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Competitive ELISA of Thiabendazole Residues in Produce Using Indirectly

Immobilized Monoclonal Antibodies
David L. Brandon a; Ronald G. Binder a; Anne H. Bates a; William C. Montague Jr a
a
Food Safety and Health Research Unit, USDA Agricultural Research Service, Western Regional Research
Center, Albany, CA, USA

Online Publication Date: 01 January 1995

To cite this Article Brandon, David L., Binder, Ronald G., Bates, Anne H. and Montague Jr, William C.(1995)'Competitive ELISA of
Thiabendazole Residues in Produce Using Indirectly Immobilized Monoclonal Antibodies',Food and Agricultural Immunology,7:2,99 —
108
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 Food & Agricultural Immunology (1995) 7, 99-108

Competitive ELISA of
Residues in
Produce Using Indirectly Immobilized Monoclonal
Antibodies
DAVID L. BRANDON, RONALD G. BINDER, ANNE H. BATES AND
WILLIAM C. MONTAGUE, JR
Food Safety and Health Research Unit, USDA Agricultural Research Service,
Downloaded By: [University of Georgia] At: 22:23 29 June 2009

Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710,

USA
(Received for publication 3 January 1995)

An enzyme-linked immunosorbent assay (ELISA), developed originally for analysis of

thiabendazole (2-[4-thiazolyl] 1H-benzimidazole, TBZ) residues in bovine liver, was used to
analyze residues of this post-harvest fungicide in the peel of apple, potato, orange,
grapefruit and banana. Residues were extracted by soaking peels overnight in 80%
methanol and filtering the decanted supernatants. A 20-fold dilution eliminated significant
matrix effects. The ELISA has a limit of detection of 0.1 ppm in peel samples, corresponding
to 10-40 ppb in the whole fruit or tuber. The ELISA employs a monoclonal antibody layer,
immobilized indirectly via a rabbit anti-mouse IgG secondary antibody, and horseradish
peroxidase-conjugated 5-succinamido-TBZ. Assay wells coated in this way could be dried
and stored for at least 6 months without detectable changes in assay parameters. The
method exceeds the required sensitivity for regulatory monitoring of TBZ residues in the
US, and offers greater speed and specificity than previously reported methods.

Keywords: Thiabendazole, ELISA, peel, extraction

INTRODUCTION
Thiabendazole (2-[4-thiazolyl]l#-benzimidazole, TBZ) is a widely used fungicide, applied
primarily as a post-harvest dip or spray to citrus, potatoes and pome fruits. Its mode of
action and toxicity have been reviewed (Davidse, 1986; Delatour & Parish, 1986; Eckert &
Ogawa, 1988). Significant quantities of TBZ have been found in the US diet (Yess et al,
1993). For example, an average 2-year-old in the US could be expected to consume
0.7 fig kg ~ '/day. This represents about 0.2% of the acceptable daily intake (ADI) of
300 fig kg ~ Vday established by the World Health Organization. A recently published report
recommended an ADI for thiabendazole of 100/Jgkg~7day (WHO, 1993).
Immunodiagnostic methods for the evaluation of food safety and quality have evolved at

0954-0105/95/020099-10 ©1995 Journals Oxford Ltd

 100 D. L. BRANDON ET AL.

a rapid rate (Samarajeewa et al, 1991; Morgan et al, 1992). Analytical methods for TBZ
in meat and produce matrices include high-pressure liquid chromatography (HPLC) and
immunoassay methods, and have been cited or published recently (Brandon et al., 1992,
1993; Mountfort et al, 1994). Some investigators have prepared samples for immunoassay
by extracting TBZ residues from produce with organic solvents (Newsome & Collins, 1987;
Mountfort et al, 1994). A previous article described an immunoassay using immobilized
TBZ conjugate that could be used to analyze aqueous extracts of apples and potatoes
without interference by peroxidase and enzyme inhibitors present in some of the extracts
(Brandon et al, 1993). However, enzyme-linked immunosorbent assay (ELISA) using
immobilized antibodies remained an appealing format because fewer steps were required.
It is hypothesized that an alternative extraction procedure might avoid the matrix effects and
limited recovery associated with aqueous extraction. In addition, the authors wished to
develop a solid-phase plate coating, which would conserve monoclonal antibodies and
permit both batch production and long-term storage of assay materials. In this paper, an
ELISA is described using 80% methanol extracts of peel from apples, potatoes, citrus and
bananas performed on polystyrene assay wells coated indirectly with monoclonal antibody
through a polylclonal rabbit anti-mouse IgG layer.
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METHODS
Samples and Treatment of Produce with TBZ
Produce samples (labeled 'organically grown') were obtained from local markets. Apples,
potatoes, grapefruit, oranges and bananas were divided into groups, and analyzed before
and after treatment with TBZ (Mertect 340-F, MSD Agvet, Rahway, NJ, USA), in
accordance with the product label. The final TBZ concentrations and duration of dip
treatments were as follows: apples, 0.57 g!" 1 , 3 min; potatoes, 1.5 g I" 1 , 20 s; citrus,
1.5 g 1 ~', 5 min; and bananas, 0.35 g I" 1 , 5 min. The dipped produce was then drained and
stored refrigerated at 4°C.

Extraction of Samples for Analysis

Produce was weighed and peeled, and the weighed peels were extracted with 80% methanol
in water (6mlg~') by shaking for 10 min followed by soaking for 16 h at 4°C. Peels
comprised the following fraction of total fruit or tuber weight: apple, 0.11 ; potato, 0.09;
orange, 0.29; grapefruit, 0.37; and banana, 0.39 (mean of at least three determinations for
each commodity). Extracts were then filtered through 0.45 fxm Teflon membranes, divided
into aliquots and either diluted for ELISA or further fractionated for HPLC.

HPLC Analysis
Previously described procedures (Brandon et al, 1993) were adapted as follows. The
methanolic extract was evaporated, taken up in 50 ml of 0.1 M-HC1 and washed three times
with 50 ml portions of ethyl acetate. The aqueous phase was then adjusted to pH 8.5-9.0
with concentrated NaOH solution and extracted three times with 50 ml of ethyl acetate. The
combined ethyl acetate extracts were dried with anhydrous Na2SC>4 and taken to dryness on
a rotary evaporator at 40°C. The residue was dissolved in 25 ml of methanol and passed
through a 0.45 /im Teflon filter.
The Chromatograph consisted of a SP8700 solvent delivery system and pump module
(Spectra-Physics, San Jose, CA, USA) and a programmable fluorescence detector (Model
1046A, Hewlett-Packard, Palo Alto, CA, USA), with excitation at 300 nm and emission
detected at 360 nm. Chromatography was generally performed on an LiChrosorb 5 pm C18
column, 200 X 4.6 mm (Hewlett-Packard), using acetonitrile:water (70:30) and a flow rate
of 0.5 ml min" 1 . Some analyses were performed on an Ultrasphere 5 fim C18 column,
250 X 10 mm (Altex, Berkeley, CA, USA), using methanol:water (55:45) and a flow rate of
 ANALYSIS OF TBZ IN PRODUCE 101

0.9 ml min ~ ' for elution. Peaks were quantified using a SP4200 computing integrator
(Spectra-Physics).

Recovery of TBZ by HPLC

The recovery of TBZ from 80% methanol extracts of produce was determined as follows.
Produce extracts, which were subsequently verified to be free of detectable TBZ, were
spiked in the range 0.5-4 ppm and processed as described above. The recovery of TBZ was
85 ± 7% (n = 6).

ELISA Plate Preparation

The ELISA was performed on immobilized monoclonal antibody 448 (Brandon et ai,
1992), which binds TBZ and 5-OH-TBZ equivalent^. ELISA plates (Immulon II, Dynat-
ech, Chantilly, VA, USA) were coated with rabbit anti-mouse IgG (Zymed, S. San
Francisco, CA, USA) at lOjUgml"1 and 100/il/well (for 4 h at room temperature or
overnight at 4°C). Plates were then drained and washed five times with water using an
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automated plate washer (Wellwash 5, Denley Instruments, Morrisville, NC, USA). Adven-
titious binding was blocked by incubation of coated wells for 1 h with 200 ^I/well of
lOmgml" 1 bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing
0.05% Tween 20 detergent (BSA-PBS-Tween). Plates were drained and washed again.
They were then coated with antibody 448 (0.2 fig ml" 1 in BSA-PBS-Tween, 100|d/well)
for 1 h and washed again. The emptied wells were then filled with 2% sucrose (200 \AI
well), incubated for 30 min, dried (40°C, 1 h) and stored desiccated at 4°C until used. Plate
stability was tested over 6 months by performing side-by-side assays on plates stored for
various times.

Analysis of Extracts
Extracts to be analyzed were diluted in BSA-PBS-Tween (or water; see discussion below)
so that the final TBZ concentration would be within the working range of the assay.
Extracts were diluted manually five-fold, and further two-fold dilutions were prepared using
a robotic pipettor (Biomek 1000, Beckman Instruments, Fullerton, CA, USA). TBZ
standards (Brandon et al., 1992) were diluted from stock in BSA-PBS-Tween to achieve
concentrations of 100, 20, 4, 0.8 and 0.16 ppb. Standards, blanks and samples were added
to triplicate wells (50 /d/well). The TBZ conjugate of horseradish peroxidase (HRP)
(Brandon et al, 1992) was diluted to its working concentration (0.3 ng ml" 1 ) in the same
diluent, and 50 ftl were added to each well. Plates were incubated with shaking for 1 h at
room temperature, then drained and washed. A one-component formulation of substrate
with tetramethylbenzidine as chromogen (ELISA Technologies, Lexington, KY, USA) was
added (100 /il/well). After an incubation of 30 min (room temperature, shaking), the
reaction was stopped by addition of 0.3 M-HC1 (100 jul/well). Absorbance was determined
at 450 nm using a microplate reader (Vmax, Molecular Devices, Menlo Park, CA, USA).
The data obtained for TBZ standards were fitted to a four-parameter logistic model (Finney,
1978) using the Softmax program (Molecular Devices, Menlo Park, CA, USA), and sample
concentrations were estimated by interpolation.

RESULTS
Time Course for Extraction
Figure 1 illustrates the time course for TBZ extraction from five commodities, evaluated by
ELISA. The TBZ residue was extracted rapidly by a simple soak in 80% methanol. Most
of the TBZ was extracted within 1 h, but an extraction time of 16 h (overnight) was chosen
 p

to H

Is
jjjj Thiabendazole(PPM) Thiabendazole (PPM)
2 »*v o o i o i n o S n o -• N u * oi

s
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11 - • • • • H PHIHIHiilH
«•a

fîg O 01 O S O — N> U *.
3 5 | I I I | | ' ' ' '

i IâkHHHI^HHl I°pi^HHHHl
5'
ft
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"7viï NoaNvaa "i -a 201

 ANALYSIS OF TBZ IN PRODUCE 103
Downloaded By: [University of Georgia] At: 22:23 29 June 2009

100

Thiabendazole (PPB)

FIG. 2. Typical standard curve for TBZ ELISA. The fitted equation is Y = (A - D)l[\ + (X/QB] + D, where
Y= A450, X = logio[TBZ], A = 1.95, B = 1.08, C= 1.57 and D = 0.0277.

as the standard procedure for this study in order to achieve uniformly high recoveries from
all matrices.

Choice of Diluent and Effect of Methanol

The effects of solvent and diluent on the IC50, the mid-point of the standard curve and the
upper asymptote {Bo) were studied. Standard curves for TBZ were obtained in different
solvents, water or BSA-PBS-Tween, with or without methanol. Figure 2 illustrates a typical
standard curve for the ELISA. Table 1 illustrates the effect of 4, 8 and 16% methanol (i.e.
20-, 10- and five-fold diluted extraction solvent) on these parameters. Sixteen percent
methanol, the highest concentration studied, desensitized the assay by increasing the IC50
and suppressing the maximal amount of conjugate bound (P < 0.05 for both parameters,
Student's Mest). All subsequent assays were performed at a final concentration of methanol
of 4% or less.

Produce Matrix Effects

Table 2 illustrates the effects of the methanol extracts of three types of peels on assay
parameters. Separate controls, performed simultaneously on the same 96-well assay plate as
TABLE 1. Effect of 80% methanol on assay parameters"
Methanol (%) Diluent IC50 (ppb) Bo (A450) n

0 BSA-PBS-Tween 1.6 ±0.33 2.5 + 0.13 6

4 BSA-PBS-Tween 2.1+0.40 2.4 + 0.49 6
4 Water 2.2 ±0.29 2.8 ±0.43 5
8 BSA-PBS-Tween 2.4 ±0.76 2.3 + 0.47 3
16 BSA-PBS-Tween 3.1 + 1.2 1.8 ±0.61 3
" Means and standard deviations.
 104 D. L. BRANDON ET AL.

TABLE 2. Effects of produce extracts on assay parameters"

Extract IC,s (ppb) IC50 (ppb) ßo (A450) Steepness parameter6 n

Gravenstein apple
Control 0.4+0.2 2.2 + 0.89 2.6 ± 0.74 1.0 ±0.2 3
5% extract 0.6 ± 0.2 2.6+1.3 2.5 ±0.34 1.2 ±0.2 3
Yellow Finnish potato
Control 0.6 ± 0.2 2.7 ± 0.30 2.4 ±0.51 1.1 ±0.2 3
5% extract 0.5 ± 0.2 2.2 ±1.3 2.7 ±0.71 1.2 ±0.2 3
Valencia orange
Control 0.8 ± 0.2 2.9 ±0.41 2.8 ± 0.09 1.2 ±0.1 4
5% extract 1.1 ±0.6 3.0 ± 0.75 2.9 ± 0.23 1.6 ±0.6 4

" Means and standard deviations.

b
Parameter B in the four-parameter logistic equation.

the experimental sample, are shown. Neither assay sensitivity, as measured by IC50, nor
binding of conjugate to immobilized antibodies, as measured by Bo, was affected by the
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20-fold diluted extracts. The IC15 value, operationally used as a limit of quantification, was
also not significantly different for the three peel/solvent matrices compared with the
matched controls. The steepness of the standard curve at its mid-point (the B coefficient in
the four-parameter equation; see Figure 2) ranged from 1.0 to 1.6 in these studies, and was
not significantly affected by the extracts.

Limit of Detection
Extracts of peels from Gravenstein apple, Yellow Finnish potato and Navel orange were
spiked with TBZ in the range 0.8^t ppb and assayed by ELISA. The results were analyzed
by linear regression of ELISA value on TBZ added. The 95% one-sided lower confidence
limits were determined to be 0.9 ppb for apple and 0.7 ppb for potato and orange. These
values corresponded to 18 and 14 ppb respectively in undiluted extracts and 108 and 84 ppb
respectively in the peel itself. The limit of detection of the HPLC method was 430 ng ml" \
corresponding to 215 ppb in the peel sample.

Limit of Quantification
This quantity was defined operationally as the IQ5 of the standard curve obtained in the
appropriate matrix: apple, 0.6 ppb; potato, 0.5 ppb; and orange, 1.1 ppb. This operational
measure has a statistical basis; for example, the 99% confidence interval for the IC15 in the
apple matrix was 0.2-0.9. Thus, for these three matrices, the limit of quantification in the
peel was =£130 ppb.

Stability of Assay Plates

Standard curves were performed on plates stored for 1 week, 3 months and 6 months. The
linear portion of the curves (standards from 0.16 to 20 ppb TBZ) for the 1 week-old plates
were fitted to a straight line, and the 95% prediction limits were calculated (Figure 3). The
assay results obtained on the plates stored for 3 or 6 months were plotted, and, as indicated
on the graph, were well within the prediction limits, indicating that no significant difference
in standard curve resulted from extended storage of the assay plates.

Analysis of Produce
Table 3 summarizes the results of analyses of oranges, apples, grapefruit, potatoes and
bananas. Each value is the average of two independent extractions and determinations. As
expected from the designation of the produce as 'organic', most samples analyzed had no
 ANALYSIS OF TBZ IN PRODUCE 105

» [; V \
\\ \
\\-\
o.o - \ \^
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11 III I I I I 1 1I I I I I I I I I III \ l I I

0.1 1 10

FIG. 3. ELIS A plate stability. Linear region of standard curve for TBZ assay, presented as absorbance divided
by absorbance in absence of analyte (B/Bo) versus log TBZ. The fitted straight line and 95% prediction
limits were derived from assays performed on plates used within 1 week of preparation. Plotted values
were obtained on stored plates: A, 3 months; • , 6 months.

detectable TBZ by either analytical method. However, four market samples were found to
be positive by both methods, and one was positive by ELISA only. Samples treated with
TBZ in the laboratory were all positive by both methods. For the linear regression of ELISA
value on HPLC value, r = 0.87 for all positive samples (n = 20). Figure 4 shows this
regression for the 18 samples with values less than 25 ppm (r = 0.97). The HPLC analysis
generally produced lower values than the ELISA (66 ±25% relative recovery compared
with ELISA).

DISCUSSION
The choice of extraction procedure in this study was motivated by a desire to minimize the
use of specialized equipment and to facilitate batch processing of multiple samples. The
overnight soaking procedure met these requirements. The resulting extracts could be
decanted and analyzed after dilution without any additional steps. Although the total
duration of the extraction was 16 h, little manual labor was involved. In this study, the use
of alternatives that could accelerate extraction of TBZ was not explored. If samples need
to be processed more rapidly, it seems likely that sample agitation (e.g. stirring or blending)
is a modification that would succeed.
Diluting 80% methanol extracts of produce samples 20-fold, to a final methanol
concentration of 4%, was adequate to eliminate matrix effects due to solvent (Table 1). It
was not necessary to limit the methanol concentration to 1% as was done in a previously
described system for TBZ ELISA (Mountfort et al, 1994). Water was adequate as a diluent
for sample extracts. The use of water versus BSA-PBS-Tween as solvent (see data for 4%
methanol), resulting in a reduction of protein concentration in the assay from 10 to
5 mg ml" 1 , did not significantly affect the assay parameters under these conditions.
Peel extracts diluted 20-fold did not affect the assay parameters studied (Table 2).
Imposing a requirement of a 20-fold dilution limits the sensitivity of the assay, but protects
 106 D. L. BRANDON ETAL.

TABLE 3. Analysis of produce forTBZ by extraction of peels with 80% methanol

Market sample TBZ-treated sample

(ppm in peel) (ppmi in peel)

Produce variety ELISA HPLC ELISA HPLC

Valencia orange (I) ND" ND 3.2 2.3

Valencia orange (II) ND ND 4.3 2.3
Navel orange 1.8 1.8 11 6.5
Pink grapefruit (I) ND ND 1.9 1.2
Pink grapefruit (II) ND ND 4.7 4.7
Gravenstein apple (I) 6.2 2.9 15 8.2
Gravenstein apple (II) ND ND 3.8 2.6
Gravenstein apple (III) ND ND 6.0 3.8
Golden Delicious apple (I) ND ND 7.1 4.2
Golden Delicious apple (II) 0.5 0.2 21 15
Mclntosh apple ND ND 7.6 5.0
Russet potato (I) 0.95 0.72 54 26
Downloaded By: [University of Georgia] At: 22:23 29 June 2009

Russet potato (II) ND ND 34 42

Yellow Finnish potato 0.4 ND 13 11
Banana ND ND 3.6 2.7
" ND, not detected.

against potential matrix effects. In an earlier study, aqueous produce extracts interfered with
the immobilized antibody format of the assay, possibly because of endogenous peroxidase
and peroxidase inhibitors, and precluded the use of this format (Brandon et al., 1993). The
use of 80% methanol instead of water eliminated these matrix effects. In addition, a higher
recovery was achieved using this method than that obtained previously in the apple peel
matrix. The present assay format has fewer steps than ELISA utilizing immobilized TBZ
conjugate (Newsome & Collins, 1987; Brandon et al, 1993), and the diluted extracts and
HRP conjugate can be added directly to the well and then mixed.
Although the ELISA values were consistently high compared with the HPLC results
(Table 3), the two sets of values were highly correlated (Figure 4). Only one market sample
was positive by ELISA (0.4 ppm) and negative by HPLC. This level is less than two-fold

25 I -7

2
2 °- /
Q^ /

<" 15 - • /
£2 / •
ÜJ • /
>. 10 - /
N mr
m jr

0L_ 1 i 1
0 5 10 15 20

TBZ by HPLC (PPM)

FIG. 4. Thiabendazole in produce peels. Linear regression of ELISA value on HPLC value for samples treated
with TBZ in the laboratory (r = 0.97).
 ANALYSIS OF TBZ IN PRODUCE 107

above the limit of detection for the HPLC method. The antibody is highly specific for TBZ
and 5-substituted derivatives (Brandon et al, 1992). Five-substituted metabolic or environ-
mental transformation products of TBZ have not been reported in plants (reviewed by
Zbozinek, 1984). The agreement between ELIS A and HPLC methods for untreated samples
(with the one exception mentioned above) rules out the possibility that matrix components
bind to the antibody. One factor contributing to the difference between the ELISA and
HPLC values is the average loss of 15% of the TBZ during the sample work-up for HPLC,
which included drying and transfer steps. In addition, the ELISA values may be high due
to a systematic error which was propagated in preparing dilution series using the robotic
pipettor. Using dye solutions diluted in a similar manner to the TBZ samples, a 15-20%
error was determined in the final dilution factor due to carry-over in the pipette tips. It
should be noted that some extracts of TBZ-treated produce were diluted over 1000-fold for
analysis by ELISA. The combination of these two factors is sufficient to account for nearly
all of the differences in the two analyses.
The TBZ concentrations found as residues in peel, whether in market or treated samples,
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were consistent with US tolerances for these commodities (lOppm for the whole fruit or
tuber). The values for laboratory-treated potato peel were especially high (54 ppm by
ELISA). Values of TBZ in potato peel in the 10 ppm range occur commonly (Friar &
Reynolds, 1991; Brandon et al, 1993). In the present study, potatoes were extracted after
only 24 h of storage and without further washing. The residues, therefore, are higher than
those typically encountered in commercial marketing channels.
The estimated limits of quantification for TBZ in apple, potato and citrus peel extracts
are all approximately 1 ppb. Since extracts were assayed following a 20-fold dilution,
20 ppb of TBZ in undiluted extracts is detectable. This corresponds to 120 ppb in the peel
itself (6 ml extradant g ~ ' peel). It had been found previously that, for apples and potatoes,
90% of the TBZ residue was in the peel, and was concentrated approximately nine-fold
compared with the content for the whole sample (Brandon et al, 1993). Thus, a typical
whole apple or potato with 15 ppb of TBZ residue (less than 0.2% of US tolerance) should
be detectable by this ELISA. Appropriate dilution of extracts can be made to adjust the
working sensitivity of the assay to the analytical need. The present ELISA is an order of
magnitude more sensitive than a recently reported method using a commercial test kit,
which employs a methyl benzimidazolecarbamate-specific antibody cross-reactive with
TBZ (Mountfort et al, 1994).
Since TBZ is used mainly as a post-harvest dip or spray for these commodities, the peel
is an appropriate target for residue screening. Use of peels for screening is subject to several
caveats, however. Treatment and storage conditions have a major influence on TBZ residue
levels (Norman et al, 1972; Ben-Arie, 1975; Cano et al, 1987). Since varietal differences
affect various post-harvest physiological processes (for example in apples, Kim et al,
1993), the relationship between peel and total residues could vary with cultivar. Since a
portion of TBZ residues can be readily removed from the surface of fruits (Norman et al,
1972), swipe samples might be analyzed using this ELISA as a quick test to distinguish
treated from untreated samples in the market-place. For this application, a coated tube
format would probably be preferable to a multi-well plate.
In conclusion, a sensitive and specific ELISA for TBZ has been developed which could
be performed without significant matrix effects in diluted methanolic extracts of produce
peel. The extracts were prepared by soaking peels and decanting, a procedure which
conserved analyst time and equipment. A novel feature of the ELISA is the utilization of
a dried monoclonal antibody indirectly immobilized through an adsorbed rabbit anti-mouse
IgG, an immunosorbent which had a shelf-life of at least 6 months.
 108 D. L. BRANDON ETAL.

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Note added in proof. While this paper was in press, two papers were published by R. J. Bushway et al.
which used a prototype commercialized kit form of the immunoassay, employing the same thiabendazole-
specific antibody: Journal of Agricultural and Food Chemistry, 43, 1407-1412 (1995) and Journal of AOAC
International, 78, 815-820 (1995).