Detection of PCP in Soil Samples
J. Agric. Food Chem., Vol. 49, No. 3, 2001 1289
10.99 (s, 1 H, broad), 10.06 (s, 1 H), 7.59 (d, 2 H, J ) 8.5 Hz),
7.19 (d, 2 H, J ) 8.5 Hz), 4.57 (s, 2 H), 3.51 (s, 2 H).
for ∼30 min, the enzymatic reactions were stopped by the
addition of 50 µL aliquots of a 3.0 M NaOH solution and the
plates were read immediately at 405 nm.
P r ep a r a tion of P CP )KLH (Im m u n ogen I). DMF (8 mL)
was added to a cold KLH solution (25 mL, 10 mg/mL) in 50
mM, pH 7.5, phosphate buffer. A solution of compound 2 (101
mg) in 2 mL of DMF was added dropwise into the KLH
solution, and the resulting solution was stirred at room
temperature overnight. The reaction solution was transferred
to a dialysis tubing with molecular weight cutoff of 2000 and
dialyzed extensively against a pH 7.5, 50 mM, phosphate
buffer. The dialyzed solution was filtered through a sterile 0.2
µm filter unit.
Extr a ction of Soil Sa m p les. Part of the soil samples were
spiked with PCP at a concentration of 4.94 µg/mL, and the
value was certified with GC-MS by Environmental Resource
Associates. Soil samples with 0.25, 0.50, 1.0, and 1.5 µg/mL of
PCP were prepared through dilution of the certified samples
with unspiked soil. All soil samples (5.0 g sample size),
including an unspiked one for use as a control, were extracted,
respectively, with a mixture of methanol/ethylene glycol (4:1,
v/v). Aliquots (∼11 µL) of the extraction solutions were directly
applied onto the OnTrak plates respectively for assay; the
recovery of the extraction was assessed with a RaPID Assays
system for PCP manufactured by Ohmicron Environmental
Diagnostics.
P r ep a r a tion of P CP )BTG (Im m u n ogen II). This im-
munogen was meant to be used as a backup and prepared
using the same procedure as outlined above for the preparation
of PCP-KLH immunogen, except that BTG was used in place
of KLH. After dialysis, the concentration of the modified BTG
was found to be 4.0 mg/mL and Lys modification 84%. The
overall protein recovery was 83%.
P r ep a r a tion of P CP )BSA Con ju ga te I. The conjugate
was made using a procedure similar to the preparation of
PCP-KLH immunogen, except that only 2 molar equiv of
compound 2 was allowed to react with BSA in a mixture of
pH 7.5, 50 mM, phosphate buffer/DMF (2.5:1, v/v).
P r ep a r a tion of P CP )BSA Con ju ga te II. The conjugate
was made using the procedure identical to the preparation of
PCP-BSA conjugate I, except that compound 4 was converted
to its NHS ester immediately before the conjugation with BSA
and, without isolation, the NHS ester formed in DMF was
directly added to a buffered BSA solution.
Ch a r a cter iza tion of th e Im m u n ogen s a n d Con ju ga tes.
The protein concentrations were determined by using the
Coomassie protein assay (7), and BSA was used as the
reference. For immunogens, the degree of PCP hapten sub-
stitution on carrier proteins was calculated on the basis of the
absorbance difference at 420 nm between the TNBS-deriva-
tized immunogen and TNBS-derivatized native carrier protein
(8, 9). On the other hand, the substitution of PCP-BSA
conjugate, which was used for further conjugation with mi-
croparticles, was estimated by using ELISA.
Develop m en t of th e P CP On Tr a k Im m u n oa ssa y. Mi-
croparticles (0.8 µm), prepared according to a previously
published procedure (6), were covalently coated with a set of
PCP-BSA conjugate and BSA mixtures using a published
procedure (11), respectively, where the ratios of PCP-BSA
conjugate versus BSA range from 1:1, 0.5:1, 0.25:1, to 0.125:
1. Each batch of the microparticles coated at a specific PCP-
BSA conjugate/BSA ratio was then mixed with various dilu-
tions (titers) of the anti-PCP antibody (antisera) and a reaction
buffer (pH 7.2, 50 mM, HEPES) to effect the agglutination
reaction. Each test result was visually inspected 3 min after
all of the reagents were mixed. The PCP-BSA conjugate/BSA
ratio and antibody titer were chosen in such a way that the
assay would give the most severe agglutination (scored 4
points) in the absence of free PCP and would give no ag-
glutination (complete inhibition, scored 0 points) once the free
PCP concentration reached g1 µg/mL in the sample tested.
With the assay parameters thus determined, a set of standard
solutions corresponding to PCP concentrations between 0 and
1.0 µg/mL produced agglutination at various degrees, which
yielded scores between 4 and 0 points.
RESULTS AND DISCUSSION
An im a l Im m u n iza tion . Four sheep and four goats were
placed on an immunization program using a method adapted
from the procedure of Erlanger (10). Each animal received
multiple site injections across the back using 1 mg of immu-
nogen I emulsified with complete Freund’s adjuvant. At the
second week, the animals received booster immunizations
containing 1 mg of the immunogen emulsified in incomplete
Freund’s adjuvant. The boost injections were repeated twice
in the following 2 weeks, followed by a monthly injection of
0.5 mg of the immunogen in incomplete Freund’s adjuvant for
a period of 6 months. The sheep and goats were then bled,
and antisera were separated from the clot by centrifugation.
Screening of the antisera was done using ELISA.
ELISA for Eva lu a tion of An tiser u m Affin ity to P CP )
BSA Con ju ga te I a n d th e Disp la cem en t of th e La tter in
th e P r esen ce of F r ee P CP . Polystyrene microplates were
coated with a conjugate I solution in phosphate-buffered saline
(PBS)/0.01% azide at a concentration of 5.0 µg/mL. The PBS/
azide buffer was made by adding 250 mg of KH2PO4, 1.38 g of
Na2HPO4, 250 mg of KCl, 9.0 g of NaCl, and 0.01% NaN3 into
1 L of water. The coating was conducted either at room
temperature for 2 h or at 4 °C overnight. The plates were then
washed three times with PBS/0.1% Tween 20, followed by the
addition of 50 µL aliquots of a 1% BSA solution (for displace-
ment evaluation, 50 µL aliquots of a 500 ng/mL solution of
free PCP in 1% BSA were added instead) and 50 µL diluted
solutions of an anti-PCP antiserum (in PBS containing 1%
BSA and 0.01% azide), respectively. The resulting plates were
incubated at 37 °C for 2 h. After five washes with PBS/Tween
20, 50 µL aliquots of a diluted solution of rabbit anti-sheep
antibody coupled with bovine alkaline phosphatase were added
to the wells. The plates were incubated at 37 °C for 2 h and
washed three times with PBS/Tween 20, and then 50 µL
aliquots of a 4-nitrophenyl phosphate solution in pH 9.8
diethanolamine buffer were added. After incubation at 37 °C
Compound 1, a close mimic of PCP in which the para-
chloro group was replaced by the oxygen, was chosen
as the hapten for conjugation to carrier proteins. The
compound was made from the reaction of 2,3,5,6-
tetrachlorohydroquinone with a large excess of 2-bromo-
acetic acid using a modified procedure of the original
method of Meyers et al. (12). The acid obtained was
converted to NHS ester by using EDC as the coupling
reagent; several byproducts were also produced in the
reaction, resulting in a low yield of the purified NHS
ester (2). The active ester (2), stable when kept desic-
cated and frozen, was readily utilized in the conjugation
with KLH and BTG. This gave immunogens I and II,
respectively, with good substitution ratios of the PCP
hapten (65 and 84%, respectively) on available Lys
residues of both carriers.
After extensive dialysis and subsequent filtration, the
immunogen I filtrate retained a KLH concentration of
3.96 mg/mL and the overall protein recovery was 85%.
The immunogen I thus prepared was given to four sheep
and four goats for immunization. After 6 months, the
animals were bled and the antisera were screened using
ELISA. It was found that the antiserum of sheep 1877
displayed the highest titer, whereas antisera of both
sheep 1877 and 1878 produced similar displacement
(∼50%) in the presence of free PCP molecules (Figure
1). The antiserum of sheep 1877 was therefore used in
the assay as the antibody reagent.
Another key reagent of the assay is the PCP-deriva-
tized microparticles. To prepare this reagent, the PCP