Chemical structure and immunoreactivity of the lipopolysaccharideSven Mu¨ller-Loennies, Lore Brade and Helmut Brade Research Center Borstel, Center for Medicine and Biosciences, Borstel,
Trang 1Chemical structure and immunoreactivity of the lipopolysaccharide
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 were determined by one- and
two-dimensional 1H-, 13C- and 31P-NMR spectroscopy
as aKdo-4P-(2fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P and
aKdo-5P-(2fi 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, respectively [Rozalski, A., Brade L., Kosma P.,
Moxon R., Kusumoto S., & Brade 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 bisphosphorylated glucosamine backbone of lipid A and Kdo-4P, whereby the latter determines the 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 major virulence factor of this
human pathogen is the type b capsule, an acidic
polysac-charide composed of ribose, ribitol and phosphate and
which is the basis of an effective conjugate vaccine [1]
Among other virulence factors is the lipopolysaccharide
(LPS) in which we are interested for various reasons: (a)
LPS is an essential component of the outer membrane in all
negative bacteria; (b) LPS is the endotoxin of
Gram-negative bacteria; (c) LPS is a major surface antigen leading
to the induction of protective antibodies; and (d) the
understanding of the biosynthesis of LPS may allow
the distinct blockage of essential steps as a new strategy
for the development of antibiotics [2,3]
The smallest LPS structure which still allows the
bacter-ium to survive was found in the mutant strain I-69 Rd–/b+
of H influenzae (referred to here as I-69) where a
single phosphorylated 3-deoxy-D
-manno-oct-2-ulopyrano-sonic acid (Kdo) residue is linked to the lipid A moiety
Helander et al have shown that the I-69 LPS was composed
of two molecular species 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 enzymes in an Escheri-chia colistrain lacking its own Kdo transferase led to the synthesis of 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 the 4- or 5-phosphorylated Kdo which was chemically synthesized and conjugated to BSA [8] In addition, we found mAb S42-10-8 which was specific for 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, in addition to a phosphorylated Kdo residue, the phosphorylated lipid A backbone As the LPS species containing the Kdo-4P or Kdo-5P could not be separated at that time and were not yet chemically synthesized, the specificity of this mAb has not yet been elucidated Here, we report on: (a) the successful separation
of the deacylated carbohydrate 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) the characterization of a new mAb recognizing a phosphorylated carbohydrate epitope
M A T E R I A L S A N D M E T H O D S
Bacteria and bacterial LPS
H influenzae I-69 Rd–/b+ was cultivated as described 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 (LPSdeac) was obtained by hydrolysis of de-O-acylated LPS in 4MKOH as reported [11] LPSdeac was further purified by preparative high performance anion exchange chromatography (HPAEC) using water as eluent A
Correspondence to H Brade, Research Center Borstel, Center for
Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany.
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 21 December 2001, accepted
3 January 2002)
Trang 2and 1Mammonium 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 : 1000, 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-dimensional1H-NMR- and 13C-NMR and
spectroscopy at 600 and 150 MHz, respectively, on a Bruker
DRX 600 Avance spectrometer; 31P-NMR spectra were
recorded on a Bruker DPX 360 Avance spectrometer at
145 MHz All spectra were recorded on a 0.5-mL solution
of 5 mg sample in D2O As reference served acetone
2.225 p.p.m (1H), dioxane 67.4 p.p.m (13C) and 85%
phosphoric acid 0 p.p.m (31P) All spectra were run at a
temperature of 300 K For31P measurements the pD was
adjusted to pD 2 Other measurements were performed at
pD 6 due to the acid labile nature of the Kdo-linkage
Two-dimensional homonuclear1H,1H-DQF-COSY was
recorded over a spectral width of 7.5 p.p.m in both
dimensions recording 512 experiments of 32 scans Four
thousand data points were recorded in F2 Zero-filling
was applied in F1 to 1000 data points Heteronuclear
1H,13C-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 was
performed using States-TPPI Prior to Fourier
transfor-mation zero-filling was applied in F1 to 512 data points
31P-NMR spectroscopy was recorded with continuous
wave decoupling during acquisition A total of 32 scans
was recorded For 1H,31P-NMR COSY a HMQC
experiment was recorded consisting of 256 experiments
and 32 scans each Two thousand data points were
collected over a spectral width of 10 p.p.m in F2 and
zero filling was applied in F1 to yield 512 data points The
spectral width was 10 p.p.m in F1
Neoglycoconjugates
The amino groups of the glucosamine residues in LPSdeac
and in the oligosaccharides obtained from LPSdeac were
activated with glutardialdehyde and conjugated to BSA as
described [12] The amount of ligand present in the
conjugates was determined by measuring the amount of
protein (Bradford assay, Bio-Rad) and glucosamine
(Table 1)
MAbs Monoclonal antibodies S42-16, S42-21 and S42-10-8 were obtained after immunization and selection as described [8] Culture supernatants were prepared in at least 100 mL quantities and antibodies were purified on protein G-Sepharose (Pharmacia/LKB) according to the supplier’s instructions Purification was ascertained 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 used Micro-titer plates were coated with the respective antigen solution
in 50 mMcarbonate buffer pH 9.2 at 4°C overnight Plates were washed twice with distilled water; further washing was carried out in NaCl/Pisupplemented with 0.05% Tween 20 (Bio-Rad) and 0.01% thimerosal (NaCl/Pi/Tween-T) Plates were then blocked with NaCl/Pi/Tween-T supplemented with 2.5% casein (NaCl/Pi/Tween-TC) for 1 h at 37°C on a rocking platform followed by two washes Appropriate antibody dilutions in NaCl/Pi/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/Pi/Tween-T, the plates were washed in substrate buffer (0.1Msodium 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 lL of a 0.1% solution) After 30 min at 37°C, the reaction was stopped by the 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/
Pi(10 mMpH 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/Pi, blocked for 1 h with blocking buffer (2.5% casein in NaCl/Pi) and then incubated for 1 h with mAb diluted in blocking buffer (50 lL) Plates were washed in NaCl/Piand incubated for
Table 1 Oligosaccharides and neoglycoconjugates used in this study For derivatization procedures see Materials and methods Molar ratio of ligand
to protein given in parentheses.
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-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)
Trang 31 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 above All tests
were set up in quadruplicate Confidence values of the means
were less than 10%
R E S U L T S
Isolation and structural analysis of the phosphorylated
carbohydrate backbone of I-69 LPS
The LPS of H influenzae I-69 was successively de-O-acylated
and de-N-acylated with hydrazine and potassium
hydrox-ide, respectively, leading to two major products as revealed
by HPAEC (Fig 1) The two peaks, compounds 1 and 2,
could be separated from each other by preparative HPAEC
with yields of 11.6 mg (21.6% of LPS) and 5.1 mg (9.5%
of LPS) for Kdo-4P-GlcN2-P2 and Kdo-5P-GlcN2-P2,
respectively
Both compounds were identified by one- and
two-dimensional NMR spectroscopy Spectra of both contained
characteristic signals of a single a-Kdo-residue, one b-linked
GlcN and one a-configured GlcN [7] In addition, three
phosphate-residues were identified by 31P-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
1H-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 the 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-GlcN2-P2 and compound 2 as Kdo-5P-GlcN2-P2 (Tables 2–4) The correct position of phosphates was finally determined by1H,31P-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 for Kdo-4P-GlcN2-P2-BSA and Kdo-5P-GlcN2
-P2-BSA, respectively Both neoglycoconjugates were used in ELISA to determine the epitope specificities of mAb LPS and LPSdeac-BSA were used for comparison, whereby the latter contained a mixture of 4- and 5-phosphorylated 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-GlcN2-P2-BSA at antibody concentrations as low as 1 ngÆmL)1 No binding
of this antibody was observed with Kdo-4P-GlcN2-P2-BSA even at highest antigen concentration (10 pmol per well) and antibody concentration (10 lgÆmL)1) (Fig 4A) The mAb S42-21 bound only to Kdo-4P-GlcN2-P2-BSA (Fig 4C) but not to Kdo-5P-GlcN2-P2-BSA (Fig 4D) The affinity of mAb S42-21 was approximately 200 times lower than that of mAb S42-16 for the homologous epitope
Fig 1 HPAEC chromatogram of deacylated LPS from H influenzae
I-69 Shown is the analytical separation of the crude mixture (A) and
the analytical chromatography of the isolated species (B and C) Peaks
1 and 2 represent 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, respectively.
Fig 2 31 P-NMR spectrum of aKdo-4P-(2 fi bGlcN-4P-(1 fi 6)-aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom).
Trang 4The generation of clone S42-10-8 has been reported
previously [8] but its epitope specificity could not be
determined so far Binding of this antibody was tested in
ELISA using various concentrations of Kdo-4P-GlcN2
-P2-BSA and Kdo-5P-GlcN2-P2-BSA, LPS or LPSdeac -BSA
Fig 3 1 H-NMR spectra 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 of tryethylamine.
Table 2 1 H-NMR chemical shift data of compounds 1 and 2 NR, not resolved.
Compound Residue
1 H-Chemical shift (p.p.m.) and coupling constants (Hz) for proton
)12; 12
2.142 5
4.141 4.507 3.736
9
3.907 3.941 13; NR 3.649
a 3 J
Trang 5As seen in Fig 4E, mAb S42-10-8 bound to
Kdo-4P-GlcN2-P2-BSA and with comparable affinity to LPS
(Fig 5A) or LPSdeac-BSA (Fig 5B) as solid phase antigen
No binding was observed with Kdo-5P-GlcN2-P2-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 the 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 epitope as no binding was observed with antigens containing Kdo-5P instead of Kdo-4P or with antigens containing nonphosphorylated Kdo
D I S C U S S I O N
Kdo is a common constituent of LPS and its presence is essential for the survival of Gram-negative bacteria Ac-cording to our present knowledge of the Kdo-lipid A region 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 one
Table 4. 31P-NMR chemical shifts of compounds 1 and 2.
Residue
31
P-Chemical shift (p.p.m.) for compound
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
Fig 4 Binding curves of mAbs S42-16 (A and B), S42-21 (C and D),
and S42-10-8 (E and 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) and 1.6 (e)
pmol ligandÆml)1and reacted with the mAb concentrations 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 concentrations used were 400 (d),
200 (m), 100 (j) 50 (r), 25 (s), 12.5 (n), 6.3 (h) and 3.2 (e) pmolÆml)1for LPS deac -BSA Due to the poor coating efficiency of LPS
2000 (d), 1000 (m), 500 (j) 250 (r), 125 (s), 63 (n), 32 (h) and 16 (e) pmolÆml)1were used for the immobilization of LPS Both were reacted with mAb concentrations indicated on the abscissa Values are the mean of quadruplicates with confidence values not exceeding 10%.
Trang 6phosphorylated 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 be
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 LPS which contained exclusively
Kdo-4P [7] As the LPS obtained from this recombinant strain
was deacylated by the same protocol as used in this study it is
apparent that the appearance of the 5P 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 preparing these two oligosaccharides in
sufficient quantities
We have performed this study not only to definitely
identify the two differently phosphorylated LPS species but
also to learn more about the recognition of charged
carbohydrate epitopes by antibodies We are interested in
this aspect to better understand protein–carbohydrate
inter-actions in general and the binding of antibodies against
bacterial LPS in particular, as some of them are able to
neutralize the endotoxic activities of LPS which are
embed-ded in the phosphorylated lipid A moiety [14] We have
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 for 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 of appropriately defined
antigens The successful separation of these oligosaccharides
described here together with a previously described
conju-gation protocol [14] allowed the characterization of the
epitope specificity of mAb S42-10-8 The binding data
obtained in ELISA unequivocally proved that this mAb
recognizes the trisaccharide
a4P-(2–6)-bGlcN-4P-(1–6)-aGlcN-1P; it does not bind to Kdo, 4P,
Kdo-5P or 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
A C K N O W L E D G E M E N T S
We thank R Moxon (Oxford, UK) for strain I-69 and V Susott and
S Cohrs for technical assistance This work was supported by the
Deutsche Forschungsgemeinschaft (grant SFB470/C1 to L B.).
R E F E R E N C E S
1 Moxon, E.R (1992) Session III: Pathogenesis of invasive
Hae-mophilus influenzae disease Molecular basis of invasive
Haemo-philus influenzae type b disease J Infect Dis 165, S77–S81.
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, G., Loppnow, H., Ulmer, A.J., Za¨hringer, U., Seydel, U., Di Padova, F., Schreier, M & Brade, H (1994) Bacterial endotoxin: Molecular relationships of structure to activity and function FASEB J 8, 217–225.
4 Helander, I., Lindner, B., Brade, H., Altmann, K., Lindberg, A.A., Rietschel, E.Th & Za¨hringer, U (1988) Chemical structure
of the lipopolysaccharide of Haemophilus influenzae strain I-69
Rd – /b + Eur J Biochem 177, 483–492.
5 White, K.A., Kalashov, I.A., Cotter, R.J & Raetz, C.R.H (1997)
A mono-functional 3-deoxy- D -manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influen-zae J Biol Chem 272, 16555–16563.
6 White, K.A., Lin, S., Cotter, R.J & Raetz, C.R.H (1999)
A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy- D -manno-octulosonic Acid (Kdo) kinase J Biol Chem.
274, 31391–31400.
7 Brabetz, W., Mu¨ller-Loennies, S & Brade, H (2000)
3-Deoxy-D -manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli.
J Biol Chem 275, 34954–34962.
8 Rozalski, A., Brade, L., Kosma, P., Moxon, R., Kusumoto, S & Brade, H (1997) Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes
in the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+
of Haemophilus influenzae Mol Microbiol 23, 569–577.
9 Zamze, S.E., Ferguson, M.A.J., Moxon, E.R., Dwek, R.A & Rademacher, T.W (1987) Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide Biochem J 245, 583–587.
10 Galanos, C., Lu¨deritz, O & Westphal, O (1969) A new method for the extraction of R lipopolysaccharides Eur J Biochem 9, 245–249.
11 Holst, O., Broer, W., Thomas-Oates, J.E., Mamat, U & Brade, H (1993) Structural analysis of two oligosaccharide biphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus-specific epitope of Chlamydia lipopolysaccharide Eur J Biochem 214, 703–710.
12 Brade, L., Holst, O & Brade, H (1993) An artificial glycoconju-gate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A binds lipid A monoclonal antibodies Infect Immun 61, 4514–4517.
13 Holst, O (1999) Chemical structure of the core region of lipo-polysaccharides In Endotoxin in Health and Disease (Brade, H., Opal, S.M., Vogel, S.N & Morrison, D.C., eds), pp 115–154 Marcel Dekker Inc., New York.
14 Di Padova, F.E., Brade, H., Barclay, G.R., Poxton, I.R., Liehl, E., Schuetze, E., Kocher, H.P., Ramsay, G., Schreier, M.H., McClelland, D.B.L & Rietschel, E.T (1993) A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharide Infect Immun 61, 3863–3872.
15 Kuhn, H.-M., Brade, L., Appelmelk, B.J., Kusumoto, S., Riets-chel, E.T & Brade, H (1992) Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid
A Infect Immun 60, 2201–2210.
16 Brade, H., Brabetz, W., Brade, L., Holst, O., Lo¨bau, S., Lucakova, M., Mamat, U., Rozalski, A., Zych, K & Kosma, P (1997) Chlamydial lipopolysaccharide J Endotoxin Res 4, 67–84.