Page 9 of 24
Journal of the American Chemical Society
103.1, 102.8, 99.1 (x2), 78.7, 77.4 (x2), 75.4, 75.3, 70.2, 69.7, 69.6, 69.1,
General Bioconjugation Procedures.
69.0, 68.9, 68.7 (x3), 68.5, 68.4, 53.0, 52.6, 51.5, 45.1, 44.9, 40.3, 40.1,
36.7, 32.4, 29.2, 26.1, 26.0, 25.9 (x2), 19.0 (x2), 17.8 (x3), 17.6, 13.9
ppm; HRMS (ESI): m/z calcd for C37H59N5O17Na [M+Na]+: 868.3798,
found: 868.3791.
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5
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Method A: BSA Conjugates. BSA (0.451 µmol) and oligosaccharide
squarate 41ꢀ46 (6.77 µmol) were dissolved in 0.5 M borate buffer pH 9
(600 µL) and stirred slowly at 21 oC for 3 days. Then, the reaction mixture
was diluted with MiliꢀQ water (5 mL), filtered through an Amicon Milliꢀ
pore filtration tube (10,000 MWCO, 4 x 10 mL), lyophilized and the BSAꢀ
conjugates 47ꢀ52 were obtained as a white foam. MALDIꢀTOF mass
spectrometry analysis indicated the conjugates had an average of 12ꢀ16
oligosaccharides per BSA.
1ꢀ[(2'ꢀAminoethylamido)carbonylpentyl 4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀ
Dꢀmannopyranosyl
mannopyranosyl
mannopyranosyl
(1→2)
(1→3)
(1→2)
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
mannopyranoside]ꢀ2ꢀbutoxycyclobuteneꢀ3,4ꢀdione (44). Analytical data
for 44: Rf = 0.20 (CH3OH/CH2Cl2, 1/4, v/v); 1H NMR (600 MHz, D2O): δ
8.18ꢀ8.24 (Z) and 7.98ꢀ8.06 (E) (m, 4H, NCHO), 4.90ꢀ5.10 (m, 4H, 4 x Hꢀ
1), 4.65ꢀ4.73 (m, 2H, ꢀCH2iꢀ), 3.76ꢀ4.20 (m, 16H, 4 x Hꢀ2, 4 x Hꢀ3, 4 x Hꢀ
4, 4 x Hꢀ5), 3.57ꢀ3.73 (m, 3H, ꢀOꢀCH2aꢀ, ꢀCH2gꢀ), 3.45ꢀ3.52 (m, 1H, ꢀOꢀ
CH2bꢀ), 3.36ꢀ3.43 (m, 2H, ꢀCH2hꢀ), 2.18ꢀ2.25 (m, 2H, ꢀCH2fꢀ), 1.74ꢀ1.82
(m, 2H, ꢀCH2jꢀ), 1.49 ꢀ1.63 (m, 4H, ꢀCH2eꢀ, ꢀCH2cꢀ), 1.38ꢀ1.48 (m, 2H, ꢀ
CH2kꢀ), 1.20ꢀ1.35 (m, 14H, ꢀCH2dꢀ, 4 x Hꢀ6), 0.87ꢀ0.96 ppm (m, 3H, ꢀ
CH3l); 13C NMR (126 MHz, D2O): δ 189.8, 189.6, 184.3 (x2), 178.5,
178.2, 178.1, 178.0, 177.9, 174.8, 174.7, 168.8, 168.7, 168.6, 165.8,
165.6, 103.3 (x2), 103.2, 102.8 (x2), 102.7, 101.7 (x2), 101.6 (x2), 99.2,
78.9 (x2), 78.4, 78.1, 77.3, 75.4, 75.3, 69.9 (x2), 69.8 (x2), 69.7, 69.3,
69.0 (x2), 68.9, 68.8 (x4), 68.6 (x2), 68.5, 68.4 (x2), 68.0, 62.5, 52.9, 52.8
(x2), 52.7, 51.8, 45.2, 45.0, 44.2, 40.6, 40.3, 40.2, 36.7, 34.4, 32.4, 29.2,
26.2, 26.1, 26.0 (x2), 25.9 (x2), 25.8, 19.3, 19.1, 19.0, 18.1, 18.0 (x2),
17.9, 17.8 (x2), 17.7 (x2), 14.0, 13.9 ppm; HRMS (ESI): m/z calcd for
C44H70N6O21Na [M+Na]+: 1041.4486, found: 1041.4484.
Method B: Tetanus Toxoid Conjugates. Oligosaccharide squarate
45ꢀ46 (0.403 µmol) was added to the solution of monomeric tetanus toxꢀ
oid (0.0133 µmol) in 0.5 M borate buffer pH 9 (1 mL) and stirred slowly
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16
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26
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32
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34
35
36
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43
44
45
46
47
48
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57
58
59
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at 21 C for 3 days. Then, the reaction mixture was subjected to buffer,
exchange with PBS, filtered through an Amicon Millipore filtration tube
(10,000 MWCO, 4 x 10 mL) and the resulting tetanus toxoidꢀconjugates
53ꢀ54 were stored in PBS buffer. The MALDIꢀTOF mass spectrometry
analysis indicated the conjugates had an average of 12ꢀ15 oligosaccharides
per tetanus toxoid.
ELISA Procedures
Antigen Coating Efficiency
Optimal antigen coating for ELISA plates was established by applying
increasing dilutions of antigens 47ꢀ52 across the rows of an ELISA plates.
Antigen solution was prepared at 10 µg/mL and successive wells across
the plate were incubated with √10 dilutions of the antigen. This resulted in
12 wells with antigen coat beginning at 10 µg/mL and ending at 0.1
ηg/mL. Coated plates were then incubated with √10 dilutions of mAb
such that each row received the same dilution of antibody. Bound antiꢀ
body was detected as described below. The resulting three dimensional
matrix showed that plates coated with antigen solutions within the range
10ꢀ1 µg/mL gave virtually identical titration results.
1ꢀ[(2'ꢀAminoethylamido)carbonylpentyl 4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀ
Dꢀmannopyranosyl
mannopyranosyl
mannopyranosyl
mannopyranosyl
mannopyranosyl
(1→2)
(1→3)
(1→2)
(1→2)
(1→2)
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
Antibodies
Murine mAbs were as previously described BM10 and
BM28,13 and YSt91 and Yst92.12 Bovine OIE international standard referꢀ
ence serum (OIESS) and high and low binding positive sera were provided
by the Animal Health Veterinary Laboratory Agency, UK. OIESS came
from cattle experimentally infected with A dominant B. abortus strain
544. The other two positive sera came from pooled field sera. Human sera
from Brucella culture positive and negative patients were supplied by Dr.
R Rennie, University of Alberta Hospital Microbiology Laboratory.
mannopyranoside]ꢀ2ꢀbutoxycyclobuteneꢀ3,4ꢀdione (45). Analytical data
for 45: Rf = 0.40 (CH3OH/CH2Cl2, 1/1, v/v); 1H NMR (700 MHz, D2O): δ
8.14ꢀ8.20 (Z) and 7.96ꢀ8.02 (E) (m, 6H, NCHO), 4.83ꢀ5.18 (m, 6H, 6 x Hꢀ
1), 4.62ꢀ4.69 (m, 2H, ꢀCH2iꢀ), 3.72ꢀ4.17 (m, 24H, 6 x Hꢀ2, 6 x Hꢀ3, 6 x Hꢀ
4, 6 x Hꢀ5), 3.66ꢀ3.69 (m, 1H, ꢀCH2gꢀ ), 3.60ꢀ3.65 (m, 1H, ꢀOꢀCH2aꢀ),
3.55ꢀ3.58 (m, 1H, ꢀCH2gꢀ ), 3.42ꢀ3.48 (m, 1H, ꢀOꢀCH2bꢀ), 3.32ꢀ3.41 (m,
2H, ꢀCH2hꢀ), 2.14ꢀ2.21 (m, 2H, ꢀCH2fꢀ), 1.71ꢀ1.79 (m, 2H, ꢀCH2jꢀ), 1.46ꢀ
1.60 (m, 4H, ꢀCH2eꢀ, ꢀCH2cꢀ), 1.36ꢀ1.44 (m, 2H, ꢀCH2kꢀ), 1.15ꢀ1.30 (m,
20H, ꢀCH2dꢀ, 6 x Hꢀ6), 0.88ꢀ0.93 ppm (m, 3H, ꢀCH3l); 13C NMR (126
MHz, D2O): δ 189.8, 189.7, 184.3, 178.5, 178.1, 177.9 (x2), 174.8, 174.7,
168.8, 165.9, 165.6, 103.3, 102.6, 101.6, 101.5, 99.2, 78.9, 78.5, 78.2,
77.8, 77.3, 75.4, 75.3, 70.8, 69.9, 69.7, 69.2, 69.0, 68.8, 68.7, 68.6, 68.5,
68.4, 57.9, 57.7, 53.0, 52.8, 52.7, 51.8, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4,
29.2, 26.1, 26.0, 25.9 (x2), 19.1, 19.0, 18.1, 18.0, 17.9 (x2), 17.8, 17.7,
13.9 ppm; HRMS (ESI): m/z calcd for C58H92N8O29Na [M+Na]+:
1387.5862, found: 1387.5864.
ELISA
Microtiter plates were coated with antigen solution (1 µg/mL for
murine and bovine ELISA and10 µg/mL for human sera 100 µL/well) by
incubation at 4° C for 18 h, then washed (5 times) with PBST (0.05%
Tweenꢀ20 in phosphate buffer saline, PBS). Plates were not subjected to a
blocking step. Serial √10 dilutions of immune sera or monoclonal antibodꢀ
ies were made in PBST containing 0.1% BSA (bovine sera were used at a
starting dilution of 1:100 and human or murine mAbs were used at a startꢀ
ing dilution of 1:1000). The solutions were distributed in triplicate on
1ꢀ[(2'ꢀAminoethylamido)carbonylpentyl 4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀ
o
coated microtiter plates and incubated at 21 C for 2 hours. Plates were
Dꢀmannopyranosyl
mannopyranosyl
mannopyranosyl
mannopyranosyl
mannopyranosyl
(1→2)
(1→2)
(1→2)
(1→2)
(1→2)
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
4,6ꢀdideoxyꢀ4ꢀformamidoꢀαꢀDꢀ
washed with PBST (5 times). Goat antiꢀmouse IgG, antiꢀbovine IgG and
antiꢀhuman IgG secondary antibodies conjugated to horseradish peroxiꢀ
dase (Kirkegaard
& Perry Laboratories; 1:5000 dilution in 0.1%
BSA/PBST; 100 ꢀL/well) were added. The mixture was then incubated
o
for 30 min at 21 C, and then washed (5 times) with PBST. Peroxidase
mannopyranoside]ꢀ2ꢀbutoxycyclobuteneꢀ3,4ꢀdione (46). Analytical data
for 46: Rf = 0.40 (CH3OH/CH2Cl2, 1/1, v/v); 1H NMR (700 MHz, D2O): δ
8.14ꢀ8.19 (Z) and 7.97ꢀ8.01 (E) (m, 6H, NCHO), 4.82ꢀ5.18 (m, 6H, 6 x Hꢀ
1), 4.62ꢀ4.70 (m, 2H, ꢀCH2iꢀ), 3.72ꢀ4.15 (m, 24H, 6 x Hꢀ2, 6 x Hꢀ3, 6 x Hꢀ
4, 6 x Hꢀ5), 3.66ꢀ3.69 (m, 1H, ꢀCH2gꢀ ), 3.60ꢀ3.65 (m, 1H, ꢀOꢀCH2aꢀ),
3.55ꢀ3.58 (m, 1H, ꢀCH2gꢀ ), 3.42ꢀ3.48 (m, 1H, ꢀOꢀCH2bꢀ), 3.31ꢀ3.41 (m,
2H, ꢀCH2hꢀ), 2.14ꢀ2.21 (m, 2H, ꢀCH2fꢀ), 1.71ꢀ1.79 (m, 2H, ꢀCH2jꢀ), 1.46ꢀ
1.59 (m, 4H, ꢀCH2eꢀ, ꢀCH2cꢀ), 1.35ꢀ1.44 (m, 2H, ꢀCH2kꢀ), 1.14ꢀ1.31 (m,
20H, ꢀCH2dꢀ, 6 x Hꢀ6), 0.87ꢀ0.93 ppm (m, 3H, ꢀCH3l); 13C NMR (126
MHz, D2O): δ 189.8, 189.6, 184.3, 178.5, 178.1, 177.9 (x2), 174.8, 174.7,
168.8, 165.9, 102.9, 101.5 (x3), 99.2 (x2), 78.5, 78.1(x2), 78.0, 75.4, 75.3,
71.4, 69.9, 69.2, 69.0, 68.9, 68.7, 68.6, 68.0, 57.8 (x2), 53.0, 52.9 (x2),
52.7, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 32.3, 32.2, 29.2, 26.1(x2), 25.9
(x2), 19.1, 19.0, 17.9 (x2), 17.8 (x2), 17.7, 13.9, 13.8 ppm; HRMS (ESI):
m/z calcd for C58H92N8O29Na [M+Na]+: 1387.5862, found: 1387.5856.
substrate, 3,3',5,5'ꢀtetramethylbenzidine (TMB) with H2O2 (1:1 mixture of
3,3',5,5'ꢀtetramethylbenzidine (0.4 g/L) and 0.02% H2O2 solution, Kirkeꢀ
gaard & Perry Laboratories) was added (100 ꢀL/well). After 15 minutes,
the reaction was quenched by addition of 1 M phosphoric acid (100
ꢀL/well). Absorbance was read at 450 nm.
End point titres are recorded as the dilution giving an absorbance 0.4
above a background ≤ 0.1.
9
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