Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine

Article abstract of DOI:10.1021/jf070948d

In this paper, a new monoclonal antibody (Mab) against sulfamethoxypyridazine (SMP) was produced, and a fluorescence polarization immunoassay (FPIA) based on the produced Mab was developed and optimized for the qualitative screening analysis of SMP. The M

Full text of DOI:10.1021/jf070948d

J. Agric. Food Chem. 2007, 55, 6871−6878 6871
Monoclonal Antibody-Based Fluorescence Polarization
Immunoassay for Sulfamethoxypyridazine and
Sulfachloropyridazine
ZHANHUI WANG,† SUXIA ZHANG,† IRINA S. NESTERENKO,‡
SERGEI A. EREMIN,‡ AND JIANZHONG SHEN*,†
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
China Agricultural University, Beijing 100094, People’s Republic of China, and Department of
Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University,
Moscow 119992, Russia
In this paper, a new monoclonal antibody (Mab) against sulfamethoxypyridazine (SMP) was produced,
and a fluorescence polarization immunoassay (FPIA) based on the produced Mab was developed
and optimized for the qualitative screening analysis of SMP. The Mab was raised from mice immunized
with SMP linked to bovine serum albumin (BSA) by carbodiimide activated ester formation, using a
succinic anhydride spacer molecule between SMP and BSA. Fluorescein labeled sulfachloropyridazine
(SCP) and SMP (tracer) were synthesized and purified by thin layer chromatography (TLC). The
developed screening FPIA method can tolerate up to 20% methanol, and satisfactory assay sensitivity
can be obtained between pH 4 and pH 8 and at lower salt concentration. The anti-SMP Mab exhibited
a high cross-reactivity with SCP. The effect of the tracer structure on the analytical characteristic of
the determination and on antigen-antibody binding constants was studied. The limits of detection
(LOD) were 0.7 ng/mL for SMP and 0.25 ng/mL for SCP in buffer, respectively, whereas negligible
cross-reactivities were exhibited by related sulfonamides. Analysis of SMP and SCP-fortified milk
samples by the FPIA showed average recoveries from 60 to 145%.
KEYWORDS: Fluorescence polarization immunoassay; monoclonal antibody; sulfamethoxypyridazine;
sulfachloropyridazine; milk
INTRODUCTION
(6). Consequently, it is proposed to include them in screening
projects to satisfy increasing public concerns about food safety.
The relatively low historical usage of the pyridazine sulfona-
mides in the food-production animal industry has resulted in
few analytical methods being reported for the detection of
residues of SMP or SCP individually in food samples. Recently,
a number of papers have reported some sulfonamide multi-
residue procedures in differing samples for SMP or SCP. These
are mostly conventional instrumental analytical methods such
as liquid chromatography (7-11), liquid chromatography-mass
spectrometry (12-14), capillary electrophoresis-mass spec-
trometry (15), and chemiluminescence detection (16). However,
instrumental analytical methods require high cost, highly skilled
workers, and time-consuming sample preparation steps and are
not suitable for routine analysis of a large number of samples.
Therefore, there is a growing demand for more rapid and more
economical methods.
Sulfamethoxypyridazine (SMP) and sulfachloropyridazine
(SCP) are examples of sulfonamide veterinary drugs and have
similar chemical structures (Figure 1 and Table 1). They are
effective antimicrobial drugs for the prevention of infections
in cattle, poultry, and swine (prophylaxis), to treat veterinary
diseases, and to promote growth (1). As a result of the extensive
use of sulfonamides in the animal industry, residues of these
drugs in food samples are a major concern because of their
contribution to the development of antibiotic resistant pathogenic
bacteria (2, 3). To minimize this risk, maximum residue limits
(MRLs) have been established for total or individual sulfona-
mides including SMP and SCP at 0.1 mg/kg in milk and meat
by China Ministry of Agriculture (No. 278, 2003.5.22), which
are similar to MRLs in the United States and Europe (4, 5).
Although not as widely used as other available sulfonamides
such as sulfamethazine and sulfaquinoxaline, SMP and SCP can
be used as an alternative, and their application is increasing
The immunoassay such as enzyme-linked immunosorbent
assay (ELISA) could be a solution to this problem (17, 18). In
previously reported immunoassays for sulfonamides, most have
shown little or no cross-reactivity for SMP or SCP, because
these antibodies were raised to specific haptens. Haasnoot et
al. reported one Mab, named Mab 21C7, raised against
* To whom correspondence should be addressed. Tel.: +86-1062732803.
Fax: +86-10-62731032. E-mail address: sjz@cau.edu.cn.
† China Agricultural University.
‡ M.V. Lomonosov Moscow State University.
10.1021/jf070948d CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/28/2007

6872 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Wang et al.
based on differences in the polarization of the fluorescence of
the fluorescent-labeled analyte in the antibody-bound and
nonbound fractions. A number of FPIAs for pesticides and
toxins have been developed in the past few years (25-27).
Several articles have been published for the determination of
sulfonamides using FPIA (28-30). Polyclonal antiserum was
used in most of these investigations, and to date, immunoassays
specific for SMP or SCP using monoclonal antibodies instead
of polyclonal antibodies have not yet been developed. In our
present paper, we produced for the first time one Mab against
SMP showing good cross-reactivity with SCP and developed
an FPIA for the detection of SMP and SCP in milk.
MATERIALS AND METHODS
Materials and Apparatus. Bovine serum albumin (BSA), fluores-
cein isothiocyanate (FITC) isomer I, SMP, SCP, and other sulfonamides
used in cross-reactivity studies, 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide (EDC), and Freund’s complete and incomplete adjuvants
were obtained from Sigma (St. Louis, MO). Polyethylene glycol (PEG
2000) was purchased from Merck-Schuchardt (Darmstadt, Germany).
Common solvents and salts were supplied by Beijing Reagent Corpora-
tion (Beijing, P.R. China). The myeloma cell line SP2/O was a gift
from Professor Jixun Zhao (China Agricultural University, Beijing, P.R.
China). Cell culture media (DMEM) was obtained from Huamei
(Beijing, P.R. China). Fetal calf serum and supplements were obtained
from GIBCO BRL (Carlsbad, CA). Pre-coated silica gel 60G F254 coated
glass plates for thin layer chromatography (TLC) were purchased from
Qingdao Haiyang (Qingdao, P.R. China). All other chemicals and
solvents were analytical reagent grade.
Figure 1. Chemical structures of sulfonamides (a) SMP and (b) SCP,
(c) immunogen, and tracers (d) SMP−FITC and (e) SCP−FITC.
Table 1. Chemical Structures of Sulfanilamide Drugs, Hapten
−Protein
Conjugate (Immunogen), and Fluorescence Labeled Analyte (Tracer)
Borate buffer (BB; 50 mM, pH 8.0) with 0.1% sodium azide was
used as working buffer for all FPIA experiments. Stock solutions (1
mg/mL) of SMP, SCP, and other analytes were prepared by dissolving
10 mg of drug in 10 mL of methanol and were stored at -20 °C.
Aqueous standard solutions of analytes in the range 0.001-100 000
ng/mL were prepared by dilution of stock solution with BB buffer.
Standard solutions were stored at 4 °C.
compound
R1
R2
SMP
SCP
OCH3
Cl
NH2
NH2
immunogen
OCH3
OCH3
Cl
NH
NH
NH
−
CO(CH2)2CO
−NH−BSA
A Cary Eclipse fluorescence spectrophotometer (Varian Corporation,
Palo Alto, CA) with a pair of polarizers was used to measure the value
of the fluorescence polarization (FP). The excitation and emission
monochromators were set at 492 and 514 nm, respectively, and the
excitation and emission slits were set at 10 nm. An IBM PC
microcomputer was used for on-line data acquisition and for data
analysis.
SMP
SCP
−
FITC
−
−
FITC
FITC
−FITC
sulfamethazine which was identified as an antibody recognizing
SMP and SCP. This MAb was used in a biosensor immunoassay
for the detection of eight sulfonamides including SCP in chicken
serum (19). A lanthanide fluoroimmunoassay for the simulta-
neous screening of 18 sulfonamides using a mutant engineering
antibody (M.3.4) was also described (20). Cliquet et al. described
one ELISA method for the determination of SCP in porcine
tissues, and the polyclonal antibody used in the study was
obtained after immunization with a sulfathiazole derivative (21).
To the authors’ knowledge, three papers have reported the
production polyclonal antibodies specifically to SMP and SCP
and developed ELISA methods to detect SMP or SCP residues
in food samples, respectively (6, 22, 23). The cross-reactivities
of all sulfonamides with ovine polyclonal antisera against
hemisuccinate derivative of SCP or SMP were less than 1%
except for SMP antisera, which showed a cross-reactivity of
55.4% with SCP (23). The polyclonal antibodies against SMP
could not recognize SCP (22), while the SCP antibodies showed
3.1% cross-reactivity with SMP (6).
Synthesis of Immunogen. The SMP hapten was synthesized as
described by Eremin et al. with some modifications (30). SMP (6 g)
and succinic anhydride (3 g) were refluxed in 60 mL of anhydrous
ethanol for 90 min. The preparation was cooled to room temperature,
and the resulting crystals were collected. This crude fraction was
redissolved in a mixture of 20 mL of ethanol and 30 mL of water,
refluxed for 30 min, and cooled to room temperature, and the crystals
were collected by filtration. The prepared succinyl hemisuccinate of
SMP (SMP-HS) was dried in a vacuum oven and stored, desiccated,
at room temperature.
Two hundred milligrams of SMP-HS was dissolved in 2 mL of
dimethylformamide, and an amount of 150 mg of EDC was dissolved
in 500 µL of water and then added to the hapten solution. The mixture
was stirred for 2 h at room temperature. The above reaction mixture
was added dropwise to 2 g of BSA in 8 mL of sodium carbonate (0.01
M, pH 7.2) and placed at 4 °C overnight. The obtained immunogen
(Figure 1) was dialyzed against phosphate-buffered saline (0.01 M,
pH 7.4) for 7 days (one change every day) and stored in freezer in
aliquots (31).
Simplifying the assay and minimizing the analysis time are
the primary goals in developing screening methods for large
numbers of samples. An alternative method to ELISA is
fluorescence polarization immunoassay (FPIA). FPIA does not
require separations or multiple steps, giving the advantages of
simplicity and high precision. The principles of FPIA have been
reviewed (24). In brief, it is a competitive immunoassay method
Synthesis of Fluorescent Conjugates (Tracers). SMP (5 mg) was
dissolved in 0.5 mL of methanol. Triethylamine (50 µL) and FITC (4
mg) were added with mixing. After overnight reaction at room
temperature, small portions (50 µL) of reaction mixture were separated
by TLC using chloroform/methanol (4:1, v/v) as the eluent. The main
yellow band at Rf 0.1 was scraped from the plate and extracted with 1

Fluorescence Polarization Immunoassay for SMP and SCP
J. Agric. Food Chem., Vol. 55, No. 17, 2007 6873
mL of methanol. SCP tracers were prepared in the same way
(Figure 1 and Table 1), and the tracers were stored in the dark at
-20 °C (30).
Production of Monoclonal Antibody. The procedures for generating
the immune response in mice and producing monoclonal antibodies
were similar to those described by Zhang et al. (32). One hundred
micrograms of the SMP-HS-BSA conjugate was emulsified with an
equal volume of Freund’s complete adjuvant and given as an intrap-
eritoneal injection to each of five female BALB/c mice, 8 weeks of
age. Booster injections were given 2, 4, and 6 weeks later with the
same immunogen in an equal volume of Freund’s incomplete adjuvant.
One week after the third injection, serum was collected from the caudal
vein of each mouse, and antibody titers were determined by the indirect
ELISA. Three days before cell fusion, the mice that produced antibody
with high titers were given another intraperitoneal booster injection of
the immunogen without adjuvant.
Immunized mouse spleen cells (1 × 106) were fused with myeloma
cells SP2/0 (6 × 106) using 1 mL of 50% PEG 2000. After fusion, the
cells were selected with HAT medium (DMEM medium with 15% fetal
calf serum, 100 units/mL penicillin, 100 µM hypoxanthine, 0.4 µM
aminopterin, and 16 µM thymidine). Aliquots of the cell suspension
(50 µL) were added to the wells of the 96-well plates and incubated at
37 °C in 5% CO2 atmosphere. After 12 h, an additional 50 µL of the
selection medium was added to the wells. After 2 days, the medium
was replaced with fresh selection medium. After day 12, aminopterin
was omitted from the medium. Between days 12 and 14, supernatants
from the 96-well plates were screened for antibody activity by the
indirect ELISA in the absence and presence of 100 ng/mL SMP.
ELISA-positive hybridoma cells were cloned by the limiting dilution
method in aminopterin-free selection medium (HT medium), and one
stable clone was obtained.
Figure 2. Dilution curves for SMP monoclonal antibody with different
tracers SMP−FITC and SCP−FITC.
Cross-reactivity (CR) was calculated according to the equation
IC50(SMP)
CR )
× 100%
IC50(sulfonamide)
where IC50 is the concentration at which 50% of the antibodies are
bound to the analyte.
Dissociation constants were calculated with Scatchard plot analysis
using FP data according to Hatzidakis et al. (33).
Fortification of SMP in Milk. Cow whole milk samples were
purchased from a local market and simply defatted by centrifugation
for 15 min (5000g at 4 °C). Aliquots (4 mL) were fortified with SMP
to at 50, 100, and 500 ng/mL and then mixed with an equal volume of
1.25% trichloroacetic acid (TCA). The mixtures were agitated on a
shaker for 1 min and then deproteinized through centrifugation for 10
min (5000 × g at 4 °C). The supernatants were diluted 1/5 in BB and
used in FPIA.
Curve Fitting and Statistical Analysis. FP was calculated according
to the formula P ) (Iv - Ih)/(Iv + Ih), where Iv and Ih are the vertical
and horizontal components of the emitted fluorescence intensities and
are expressed in “millipolarization” units (mP).
The four-parameter logistic equation as defined below was used to
fit the immunoassay data
A - D
Y )
[1 + (X/C)B + D]
RESULTS AND DISCUSSION
where A ) response at high asymptote, B ) slope factor, C )
concentration corresponding to 50% specific binding (IC50), D )
response at low asymptote, and X ) calibration concentration.
The IC50 value, the assay dynamic range, and the limit of detection
(LOD) serve as criteria to evaluate the FPIA. These characteristics
represent the analyte concentrations that provide a tracer binding
inhibition in the FP assay of 50%, between 20% and 80%, and of 90%,
respectively (28).
Assay Optimization. Several physicochemical factors influencing
the immunological reaction were studied in the FPIA, including pH,
salt concentration, and organic solvent. Modification of mPmax
(mPmax is the maximum FP value of the inhibition curve) and IC50
parameters of the standard curves was evaluated under different
conditions.
Antibody Dilution Curve. Mabs were serially diluted 1/100, 1/400,
..., 1/819 200 in BB. Then 1 mL of diluted antibody solution was added
to 1 mL of tracer in BB. Tracer concentration was selected such that
the total final fluorescence intensity was 10 times higher than the
background of BB (the excitation and emission slits were set at 5 nm
for these measurements). The reaction mixtures were incubated for 2
min, and then polarization readings were made.
Competitive Fluorescence Polarization Immunoassay (FPIA)
Procedure. FPIA calibration curves were obtained by adding to a
cuvette 700 µL of standard solutions or sample, 700 µL of tracer in
BB, and 700 µL of the optimal dilution of antibody. The reaction
mixture was vortex-mixed, allowed to stand for 5 min, and then
measured in the fluorescence spectrophotometer.
Selection of Reagent (Tracer) Concentration. Limiting
concentrations of antibody and tracer immunoreactants are
required for a competitive immunoassay to obtain the required
analytical sensitivity. In particular, tracer concentration dictates
the extent of competition allowed to be measured by the analyte
and therefore the ultimate assay sensitivity. The lowest possible
tracer concentration, which allows the reliable detection of label
and does not affect the competition, is desirable for highest
sensitivity. The lowest tracer concentration giving a signal must
be approximately 10 times higher than the background signal
from BB (total fluorescence intensity) (30). The antibody titer
is usually assessed by FPIA using serially diluted antibody with
a fixed concentration of the fluorescence-labeled tracer. In this
study, antibody titer was determined using SMP-FITC and
SCP-FITC. Antibody titration curves with these two tracers,
both of which gave satisfactory binding, are shown in Figure
2. Here, sufficient antibody concentration was used to observe
binding to both SMP-FITC and SCP-FITC. The highest
possible sensitivity for the SMP analyte was experimentally
determined in the competitive FPIA by then varying the antibody
concentration within the optimal range, to give the lowest IC50
value. This effect of antibody concentration on the immunoassay
sensitivity (IC50) was studied. Different concentrations of anti-
SMP antibody were used to reach the more optimal IC50 using
SMP-FITC and SCP-FITC tracers. The relationship between
the antibody concentration and the IC50 is shown in Figure 3,
To normalize the FP value, the ratio mP/mPmax (where mPmax is the
maximum FP value of the inhibition curve and mP is the current value)
resulting in relative units was used.

6874 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Wang et al.
Table 2. Analytical Parameters of Standard Curves for SMP and SCP
by FPIA Using the Same MAb and Two Different Tracers SMP−FITC
and SCP
−FITC
IC50
(ng/mL)
working range
(ng/mL)
LOD
(ng/mL)
tracer
dilution
SMP
SCP
SMP
2.3 82
49
SCP
SMP
SCP
SMP
SCP
−
FITC
FITC
1/8000
1/10000
10.6
11.5
38.7
29
−
1.8−364
0.8
0.7
0.3
0.25
−
2
−
2
−179
affects these assay characteristics. Theoretically, a sensitive
competitive immunoassay for a small molecule, such as a
veterinary drug, can be achieved when the antibody affinities
for tracer and analyte are comparable. However, in practice the
affinity for the labeled antigen is generally higher than for
analyte (34). Both the structural features of the tracer hapten
itself and the structure and length of the bridge between the
hapten molecule and the fluorescein label markedly influence
recognition of the tracer by the antibody, and under conditions
of competitive interaction with the analyte, assay sensitivity is
modified (35). Labeled antigens that are either structurally
similar or minorly different from the primary target analytes
were investigated in this study. In agreement with previous
studies, some FPIA systems were found to be more sensitive
when structurally more different hapten tracers were used (36).
To study the influence of tracer structure on assay sensitivity,
both SMP-FITC and SCP-FITC tracers were used in the
following investigation. These were selected because SMP and
SCP are similar molecules, both containing a pyridazine group
in the N1 position of the aromatic sulfonamido nucleus which
is different from other sulfonamides. The structures of the two
tracers are show in Figure 1 and Table 1. As can been seen in
Table 2, more slightly satisfactory IC50 values (11.5 and 29
ng/mL for SMP and SCP) and LOD (0.7 and 0.25 ng/mL for
SMP and SCP) were observed when SCP-FITC was used,
although SMP-FITC also presented with satisfactory analytical
parameters (IC50 of 10.6 and 38.7 ng/mL, LOD values of 0.8
and 0.3 ng/mL for SMP and SCP).
Figure 3. Relationship curve between IC50 and antibody dilution.
The developed FPIA was specific for SMP with cross-
reactivity for SCP of 40% and 27%, using SCP-FITC and
SMP-FITC tracers, and sulfamethizole of 1% using SCP-
FITC, respectively. No significant cross-reactivity was observed
with other sulfonamides (sulfamerazine, sulfamethazine, sulfa-
diazine sulfamethoxazole, sulfaphenazole, sulfadimethoxine,
sulfamonomethoxine, sulfisoxazole, sulfaquinoxaline, sulfame-
ter, sulfamoxol, sulfamethizole, sulfapyridine, and sulfanil-
amide). Standard curves for SMP and SCP obtained using both
the SMP-FITC and the SCP-FITC tracers are shown in Figure
4. The tracer SCP-FITC was used in all subsequent studies.
Martlbauer et al. (22) produced polyclonal antibodies to SMP
and homologous tracer SMP-enzyme using glutaraldehyde as
the coupling reagent. Their ELISA showed an LOD of 2.8 ng/
mL for SMP and a cross-reactivity below 1% for SCP. Spinks
et al. (6) synthesized an SCP-immunogen conjugate to raise SCP
antisera using 2-fluoro-1-methypyridinium-p-toluenesulfonate
(FMP) as a cross-linking reagent and a heterologous coating
antigen using hemisuccinate bridge, having the same chemical
structure as with the immunogen in our studies (Figure 1). The
developed ELISA was highly specific for SCP and had a LOD
of 0.65 ng/mL in assay buffer and showed cross-reactivity with
SMP and sulfamethizole 3.1% and 1.9%, respectively. With the
aim of obtaining sulfonamide group-specific antibodies, several
authors have described the production of polyclonal antibodies
and the isolation of one monoclonal antibody using different
Figure 4. FPIA calibration curves for SMP and SCP using the same
Mab and two different tracers: (a) SMP−FITC and (b) SCP−FITC. FPIA
calibration curve and the SCP and SMP standard drug were dissolved in
BB buffer. FP values, which are means of three replicates, were plotted
against analyte concentration.
where we observed that 1/8000 would provide the best IC50
value. Calibration curves obtained under these optimal condi-
tions are presented in Figure 4. The analytical characteristics
of the FPIA developed are listed in Table 2.
Competitive FPIA. The antibody is a key determinant of
both analytical specificity and analytical sensitivity in immu-
noassay methods. However, the structure of the tracer also

Fluorescence Polarization Immunoassay for SMP and SCP
J. Agric. Food Chem., Vol. 55, No. 17, 2007 6875
Figure 5. Influence of the (a) pH and (b) salt concentration of assay
Figure 6. Effect of (a) methanol and (b) acetonitrile on SMP FPIA
buffer on the analytical characteristics of SMP competitive standard
performance: (9) value of IC50 for SMP and (0) mP value in the absence
curve: (9) value of IC50 for SMP and (0) mP value in the absence of
of SMP (mPmax). Values refer to the final concentrations of solvents (v/v)
in the competitive assay solution. Each point represents the mean of three
replicates.
SMP (mPmax). All reagents were prepared in BB at the appropriate pH
and salt concentration. Each point represents the mean of three replicates.
group-specific haptens in the past few years (31, 37, 38). The
group-specific polyclonal antibodies obtained following im-
munization with N-sulfanil-4-aminobutyric acid (SAB) linked
to soybean trypsin inhibitor (STI) showed sufficiently high cross-
reaction to SMP and SCP (75% for SMP and 28% for SCP)
and the group-specific monoclonal raised from N-sulfanilyl-4-
aminobenzoic acid (SUL) linked to keyhole limpet hemocyanin
(KLH) exhibited little or no ability to recognize SMP or SCP
(1.55% for SCP) (36, 37). Similarly, polyclonal antibodies
against sulfacetamide (SAM) linked to bovine thyroglobulin
(BTG) showed no binding with any sulfonamides (31).
Effects of Physicochemical Conditions on Assay Perfor-
mance. Immunoassay performance is often influenced by
chemical parameters such as salt concentration, pH, and organic
solvent concentration. The effects of these parameters were
assessed by comparing IC50 and mPmax obtained under various
conditions: the maximum polarization (mPmax), reflecting the
Mab recognition of the tracer in the absence of analyte, and the
IC50 for SMP, reflecting the Mab affinity for the analyte itself.
To study the influence of pH in the assay system, competition
curves for SMP using SCP-FITC as the tracer were obtained
at different pH values from 2 to 12. The relationship of these
parameters as a function of pH is shown in Figure 5. Deleterious
effects on IC50 were observed at lower or higher pH, but no
significant changes were observed in the range pH 4-8. The
results indicated that the assay sensitivity decreased at a more
acidic or basic pH value.
Table 3. Constant of Affinity for Monoclonal Antibody
-1
-1
tracer
SCP FITC
SMP FITC
K
aff for SCP (
×
109
M
)
K
aff for SMP (
×
109
M
)
−
−
2.1
2.3
1.1
1.1
higher salt concentration greatly reduced mPmax, whereas the
IC50 rose dramatically. An almost threefold increase in the IC50
value occurred as a result of 800 mM salt concentration, while
a 20% decrease in the mPmax value was seen. Because the
primary effect of increased salt ion concentration is to reduce
hydrophobic bond formation and stability, the interaction of
antibody-analyte will decline under conditions of increasing
salt concentration. The results obtained were consistent with
the study of the polar pesticide TCP by ELISA (39), in contrast
to the case of the polar 4-nitrophenol ELISA, wherein increasing
salt concentration increased the antibody affinity for 4-nitro-
phenol and improved the assay sensitivity (40). Our studies
indicated that the ability of the antibody to recognize both the
tracer (mPmax) and the analyte (IC50) follows a similar trend;
that is, when antibody recognition of analyte increases, the value
of mPmax will decrease accordingly because of the analyte and
tracer possessing almost the same chemical immuno-recognition
moiety.
In addition, the effects of methanol and acetonitrile were
studied because these solvents are water-miscible and commonly
used in sample extraction procedures. The mPmax and IC50
changes were recorded, and results are shown in Figure 6. In
general, IC50 increased and mPmax also slightly increased
Because the ionic strength of the assay system can affect
antibody binding, the salt concentration of working buffer was
varied from 0 to 800 mM (Figure 5). For the FPIA method,

6876 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Wang et al.
Table 4. Percentage Recovery of SMP and SCP Fortified Milk
Samples by FPIA Using Tracer SCP FITC (n 3)
−
)
mean recovery
(%)
found (ng/mL)
type of
added
deproteinization
(ng/mL)
SMP
SCP
SMP
SCP
acetonitrile
1.25% TCA
50
100
500
50
100
500
34
87
726
32
70
489
±
±
±
±
±
2.7
9.6
65
3.6
7.5
30
96
600
32
78
513
±
±
±
±
±
3.5
8.3
62
4.1
7.5
68
87
145
64
70
98
60
96
120
64
78
103
±
43
±
46
Table 5. Influence of the Milk Sample Matrix on the FPIA Standard
Curves for SMP and SCP Using SCP−FITC as Tracer after Protein
Precipitation
IC50 (ng/mL)
mPmax
sample
precipitate content
SMP
SCP
SMP
SCP
buffer
milk
11.5
17
15
29
44
39
74
167
148
151
165
143
153
acetonitrile
1.25% TCA
2.5% TCA
30
Sample Preparation. The optimized FPIA method was used
to detect SMP and SCP spiked in milk. The analytical
characteristics of an immunochemical technique can be signifi-
cantly influenced by the organic solvent and the various
components existing in complicated matrices, such as protein
and fat (41). FPIA is susceptible to interference by different
components existing in milk. As some papers have reported
previously, the most common ways to reduce such matrix effects
are solid-phase extraction (SPE) techniques or dilution after
selective extraction (cleanup) to bring the interfering substances
below a concentration that would affect the assay. The SPE
approach is relatively time-consuming and expensive and would
counteract the advantage of the FPIA method in being a rapid
and simple screening application. Thus, in the present study,
the extracts were diluted to reduce matrix influences before
analysis.
The impact on FPIA of several common organic solvents has
been reported (42, 43). In these studies, methanol had been
selected as the preferred extraction reagent, not only because
of the efficiency of drug extraction and precipitation properties
but also because the methanol extract has the least influence
on the immunoassay. In the present recovery studies, negative
milk spiked with SMP and SCP (50, 100, and 500 ng/mL) was
extracted using three organic solvents, methanol, acetonitrile,
and 1.25% TCA with the same sample preparation protocol,
and recoveries of SMP and SCP from the milk were determined.
Samples for the recovery studies were prepared by extraction
of 4 mL of spiked milk with an equal volume of three extraction
reagents, followed by centrifugation to remove the protein and
dilution of 1 mL of supernatant in 4 mL of BB. The results are
represented in Table 4. The data obtained from methanol were
not presented because the extract was white (containing a lot
of protein) and could not be measured by the FPIA machine.
In a separate experiment for evaluation of matrix interference,
4 mL of unspiked milk was treated similarly, and the protein
was precipitated by 4 mL of methanol, acetonitrile, and 1, 1.25,
2.5, and 5% TCA, respectively. SMP standards were prepared
in the diluted extract to obtain standard curves. The mPmax and
IC50 for each standard curve are presented in Table 5. The mPmax
and IC50 values were significantly affected by methanol and
Figure 7. Scatchard plot for Kaff calculation. I: (a) tracer SCP−FITC,
antigen SMP, Y ) −0.903X
SCP, Y ) −0.433X 9.121. II: (c) tracer SMP
) −0.891X 20.716; (d) tracer SMP FITC, antigen SCP, Y ) −0.478X
9.488.
+
19.442; (b) tracer SCP−FITC, antigen
+
−FITC, antigen SMP, Y
+
−
+
gradually as the concentration of methanol and acetonitrile
increased. It can be observed that tolerance of methanol was
greater than that of acetonitrile. Acetonitrile concentrations
exceeding 5% (final concentration) were not tolerated, whereas
for methanol a concentration of 20% was still tolerated under
the conditions used for the FPIA, because in this situation, no
significant increase of IC50 and decrease of mPmax were
observed. The presence of 30% methanol resulted in a 37%
rise in IC50 value, but the mPmax, however, did not change
appreciably. Therefore, solvent type and concentration should
be carefully controlled for a reproducible FPIA procedure.
Measurement of Affinity Constant (Kaff). To explain the
difference in sensitivity, the Kaff values of monoclonal antibody
with two tracers were calculated. The results from Scatchard
plots are summarized in Table 3. The Scatchard graphs are
shown in Figure 7. From Table 3 and Figure 7 we observed
that the antibody affinity for SMP is the similar when SCP-
FITC and SMP-FITC were used; however, antibody affinity
for SCP using SMP-FITC (2.3 × 109 M-1) is slightly higher
than that of SCP-FITC used (2.1 × 109 M-1). This difference
in affinity maybe result in that the FPIA presents slightly better
sensitivity by SCP-FITC than SMP-FITC for SCP. The
competition of SCP was more effective in the case of tracer
SCP-FITC giving thus 25% better IC50 and consequently better
sensitivity in the assay.

Fluorescence Polarization Immunoassay for SMP and SCP
J. Agric. Food Chem., Vol. 55, No. 17, 2007 6877
(7) Berzas Nevado, J. J.; Castaneda Penalvo, G.; Guzman Bernardo,
F. J. Simultaneous determination of sulfamethoxypyridazine,
sulfamethoxazole, sulfadimethoxine and their associated com-
pounds by liquid chromatography. Anal. Chim. Acta 2001, 442,
241-248.
(8) Msagati, T. A. M.; Nindi, M. M. Multiresidue determination of
sulfonamides in a variety of biological matrices by supported
liquid membrane with high pressure liquid chromatography-
electrospray mass spectrometry detection. Talanta 2004, 64, 87-
100.
(9) Gratacos-Cubarsi, M.; Castellari, M.; Valero, A.; Garcia-
Regueiro, J. A. A simplified LC-DAD method with an RP-C(12)
column for routine monitoring of three sulfonamides in edible
calf and pig tissue. Anal. Bioanal. Chem. 2006, 385, 1218-
1224.
TCA concentrations above 2.5 or below 1% in the final extract
(data not shown). Although successfully applied to extract some
sulfonamides from milk and other drugs in food samples by
ELISA or FPIA (6, 29), methanol was not an effective protein
precipitation reagent without further purification processes in
this experiment. The TCA proved to be a strong protein
precipitation reagent (29). A 1.25% solution was an effective
concentration of TCA to completely remove the protein in milk
with only a slight effect on mPmax and IC50. The mean recoveries
of three spiked concentrations at 50, 100, and 500 ng/mL were
60-98% for SMP and 64-103% for SCP, respectively. A
concentration of TCA below 1% was ineffective in removing
interfering protein from the milk sample, and conversely, a
concentration of TCA exceeding 2.5% reduced antibody interac-
(10) Yang, S.; Cha, J.; Carlson, K. Simultaneous extraction and
analysis of 11 tetracycline and sulfonamide antibiotics in
influent and effluent domestic wastewater by solid-phase
extraction and liquid chromatography-electrospray ionization
tandem mass spectrometry. J. Chromatogr., A 2005, 1097, 40-
53.
(11) Wen, Y.; Zhang, M.; Zhao, Q.; Feng, Y. Q. Monitoring of five
sulfonamide antibacterial residues in milk by in-tube solid-
phase microextraction coupled to high-performance liquid
chromatography. J. Agric. Food Chem. 2005, 53, 8468-
8473.
(12) Maudens, K. E.; Zhang, G. F.; Lambert, W. E. Quantitative
analysis of twelve sulfonamides in honey after acidic hydrolysis
by high-performance liquid chromatography with post-column
derivatization and fluorescence detection. J. Chromatogr., A
2004, 1047, 85-92.
(13) Pang, G. F.; Cao, Y. Z.; Zhang, J. J.; Jia, G. Q.; Fan, C. L.; Li,
X. M.; Liu, Y. M.; Li, Z. Y.; Shi, Y. Q. Simultaneous
determination of 16 sulfonamides in honey by liquid chroma-
tography/tandem mass spectrometry. J. AOAC Int. 2005, 88,
1304-1311.
(14) Qin, Y.; Zhang, M.; Lin, H. Qualification and quantification of
10 sulfonamides in animal feedstuff by high performance liquid
chromatography-electrospray tandem mass spectrometry. Sepu
2005, 23, 397-400.
(15) Santos, B.; Lista, A.; Simonet, B. M.; Rios, A.; Valcarcel, M.
Screening and analytical confirmation of sulfonamide residues
in milk by capillary electrophoresis-mass spectrometry. Elec-
trophoresis 2005, 26, 1567-1575.
(16) Pasekova´, H.; Pola´sˇek, M.; Cigarro, J. F.; Dolejsˇova´, J. Deter-
mination of some sulphonamides by sequential injection analysis
with chemiluminescence detection Anal. Chim. Acta 2001, 438,
165-173.
(17) Bonwick, G. A.; Smith, C. J. Immunoassays: their history,
development and current place in food science and technology.
Int. J. Food Sci. Technol. 2004, 39, 817-827.
(18) Morozova, V. S.; Levashova, A. I.; Eremin, S. A. Determination
of pesticides by enzyme immunoassay. J. Anal. Chem. (Engl.
Transl.) 2005, 60, 202-217.
(19) Haasnoot, W.; Bienenmann-Ploum, M.; Kohen, F., Biosensor
immunoassay for the detection of eight sulfonamides in chicken
serum. Anal. Chim. Acta 2003, 483, 171-180.
(20) Korpimaki, T.; Hagren, V.; Brockmann, E. C.; Tuomola, M.
Generic lanthanide fluoroimmunoassay for the simultaneous
screening of 18 sulfonamides using an engineered antibody. Anal.
Chem. 2004, 76, 3091-3098.
(21) Cliquet, P.; Cox, E.; Haasnoot, W.; Schacht, E.; Goddeeris, B.
M. Extraction procedure for sulfachloropyridazine in porcine
tissues and detection in a sulfonamide-specific enzyme-linked
immunosorbent assay (ELISA). Anal. Chim. Acta 2003, 494, 21-
28.
tions and showed an increase in IC50 and a decrease in mPmax
.
At 5% TCA, there was a 12-fold increase in IC50 and 33%
decrease in mPmax, compared to that of the FPIA test result
obtained using BB. Similar recovery results were observed using
acetonitrile for interfering protein precipitation; however, the
presence of acetonitrile in the final extract solution resulted in
an 11% and 13% drop in mPmax and 34 and 37% rise in IC50
for SMP and SCP, respectively. Recoveries using acetonitrile
were 68-145% for SMP and 60-120% for SCP, and the mean
inter-assay coefficients of variation found for the assay for SMP
and SCP were 9.3 and 10.1%.
Conclusion. A sensitive, specific FPIA based on a Mab for
determination of SMP and SCP was developed. The Mab was
generated using an SMP hapten with a hemisuccinate bridge
spacer arm at the N4 position and conjugated to BSA. The IC50
values of the optimized FPIA were 11.5 and 29 ng/mL with a
detection limit of 0.7 and 0.25 ng/mL for SMP and SCP, and
the cross-reactivities with SMP and SCP were 100% and 40%,
respectively. The antibody showed negligible cross-reactivity
to other sulfonamides. SMP and SCP spiked in milk were
analyzed with satisfactory recovery. It was found that acetonitrile
and 1.25% TCA were optimal regents to remove the protein in
milk, and dilution of the extract was an effective means to reduce
the matrix and organic reagent interference. However, further
work will be needed to validate this assay for other applications.
The major advantages of FPIA are rapidity, simplicity, and
convenience of use in routine screening analysis.
LITERATURE CITED
(1) Dost, K.; Jones, D. C.; Davidson, G. Determination of sulfona-
mides by packed column supercritical fluid chromatography with
atmospheric pressure chemical ionisation mass spectrometric
detection. Analyst 2000, 125, 1243-1247.
(2) Johnson, D. K.; Combs, S. M.; Parsen, J. D.; Jolley, M. E. Lead
analysis by anti-chelate fluorescence polarization immunoassay.
EnViron. Sci. Technol. 2002, 36, 1042-1047.
(3) Blanchflower, W. J.; Rice, D. A. Extraction of sulfamethazine
from feed samples. J. AOAC Int. 1988, 71, 302-303.
(4) Muldoon, M. T.; Buckley, S. A.; Deshpande, S. S.; Holtzapple,
C. K.; Beier, R. C.; Stanker, L. H. Development of a monoclonal
antibody-based cELISA for the analysis of sulfadimethoxine. 2.
Evaluation of rapid extraction methods and implications for the
analysis of incurred residues in chicken liver tissue. J. Agric.
Food Chem. 2000, 48, 545-550.
(5) Haasnoot, W.; Cazemier, G.; Du Pre, J.; Kemmers-Voncken, A.;
Bienenmann-Ploum, M.; Verheijen, R. Sulphonamide antibod-
ies: from specific polyclonals to generic monoclonals. Food
Agric. Immunol. 2000, 12, 15-30.
(22) Martlbauer, E.; Meier, R.; Usleber, E.; Ierpian, G. Enzyme
immunoassay for the detection of sufamethazine, sulfadiazine,
sulfamethoxypyridazine and trimethoprim in milk. Food Agric.
Immunol. 1992, 4, 219-228.
(6) Spinks, C. A.; Schut, C. G.; Wyatt, G. M.; Morgan, M. R.
Development of an ELISA for sulfachlorpyridazine and inves-
tigation of matrix effects from different sample extraction
procedures. Food Addit. Contam. 2001, 18, 11-18.

6878 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Wang et al.
(23) Jackman, R.; Brown, A. J.; Dell, A. N.; Everest, S. J. Production
of antisera to 12 sulphonamides and their use in enzyme
immunoassays. In Proceedings of EuroResidues II conference,
Veldhoven, The Netherlands; Haagsma, N., Ruiter, A., Czedik-
Eysenberg, P. B., Eds.; 1993; pp 391-394.
(24) Eremin, S. A.; Smith, D. S. Fluorescence polarization immu-
noassays for pesticides. Comb. Chem. High Throughput Screen-
ing 2003, 6, 257-266.
(25) Kolosova, A. Y.; Park, J. H.; Eremin, S. A.; Kang, S. J.; Chung,
D. H. Fluorescence polarization immunoassay based on a
monoclonal antibody for the detection of the organophosphorus
pesticide parathion-methyl. J. Agric. Food Chem. 2003, 51,
1107-1114.
(26) Yakovleva, J. N.; Lobanova, A. I.; Panchenko, O. A.; Eremin,
S. A. Production of antibodies and development of specific
polarization fluoroimmunoassay for acetochlor. Int. J. EnViron.
Anal. Chem. 2002, 82, 851-863.
(27) Yakovleva, J.; Knopp, D.; Niessner, R.; Eremin, S. A. Develop-
ment of a polarization fluoroimmunoassay for herbicide met-
sulfuron-methyl. Food Agric. Immunol. 2002, 14, 217-229.
(28) Murtazina, N. R.; Eremin, S. A.; Mozoleva, O. V.; Everest, S.
J.; Brown, A. J.; Jackman, R. Fluorescent polarization immu-
noassay for sulphadiazine using a high specificity antibody. Int.
J. Food Sci. Technol. 2004, 39, 879-889.
(29) Eremin, S. A.; Murtazina, N. R.; Ermolenko, D. N.; Zherdev,
A. V.; Mart’ianov, A. A.; Yazynina, E. V.; Michura, I. V.;
Formanovsky, A. A.; Dzantiev, B. B. Production of polyclonal
antibodies and development of fluorescence polarization immu-
noassay for sulfanilamide. Anal. Lett. 2005, 38, 951-969.
(30) Eremin, S. A.; Landon, J.; Smith, D. S. Development of a
polarization fluoroimmunoassay for sulphamethazine using an
automated analyzer. Analyst 1994, 119, 2723-2726.
(36) Choi, M. J.; Lee, J. R.; Eremin, S. A. Development of single
reagent for fluorescence polarization immunoassay of atrazine.
Food Agric. Immunol. 2002, 14, 107-120.
(37) Eremin, S. A.; Knopp, D.; Niessner, R.; Hong, J. Y.; Park, S.
J.; Choi, M. J. High throughput determination of BTEX by a
one-step fluorescence polarization immunoassay. EnViron. Chem.
2005, 2, 227-234.
(38) Muldoon, M. T.; Font, I. A.; Beier, R. C.; Holtzapple, C. K.;
Young, C. R.; Stanker, L. H. Development of a cross-reactive
monoclonal antibody to sulfonamide antibiotics: evidence for
structural conformation-selective hapten recognition. Food Agric.
Immunol. 1999, 11, 117-134.
(39) Manclus, J. J.; Montoya, A. Develpoment of an enzyme-linked
immunosorbent assay for 3,5,6-trichloro-2-pyridinol. 1. produc-
tion and characterization of monoclonal antibodies. J. Agric.
Food Chem. 1996, 44, 3703-3709.
(40) Li, Q. X.; Zhao, M. S.; Gee, S. J.; Kurth, M. J.; Seiber, J. N.;
Hammock, B. D. Development of enzyme-linked immunosorbent
assays for 4-nitrophenol and substituted 4-nitrophenols. J. Agric.
Food Chem. 1991, 39, 1685-1692.
(41) Scortichini, G.; Annunziata, L.; Haouet, M. N.; Benedetti, F.;
Krusteva, I.; Galarini, R. ELISA qualitative screening of
chloramphenicol in muscle, eggs, honey and milk: method
validation according to the Commission Decision 2002/657/EC
criteria. Anal. Chim. Acta 2005, 535, 43-48.
(42) Eremin, S. A.; Ryabova, I. A.; Yakovleva, J. N.; Yazynina, E.
V.; Zherdev, A. V.; Dzantiev, B. B. Development of a rapid,
specific fluorescence polarization immunoassay for the herbicide
chlorsulfuron. Anal. Chim. Acta 2002, 468, 229-236.
(43) Shim, W. B.; Kolosova, A. Y.; Kim, Y. J.; Yang, Z. Y.; Park,
S. J.; Eremin, S. A.; Lee, I. S.; Chung, D. H. Fluorescence
polarization immunoassay based on a monoclonal antibody for
the detection of ochratoxin A. Int. J. Food Sci. Technol. 2004,
39, 829-837.
(31) Spinks, C. A.; Wyatt, G. M.; Lee, H. A.; Morgan, M. R.
Molecular modeling of hapten structure and relevance to broad
specificity immunoassay of sulfonamide antibiotics. Bioconjugate
Chem. 1999, 10, 583-588.
(32) Zhang, S. X.; Wang, Z. H.; Nesterenko, I. S.; Eremin, S. A.;
Shen, J. Z. Fluorescence polarisation immunoassay based on a
monoclonal antibody for the detection of sulphamethazine in
chicken muscle. Int. J. Food Sci. Technol. 2007, 42, 36-44.
(33) Hatzidakis, G. I.; Tsatsakis, A. M.; Krambovitis, E. K.; Spyros,
A.; Eremin, S. A. Use of L-lysine fluorescence derivatives as
tracers to enhance the performance of polarization fluoroimmu-
noassays. A study using two herbicides as model antigens. Anal.
Chem. 2002, 74, 2513-2521.
(34) Krasnova, A. I.; Eremin, S. A.; Natangelo, M.; Tavazzi, S.;
Benfenati, E. A polarization fluorescence immunoassay for the
herbicide propanil. Anal. Lett. 2001, 34, 2285-2301.
(35) Colbert, D. L.; Eremin, S. A.; Landon, J. The Effect of
Fluorescein Labels on the Affinity of Antisera to Small Haptens.
J. Immunol. Methods 1991, 140, 227-233.
Received for review April 2, 2007. Revised manuscript received June
20, 2007. Accepted June 21, 2007. Financial support was obtained from
the China-Russia grant “The investigation of antigen and antibody
interaction for veterinary drug avermectins and sulfonamides and
development of new immunoassay for environmental monitoring”
(RFBR-GFEN 03-03-39004), the Grant of Russian Foundation for Basic
Research “Influence of structural peculiarities of reactants on the
specificity of immunochemical assay of sulfonamides and development
of class-specific techniques for their detection” (RFBR-GFEN 06-03-
33015), and the National Natural Science Foundation of China (NSFC)
grant “Fluorescence polarization immunoassay for sulfonamides in
biological samples” (30471305).
JF070948D