ELISA for Fenarimol
J. Agric. Food Chem., Vol. 52, No. 24, 2004 7207
1.41 (OCH2CH2CH2CH2CH2COOH), 1.27 (2H, OCH2CH2CH2CH2CH2-
COOH); 13C NMR (600 MHz, DMSO-d6) δ 177.568, 157.094, 156.062,
139.572, 138.073, 136.658, 132.975, 132.797, 132.219, 130.849,
130.769, 129.844, 128.768, 127.804, 83.216, 64.115, 29.279, 25.818,
25.395; low FAB (+)-MS, m/z 445 [M + H]+.
Hapten-2 (Compound 5), (()-5-[(2-Chlorophenyl)-(4′-chlorophenyl)-
pyrimidin-5-yl-methoxy]pentanoic Acid. NaH was added slowly to
fenarimol (99.36 mg, 0.3 mmol) dissolved in 5 mL of dry DMF at 0
°C until no more hydrogen gas was generated. The mixture was stirred
at room temperature for 5 h to form the sodium salt (compound 1).
Ethyl 5-bromovalerate (156.8 mg, 0.75 mmol) in 2 mL of DMF was
reacted with the resulting oxygen nucleophile. After 48 h of stirring at
60 °C, DMF was removed on a rotary evaporator under reduced pressure
(compound 3). The subsequent hydrolysis and purification procedures
of compound 3 were the same as those for hapten-1 (Figure 1): 1H
NMR (CDCl3) δ 9.11 (s, 1H, pyrimidine H), 8.84 (s, 2H, pyrimidine
H), 7.18-7.37 (m, 8H, 2Ar), 3.06 (t-like, 2H, OCH2CH2CH2CH2-
COOH), 2.22 (m, 2H, OCH2CH2CH2CH2COOH), 1.74 (m, 4H,
OCH2CH2CH2CH2COOH); low FAB (+)-MS, m/z 431 [M + H]+.
Conjugation of Carboxylic Acid Haptens to Carrier Proteins.
Haptens were conjugated to carrier proteins (BSA, KLH, and OVA)
using the activated ester method (11). That is, hapten-1 was coupled
to KLH and BSA. Meanwhile, hapten-2 was conjugated to KLH, BSA,
and OVA. Instead, 4,4′-DDA was conjugated only to BSA. The
carboxylic acid haptens (0.04 mmol, each) were dissolved in 0.2 mL
of dry DMF with equimolar N-hydroxysuccinimide (NHS) and a 10%
molar excess of 1,3-dicyclohexylcarbodiimide. After 5 h of stirring at
22 °C, the precipitated dicyclohexylurea was removed by filtration,
and the resulting activated ester was added slowly to the protein solution
(10 mg of protein in 1 mL of 0.05 M borate buffer of pH 8) with
vigorous stirring. The reaction mixture was stirred gently at 4 °C for
24 h to complete the conjugation and then dialyzed exhaustively against
normal-strength PBS, which was changed twice a day for 5 days.
Finally, the conjugates were dispensed into 2 mL cryogenic vials, stored
at -80 °C, and used when needed.
Protein Determination. The protein contents of the hapten-protein
conjugates were determined spectrophotometrically at 595 nm according
to the Bio-Rad protein assay based on the method of Bradford (12).
Determination of the Coupling Density. The free amino groups
of the hapten-protein complex were determined according to a
modification of the trinitrobenzenesulfonic acid (TNBSA) method (13).
That is, 0.5 mg/mL of protein or 0.5 mg/mL of protein conjugate
solution in 1× PBS, 1.0 mL of carbonate buffer (1.59 g/L Na2CO3,
2.93 g/L NaHCO3, pH 9.6), and 1.0 mL of 0.1% TNBSA was reacted
at 40 °C for 2 h. After that, 1.0 mL of 10% sodium dodecyl sulfate
(SDS) was added, and the reaction was terminated with 0.5 mL of 1
M HCl. After thorough mixing, the absorbance was measured at 335
nm. The coupling density was estimated by comparing the absorbance
with the corresponding values of hapten-free proteins.
Figure 1. Procedure for the synthesis of haptens.
performed on 96-well microtiter plates (Nunc-Immuno plate, MaxiSorp
surface, Roskilde, Denmark) and read spectrophotometrically with a
microplate reader, Bio-Rad model 550 (Hercules, CA).
Buffer Solutions. Buffer solutions used for the immunoassay include
normal strength phosphate-buffered saline (1× PBS; 8 g/L NaCl, 0.2
g/L KH2PO4, 1.2 g/L Na2HPO4, and 0.2 g/L KCl, pH 7.5), PBST (1×
PBS containing 0.05% Tween 20, pH 7.5), 0.1× PBST (0.1× PBS
containing 0.05% Tween 20), carbonate buffer (1.59 g/L Na2CO3, 2.93
g/L NaHCO3, pH 9.6), and 0.05 M borate buffer (19.1 g/L Na2B4O7‚
10H2O, pH 8).
Immunization. Female New Zealand white rabbits weighing 3 kg
were used for raising polyclonal antibodies. Routinely, 100 µg (protein
equivalent) of each immunogen (hapten-KLH conjugate) in 0.5 mL
of 0.85% saline was thoroughly emulsified with an equal volume of
Freund’s adjuvant. The emulsion was subcutaneously injected at five
different sites on the neck and back of each rabbit. Freund’s complete
adjuvant was used in the first injection, and Freund’s incomplete
adjuvant was used for the subsequent boost injections. Boosts were
given at 3-week intervals by the same method. On the seventh day
after each boost, ∼10 mL blood samples were taken from an ear vein
to check the titer of the antisera. The blood samples were allowed to
coagulate for ∼2 h at room temperature and then kept in a refrigerator
overnight. The serum was decanted and centrifuged at 800g, and the
supernatant was dispensed into cryogenic vials and stored at -80 °C.
Boosting injections and bleedings thereafter were done five times.
Checkerboard Titration. A checkerboard titration (14) was per-
formed for the antiserum collected from each rabbit by the homologous
indirect ELISA to select the antisera showing an absorbance of 0.5-
1.0. The coating antigen concentration ranged from 0.01 to 1 µg/mL,
and the antiserum dilution was between 1:16000 and 1:256000. Rabbits
A-C were immunized against hapten-1-KLH and rabbits D-F against
hapten-2-KLH (Table 1). Titers of the antisera raised by six rabbits
Hapten Synthesis and Verification. Hapten-1 (Compound 4), (()-
6-[(2-Chlorophenyl)-(4’-chlorophenyl)pyrimidin-5-yl-methoxy]hexano-
ic Acid. Sodium hydride (NaH) was added slowly to fenarimol (99.36
mg, 0.3 mmol) dissolved in 5 mL of dry dimethylformamide (DMF)
at 0 °C until no more hydrogen gas was generated. The mixture was
stirred at room temperature for 5 h to form the sodium salt (compound
1). Then, ethyl 6-bromohexanoate (167.34 mg, 0.75 mmol) in 2 mL of
DMF was reacted with the resulting oxygen nucleophile. After 48 h of
stirring at 60 °C, DMF was removed on a rotary evaporator under
reduced pressure to obtain compound 2, which was hydrolyzed with
1.5 mL of 1 N NaOH at 65 °C for 1.5 h. The reaction mixture was
cooled and poured into 50 mL of distilled water. The aqueous solution
was washed with 50 mL of ethyl acetate to remove some impurities,
acidified to pH 2 with 6 N hydrochloric acid, and then extracted with
ethyl acetate (2 × 50 mL). The combined organic layer was dried over
anhydrous sodium sulfate and concentrated on a rotary evaporator under
reduced pressure. The residue was purified by silica gel flash chro-
matography using ethyl acetate/n-hexane as an eluent to obtain hapten-1
(compound 4), a yellow solid (Figure 1): 1H NMR (DMSO-d6) δ 9.06
(s, 1H, pyrimidine H), 8.76 (s, 2H, pyrimidine H), 7.38-7.75 (m, 8H,
2Ar), 2.94 (m, 2H, OCH2CH2CH2CH2CH2COOH), 2.03 (t, 2H, OCH2-
CH2CH2CH2CH2COOH), 1.57 (2H, OCH2CH2CH2CH2CH2COOH),