4502 J. Agric. Food Chem., Vol. 54, No. 13, 2006
Zhang et al.
mmol) was used. The residue was a yellow powder. Further purification
in silica gel columns using methanol/chloroform/ice acetic acid (1:20:
0.04) as eluant finally gave the product D2 (0.1564 g, 25.2% yield,
yellowish powder): TLC [methanol/chloroform/ice acetic acid (5:5:
Antibody Production. Antibodies were produced in rabbits as
described by Wang et al. (13). Six white rabbits (two for each
immunogen) were immunized by intradermal and intramuscular injec-
tions of the immunogens D1-KLH, D3-KLH, and D4-KLH. IgG
from the antisera was purified by Protein A-Sepharose 4B affinity
chromatography.
Screening of Antisera. Antisera titers, antibody coating quantity,
and enzyme conjugate dilution factors were optimized to give absorb-
ance values ranging from 0.7 to 1.2 in the absence of analytes using a
checkerboard titration. The antisera titers were tested for D1-KLH on
D1-OA coating, for D3-KLH on D3-OA coating, and for D4-KLH
on D4-OA coating using antigen-coated indirect ELISA format, and
the antisera were diluted from 1/1000 to 1/200000 to microtiter plates
coated with 1 µg per well of the hapten-OA conjugate. Similarly,
antibody coating quantity and enzyme conjugate dilution factors were
tested on an antibody-coated direct ELISA format using enzyme
conjugate dilutions from 1/1000 to 1/100000 and plates coated with
0.5, 1, or 1.5 µg per well of the antibodies
Indirect ELISA. Flat-bottom polystyrene ELISA plates were coated
with OA-hapten (D1-D5) conjugates at 1 µg per well in 100 µL of
CB and incubated overnight at room temperature. Plates were then
washed three times with 10 mmol L-1 PBS-T, and unbound active sites
were blocked with 200 µL of 1% BSA/PBS per well for 1 h. After the
plate had been washed four times, 100 µL of the appropriate sera
dilution in PBS was added per well and incubated for 1 h at room
temperature. After four washings, plates were incubated for 1 h with
peroxidase-labeled goat anti-rabbit immunoglobulins diluted 1:10000
in PBS (100 µL per well). After five washings, the HRP tracer activity
was measured by adding 150 µL per well of TMB substrate solution.
The enzymatic reaction was stopped after 30 min by adding 2.5 mol
L-1 H2SO4 (50 µL per well), and the absorbance was read in dual-
wavelength mode (450 nm as test and 650 nm as reference).
Direct ELISA. Polystyrene ELISA plates were coated with purified
antibodies at 0.5, 1, or 1.5 µg per well in 100 µL of CB, incubated
overnight at room temperature. Plates were then washed three times
with 10 mmol L-1 PBS-T, and unbound active sites were blocked with
200 µL of 1% BSA/PBS per well for 1 h. After the plate had been
washed four times, for competitive assays, 100 µL of standards in PBS
and 100 µL of HRP-haptens in PBS were then added to each well
and incubated for 1 h at room temperature. After five washings, the
HRP tracer activity was measured by adding 150 µL per well of TMB
substrate solution. The enzymatic reaction was stopped after 30 min
by adding 2.5 mol L-1 H2SO4 (50 µL per well), and the absorbance
was read in dual-wavelength mode (450 nm as test and 650 nm as
reference).
1
0.04)] Rf ) 0.76; H NMR (400 MHz, DMSO-d6) δ 12.167 (br, 1H,
COOH), 8.581 (s, 1H, SO2NH), 8.548 (d, 1H, J ) 4.4 Hz, CHar-N),
8.057 (d × d, 1H, J ) 8.8 Hz, 2.4 Hz, CHar-CCO), 7.537 (d, 2H, J )
8.8 Hz, CHar), 7.070 (d, 1H, J ) 8.8 Hz, CHar-CNH), 6.532 (d, 2H, J )
8.8 Hz, CHar), 6.029 (s, 2H, NH2); MS (ESI) [found: m/z 294.17 [M
+ H]+, calcd for C12H11N3O4S, M, 293]; IR (KBr, cm-1) 3474, 3375,
3234, 1707, 1635, 1595, 1538, 1505, 1383, 1298, 1276, 1139, 1058,
959, 837, 770, 681, 666, 583, 554.
Synthesis of Hapten 4-(4-Aminobenzenesulfonylamino)benzoic Acid
(D3). The above-described general procedure was applied to prepare
the hapten D3. For that, 0.2900 g of 4-aminobenzoic acid (2.12 mmol)
was used. The residue was a yellow oil, and further purification in
silica gel columns using methanol/chloroform/ice acetic acid (1:40:
0.08) as eluant finally gave the product D3 (0.4445 g, 71.8% yield,
yellow powder): TLC [methanol/chloroform/ice acetic acid (1:20:0.04)]
1
Rf ) 0.25; H NMR (400 MHz, DMSO-d6) δ 7.803 (d, 2H, J ) 8.0
Hz, CHar), 7.479 (d, 2H, J ) 8.0 Hz, CHar), 7.162 (d, 2H, J ) 8.0 Hz,
CHar), 6.575 (d, 2H, J ) 8.4 Hz, CHar), 6.071 (s, 2H, NH2); MS (ESI)
[found: m/z 606.86 [2M + Na]+, calcd for C13H12N2O4S, M, 292]; IR
(KBr, cm-1) 3494, 3464, 3391, 3373, 3167, 2930, 1698, 1633, 1608,
1595, 1505, 1460, 1407, 1324, 1291,1235, 1187, 1148, 1092, 1018,
912, 834, 769, 718, 678, 658, 578, 548.
Synthesis of Hapten 6-(6-Aminobenzenesulfonylamino)hexanoic Acid
(D4). The above-described general procedure was applied to prepare
the hapten D4. For that, 0.2777 g of 6-aminohexanoic acid (2.12 mmol)
was used. The residue was a yellow oil and purified in silica gel columns
using methanol/chloroform/ice acetic acid (1:20:0.04 and 1:10:0.02)
as eluant, finally giving the product D4 (0.2158 g, 35.6% yield, milky
powder): TLC [methanol/chloroform/ice acetic acid (1:10:0.02)] Rf )
0.61; 1H NMR (300 MHz, DMSO-d6) δ 11.957 (br, 1H, COOH), 7.403
(d × t, 2H, J ) 8.7 Hz, 2.4 Hz, CHar), 7.065 (t, 1H, J ) 6.0 Hz,
SO2NH), 6.607 (d × t, 2H, J ) 8.7 Hz, 2.3 Hz, CHar), 5.923 (s, 2H,
NH2), 2.519 (d × d, 2H, J ) 6.8 Hz, 6.3 Hz, NHCH2), 2.156 (t, 2H,
J ) 7.4 Hz, CH2COOH), 1.471-1.159 (m, 6H, CH2CH2CH2); MS(ESI)
[found: m/z 595.10 [2M + Na]+, calcd for C12H18N2O4S, M, 286];
IR(KBr, cm-1) 3413, 3326, 3126, 2937, 2859, 1699, 1627, 1596, 1502,
1467, 1429, 1304, 1250, 1231, 1197, 1143, 1095, 1036, 1013, 985,
936, 846, 678, 581, 563, 546.
Synthesis of Hapten 6-(4-Aminobenzenesulfonylamino)butanoic Acid
(D5). The above-described general procedure was applied to prepare
the hapten D5. For that, 0.2184 g of 4-aminobutanoic acid (2.12 mmol)
was used. The residue was a yellow oil and purified in silica gel columns
using methanol/chloroform/ice acetic acid (1:20:0.04, 1:10:0.02 and
1:5:0.01) as eluant, finally giving the product D5 (0.1538 g, 28.1%
yield, yellowish powder): TLC [methanol/chloroform/ice acetic acid
(1:10:0.02)] Rf ) 0.26; 1H NMR (300 MHz, DMSO-d6) δ 12.109 (br,
1H, COOH), 7.423 (d, 2H, J ) 8.7 Hz, CHar), 7.151 (t, 1H, J ) 5.7
Hz, SO2NH), 6.630 (d, 2H, J ) 8.7 Hz, CHar), 5.957 (s, 2H, NH2),
2.683 (d × d, 2H, J ) 6.0 Hz, 6.8 Hz, 6.3 Hz, NHCH2), 2.226 (t, 2H,
J ) 7.2 Hz, CH2COOH), 1.594 (m, 2H, CH2CH2CH2); MS (ESI)
[found: m/z 258.87 M + H]+, calcd for C10H14N2O4S, M, 258]; IR
(KBr, cm-1) 3413, 3336, 3127, 2950, 2622, 1907, 1699, 1633, 1596,
1501, 1432, 1305, 1261, 1216, 1188, 1147, 1090, 1012, 965, 896, 841,
682, 577, 548, 509.
Preparation of Enzyme and Protein Conjugates. The sulfonamide
derivatives were attached to the carrier proteins (KLH, OA) or HRP
using a mixed anhydride method using EDC without isolation of
intermediates. Haptens D1, D3, and D4 were coupled to KLH for use
as immunogens. All five haptens were coupled to OA as coating
proteins, coupled to HRP as enzyme tracers, respectively.
The carboxylic acid of the hapten (0.025 mmol) and 0.05 mmol of
EDC were dissolved in 1 mL of dry DMF. Then mixture was added
dropwise with mixing to 2 mL of solution of 10 mg of protein (KLH,
OA, or HRP) in 130 mmol L-1 sodium bicarbonate (pH 8.1). After
4-6 h of mixing at room temperature on a mixing wheal, another 4.7
mg of EDC was added. After mixing overnight at 4 °C on the mixing
wheel, the mixture was dialyzed against PBS.
Sample Preparation. Milk samples were bought from local markets.
Before the spike and recovery studies, each test sample was verified
to contain sulfonamides at <10 ng mL-1 by HPLC. For a spiking study,
20 mL milk samples were spiked with a single sulfonamide dissolved
in methanol at different levels, thoroughly mixed, and then centrifugated
for 10 min at 3000 rcf. After the fat layer had been discarded, 20 mL
of acetone was added and shaken by hand, then centrifugated for 10
min at 3000 rcf. The upper clear liquid was diluted with PBS and
analyzed.
The sample extracts were appropriately diluted before ELISA
analysis, and 1% BSA, 0.5% FG, or 0.05% Tween 20 was added to
the PBS to avoid the sample matrix effect.
Instrumentation for HPLC Analysis. The test sample was verified
using a Shimadzu (Tokyo, Japan) HPLC equipped with an LC-10AT
vp pump with a Hamilton injector (25 µL loop), a DGU-12A online
degasser, and a CTO-10AS vp column oven. A C18 reversed-phase
column (15 cm × 4.6 mm i.d., 5 µm) was used. The analyses were
performed at 270 nm, and the mobile phase was methanol/water (28:
72) (water pH value was adjusted to 3.5 before it was mixed with
methanol) at a flow rate of 1.0 mL min-1. The temperature of the
column oven was 35 °C.
RESULTS AND DISCUSSION
Hapten Synthesis. Five different sulfonamide haptens (D1-
D5) were previously reported for the detection of sulfonamides