Quantitative Determination of Quizalofop-p-ethyl
J. Agric. Food Chem., Vol. 54, No. 23, 2006 8683
Figure 1. Synthetic route for the preparation of the hapten.
(s), 1001 (m). Mass spectrum found (70 eV, two most intense ions
each 14 mass units above m/e 48): 50(7), 56(6), 63(5), 64(7), 76(9),
81(6), 91(10), 100(19), 109(8), 110(11), 119(5), 124(8), 136(35), 138-
(11), 149(8), 150(3), 163(51), 165(17), 173(1), 179(3), 181(3), 191-
(3), 192(14), 208(6), 215(5), 216(3), 217(2), 243(38), 255(12), 257(5),
271(12), 272 (11), 285(2), 287(1), 299(74), 301(34), 344(100), 346-
(32).
Preparation of Hapten-Protein Conjugates. ActiVe Ester Method
(21). The quizalofop acid hapten (68.9 mg) was dissolved in DMF (1
mL), and then a solution of N-hydroxysuccinimide (NHS, 69 mg) and
N,N-dicyclohexylcarbodiimide (DCC, 61.8 mg) in DMF (2 mL) was
added. The mixture was stirred for 12 h at room temperature, filtered,
and one-half of the filtrate was added to BSA (40 mg) in 4 mL of PBS
(pH 7.4) with stirring, and then the reaction mixture was stirred at
4 °C overnight. After that, the solution was dialyzed in PBS at 4 °C
for 72 h and stored at -20 °C. Characterization of the conjugate was
done using spectroscopy (21). The conjugate was applied as an
immunogen (Hapten-BSA).
Mixed Anhydride Reaction (22). The hapten was coupled to OVA
via a mixed anhydride reaction. The hapten (34.5 mg) and tri-n-
butylamine (75 µL) were dissolved in dimethyl formamide (DMF, 1
mL). To this solution was added isobutylchlorocarbonate (20 µL), the
formation of the mixed anhydride was allowed to proceed for 20 min,
and the mixture was stirred at room temperature. The solution was
then added dropwise to OVA (60 mg) in 5 mL of PBS (pH 7.4) with
stirring. The mixture was stirred for 2 h and then dialyzed in PBS at
4 °C for 72 h and stored at -20 °C. Characterization was finished
using spectroscopy (21). The hapten was covalently attached to OVA
as coating antigen.
Immunization of Rabbits and Screening of Antisera. The method
of producing antibody was described before (23); three New Zealand
white rabbits (about 2 kg) were used to immunize with Hapten-BSA.
The antigen (1.0 mg of Hapten-BSA) dissolved in PBS (1.5 mL) was
emulsified with Freund’s complete adjuvant (1:1 volume ratio) and
injected intradermally at multiple sites (about 40-50 µL per site, 20-
25 sites) on the back of each rabbit. Three weeks later, the animals
were boosted with an additional 1.0 mg of immunogen (Hapten-BSA)
that was emulsified with Freund’s incomplete adjuvant (1:1 volume
ratio). Three booster injections were conducted on a 2-week interval.
The rabbits were bled 7-10 days later after each boosting to screen
antisera. After this, the last boosting was performed with another
additional 2.0 mg (immunogen Hapten-BSA) of the conjugate and
physiological saline (1:1 volume ratio). The serum was isolated by
centrifugation (10 000g, 10 min, 4 °C), and sodium azide (NaN3) was
added as a preservative at a final concentration of 0.02%. Antiserum
was then aliquotted and stored at -20 °C.
quizalofop-p-ethyl. In this paper, we developed an ELISA for
quizalofop-p-ethyl, and the evaluation of the assay’s perfor-
mance in water and soil is described.
MATERIALS AND METHODS
Reagents. All reagents and solvents were analytical grade. DCC,
dry pyridine, Tween-20, tri-n-butylamine, DMSO, isobutylchlorocar-
bonate, NHS, and DMF were obtained from Sino-American Biotech-
nology Co. (Henan, China). BSA, OVA, Freund’s complete and
incomplete adjuvants, goat antirabbit IgG-horseradish peroxidase, and
TMB were purchased from Sigma Chemical Co. (Shanghai, China).
96-Well polystyrene microplates (Maxisorp) were obtained from Nunc
(Roskilde, Denmark). NaN3 and concentrated HCl were purchased from
Chemical Reagent Co. (Shanghai, China).
Buffers: 0.01 mol/L of PBS (pH 7.4, 1.42 g of Na2HPO4, 0.27 g of
KH2PO4, 8.01 g of NaCl, 0.2 g of KCl in 1 L of distilled water); 0.05
mol/L of CBS (pH 9.6, 1.59 g of Na2CO3, 2.93 g of NaHCO3 in 1 L
of distilled water); PBST (0.01 mol/L of PBS plus 500 µL of Tween-
20).
Pesticide Standards. For the development of enzyme immunoassays
and specificity studies, the following analytical standards were obtained
from the United States or the Jiangsu Branch of the National Pesticide
R&D South Center, China: quizalofop-p-ethyl, fenoxaprop-p-ethyl,
benazolin-ehtyl, quinclorac, cyhalofop-butyl, (R)-haloxyfop, and
imazezethapyr. The quizalofop-p-ethyl standard sample was dissolved
in methanol to 8 µg/mL for stock solution, which was diluted into the
working range of this assay with PBS in volumetric flasks, and these
solutions were used within 30 min to avoid pesticide loss through
adhesion to glass surface. The organic solvent concentration in PBS is
negligible.
Instruments. The NMR spectrum was obtained via an AV300
spectrometer (Bruker, Switzerland). Chemical shift values are given
in parts per million (ppm) downfield from internal standard tetramethyl
silane. Coupling constants are expressed in hertz, and the abbreviations
s, d, t, and q represent singlet, doublet, triplet, and quartet, respectively.
MS was obtained by a HP-5973 (Agilent, USA). The IR spectrum was
recorded by Bruker Vertex 70FT-IR (Germany). The characterization
of hapten with protein conjugate (UV spectra) was recorded on a
Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). ELISA
plates were washed with a Wellwash 4 MK 2 (Labsystems, Finland).
ELISA experiments were performed in 96-well polystyrene microplates
(Maxisorp), and the absorbances were read by a Multiskan Ascent
microtiter plate reader (Labsystems, Finland) at 450 nm.
Hapten Synthesis. Synthesis of the hapten was carried out as
outlined in Figure 1. The reaction was straightforward, and the yield
was high.
The titer of the sera from each animal was screened by measuring
the binding of serial dilutions to microtiter plates coated with several
concentrations of Hapten-OVA. Optimal concentrations for coating
antigen and antisera dilution were determined by performing two-
dimensional titration experiments (24).
Quizalofop-p-ethyl (0.02 mol) was added to 8.4 mL of 10% NaOH
(0.021 mol) liquid solution (1:1.05 mol ratio). The mixture was stirred
at room temperature for 2 h and then heated to 60 °C for 1 h. The
mixture was heated again to reflux for 6 h until the liquid was clear.
If there were some indiscernible solids, the mixture was filtered. The
filtrate was treated with concentrated HCl to adjust the pH to 1-2.
Thin yellow sedimentation was collected and filtered. The residue was
dried under infrared light. The hapten was obtained as a straw yellow
solid.
The straw yellow solid was analyzed via NMR, IR, and MS (92%
yield). 1H NMR (DMSO): δ 1.52 (d, 3-CH3), 4.85 (q, 1, CH), 6.94 (d,
2, 2-H, 6-H), 7.24 (d, 2, 3-H, 5-H), 7.73 (s, 2, 7′-H, 8′-H), 8.13 (s, 1,
3′-H), 8.88 (s, 1, 5′-H). IR (solid, KBr): cm-1 3000-2622 (m), 1718
(vs), 1569 (s), 1504 (vs), 1446 (s), 1399 (s), 1213 (s), 822 (s), 1131
Indirect Competitive ELISA. The coated-antigen assays under the
optimized conditions were performed as follows.
Serial dilutions of the analyte standard in PBS and the antiserum
dilution (1/100 000) were individually prepared. Micro-titer plates were
coated overnight at 4 °C with Hapten-OVA (2.05 µg/mL, 100 µL per
well) in CBS. After the coated plates were washed three times with
PBST (PBS containing 0.05% Tween-20, pH 7.4), 200 µL of blocking
solution (1% OVA in PBS) was added and incubated for 1 h at 37 °C.
After another washing step, 50 µL/well of inhibitor solution and 50
µL/well of the antiserum were added and shaken for 1 min, and the