into
two pure oligosaccharides containing either Kdo-4P or
Kdo-5P; (b) the structural analysis of both oligosaccharides
by NMR; and (c) t he characterization of a n ew mAb

recognizing a phosphorylated carbohydrate epitope.
MATERIALS AND METHODS
Bacteria and bacterial LPS
H. influenzae I-69 Rd
–
/b
+
was cultivated a s d escribed
previously [9]. Bacteria were washed with ethanol, acetone
(twice), and ether, and dried. LPS was extracted from dry
bacteria by the phenol/chloroform/petroleum ether method
[10] in a yield of 4.4% of dry bacteria. De-O-acylated LPS
was prepared after hydrazine treatment of LPS for 30 min
at 37 °C (yield: 81% based on the glucosamine content),
and deacylated LPS ( LPS
deac
) w as ob tained by hydrolysis of
de-O-acylated LPS in 4
M
KOHasreported[11].LPS
deac
was further purified by preparative high performance anion
exchange chromatography(HPAEC) u sing water as e luent A
Correspondence to H. Brade, Research Center Borstel, Center for
Medicine and Biosciences, Parkallee 22, D-2 3845 Bor stel, Ge rmany.
Fax: + 49 4537 188419, Tel.: + 49 4537 188474,
E-mail: hbrade@ fz-borstel.de
Abbreviations: HPAEC, high performance anion exchange chroma-
tography;Kdo,3-deoxy-
D

-manno-oct-2-ulopyranosonic acid; LPS,
lipopolysaccharide; LPS
deac
, deacylated LPS.
Note:S.Mu
¨
ller-Loennies and L. Brade contibuted equally to this
work.
(Received 8 August 2001, revised 2 1 December 2001, accepted
3 January 2002)
Eur. J. Biochem. 269, 1237–1242 (2002) Ó FEBS 2002
and 1
M
ammonium acetate as eluent B and a gradient of
1% to 99% over 80 min. Desalting was achieved by gel
filtration on a column of 100 · 1.5 cm Sephadex G10 in
pyridine/acetic acid/water (4 : 10 : 10 00, v/v/v) at a flow rate
of 1 mLÆmin
)1
. Fractions 1 and 2 were obtained in pure
form in yields of 21.6 and 9.5%, respectively, based on the
glucosamine content.
NMR spectroscopy
The deacylated LPS from H. influenzae I-69 was investi-
gated by one-dimensional
1
H-NMR- and
13
C-NMR and
spectroscopy at 600 and 150 MHz, r espectively, on a Bruker

DRX 600 A vance spectrometer;
31
P-NMR spectra were
recorded on a Bruker D PX 360 Avance spectrometer a t
145 MHz. All spectra were recorded on a 0.5-mL solution
of 5 mg s ample i n D
2
O. As reference served acetone
2.225 p.p.m. (
1
H), d ioxane 67.4 p.p.m. (
13
C) and 85%
phosphoric acid 0 p.p.m. (
31
P). All spectra were run at a
temperature of 300 K. For
31
P measurements the pD was
adjusted to pD 2. Other measurements were performed at
pD 6 due to the a cid labile nature of the Kdo-linkage.
Two-dimensional homonuclear
1
H,
1
H-DQF-COSY was
recorded over a sp ectral width of 7.5 p.p.m. in both
dimensions recording 512 experimen ts of 32 s cans. Four
thousand data points were recorded in F2. Zero-filling
was applied in F1 to 1000 data points. Heteronuclear

1
H,
13
C-NMR correlation spectroscopy was recorded as
HMQC. Two thousand data points were recorded in F2
over a spectral width of 10 p.p.m. and 256 experiments
consisting of 24 scans per increment. Phase cycling w as
performed using States-TPPI. Prior to Fourier transfor-
mation zero-filling was applied in F1 to 512 data points.
31
P-NMR spectroscopy was recorded with continuous
wave decoupling during acquisition. A total of 32 scans
was recorded. For
1
H,
31
P-NMR COSY a HMQC
experiment was recorded consisting of 256 experiments
and 32 scans each. Two thousand data points were
collected ove r a spectral w idth of 10 p.p.m. in F2 and
zero filling was applied in F1 to yield 512 data points. The
spectral width w as 10 p.p.m. in F1.
Neoglycoconjugates
The amino groups of the glucosamine residues in LPS
deac
and in the oligosaccharides obtained from LPS
deac
were
activated with glutardialdehyde and conjugated to BSA as
described [12]. The amount of ligand present in the

conjugates was d etermined by measuring the amount of
protein (Bradford assay, Bio-Rad) and glucosamine
(Table 1).
MAbs
Monoclonal antibodies S 42-16, S42-21 and S42-10-8 were
obtained after immunization and selection as described [8].
Culture s upernatants were p repared in at least 100 mL
quantities and antibodies were purified on protein
G-Sepharose (Pharmacia/LKB) according to the supplier’s
instructions. Purification was a scertained by SDS/PAGE
and protein concentrations were determined by the bicin-
choninic acid assay (Pierce).
Serology
For ELISA, neoglycoconjugates were coated onto Maxi-
Sorp microtiter plates (U-bottom, Nunc). Antigen solutions
were adjusted to equimolar concentrations based on the
amount of ligand present in the respective glycoconjugate.
Unless stated otherwise, 50 lL volumes were u sed. Micro-
titer plates w ere coated with the respective antigen solution
in 50 m
M
carbonate buffer pH 9 .2 at 4 °C overnight. Plates
were washed twice with distilled wate r; further washing was
carried out in NaCl/P
i
supplemented with 0.05% Tween 20
(Bio-Rad) and 0.01% thimerosal (NaCl/P
i
/Tween-T). Pla tes
were then blocked w ith NaCl/P

i
/Tween-T supplemented
with 2.5% casein (NaCl/P
i
/Tween-TC) f or 1 h a t 37 °Cona
rocking platform f ollowed by t wo washes. A ppropriate
antibody dilutions in NaCl/P
i
/Tween-TC supplemented
with 5% BSA were added and incubated for 1 h at 37 °C.
After washing, peroxidase-conjugated goat anti-(mouse
IgG) Ig (heavy and light chain specific; Dianova) was
added (diluted 1 : 1000) and incubation was continued for
1 h at 37 °C. After three washes in NaCl/P
i
/Tween-T, the
plates were wash ed in substrate buffer ( 0.1
M
sodium citrate,
pH 4.5). Substrate solution was freshly prepared and was
composed of azino-di-3-ethylbenzthiazolinsulfonic acid
(1 mg) dissolved in substrate buffer ( 1 mL) with sonication
in an ultrasound water bath for 3 min followed by the
addition of hydrogen peroxide (25 lLofa0.1%solution).
After 3 0 min at 3 7 °C, the reaction w as stopped b y t he
addition of 2% aqueous oxalic acid and the plates were read
with a microplate reader (Dynatech MR 700) at 405 nm.
For ELISA using LPS as a solid-phase antigen another
protocol was used. Polyvinyl microtiter plates (Falcon 3911)
were coated with various amounts of LPS dissolved in NaCl/

P
i
(10 m
M
pH 7.3, 0.9% NaCl, 50 lL) at 4 °C overnight or
at 37 °C for 1 h. All following steps were performed at 37 °C
with gentle agitation and all washing steps were performed
four times. Coated plates were washed in NaCl/P
i
, blocked
for 1 h with blocking b uffer (2.5% casein in NaCl/P
i
)and
then incubated f or 1 h with mAb diluted in blocking buffer
(50 lL). Plates were washed in NaCl/P
i
and incubated for
Table 1. Oligosaccharides and neoglycoconjugates used in this study. For derivatization procedures see Materials and methods. Molar ratio o f ligand
to protein given in parentheses.
Chemical structure Abbreviation
Amount of ligand
(nmolÆmg
)1
)
aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P Kdo4PGlcN
2
P
2
aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P Kdo5PGlcN
2

P
2
aKdo-4/5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA LPS
deac
-BSA 33 (2.4)
aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA Kdo4P-GlcN
2
P
2
-BSA 16 (1.1)
aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA Kdo5P-GlcN
2
P
2
-BSA 15 (1.0)
1238 S. Mu
¨
ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002
1 h with peroxidase-conjugated goat anti-(mouse IgG) Ig or
goat anti-(rabbit IgG) Ig (heavy and light chain specific,
Dianova; diluted 1 : 1000 in blocking buffer, 50 lL). Further
development of the reaction was as described a bove. All tests
were set up in quadruplicate. Confidence v alues of the means
were less than 10%.
RESULTS
Isolation and structural analysis of the phosphorylated
carbohydrate backbone of I-69 LPS
The LPS of H. influenzae I-69 was s uccessively de-O-acylated
and de-N-acylated with hydrazine and potassium hyd rox-
ide, respective ly, leading to two major products as revealed

by HPAEC (Fig. 1). The two peaks, compounds 1 and 2,
could be separated from each other b y preparative HPAEC
with yields of 11.6 mg (21.6% of LPS) and 5.1 mg (9.5%
of LPS) for Kdo-4P-GlcN
2
-P
2
and K do-5P-GlcN
2
-P
2
,
respectively.
Both compounds were identified by one- and two-
dimensional NMR spectroscopy. Spectra of both contained
characteristic signals o f a single a-Kdo-residue, one b-linked
GlcN and one a-configured GlcN [7]. In addition, three
phosphate-residues were identified by
31
P-NMR spectro-
scopy (Fig. 2). With respect to the carbohydrate and
phosphate composition the two compounds were identical
and was reflected by almost identical one-dimensional
1
H-NMR spectra (Fig. 3, Table 2). As expected the com-
pounds differed in their phosphate substitution (Fig. 3,
Table 4). Both compounds contained one glycosidic phos-
phate linked to the a-GlcN (A) of the lipid A backbone
leading to a splitting of the signal of its anomeric proton and
another phosphate linked to the 4-position of the b-config-

ured GlcN (B). The far downfield position of the chemical
shifts of proton H-4 and carbon C-4 of the Kdo-residue (C)
of compound 1 and the downfield shift to t he same
frequencies of proton H-5 and carbon C-5 of the Kdo-resi-
due (C) of compound 2 identified compound 1 as Kdo-4P-
GlcN
2
-P
2
and compound 2 as Kdo-5P-GlcN
2
-P
2
(Tables 2 –4). The correct position o f p hosphates was finally
determined by
1
H,
31
P-HMQC spectroscopy.
Serology
Both oligosaccharides were activated with glutardialdehyde
and conjugated to BSA as described [ 12]. Chemical analyses
indicated a molar ratio of protein to ligand of 1 : 1.1 and
1 : 1.0 f or Kdo-4P-GlcN
2
-P
2
-BSA and Kdo-5P-GlcN
2
-

P
2
-BSA, r espectively. Both neoglycoconjugates w ere used in
ELISA to determine the epitope specificities of mAb. LPS
and LPS
deac
-BSA were used for comparison, whereby the
latter contained a mixture of 4- and 5-phosp horylated Kdo
in the ratio as it occurs in natural LPS. Clone S42-16 and
S42-21 were confirmed to be specific for Kdo-5P and
Kdo-4P, respectively. As seen in Fig. 4B clone S42-16
bound over a wide range of antigen coating concentrations
(10–0.08 pmol per well) to Kdo-5P-GlcN
2
-P
2
-BSA at
antibody concentrations as low as 1 ngÆmL
)1
. No binding
of this antibody was observed with Kdo-4P-GlcN
2
-P
2
-BSA
even at highest antigen concentration (10 pmol per w ell)
and antibody concentration ( 10 lgÆmL
)1
) (Fig. 4A). The
mAb S42-21 bound only to Kdo-4P-GlcN

2
-P
2
-BSA
(Fig. 4 C) but not to Kdo-5 P-GlcN
2
-P
2
-BSA (Fig. 4D)
The affinity of mAb S42-21 was approximately 200 times
lower t han t hat o f m Ab S42-16 for the homologous epitope.
Fig. 1. HPAEC chromatogram of deacylated LPS from H. influenzae
I-69. Shown is the analytical separatio n of the crude mixture (A) and
the a nalytical chromatography of the isolated species (B a nd C). Peaks
1and2representaKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P
and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P, respectively.
Fig. 2.
31
P-NMR spectrum of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-
aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P
(bottom).
Ó FEBS 2002 Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1239
The generation of clone S42-10-8 has been reported
previously [8] but its e pitope specificity c ould not be
determined so far. Binding of this antibody was tested i n
ELISA using various concentrations of Kdo-4P-GlcN
2
-
P
2

-BSA and Kdo-5P-GlcN
2
-P
2
-BSA, LPS or LPS
deac
-
BSA.
Fig. 3.
1
H-NMR spectr a of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom).
The asterisk indicates signals o f tryethylamine.
Table 2 .
1
H-NMR chemical s hift data of compounds 1 and 2. NR, not resolved.
Compound Residue
1
H-Chemical shift (p.p.m.) and coupling constants (Hz) for proton
H-1 H-2 H-3ax H-3eq H-4 H-5 H-6a H-6b H-7 H-8a H-8b
1Afi6aGlcN1P 5.659 3.380 3.873 3.448 4.123 3.852 4.248
4; 8
a
10 10 10 12; 9 4
B fi 6bGlcN4P 4.908 3.072 3.859 3.859 3.740 3.495 3.698
810
CaKdo4P 1.925 2.149 4.514 4.179 3.766 3.926 3.918 3.652
)12; 12 6 6 9 12; NR 6
2Afi6aGlcN1P 5.654 3.364 3.889 3.426 4.124 3.861 4.229
4; 7
a

11 10 10 12; 9 4
B fi 6bGlcN4P 4.902 3.074 3.854 3.840 3.727 3.471 3.714
81010
CaKdo5P 1.919
)12; 12
2.142
5
4.141 4.507 3.736
9
3.907 3.941
13; NR
3.649
a3
J
H-1,P
.
1240 S. Mu
¨
ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002
As seen in Fig. 4E, mAb S42-10-8 bound to Kdo-4P-
GlcN
2
-P
2
-BSA and with comparable a ffinity to LPS
(Fig. 5A) or LPS
deac
-BSA (Fig. 5B) as solid phase antigen.
No binding was observed with Kdo-5P-GlcN
2

-P
2
-BSA
(Fig. 4F).
The data show, together with those published earlier [8],
that mAb S42-10-8 binds to a complex epitope composed of
Kdo-4P linked to t he bisphosphorylated glucosamine
backbone of the LPS of H. influenzae I-69.
Although, Kdo-4P alone is not bound to the antibody,
the position of the phosphate group strictly determines the
specificity of the e pitope as no binding was observed with
antigens containing Kdo-5 P instead o f Kdo-4P or with
antigens containing nonphosphorylated Kdo.
DISCUSSION
Kdo is a common constituent of LPS and its presence is
essential for the survival of Gram-negative bacteria. A c-
cording t o our present knowledge of the Kdo-lipid A r egion
one Kdo residue is linked to position 6¢ of the glucosamine
disaccharide backbone of lipid A and is substituted at
position 5 by another sugar and at position 4 by another
sugar or phosphate [13]. The LPS of the deep rough mutant
I-69 of H. influenzae is unique in being composed of only o ne
Table 4.
31
P-NMR chemical shifts of compounds 1 and 2.
Residue
31
P-Chemical shift (p.p.m.) for compound
12
A)1.41 )1.64

B 0.58 )0.01
C 0.68 1.65
Table 3.
13
C-NMR chemical shift data of compounds 1 and 2. ND, not determined.
Compound Residue
13
C-Chemical shift ( p.p.m.) of carbon
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8
1Afi6aGlcN1P 91.10 54.45 69.76 70.07 72.69 69.36
B fi 6bGlcN4P 99.14 55.74 72.08 74.44 74.20 62.40
C aKdo4P ND ND 33.77 70.98 65.88 71.68 69.77 63.47
2Afi6aGlcN1P 91.16 54.45 69.61 70.12 72.68 69.20
B fi 6bGlcN4P 99.02 55.76 72.02 74.52 74.09 62.60
C aKdo5P ND ND 34.92 65.99 71.00 71.64 69.65 63.07
Fig. 4. Binding curves of mAbs S42-16 (A and B), S42-21 (C and D),
and S42-10-8 (E a nd F) to Kdo4P-GlcN
2
P
2
-BSA (A, C and E) and
Kdo5P-GlcN
2
P
2
-BSA (B, D and F). ELISA plates were coated with 200
(d), 100 (m), 50 (j)25(r), 12.5 (s), 6.3 (n), 3.2 (h)and1.6(e)
pmol ligandÆml
)1
and reacted with the mAb c oncen tratio ns indicated

on the abscissa. Values are the mean of quadruplicates with confidence
values not exceeding 10 %.
Fig. 5. Binding curve of mAb S42-10-8. The ligands were I-69 LPS (A)
and LPS
deac
-BSA (B). The coating concentrat ions used were 400 (d),
200 (m), 100 (j)50(r), 25 (s), 12.5 (n), 6.3 (h)and3.2(e)
pmolÆml
)1
for LPS
deac
-BSA. D ue to the poor c oating effi ciency of LPS
2000 (d), 1000 (m), 500 (j)250(r), 125 (s), 63 (n), 32 (h)and16
(e)pmolÆml
)1
were used for the immobilization of LPS. Both were
reacted with mAb concentrations indicated o n the abs cissa. Values are
the mean of quadruplicates with confidence values not exceeding 10%.
Ó FEBS 2002 Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1241
phosphorylated Kdo residue in addition to lipid A whereby
the Kdo is phosphorylated either at position 4 or 5. There
was some uncertainty in the beginning whether the Kdo-5P
was the result of phosphate migration [4], however, when
mAbs specific for the 4- or 5-P became available it could b e
shown that both antibodies bound to native bacteria [8]. The
final proof that both phosphates are made by the bacterium
was provided recently when we coexpressed the monofunc-
tional Kdo transferase and a phosphokinase of H. influenzae
in E. coli resulting in L PS whic h c ontained exclusively Kdo-
4P [7]. As the LPS obtained from this recombinant strain

was deacylated by the same pro tocol as used in this study it is
apparent that the appearance of the 5 P is not the result
of phosphate migration. Therefore, we conclude that
H. influenzae possesses two independent phosphokinases
attaching phosphate to position 4 or 5 whereby the 5 -kinase
has not yet been identified. With the results presented here
the complete structures of the phosphorylated carbohydrate
backbones of both LPS species made by H. influenzae I-69
are uniquivocally established and we have presented a
protocol for p reparing these two oligosaccharides in
sufficient quantities.
We have performed this s tudy not only t o d efinitely
identify the two differently phosphorylated LPS species bu t
also to learn more about the recognition of charged
carbohydrate epitopes b y antibodies. W e are interested in
this aspect to better understand protein–carbohydrate inter-
actions in general and the b inding of antibodies against
bacterial LPS in particular, as some of them are able to
neutralize the endotoxic activities of LPS which are embed-
ded i n t he phosphorylated lipid A moiety [ 14]. W e h ave
already characterized antibodies against the isolated lipid A
moiety [15] or against Kdo [16] or Kdo- P [8]. In this context
mAb S42-10-8 against I-69 LPS was of specific interest f or us
as it binds to an epitope composed of Kdo-P and lipid A;
however, its detailed epitope specificity could not be inves-
tigated so far due to the lack o f a ppropriately defined
antigens. The successful separation of these o ligosaccharides
described here together w ith a previously described conju-
gation protocol [14] allowed the characterization of the
epitope specificity of mAb S42-10-8. The binding data

obtained i n ELISA unequivocally proved that this m Ab
recognizes the trisaccharide aKdo-4P-(2–6)-bGlcN-4P-
(1–6)-aGlcN-1P; it does not bind to Kdo, Kdo-4P,Kdo-
5Por aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-aGlcN-1P.
The availability of both oligosaccharides as free ligands and
as neoglycoconjugates now enables us to investigate further
this antibody by NMR and crystallography.
ACKNOWLEDGEMENTS
We thank R . Moxon (Oxford, UK) for strain I-69 and V. Susott and
S. Cohrs for technical assistance. This w ork was supported by the
Deutsche Forschungsgemeinschaft (grant SFB470/C1 to L. B.).
REFERENCES
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mophilus influenzae disease. Molecular basis of invasive Haemo-
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2. Raetz, C.R.H. (1990) Biochemistry of endotoxins. Ann. Rev.
Biochem. 59, 129–170.
3. Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt,
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¨
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