3332 J. Agric. Food Chem., Vol. 46, No. 8, 1998
Wang et al.
bp 54 °C at 0.05 mmHg (bplit 62-63 °C at 0.4 mmHg, Wie et
by ethyl 4-bromobutyrate, followed by reaction with 2,6-
difluorobenzoyl isocyanate. The resultant ethyl ester was
hydrolyzed to provide hapten III. The active ester synthesis
of this hapten was similar to that above for hapten II.
1
al., 1982); H NMR (CDCl3) δ 7.01 (t, J ) 8.3 Hz, 2H), 7.48-
7.58 (m, 1H).
1-(4-Nitrophenyl)-3-(2,6-difluorobenzoyl)urea. A solution of
4.96 g (27.1 mmol) of 2,6-difluorobenzoyl isocyanate in 15 mL
of dry benzene was added to a stirred solution of 3.36 g (24.3
mmol) of 4-nitroaniline in 50 mL of dry benzene. The mixture
was stirred overnight at room temperature, the solvent was
removed by evaporation under reduced pressure, and the
residue was recrystallized from ethyl acetate to give yellow
crystals in 88% yield: mp 256-258 °C (mplit 260 °C, Yu and
Kuhr, 1976); TLC Rf 0.49 (ethyl acetate/toluene 1:2); 1H NMR
(CDCl3) δ 7.02 (t, J ) 8.3 Hz, 2H, aromatic), 7.44-7.55 (m,
1H, aromatic), 7.78 (d, J ) 9.2 Hz, 2H, aromatic), 8.23 (d, J )
9.2 Hz, 2H, aromatic), 10.95 (s, 1H, NH), 11.14 (s, 1H, NH).
1-(4-Aminophenyl)-3-(2,6-difluorobenzoyl)urea (Hapten I). A
solution of 5 g of 1-(4-nitrophenyl)-3-(2,6-difluorobenzoyl)urea
(15.6 mmol) in 85 mL of 1-methyl-2-pyrrolidone was reduced
with hydrogen over 10% palladium on activated carbon (0.5
g) under 1 atm at room temperature. No more hydrogen was
absorbed after overnight treatment. The reaction mixture was
then filtered through Celite, and 1-(4-nitrophenyl)-3-(2,6-
difluorobenzoyl)urea was precipitated as a yellow powder by
adding water. The pure product was obtained by recrystalli-
zation from ethyl acetate (3.9 g, 86%): mp 210-212 °C (dec);
found: C, 57.81%, H, 3.92%, N, 13.81%; requires: C, 57.73%,
H, 3.81%, N, 14.43%; TLC Rf 0.74 (ethyl acetate); 1H NMR
(acetone-d6) δ 6.67 (d, J ) 8.8 Hz, 2H, aniline), 7.17 (t, J )
8.3 Hz, 2H, aromatic), 7.31 (d, J ) 8.8 Hz, 2H, aniline), 7.59-
7.69 (m, 1H, aromatic); 13C NMR (acetone-d6) δ 112.82 (dd, J
) 3.1, 22.5 Hz, C3′ and C5′), 114.51 (t, J ) 21.6 Hz, C1′), 122.47
(s, C2 and C6 or C3 and C5), 122.60 (s, C3 and C5 or C2 and C6),
128.13, (s, C1 or C4), 134.02 (t, J ) 10.1 Hz, C4′), 146.24 (s, C4
or C1), 150.59, (s, NHCONH), 160.34 (dd, J ) 6.8, 250.9 Hz,
C2′ and C6′), 163.09 (s, aromatic CONH); mass spectrum, m/e
292 (M + 1, 88%), 158 (100), 141 (6), 135 (71), 107 (5).
Hemisuccinate of Hapten I (Hapten II). Seven hundred
milligrams of 1-(4-aminophenyl)-3-(2,6-difluorobenzoyl)urea
(2.4 mmol) and 720 mg of succinic anhydride (7.2 mmol) in
pyridine (25 mL) were refluxed for 3 h. The pyridine was
evaporated under reduced pressure, and the resulting black
oil was stirred with 30 mL of ethyl acetate. The precipitated
solid was recrystallized from ethyl acetate several times to give
hapten II as white needles (197 mg, 21%): mp 278-280 °C
(dec); found: C, 55.38%, H, 3.81%, N, 10.53%; requires: C,
55.25%, H, 3.86%, N, 10.47%); TLC Rf 0.73 (ethyl acetate/
P r ep a r a tion of Im m u n ogen s. Three haptens were con-
jugated to ovalbumin (OA) and keyhole limpet hemocyanin
(KLH). For hapten I, the diazo conjugate method (Sheth and
Sporns, 1991) was used with some modifications. Briefly,
hapten I (15.5 mg) dissolved in dry dimethylformamide (DMF)
was added to 2 mL of 3.5 N HCl, followed by 0.8 mL of 1%
sodium nitrite. After 10 min of stirring at 0 °C, 39.2 mg of
ammonium sulfate was added, and the reaction was stirred
for another 10 min at 0 °C. The diazonium salt was then
reacted with proteins (OA and KLH) in PBS buffer. The pH
of the reaction was adjusted to 9, and the reaction mixture
was kept stirring at 4 °C overnight and then dialyzed
extensively in phosphate-buffered saline (PBS, 50 mM sodium
phosphate-0.9% NaCl, pH 7.2) containing 0.01% sodium azide
and stored at 4 °C. For linking hapten I to OA through two
homolinkers 1,4-butanediol diglycidyl ether (BDE) and disuc-
cinimidyl suberate (DSS), the methods were adapted from
previously reports (Lommen et al., 1995; Paek et al., 1993).
For BDE, 50 mg of BDE was added to 20 mg of protein in 1
mL of 50 mM sodium carbonate buffer, pH 9.6, and the
solution was mixed overnight at room temperature. The
solution was then desalted on a PD-10 column (Pharmacia,
Uppsala, Sweden), and 10 mg of hapten I in dry dimethyl
sulfoxide (DMSO) was added. After mixing overnight at room
temperature, the mixture was dialyzed against PBS exten-
sively. For DSS, the hapten solution (10 mg/mL in dry DMSO)
was added to the cross-linker solution (20 mg/mL in dry
DMSO) such that there was a 2 molar excess of cross-linker.
This mixture was stirred for 30 min at room temperature. The
mixture was then added to 10 mg of protein in 2 mL of 10
mM phosphate buffer at pH 7, and a 20 molar excess of hapten
was used. After incubation for 2 h at room temperature, the
mixture was dialyzed against PBS as described above. The
conjugation method for the NHS activated ester of haptens II
and III was adapted from that of McAdam et al. (1992). The
active ester dissolved in dry DMF was slowly added to a
precooled buffer solution (50 mM K2HPO4, pH 9.1) containing
the protein, and the reaction solution was gently mixed by
hand and then left at 4 °C overnight. The next day the
conjugates were transferred to dialysis tubing and dialyzed
extensively. The concentration of the coupled protein was
determined using the Coomassie Brilliant Blue G-250 dye-
binding assay method (Bradford, 1976).
1
methanol/acetic acid 10:1:0.1); H NMR (methanol-d4) δ 2.65
(s, 4H, CH2CH2), 7.12 (t, J ) 8.2 Hz, 2H, aromatic), 7.49 (d, J
) 9.2 Hz, 2H, aniline), 7.55 (d, J ) 9.2 Hz, 2H, aniline), 7.48-
7.59 (m, 1H, aromatic); 13C NMR (methanol-d4) δ 30.37, 32.35
(s, CH2CH2), 113.03 (dd, J ) 3.1, 22.4 Hz, C3′ and C5′), 114.64
(t, J ) 20.5 Hz, C1′), 121.75 (s, C2 and C6 or C3 and C5), 122.08
(s, C3 and C5 or C2 and C6), 134.35 (t, J ) 10.3 Hz, C4′), 134.38
(s, C1 or C4), 136.61 (s, C4 or C1), 152.17 (s, NHCONH), 160.84
(dd, J ) 6.5, 251.8 Hz, C2′ and C6′), 164.15 (s, aromatic CONH),
172.71 (s, NHCOCH2), 176.27 (s, COOH); mass spectrum, m/e
392 (M + 1, 68%), 374 (23), 292 (32), 184 (100), 158 (62), 141
(52).
Preparation of Active Ester of Acid Derivative. One hundred
and thirty-one milligrams of hapten II (0.33 mmol) and 42.3
mg of N-hydroxysuccinimide (NHS; 36.7 mmol) were dissolved
in 4 mL of freshly distilled tetrahydrofuran (THF). To this
solution was added 75.8 mg of N,N-dicyclohexylcarbodiimide
(DCC; 36.7 mmol) with stirring. The reaction was stirred at
room temperature under N2 for 3.5 h. The precipitate was
filtered off and washed with THF, and the combined THF
filtrates were evaporated under reduced pressure. The result-
ing white solid residue was purified by flash chromatography
(ethyl acetate/acetone 2:1) to yield a white solid. The structure
was confirmed by its 1H NMR spectrum with succinimide
protons at δ 2.78 (CDCl3) and mass spectrum m/e 489 (M + 1,
4%), 391 (7), 374 (100), 348 (8), 331 (55), 315 (14).
P r ep a r a tion of En zym e Tr a cer s. HRP was used for the
preparation of enzyme conjugates. The methods were similar
to those described for the conjugation of hapten to the protein,
except that the diazo method was not chosen for conjugating
hapten I to the enzyme. The concentration of coupled enzyme
in solution was determined using an extinction coefficient,
1cm
403
E
) 2.25 for 1 mg/mL solution (McAdam et al., 1992).
An tibod y P r od u ction . Female New Zealand white rabbits
were immunized by intradermal and intramuscular injections
of haptens conjugated to KLH and OA. The initial immunizing
dose consisted of 1 mg of hapten-protein conjugate in 0.5 mL
of 0.9% NaCl (saline) and 0.5 mL of Freund’s complete
emulsion. Subsequent booster injections (0.5 mg of conjugate
in 0.5 mL of saline-0.5 mL of Freund’s incomplete emulsion)
were performed 2, 4, and 6 weeks later and then at monthly
intervals. Blood was collected from the marginal ear vein 9
days after each monthly injection. Antiserum was purified
using affinity chromatography on protein G-Sepharose (Ak-
erstrom et al., 1985). The concentration of the purified
antibody was determined by Beer-Lambert’s law using the
extinction coefficient of 1.35 for 1 mg/mL rabbit IgG and then
stored at 4 °C after 0.1% (w/v) sodium azide was added. Two
rabbits were immunized with each hapten-protein conjugate,
and the results shown were obtained from the fourth bleed of
individual rabbits.
The preparation of hapten III with handle attachment at
the middle nitrogen was carried out according to the method
of Wie et al. (1982). Briefly, 4-chloroaniline was derivatized
Dir ect Com p etitive ELISAs. The microwell plates were
coated with purified anti-BPU IgG at 1 µg per well in 100 µL