Synthetic glycoconjugates characterize the fine specificity of: Brucella A and M monoclonal antibodies

Article abstract of DOI:10.1039/c7ob00445a

The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of the smooth lipopolysaccharide. Infection by various Brucella biovars causes abortions and infertility in a wide range of domestic and wild animals and debilitating disease in humans. Diagnosis relies on the detection of antibodies to the A and M antigens expressed in the O-polysaccharide. This molecule is a homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-d-mannopyranose (Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides only available by chemical synthesis and conjugated via reducing and non-reducing residues to bovine serum albumin have revealed the structural basis of the fine specificity that allows the discrimination of these closely related A and M epitopes. All three M specific monoclonal antibodies (mAbs) are inferred to possess groove type binding sites open at each end, and recognize an α1,3 linked Rha4NFo disaccharide as a part of a trisaccharide epitope, which in two mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is sufficiently large to engage up to six Rha4NFo residues and involves weak recognition of α1,2 linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M tetrasaccharide. Two A specific mAbs also possess groove type binding sites that accommodate six and four α1,2 linked Rha4NFo residues.

Full text of DOI:10.1039/c7ob00445a

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Synthetic Glycoconjugates Characterize the Fine Specificity of Brucella A and M
Monoclonal Antibodies
Satadru Sekhar Mandal, N. Vijaya Ganesh, Joanna M. Sadowska and David R. Bundle
The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of smooth
lipopolysaccharide. Infection by various Brucella biovars cause abortions and infertility in a wide
range of domestic and wild animals and a debilitating disease in humans. Diagnosis relies on
detection of antibodies to A and M antigens expressed in the O-polysaccharide. This molecule is a
homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-D-mannopyranose
(Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence
Received 00th January 20xx,
capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides
Accepted 00th January 20xx
only available by chemical synthesis and conjugated via reducing and non-reducing residues to
bovine serum albumin have dissected the structural basis of the fine specificity that allows
DOI: 10.1039/x0xx00000x
www.rsc.org/
discrimination of these closely related A and M epitopes. All three M specific monoclonal
antibodies (mAbs) are inferred to possess grove type binding sites open at each end, and
recognize an α1,3 linked Rha4NFo disaccharide as part of a trisaccharide epitope which in two
mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is
sufficiently large to engage upto six Rha4NFo residues and involves weak recognition of α1,2
linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M
tetrasaccharide. Two A specific mAbs also possess grove type binding sites that accommodate six
and four α1,2 linked Rha4NFo residues.
[α-Rha4NFo-2α-Rha4NFo-3α-Rha4NFo-2α-Rha4NFo]n –
[2α-Rha4NFo]m.
Introduction
The fine structural difference (Figure 1) between the A and M
Brucellosis causes abortions and infertility in a wide range
antigens, a single 1,3 linkage in an otherwise 1,2 linked
of domestic and wild animals and undulant fever in humans, a
polysaccharide, requires A and M specific antibodies to
grave disease that requires a long and costly antibiotic
discriminate antigenic determinants with similar structural
therapy.1 The WHO ranks it among the seven most neglected
motifs and overlapping topology. Several monoclonal
zoonoses.2 It is the cause of significant economic loses
antibodies (mAbs) have been described that possess high
especially in those countries that lack the infrastructure for
selectivity for the two antigenic determinants.9-11
diagnosis and control.3 Diagnosis relies upon detection of
antibodies to the cell wall lipopolysaccharide (LPS) O-antigen
or O-polysaccharide.4 In Brucella abortus, B. melitensis and B.
suis this molecule is composed of
a
single rare
monosaccharide, 4-formamido-4,6-dideoxy-D-mannopyranose
(Rha4NFo)5,6 and since the 1930s two inseparable antigenic
determinants identified as A and M antigens have been
known.7 The structural basis of two epitopes embedded in a
single polysaccharide arise because unlike most gram negative
bacteria that produce O-antigens with a regular repeating unit
sequence, Brucella species synthesizes an O-antigen that is
best described as
a block co-polymer of two distinct
homopolysaccharide sequences;8
Figure 1.
tetrasaccharide containing
The structure of the Brucella O-antigen with the capping
1,3 linkage.8 The percentage of 1,3 linkages
a
compared to 1,2 ranges from a high of 20% to as low as 1% in different Brucella.
In the prototypical M biovar B. melitensis 16M (~20% 1,3 linkages) there are
several repeats of this tetrasaccharide m>1. In B. abortus A type biovars, often
with as little as 1% 1,3 linkages m = 1. B. suis biovars 2 lacks a capping
tetrasaccharide and is exclusively 1,2 linked.12
a.Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2,
Canada
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
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Figure 2
Oligosaccharides used in this study as bovine serum albumin (BSA) conjugates.
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A vaccine that generates antibodies against an A epitope monosaccharide,
-Rha4NFo, is responsible for creating both
but not the M epitope used for serodiagnosis would allow the internal and terminal epitopes and 1,2 linkages are known
vaccinated animals to be distinguished from those with to expose internal residues so that internal pyranose residues
brucellosis.13 Binding studies of A and M specific mAbs can may present as the “immunochemical equivalent” of terminal
provide insights on the subtle recognition that permits this monosaccharides.17
discrimination. Repeated attempts to prepare homogeneous
Fab fragments from three M and two A specific mAbs for the previously synthesized M tetrasaccharide
crystallographic studies of antigen-antibody complexes using component trisaccharides and as well as the 1,3 linked
various proteolytic enzymes were unsuccessful with the disaccharide 4 13
To distinguish the terminal -Rha4NFo
exception of one A specific mAb (Yst9.1) for which a crystal monosaccharide a range of compounds in which the terminal
structure without bound ligand was obtained.14 The mAbs residue was masked by O-methyl groups
or replaced by
comprised two groups previously reported by us BM10,9 mannopyranose
10 were synthesized. To further
In order to probe the M antigenic determinant we utilized
1
and its
2
3
.

5
-
7
-
8
-
Bm28,9 Yst9.110 and Yst9.2.10 In addition we have studied a differentiate binding to tip epitopes present in the M antigenic
third M specific mAb BM40, which is described in the determinant from recognition of internal sequences, tri- and
literature.11 In order to characterize the fine specificity of the tetrasaccharides 11 and 12 were synthesized with a tether
three
oligosaccharides
(ESI FigureS1).
M
specific mAbs we have used
a
panel of attached to a terminal α-
12 (Figure 2) as their BSA glycoconjugates than by the conventional approach of conjugation via a tether
attached to the anomeric centre of the sugar at the
D-rhamnopyranosyl residue rather
1-
Antibodies frequently recognize the distal tip of an O- oligosaccharide reducing-end.
antigen, which is the most exposed element of the
We report the syntheses of oligosaccharides
5
-
12 and the
polysaccharide on the bacterial surface to be sensed by the conjugation of 1-12 to BSA using disuccinimidyl glutarate (DSG)
immune system.15,16 In Brucella difficulties can arise in or dibutyl squarate. These glycoconjugates were then used to
identifying antibody specific for the capping residue: a single
Scheme 1
Reagents and conditions: a) TMSOTf, NIS, 4 Ǻ MS, CH2Cl2 -20° C to rt, 3 h; b) NaOMe, MeOH, rt, 6 h; c) MeOTf, 4 Ǻ MS, CH2Cl2, 0° C to rt, 3 h; d)
TMSOTf, NIS, 4 Ǻ MS, CH2Cl2 -10° C to rt, 3 h.
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probe previously reported monoclonal antibodies BM10, precise structural terms.13,24 Here we concentrate on the M
BM289 and BM4011 via indirect ELISA (iELISA) and inhibition of epitope and compare the structural requirements to bind
iELISA. The longer inner sequence of
constitute the antigen and this is capped by
tetrasaccharide sequence that creates the M antigen, which

1,2 linked residues three M specific mAbs and compare them to the binding
A
a
profiles of two A specific reagents.
consists of one or several tetrasaccharide repeats (Figure 1). Results
Three general categories have been used to define antibody
specificity to Brucella antigens; A, M, and epitopes shared by described.13
both A-dominant and M-dominant strains, which have been
Oligosaccharides 1-4 were synthesized as previously
Synthesis of O-methylated oligosaccharides
named common (C) epitopes.18,19 The C epitope was recently
inferred to be a pentasccharide containing a central 1,3 linkage
bounded by 1,2 linked residues.20
Defined oligosaccharide sequences are only available
through challenging chemical synthesis and although there
have been many attempts to define A and M epitopes,9,21-23
there has been only limited work to describe the specificity in
The synthesis of the oligosaccharides capped by O-
methylated α-Rha4NFo residues utilized methylated glycosyl
donors (described in electronic supplementary information, ESI)
and
Glycosylation with di-O-methyl thioglycoside 13 and the mono-
O-methyl thioglycoside 14 gave disaccharides 20 and 21
a
common glycosyl acceptor S15 (Scheme 1).25
.
Throughout the synthetic schemes used here ester protecting
groups were always removed from the final, protected target
compounds
Scheme 2
Reagents and conditions: a) TMSOTf, 3Å MS, CH2Cl2, rt, 1 h; b) NaOMe, MeOH, rt, 4 h; c) MeOTf, 3Å MS, CH2Cl2, rt, 64 h.
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to permit a common deprotection strategy described later.
M-type tetrasaccharide 31 and after de-O-benzoylation
A mono-O-methylated trisaccharide corresponding to the compound 32
terminal trisaccharide of the M tetrasaccharide sequence was
.
Utilizing a previously reported pentasaccharide acceptor
synthesized by glycosylation of S15 by thioglycoside 14. After 3013 glycosylation by S2026 afforded a mannose capped A-type
removal of the benzoate from the product 23, the disaccharide hexasaccharide glycoside 34 and subsequently deacylated
alcohol was glycosylated by 14 to yield the trisaccharide 25 hexasaccharide glycoside 35
.
and after transesterification compound 26
.
Oligosaccharides with a non-reducing Terminal Tether
Synthesis of Mannose capped Oligosaccharides
As described in the preceding two sections, the usual
approach to synthesizing glycoconjugates involves placing a
tether at the anomeric centre of the terminal reducing sugar.
To eliminate terminal residue specific antibodies from binding
to a subset of our screening panel, we created two M-type
epitopes 11 and 12 with a linker attached to the terminal non-
reducing end of the oligosaccharide. This necessitated the
synthesis of a glycosyl donor for the final chain extension
reaction that was pre-formed with attached tether. The
rhamopyranosyl thioglycoside 19 was recently used in our
laboratory to synthesize A-type oligosaccharide vaccines and is
used here (scheme 3).27
The known per-O-benzylated mannose trichloro-
acetimidate donnor S2026 served to provide three mannose
capped oligosaccharides (Scheme 2). Glycosylation of acceptor
S1525 gave
the protected M-type disaccharide 27 and after
transesterication the selectively deprotected alcohol 28. The
selectively protected thioglycoside alcohol S1422,23 was
converted to a disaccharide donor 29 by glycosylation with S20
and this disaccharide thioglycoside in turn glycosylated
disaccharide 30 to afford the fully protected mannose capped
Scheme 3
Reagents and conditions: a) TMSOTf, NIS, 4 Å MS, CH2Cl2 -10° C to rt, 3 h; b) NaOMe, MeOH, rt, 6 h.
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The selectively protected methyl glycoside S1821 was Deprotection of Oligosaccharides and Conjugation to Protein
glycosylated by thioglycoside S1621 to yield the 1,3 linked
The target oligosaccharides 1-12 were obtained from their
disaccharide 36. Transesterification yielded the disaccharide
alcohol 37 and this compound was glycosylated by the
rhamopyranosyl thioglycoside 1927 to give the protected target
protected precursors by a common deprotection strategy that
we have previously described (Scheme 4). Briefly, the azido
groups were reduced to amine and immediately formylated by
formic anhydride and then the persistent benzyl protecting
groups were removed by a hydrogenolysis step.
trisaccharide 38
.
In order to access tetrasaccharide 12 the methyl glycoside
S16 was glycosylated by S17 to provide 1,2 linked
a
disaccharide 40 with a temporary protecting group at O-3'.
Transesterification afforded 41 which was glycosylated by
thioglycoside 15 to yield trisaccharide glycoside 42
.
Transesterification of 42 gave the trisaccharide alcohol 43 and
it was glycosylated by thioglycoside 1927 to yield the protected
Scheme 4
Reagents and conditions: a) H2S, Py/H2O, 40º C, 16 h; b) (HCO)2O,
target tetrasaccharide 45
.
MeOH, -20° C, 3 h; c) H2, Pd(OH)2, MeOH/H2O, rt, 16 h.
Scheme 5
Reagents and conditions: a) NH2CH2CH2NH2, 20° C, 48 h; b) DSG, DMF:PBS Buffer (pH7.3) = 4:1, rt, 4 h; c) 0.1M PBS Buffer (pH9.0), rt, 72 h;. Conditions:
a) Dibutyl squarate, EtOH/H2O, rt, 2 h; b) 0.1M PBS Buffer (pH9.0), rt, 72 h.
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Oligosaccharides possessing a tether attached to the
anomeric centre were conjugated to BSA using the
homobifunctional linkers disuccinimidyl glutarate (DSG) or
dibutyl squarate. Oligosaccharides
1-10 were first converted to
the corresponding amides 46 55 by reaction with ethylene
-
diamine (Schemes 5A and 5B). A 15 molar equivalent of DSG
was used to prepare the oligosaccharide succinimide ester
from amides 46
repeated washing
-
47
,
50
-
52 and excess reagent was removed by
with ethyl acetate. Activated
oligosaccharide was reacted with BSA over 3 days and after
dialysis and lyophilization the degree of oligosaccharide
incorporation was measured by MALDI-TOF mass
spectrometry. Amides 48
using dibutyl squarate as previously described to afford first
the half esters and after reaction with BSA conjugates 60 61
65 67 70 71 and 75 77 (Scheme 5B).
Dibutyl squarate was employed for conjugation of 11 and
-49, 53-55 were conjugated to BSA
-
,
-
,
-
-
12 to protein. After reaction with each oligosaccharide for 1 h
the reaction was monitored by mass spectrometry and the half
esters 56 and 57 purified by reverse phase HPLC and
lyophilized. The activated oligosaccharide was allowed to react
with BSA in buffer for 3 days and conjugates were isolated by
dialysis and lyophilization to yield 78 and 79 (Schemes 5C).
Indirect Elisa Screening of mAbs
The three M specific mAbs studied here were generated by
immunization of mice with killed whole cells of B. melitensis
16M.9,11 The A specific mAbs were produced from mice
immunized with Y. enterocolitica O:9.10 Two of three M specific
mAbs have been studied previously in direct competitive ELISA
where Bm10 and Bm28 were shown to exhibit ~400 fold
preference for binding LPS from M>A type Brucella over the
LPS from A> M type LPS.9 Bm40 generated by others11 was not
investigated by us prior to this work. Bm10 and Bm28 had
shown weak inhibition with two M type pentasaccharides
neither of which had the 1,3 linkage in the relative position
characteristic of the recently revised structure of the M
antigen,8 but rather at the reducing and non-reducing
positions which does not correspond to the native sequence.8
In the same study the two A type mAbs, Yst9.1 and Yst9.2
exhibited strikingly different properties. Yst9.2 exhibited equal
affinity of M, A and Y. enterocolitica O:9 O-antigens. Yst9.1
showed a strong 1000 fold preference for A>M Brucella LPS
and a 10 fold preference for A type LPS over Y. entercolitica
O:9. The two antibodies were inhibited by oligomers
Figure 3
A) End point titrations of Bm10 against glycoconjugates 68
76; B) End point titrations of Bm28 against glycoconjugates 68
71; C) End point titrations of Bm40 against glycoconjugates 68
71 S27 and S28
-
70 and
-
-
,
.
composed exclusively of l,2-linked residues. Whereas Yst9-1
required at least five, and possibly six, contiguous residues,
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Yst9-2 was effectively inhibited by a tetrasaccharide ligand.9 To
refine our understanding of the binding profiles of these five
antibodies we used the conjugates (Scheme 5) derived from
the oligosaccharides (Figure 2) with additional glycoconjugates
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DOI: 10.1039/C7OB00445A
S27-S31 already reported by us (ESI Figure S1). Microtitre
plates were coated with these glycoconjugates and each of the
three M and two A specific mAbs were titred to their end
point.
When conjugates are presented on solid surfaces their
degree of multivalency can mask subtle differences in intrinsic
affinities. We also conducted inhibition experiments with the
soluble BSA glycoconjugates 68
selected group of previously reported conjugates S27
-
79 (Scheme 5) as well as a
S31
-
(Figure S1). Under conditions of antigen excess multivalency of
antigen and cross linking in antigen-antibody complexes will be
disfavoured and 1:1 conjugate-antibody complexes should
predominate and permit relative ranking of inhibitors.
The titration of Bm10 against the glycoconjugates
immediately shows that the antibody recognizes the internal
trisaccharide sequence with 1,3:1,2 linkages, conjugate 70
(derived from
linked trisaccharide compound 69
The terminal non reducing Rha4NFo is clearly not essential for
binding since the mannose capped tetrasaccharide 76 ) binds
almost as well as the native tetrasaccharide sequence 68 ).
Inhibition data for Bm10 show that the terminal M
tetrasaccharide repeat 68 ) and a pentasaccharide (S27) that
corresponds to a terminal sequence share related IC50 values
with trisaccharide 70 ). This implies that Rha4NFo residues
that extend trisaccharide at either end contribute no
3
) (Scheme 5) and that the terminal 1,2:1,3
(2
) is hardly bound by Bm10.
(9
(1
(1
(3
3
additional significant binding energy. However, the S27
inhibition data also imply a binding site that that can
accommodate these adjacent residues without steric clashes
that reduce binding energy.
Bm28 exhibits strong specificity for the chain end
1,2:1,3 linked trisaccharide 69
adjacent trisaccharide sequence 70
68 ) binds strongest and the 1,3 linked disaccharide 71
binds well. Inhibition data clarifies the recognition indicating
the strength of binding is 2>1 S27 S28>> . The M type
pentasaccharide S27 and the hexassacharide S28 are almost as
effective inhibitors as tetrasaccharide 68 ). This again
suggests that the primary recognition unit is a trisaccharide
corresponding to and its constituent 1,3 linked disaccharide.
(2
) and weak binding to the
(3
). The M tetrasaccharide
(1
(4)

>
4
(1
2
Adjacent residues neither contribute to nor detract
significantly from the binding energy. We conclude therefore
that Bm28 and Bm10 have relatively open grove type binding
sites. The former recognizes the terminal trisaccharide
Figure 4
Inhibition of Brucella mAbs binding to microtitre plates coated
with the
M type hexasaccharide glycoconjugate S28:- A)
Inhibitors glycoconjugates 68
glycoconjugates 68 69, 71
glycoconjugates 68 69 S27 and S28;.
,
70
,
S27 and S28; B) Inhibitors
sequence while Bm10 recognizes the internal trisaccharide
the M tetrasaccharide epitope
Bm40 titration curves show a pattern of recognition similar
to that of Bm28, again with the terminal trisaccharide and
disaccharide as important elements of the epitope. Inhibition
data reveal that epitopes S27 S28 fall within a narrow 4
3 of
,
,
S27 and S28;
C) Inhibitors
,
,
1
.
S27 and hexasaccharide S28 are slightly better inhibitors. This
suggests that the primary recognition element is the terminal
trisaccharide
upto a hexasaccharide with small contributions to binding
strength from the 1,2 linked Rha4NFo residues of the A
2
4
2 and that the antibody site can accommodate
1
,
2
,
,
fold range of IC50 values. However the larger pentasaccharide
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antigen to which the M tetrasaccharide is attached (Figure 1). consistent with the suggestion that internal 1,2 linked residues
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In support of a binding site recognizing segments of 1,2 linked can function as the equivalent of chain end residues. A
oligomers Bm40 binds to glycoconjugate S31 an exclusively 1,2 terminal hexasaccharide bearing a 1,3 linkage cannot bind
linked heptasaccharide attached to protein via it non reducing although frame shifting presumably allows the 1,3 linkage to
residue (ESI Figure S2).
lie outside the site.
We noted in the introduction that internal residues of 1,2
Yst9.2 was previously reported to have a binding site
linked polysaccharides are known to expose pyranose residues capable of binding Brucella A and M antigens with equal
such that they act as the “immunochemical equivalent” of avidity.9 Its ability to bind conjugates S29
-S31 is consistent
terminal monosaccharides.17 Two strategies were adopted to with this interpretation and no additional conclusions can be
determine the involvement of the terminal residues in binding, inferred from data collected here.
introduction of O-methyl groups on hydroxyl groups
Conclusion
potentially involved in sugar protein interactions and
Synthetic glycoconjugates provide powerful insights and
replacement of Rha4NFo by Man. The bulk of the former
allow inferences that highlight three slightly different modes of
would prevent close sugar protein interaction at the site of O-
binding of the terminal M epitope. In all three cases the
methylation.28 Introduction of mannose involves a bulky
distinguishing 1,3 linked disaccharide
4 is the key recognition
hydroxymethyl group at C5 and loss of the formamido group.
The fine specificity of Bm10 shows that the mannose
capped tetrasaccharide exhibits a small reduction in activity
element. While the adjacent internal Rha4NFo residue is
crucial for Bm10 interactions, the terminal residue plays a
more subtle role in binding to Bm28 and to Bm40, where the
inference is made for displacemt of water at the periphery of
the binding site.29 This appears to be the extent of crucial
interactions for Bm28 but Bm40 is able to interact with a larger
epitope including several 1,2 linked Rha4NFo residues.
consistent with the conclusion that trisaccharide
recognition element. The interaction of Bm28 and Bm40 with
tetrasaccharide 76 capped by mannose shows small
3 is the major
a
reduction in binding but titres remain in an intermediate range
and inhibitory data show 5 and 10 fold drops in inhibitory
activity relative to 68
and Bm 40 against the
conjugate 74 ) the corresponding hydroxyl group is not have noted in previous work that the 1,2 linked oligomers can
involved in key polar interactions with the binding sites of adopt a rod like conformation that exposes formamido groups
either antibody. Methylated disaccharide conjugate 72 ) also at its outer edge and that a 1,3 linkage creates a significant
shows only a small loss in binding (ESI Figure S3) suggesting change in this topography.13,30 So despite the
that O-3 of disaccharide is not involved in polar inactions homopolysaccharide nature of the Brucella A and M antigens,
with the binding site for these two antibodies. Conjugates 78 the introduction of a 1,3 linkage creates a kink in a rod like
11) and 79 12) also shed light on the fine specificity of the polysaccharide that is distinct and able to generate a subset of
(1
). Since titres remain high for Bm28
The binding sites of all 5 mAbs are deduced to be groves
2
''-O-methylated trisaccharide and for Yst9.1 this is consistent with a crystal structure.14 We
(7
(5
'
5
(
(
three M specific mAbs. These conjugates are attached to antibodies that do not bind the A antigen.
protein via a terminal non reducing D-rhamopyranose residue
In an attempt to obtain Fab fragments that will allow
to which a linker is attached at O-4. All three mAbs bind well to
conjugate 79 12) (ESI Figure S4). This suggests the groove type
binding site of all three can tolerate the bulky protein and
linker adjacent to the site. Bm10 although it does no bind the
crystallographic studies of Bm10, Bm28 and Yst9.2 with bound
antigen, the genes for the Fab fragments will be expressed in
mammalian cells.
(
terminal residue has weak to no binding to conjugate 78 (11)
suggesting that the third RhaNFo residue is very important to
binding. Conversely Bm28 and Bm40 show almost no loss in
Experimental
Synthetic Procedures
binding with 78
of the 2''-O-methylated trisaccharide conjugate 74
mannose capped tetrasaccharide 76 ). These observations
(11) which is consistent with the good binding
Full details of all syntheses and characterization of
products are provided as electronic supplementary information.
(7
) and
(9
suggest a role for the terminal residue that might involve
displacement of water29 from this area of the sites of Bm28
and Bm40 rather than any specific polar interactions.
ELISA Procedures
Antibodies
The solved crystal structure of Yst9.1 Fab shows a grove
shape site capable of accommodating six Rha4NFo residues.13
This is consistent with the binding profiles observed for
hexassacharide conjugate S30 and the weaker binding to
trisaccharide conjugate S29 (ESI Figure S5). The antibody binds
internal and end terminal hexasaccharides S30 and S31 with
equal avidity suggesting that the grove shaped binding site is
open at either end (ESI Figure S2). The comparable avidity for
chain end and internal 1,2 linked oligomers would also be
Murine mAbs were as previously described Bm10 and
Bm28,9 and YSt91 and Yst92.11 Bm4012 ascites fluid was a gift
from Dr. J. McGiven (FAO/WHO Collaborating Centre for
Brucellosis, OIE Brucellosis Reference Laboratory, Department
of Bacteriology, Animal & Plant Health Agency, United
Kingdom).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
Page 10 of 10
ARTICLE
ELISA
Journal Name
5
6
M. Caroff, D. R. Bundle, M. B. Perry, J. W. Cherwonogrodsky,
and J. R. Duncan. Infect. Immun., 1984D, O46I:,1308.1403.V9ie/Cw7AOrtBic0le0O4n4li5nAe
P. J. Meikle, M. B. Perry, J. W. Cherwonogrodzky, and D. R.
Bundle. Infect. Immun. 1989, 57, 2820.
Microtiter plates were coated with antigen solution (1
µg/mL PBS) by incubation at 4 C for 18 h, then washed (5
times) with PBST (0.05% Tween-20 in phosphate buffer saline,
PBS). Plates were not subjected to a blocking step. Serial 10
dilutions of monoclonal antibodies were made in PBST. The
solutions were distributed in triplicate on coated microtiter

7
8
G. S. Wilson and A. A. Miles. Brit J. Exptl Pathol., 1932, 13, 1.
J. Kubler-Kielb, and E. Vinogradov. Carbohydr. Res., 2013,
378, 144.

9
D. R. Bundle, J. W. Cherwonogrodzky, M. A. J. Gidney, P. J.
Meikle, M. B. Perry and T. Peters. Infect. Immun., 1989, 57
,
o
plates and incubated at 21 C for 2 hours. Plates were washed
2829.
10 D. R. Bundle, M. A. J. Gidney, M. B. Perry, J. R. Duncan, and J.
W. Cherwonogrodzky. Infect. Immun., 1984, 46, 389.
11 I. Greiser-Wilke and V. Moennig. Ann. Inst. Pasteur Microbiol.
1987, 138, 549.
with PBST (5 times). Goat anti-mouse IgG antibody conjugated
to horseradish peroxidase (Kirkegaard & Perry Laboratories;
1:5000 dilution in PBST; 100
was then incubated for 1h at 21 oC, and then washed (5 times)
with PBST. Peroxidase substrate, 3,3',5,5'-
L/well) were added. The mixture
12 M. V. Zaccheus, T. Ali, A. Cloeckaert, M. S. Zygmunt, A.
Weintraub, M. Iriarte, I. Moriyón and G. Widmalm. PLoS One,
2013, 8:e53941.
tetramethylbenzidine (TMB) with H2O2 (1:1 mixture of 3,3',5,5'-
tetramethylbenzidine (0.4 g/L) and 0.02% H2O2 solution,
13 V. N. Ganesh, J. M. Sadowska, S. Sarkar, L. Howells, J.
McGiven and D. R. Bundle. J. Am. Chem. Soc., 2014, 136
,
Kirkegaard & Perry Laboratories) was added (100
L/well).
16260.
After 15 minutes, the reaction was quenched by addition of 1
14 D. R. Rose, M. Przybylska, R. J. To, C. S. Kayden, R. P. Oomen,
M phosphoric acid (100
nm.
L/well). Absorbance was read at 450
E. Vorberg, N. M. Young and D. R. Bundle. Protein Science,
1993, 2, 1106.
15 Z. Lu, M. J. Rynkiewicz, C.-Y. Yang, G. Madico, H. M. Perkins,
Q. Wang, C. E. Costello, J. Zaia, B. A. Seaton and J. Sharon. J.
Immunol. 2013, 140, 374.
Inhibition ELISA
Inhibition experiments performed with the M specific
mAbs Bm10, Bm28 and Bm40 were performed on plates
coated by the hexasaccharide conjugate S28 antigen solution
(1 µg/mL). Inhibition experiments with Yst9.1 and Yst9.2
employed plates coated with the hexasaccharide conjugate
S30 antigen solution (1 µg/mL).
16 S. Villeneuve, A. Boutonnier, L. A. Mulard and J. M. Fournier.
Microbiology, 1999, 145, 2477.
17 K. Jann and O. Westphal, “Microbial polysaccharides”. in The
Antigens (M. Sela, ed), 1975 Vol.
Press, Inc., New York.
3, pp. 1–125, Academic
18 V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel,
J. Godfroid, J. N. Limet and J. J. Letesson. Infect. Immun.,
1997, 65, 1939.
19 A. Cloeckaert, V. Weynants, J. Godfroid, J. M. Verger, M.
Grayon and M. S. Zygmunt. Clin Diagn Lab Immunol., 1998, 5,
862.
20 M. S. Zygmunt, D. R. Bundle, N. V. Ganesh, J. Guiard and A.
Cloeckaert, Clin. Vaccine Immunol. 2015, 22, 979.
21 D.R. Bundle, M. Gerken and T. Peters. Carbohydr. Res., 1988,
174, 239.
Monoclonal antibody was diluted to a concentration twice
that of the mid-point of the respective titration curve (typically
a dilution of mouse ascites of ~1:50,000). Antibody solution
was mixed with inhibitor dissolved in PBST at varying
concentrations and the resulting solutions were added to the
coated microtitre plate in triplicate and incubated at room
temperature for 2 h. Plates were washed with PBST (5 times)
and developed as described above.
22 T. Peters and D. R. Bundle. Can. J. Chem., 1989, 67, 491.
23 T. Peters and D. R. Bundle. Can. J. Chem., 1989, 67, 497.
24 J. Guiard, E. Paszkiewicz, J. M. Sadowska and D. R. Bundle.
Angew. Chem. Int. Ed., 2013, 52, 7181.
Acknowledgements
This work was supported by a Discovery grant from the
Natural Science and Engineering Research Council of Canada
(NSERC) and in part by Bill and Melinda Gates Grand Challenges
Round 11 funding.
́
25 R. Saksena, A. Chernyak, and P. Kovac. Carbohydr. Res.,
2008, 343, 1693.
26 F. Bien and T. Ziegler. Tetrahedron: Asymmetry 1998, 9, 781.
27 S. S. Mandal, L. Duncombe, N. V. Ganesh, S. Sarkar, L.
Howells, P. Hogarth, D. R. Bundle and J. McGiven, ACS Cent.
Sci., accepted Feb 15 2017.
28 P. V. Nikrad, H. Beierbeck and R. U. Lemieux. Can. J.
Chem.,1992, 70, 241.
29 R. U. Lemieux. Acc. Chem. Res., 1996, 29, 373.
30 D. R. Bundle, J. W. Cherwonogrodzk and M. B. Perry.
Biochemistry, 1987, 26, 8717.
Notes and references
Department of Chemistry, University of Alberta, Edmonton,
Alberta, T6G 2G2
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
a
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M. J. Corbel, S.S. Elberg and O. Cosivi. Brucellosis in humans
and animals. 2006, World Health Organization, Geneva
I. Maudlin and S. Weber. The control of neglected zoonotic
diseases: a route to poverty alleviation. 2006, WHO/SDE/
FOS/2006.1. Geneva
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B. B. Singh, N. K. Dhand, J. P. Gill. Prev. Vet. Med. 2015, 119,
211.
K. Nielsen. Vet. Microbiol. 2002, 90, 447.
10 | J. Name., 2012, 00, 1-3
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