3698 J. Agric. Food Chem., Vol. 51, No. 13, 2003
Lee et al.
TLC (methanol/methylene chloride (1:9, v/v; 1.5% acetic acid)): Rf,
0.40. H NMR (CDCl3): δ 1.71 (m, 4H, CH2), 2.39 (m, 4H, CH2),
subsequent boost injections. Boosts were given every 3 weeks in the
same manner. On the seventh day after each boost, about 10 mL of
blood sample was taken from an ear vein to check the titer of the
polyclonal antibody. The blood sample was allowed to coagulate for
about 2 h at room temperature and then stored overnight in a
refrigerator. The serum was decanted and centrifuged (800g), and the
supernatant was stored in conveniently sized aliquots at -80 °C. Boosts
were given six times.
1
2.38 (d, J ) 16.4 Hz, 3H, CH3S), 3.86 (d, J ) 13.3 Hz, 3H, CH3O),
9.14 (bd, 1H, NH). 13C NMR (CDCl3): δ 12.0 (d, J ) 3.6 Hz), 23.88,
23.93, 33.4, 36.2 (d, J ) 6.9 Hz), 53.7 (d, J ) 6.6 Hz), 174.7, 177.6.
The product was soluble in water. A 5 mg sample treated with NaHCO3
solution evolved gas. Acidification and extraction with ether made it
return to the acid showing no change on TLC.
For the synthesis of hapten-4 (19) 1454.4 mg (7.05 mmol) of DCC
in 10 mL of methylene chloride was added to a stirred solution of 665
mg (4.7 mmol) of methamidophos, 13, and 828.08 mg (5.17 mmol) of
mono-tert-butyl malonate, 15, in 15 mL of methylene chloride chilled
in an ice bath. A white precipitate formed almost immediately. Stirring
was continued at 0 °C for 1 h and at room temperature overnight. The
urea precipitate was filtered and washed with 10 mL of methylene
chloride. The filtrate was washed once with water, and the organic
layer was evaporated. The resulting yellow mixture was extracted with
reagent grade acetone, and the acetone solution was evaporated. The
concentrate was purified by silica gel flash chromatography using 100
mL of n-hexane, 100 mL of n-hexane/ethyl acetate/acetic acid (2:1:
0.1, v/v/v), and 100 mL of n-hexane/ethyl acetate/acetic acid (5:5:0.1,
v/v/v). Fractions containing pure product were stripped, and the residual
acetic acid was removed by the successive addition and removal of 40
mL of toluene, 20 mL of ethyl acetate, and 20 mL of methylene
chloride. Stripping the residue under high vacuum gave 300 mg of the
oily ester 17 (tert-butyl 3-(O,S-dimethylthiophosphoramido)-3-oxopro-
panoate). TLC (n-hexane/ethyl acetate/acetic acid (2:1:0.1, v/v/v)): Rf,
0.46 (twice developed). 1H NMR (CDCl3): δ 9.15 (bd, 1H, NH), 3.90
(d, J ) 13.3 Hz, 3H, CH3O), 3.32 (s, 2H, COCH2) 2.40 (d, J ) 16.1
Hz, 3H, CH3S), 1.40 (s, 9H, CH3).
Checkerboard Titration. Titers of the eight antisera were evaluated.
Rabbit nos. 8377, 8390, and 8391 were immunized against hapten-1-
KLH, nos. 301 and 302 were immunized against hapten-2-KLH, and
nos. 11425 and 11427 were immunized against hapten-3-KLH,
respectively. To check the titers of the antisera by the homologous
indirect ELISA, each antiserum was diluted 256 000-fold. A checker-
board titration (19) was performed with the antisera collected from
each rabbit. The checkerboard assay selected the combination of
antiserum dilution and coating antigen concentrations (hapten-BSA
conjugate) that demonstrated the greatest binding. The tested ELISA
for acephate used a coating antigen concentration between 0.01 and 1
µg/mL and an antiserum dilution between 16 000 and 256 000.
Indirect ELISA, Competitive Inhibition ELISA, and Cross-
Reactivities. Indirect ELISA and competitive indirect ELISA were
performed according to the method of Voller et al. (20) as modified
by Harrison et al. (21). For the checkerboard titration, an indirect ELISA
was conducted. That is, 96 well microtiter plates were coated with 100
µL/well of the hapten-BSA conjugate in a carbonate buffer and allowed
to stand overnight at 4 °C. On the following day, the plates were washed
five times with 0.1 × PBST and thoroughly tapped dry. Sites not coated
with the conjugate were blocked with 200 µL/well of 3% (w/v) skim
milk in 1 × PBS. After they were incubated at 37 °C for 1 h, the
plates were washed as described above. Antiacephate antiserum diluted
with 1 × PBS (100 µL/well) was added, and the plates were incubated
at room temperature for 1 h. After the plate was washed, 100 µL/well
of a secondary antibody, goat antirabbit IgG conjugated with horseradish
peroxidase diluted 1:10 000 with 1 × PBST, was added and the plates
were incubated for 1 h at room temperature. The plate was washed,
and 100 µL/well of a substrate solution (0.1 mL of 1% hydrogen
peroxide and 0.4 mL of 0.6% 3,3′,5,5′-tetramethylbenzidine in dimethyl
sulfoxide (DMSO) added to 25 mL of citrate-acetate buffer, pH 5.5)
was added to each well. After 15 min at room temperature, the reaction
was stopped by adding 50 µL/well of 4 N sulfuric acid. The yellow-
colored plate was read spectrophotometrically in a dual wavelength
mode at 450 nm using a reference wavelength of 655 nm. The amount
of the enzyme bound, as indicated by the change of the colorless
substrate to blue-colored product, is directly related to the amount of
the rabbit antihapten antibody bound to the plate-coating antigen.
A competitive inhibition ELISA was used to assess the specificity
of the antibody to free acephate and the cross-reactivities of structurally
related compounds to the antibody. For competition, 50 µL of standards
in assay buffer or diluted samples was placed in the wells and 50 µL
of the antiacephate antiserum diluted with assay buffer was added to
it. After it was mixed for 30 s and incubated at room temperature for
1 h, the plate was washed. The subsequent procedure was followed as
described for the indirect ELISA. With the inhibition ELISA format,
analytes that do not react with the antibody would produce absorbances
near 100% of the zero analyte control; conversely, analytes that do
react with the antibody would decrease the absorbance. Standard curves
were calculated by mathematically fitting experimental points to a four
parameter logistic equation (22) using a commercial software package
(Origin, Microcal).
This ester was readily converted to 200 mg of the oily acid, hapten-4
(19), by treatment with TFA as described above for 11. TLC (methanol/
methylene chloride/acetic acid (1:9:1.5, v/v/v)): Rf, 0.7 (three times
1
developed). H NMR (CDCl3): δ 9.15 (bd, 1H, NH), 3.90 (d, J )
13.3 Hz, 3H, CH3O), 3.41 (s, 2H, COCH2), 2.40 (d, J ) 16.1 Hz, 3H,
CH3S). Low FAB(+)-MS: m/z 227.9 [M + H]+.
Conjugation of Haptens with Carrier Proteins. The four haptens,
hapten-1, -2, -3, and -4, were conjugated with proteins (BSA and KLH)
by the active ester method (17), and hapten-5 (13), methamidophos,
was conjugated with a protein (BSA) by the diazotization method (18).
N-Hydroxysuccinimide (NHS) ActiVe Ester Method. The haptens,
except for hapten-5, were coupled covalently with the lysine moieties
of the carrier proteins such as KLH and BSA according to the activated
ester method. That is, each hapten (0.04 mmol) was dissolved in 0.2
mL of dry N,N-dimethylformamide (DMF) with equimolar NHS and a
10% molar excess of DCC. After the mixture was stirred at 22 °C for
5 h, the precipitated DCU was removed by filtration, and about 0.2
mL of the active ester was added slowly to a solution of the protein
(10 mg of protein in 1 mL of 0.05 M borate buffer at pH 8) with
vigorous stirring. The reaction mixture was stirred gently at 4 °C for
24 h to complete the conjugation and then dialyzed against normal
strength PBS, which was changed with fresh one twice a day for 5
days. Finally, the conjugates were dispensed in 2 mL cryogenic vials
and stored at -80 °C.
Diazotization Method. All steps of the preparation of the methami-
dophos-BSA conjugate were performed at 0 °C with stirring. A 1%
aqueous solution of sodium nitrite (NaNO2) was added dropwise to a
solution of methamidophos (200 mg in 15 mL of 0.1 N HCl) until a
positive starch iodide test was confirmed. After 30 min of additional
stirring, 5 mL of the diazotization mixture was added slowly to BSA
(5 mg/mL in the borate buffer, pH 9.0). The pH was maintained by
addition of 5 N NaOH. The mixture was stirred for 2 h and then
dialyzed as described above.
Because the cross-reactivity is defined as the ability of compounds,
structurally related to the target analyte, to bind to the specific antibody
raised against the analyte, some organophosphorus insecticides and a
major metabolite of acephate were tested for selectivity of the ELISA
by determining their respective IC50 values in the competitive assays.
Cross-reactivity values were calculated as the ratio of the IC50 of the
acephate standard to the IC50 of the test compounds and expressed as
a percentage.
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 a rabbit. Three rabbits were
used for each immunogen. Freund’s complete adjuvant was used in
the first injection, and Freund’s incomplete adjuvant was used for
Analyses of Some Acephate-Fortified Samples. Tap Water. For
the recovery test, five concentrations (2, 5, 25, 50, and 100 ng/mL) of
acephate in tap water were prepared with the acephate stock solution