Design and synthesis of immunoconjugates and development of an indirect ELISA for rapid detection of 3, 5-dinitrosalicyclic acid hydrazide

Article abstract of DOI:10.3390/molecules13092238

In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acid-ovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a "substructural coating antigen" to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the "substructural coating antigen" concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC₅₀ of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue.

Full text of DOI:10.3390/molecules13092238

Molecules 2008, 13, 2238-2248; DOI: 10.3390/molecules13092238
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.org/molecules
Article
Design and Synthesis of Immunoconjugates and Development of
an Indirect ELISA for Rapid Detection of 3, 5-Dinitrosalicyclic
Acid Hydrazide
Yu-Dong Shen 1, Shi-Wei Zhang 1, Hong-Tao Lei 1, Hong Wang 1, Zhi-Li Xiao 1,
Yue-Ming Jiang 2,* and Yuan-Ming Sun 1,*
1
College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China;
E-mails: sydscau@yahoo.cn (Y-D. S), zsw_8506@163.com (S-W. Z), hongtao@scau.edu.cn (H-T.
W); gzwhongd@163.com (H. W.); scauxzl@scau.edu.cn (Z-L. X)
2
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P. R. China
* Authors to whom correspondence should be addressed; E-mails: ymsun@scau.edu.cn (Y-M. S) or
ymjiang@scib.ac.cn (Y-M. J).
Received: 22 August 2008; in revised form: 14 September 2008 / Accepted: 16 September 2008 /
Published: 23 September 2008
Abstract: In this study novel immunoconjugates were designed, synthesized and then used
to develop a rapid, specific and sensitive indirect ELISA method to directly detect
residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol
present in chicken tissues. The hapten DNSHA was first designed and used to covalently
couple to BSA to form an immunogen which was immunized to rabbits to produce a
polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acid-
ovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was
designed and used as a “substructural coating antigen” to improve the sensitivity of an
indirect ELISA analysis for a direct DNSH detection. Based on the “substructural coating
antigen” concept, an optimized indirect ELISA method was established that exhibited good
specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than
0.1% (excluding the parent compound nifursol), IC50 of 0.217 nmol/mL and detection limit
of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken
tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%,

Molecules 2008, 13
with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA
2239
method exhibited the potential for a rapid detection of DNSH residues in tissue.
Keywords: Nifursol, DNSH, Hapten design, Indirect ELISA
Introduction
Nifursol is a member of the nitrofuran antibiotics family (Figure 1), which is used extensively as a
feed additive for the prevention of histomoniasis. Due to the potentially harmful effects of nifursol on
human health, its use in food-producing animals has been banned in the European Union since 2002
[1]. As nifursol was rapidly metabolized to form the metabolic marker 3,5-dinitrosalicyclic acid
hydrazide (DNSH, Figure 1) which can persist for a long time in vivo, several laboratories have
focused on development of DNSH detection methods [2-4]. At present, the detection of DNSH is
mainly at the μg/kg level by HPLC-MS methods, which are based on the analysis of the DNSH
derivative 2-hydroxy-3,5-dinitro-N'-(2-nitrobenzylidene)benzohydrazide (NPDNSH, Figure 1) [2-4].
Disadvantageously, these analytical methods not only require a time-consuming and troublesome
derivatization step and expensive apparatus but also have low sample throughput.
Immunoassay is an efficient screening technique for monitoring of illegal and harmful chemicals in
food and the environment, which exhibits the great advantages of speed and high sample throughput
[5]. To the best of our knowledge, there have been no reports on the development of a specific
antibody to DNSH and an immunoassay for a direct detection of DNSH. This study reports for the first
tme the synthesis of immunoconjugates for DNSH, the preparation of a polyclonal antibody against
DNSH and the development of an indirect competitive enzyme-linked immunosorbent assay (indirect
ELISA) for rapidly analyzing DNSH residues present in chicken tissue. At the same time, this study
developed a “substructural coating hapten” strategy to improve the precision of indirect ELISA for
detecting DNSH.
Figure 1. The structures of nifursol, DNSH, NPDNSH, DNSA and DNSHA [2-4].

Molecules 2008, 13
2240
Results and Discussion
Immunogen design and synthesis
The structural design of the hapten is an important step in the development of immunoassays for
small organic analytes. As the structure of the hapten used to form an immunogen can remarkably
affect the sensitivity and specificity of the antibody, the hapten should be a near perfect mimic of the
structure of the target analyte [6-8]. On the other hand, as organic molecules of less than 1,000 Da
possess no antigenicity, they must be covalently conjugated to a carrier protein by introducing a linker
with an active terminal group on its organic molecular structure to form a complete antigen to
smoothly elicit a specific immune response. Furthermore, the arm used to couple to a carrier protein
was another key role in hapten design, which should not elicit antibody recognition in itself [8].
Generally, an appropriate linear hydrocarbon handle is necessary. Our previous studies showed that a
short unsaturated linear chain which was attached to target molecule can remarkably improve the
capability to evoke a specific immune response [9]. In this study, a short unsaturated arm based on
glyoxalic acid was directly introduced into the target analyte to form a DNSHA hapten which was
covalently linked to the carrier protein bovine serum albumin (BSA) to produce a DNSHA-BSA
immunogen (Figure 2).
Figure 2. Synthesis of immunogen DNSHA-BSA [10-13].
O
O
O
O2N
O2N
NH2
O2N
OH
OH
N
O
Methol
H2SO4
N2H5HSO4
NH3
H
OH
OH
NO2
NO2
NO2
DNSH
MDNSA
DNSA
O
O
O
O
O2N
N
BSA
O2N
N
N
N
N
OH
1.DCC, NHS
2. BSA
Glyoxalic acid
Methol
H
H
H
OH
OH
NO2
NO2
DNSHA
DNSHA-BSA
NMR and MS analyses, as indicated by in the Experimental, confirmed that the DNSHA hapten
was prepared. The DNSHA-BSA immunogen was synthesized by the active ester method [13]. UV
spectra showed qualitative differences between the carrier protein and conjugate in the maximum
absorbance of hapten, which suggested the presence of DNSHA-BSA. Furthermore, the ratio of
DNSHA coupling to BSA was determined to be 21:1 by the TNBS method [14].
Design, synthesis and choice of coating antigens
The structure of the hapten used to form a coating antigen has a direct effect on the immunoassay
[15]. Generally, immunohapten and target analyte have a similar structure and their affinities for an

Molecules 2008, 13
2241
antibody are almost identical. Thus, homologous coating using an immuno-hapten-OVA conjugate as a
coating antigen usually results in a similar or weaker recognition of the antibody towards the target
analyte. The “substructural coating hapten”, a major structure of which was one of the fragments of
target analyte structure, can improve remarkably the competitive recognition sensitivity of antibody to
a target analyte, which could result from less interaction regions or points of the “substructural coating
hapten” with the pocket area of the antibody than those of the analyte. Based on this concept, DNSA
was designed as a “substructural coating hapten” for detecting DNSH by indirect ELISA. In addition,
in order to prove the validity of the “substructural coating hapten” concept, standard curves of DNSH
were established under optimum conditions using DNSA-OVA as coating antigen and DNSHA-OVA
as a control, respectively. As we had expected, the result shows that the IC50 and detection limit of the
indirect ELISA with DNSA-OVA as coating antigen were much better than those of the indirect
ELISA with DNSHA-OVA (Table 1), which suggested that the “substructural coating hapten” strategy
for indirect ELISA improvement was rational and efficient.
Figure 3. UV spectra of BSA, OVA, DNSHA-BSA, DNSHA-OVA and DNSHA.
2
OVA
DNSHA-BSA
DNSHA-OVA
BSA
300
DNSHA
500
0
250
350
400
450
Wavelength
Figure 4. UV spectra of OVA, DNSA-OVA and DNSA.
2
DNSA
OVA
DNSA-OVA
0
250
300
350
400
450
500
Wavelength

Molecules 2008, 13
2242
Coating antigens were also synthesized by the active ester method with OVA instead of BSA [13].
UV spectra show qualitative differences between OVA and conjugates (DNSHA-OVA or DNSA-OVA)
in the maximum absorbance of corresponding haptens (DNSHA or DNSA), as shown in Figures 3 and
4, which suggested the presence of DNSHA-OVA and DNSA-OVA. The molar ratio of DNSHA and
DNSA coupling to OVA were determined to be 11:1 by the TNBS method [14].
Table 1. Indirect ELISA evaluation for DNSH with different coating antigens.
Immunogen
Coating antigen
DNSHA-OVA
DNSA-OVA
IC50 (nmol/mL)
10.328
Detection limit (nmol/mL)
0.892
0.018
DNSHA-BSA
0.217
Table 2. Cross-reactivity of the antibody with various chemicals.
Competitor IC50 (nmol/mL) Cross-reactivity (%)
0.217
0.069
100
314.5
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
3,5-Dinitrosalicyclic acid hydrazide (DNSH)
Nifursol
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3-Amino-2-oxazolidinone (AOZ)
5-Methylamorfolino-3-amino-2-oxazolidone (AMOZ)
1-Aminohydantoin (AHD)
Semicarbazide (SEM)
Ciprofloxacin
Enrofloxacin
Clenbuterol
Salbutamol
Ractopamine
Malachite green
Evaluations of antibody specificity and indirect ELISA
A DNSH standard curve (Figure 5) was prepared using DNSA-OVA as coating antigen, with a
coating concentration of 1000 ng/mL, antibody dilution of 1:2000, competitive time of 1 h, sheep anti-
rabbit IgG dilution of 1:10000 and reaction time of 20 min. The IC50 (concentration causing 50%
inhibition of binding) was 0.217 nmol/mL. The limit of detection (the concentration causing 90%

Molecules 2008, 13
2243
inhibition of binding) was 0.018 nmol/mL. The linear detection range (the concentration
correspondong to 20% to 80% inhibition of binding) was 0.046 to 1.029 nmol/mL. In addition, under
the optimized indirect ELISA conditions, the specificity of the antibody was estimated using DNSA-
OVA as coating antigen and ten frequently-used chemicals as competitors (Table 2). The results show
that the cross-reactivity rates for the above-mentioned ten competitors, excluding nifursol (the parent
compound of the analyte), were all less than 0.1%, which suggested the antibody possessed a high
specificity for detection of DNSH and the parent compound nifursol (Table 2). However, nifursol
could be quite rapidly metabolized into DNSH in vivo, so its high cross-reactivity rate didn’t affect the
indirect ELISA’s application to DNSH in real samples. The study indicated that the indirect ELISA
method could be practical for development of commercial ELISA kits for monitoring DNSH residues
in chicken tissues.
Figure 5. Standard curve of indirect ELISA for DNSH (n=3).
100
80
60
40
20
0
1E-3
0.01
0.1
1
10
DNSH (nmol/mL)
Sample analysis
Pretreatment of samples. Generally, the DNSH present is bonded with animal tissues in vivo and it
appears in a free form by acid hydrolysis [4]. The chicken tissues mixed with DNSH were extracted
with water. Ether was used to degrease the samples because of insolubility of DNSH in ether, while
methanol was used to remove tissue proteins from aqueous solution with DNSH. A pretreatment
period of less than 50 min was used in this study.
Analysis of samples. The indirect ELISA method was used to detect DNSH present in chicken samples.
The analysis results are summarized in Table 3. All coefficients of variation were below 18.4% and the
average recovery was 82.3%, which suggested that good precision was obtained in this study.

Molecules 2008, 13
Table 3. Analysis of DNSH present in chicken tissues by indirect ELISA (n=3).
2244
Fortification
Mean ± S.D.
(nmol/mL)
0.076 ± 0.014
0.17 ± 0.03
0.69 ± 0.09
1.64 ± 0.24
Recovery
(%)
CV
(%)
18.4
17.6
13.0
14.6
15.9
Level (nmol/mL)
0.10
0.20
76.0
85.0
0.80
86.2
2.00
82.0
Means
82.3
Conclusions
Based on the concept of “substructural coating antigen”, an indirect ELISA for direct DNSH
detection, with an IC50 value of 0.217 nmol/mL and a limit of detection of 0.018 nmol/mL, was
developed in this study. The indirect ELISA was applied for DNSH determination in chicken samples,
with a pretreatment and detection period of less than 2.5 h. Coefficients of variation were about 18.4%
and the mean recoveries were 82.3%. Thus, this indirect ELISA with high sensitivity and good
stability could potentially be used to develop commercial ELISA kits for a sensitive and rapid
detection of DNSH residues.
Experimental
Chemicals and reagents
3,5-Dinitrosalicylic acid (DNSA), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC),
3,3’,5,5’-tetramethylbenzidine (TMB), bovine serum albumin (BSA) and ovalbumin (OVA) were
purchased from Sigma Chemical Company. N,N-Dimethylformamide (DMF), Tween-20 and methanol
were obtained from Damao Co. Ltd (Tian Jin, P.R. China). Thin-layer chromatography (TLC) was
done using 0.25-mm precoated silica gel GF254 on aluminum sheets purchased from Qingdao Haiyang
Chemical Co. Ltd. (Qingdao, P.R. China). New Zealand white rabbits were supplied by the
Guangdong Medical Experimental Animal Centre. Enzyme immunoassay grade horseradish
peroxidase (HRP), peroxidase-labeled goat antirabbit IgG were obtained from Wuhan Boster Biotech
Co. The other chemicals were purchased from Alfa Corporation. Polystyrene ELISA plates were
obtained from Guangzhou Jiete Biotech Co. and then treated with a Multiskan MK2 microplate washer
(Thermo Labsystems). Absorbance in ELISA analysis was recorded with a Multiskan MK3 microplate
reader (Thermo Labsystems). HPLC-MS analyses were performed using an Agilent HP1100 series
(Agilent, Palo Alto, CA). Ultraviolet-visible (UV) spectra were obtained on a UV-160A Shimadzu
1
spectrophotometer (Kyoto, Japan) and H-NMR spectra were recorded using a DRX-600 NMR
spectrometer (Bruker, Germany-Switzerland). The buffers used in this study were as follows: 0.1
mol/L phosphate buffer saline (PBS) containing 2 mol/L NaOH for sample neutralization, 50 mmol/L
carbonate buffer (pH 9.6) for coating, 10 mmol/ L phosphate buffer saline (PBS) with 0.1% Tween-20
for washing, 0.1 mol/L sodium acetate (pH 5.5) for substrate buffer and 2 mol/L H2SO4 as the stopping

Molecules 2008, 13
2245
reagent. In addition, the substrate solution for horseradish peroxidase was prepared by addition of 10
mL of above-mentioned PBS substrate buffer and 150 μL of 1% (w/v) TMB solution into DMF and
2.5 μL of 6% (w/v) H2O2.
Synthesis of methyl 3,5-dinitrosalicylate (MDNSA)
DNSA (2.28 g) was dissolved in methanol (50 mL), and the solution was heated under reflux with
stirring for 24 h after the addition of 98% H2SO4 (0.5 mL). About half of the solution was removed
under vacuum before Na2CO3 was added until no gas bubbles were observed. The obtained residue
was dissolved in water (100 mL) and MDNSH (2.1 g) was separated by filtration and washed 3 times
1
with water. APCI-MS analysis (negative) m/z 241 [M-H]- ; H-NMR (600 MHz, CDCl3 and TMS): δ
9.03 (d, J=3.0 Hz, 1H, ArH), 9.01 (d, J=3.0 Hz, 1H, ArH) and 4.12 (s, 3H, CH3).
Synthesis of 3,5-dinitrosalicyclic acid hydrazide (DNSH)
MDNSA (1 g) was reacted with hydrazine sulphate (0.8 g) and then suspended in 30% ammonia
(200 mL). The mixture was allowed to stand for 24 h at 60 ºC and then filtered. The filtered residue
was washed with ethanol and then recrystallized from methanol to give DNSH (0.7 g, 70% yield) as
1
yellow crystals. APCI-MS analysis (negative) m/z was 241 [M-H]- while H-NMR (600 MHz, d6-
DMSO and TMS): δ 8.92 (d, J=3.0Hz, 1H, ArH) and 8.72 (d, J=3.0 Hz, 1H, ArH).
Synthesis of 2-(2-(2-hydroxy-3,5-dinitrobenzoyl)hydrazono) acetic acid (DNSHA)
To a methanol solution (10 mL) containing glyoxalic acid (0.11 g), DNSH (0.24 g) was added. The
mixture was allowed to stand for 3 h at about 25 ºC and then filtered. The filtered residue was washed
three times with ethanol to give DNSHA (0.21 g, 72% yield) as a yellow powder. APCI-MS analysis
1
(negative) m/z 297 [M-H]-; H-NMR (600 MHz, d6-DMSO and TMS): δ 14.13 (s, 1H, OH); 8.78 (d,
J=3.0 Hz, 1H, ArH), 8.58 (d, J=3.0 Hz, 1H, ArH), 7.95 (s, 1H, N=CH) and 7.70 (s, 1H, NH).
Preparation of immunizing conjugates
DNSHA (or DNSA) (10 μmol), NHS ester (10 μmol) and DCC (10 μmol) were dissolved in DMF
solution (1000 μL). The mixture was gently stirred overnight at 4 ºC and then centrifuged (2,500 rpm
for 10 min) to remove precipitated urea. The clear supernatant phase was collected and 900 μL of the
supernatant was added to BSA or OVA (60 mg) in PBS (pH 7.4, 9 mL). The reaction mixtre was
stirred overnight at 4°C and then dialyzed against 0.9% NaCl solution (1,000 mL) for 3 days at 4°C
with three changes per day to give the DNSHA-BSA, DNSHA-OVA and DNSA-OVA immuno-
conjugates. They were respectively diluted to 1 mg/mL with 0.9% NaCl solution and then stored at -
20°C until used. The ratios of DNSHA and DNSA to carrier proteins were determined by the TNBS
method.

Molecules 2008, 13
2246
Preparation of polyclonal antibody to DNHS
For booster immunizations, immunogen (1 mg) was dissolved in 0.01 mol/L PBS (pH 7.4, 0.5 mL)
and emulsified with Freund’s incomplete adjuvant (0.5 mL). The emulsion was then injected
subcutaneously. The booster immunizations were repeated every three weeks. The New Zealand white
rabbit was bled through ear vein one week after each injection. To obtain antiserum, blood samples
were left to coagulate for 1 h at about 25 ºC and overnight at 4 ºC, followed by centrifugation for 10
min at 10,000 rpm. The clear supernatant phase was carefully collected. The polyclonal antibody was
purified from antiserum by the ammonium sulfate precipitation method [16], then divided into aliquots
and finally stored at -20 ºC until used.
Indirect ELISA procedure
Standard DNSH and its structurally related compounds were diluted to various concentrations with
PBS before the ELISA assay. The checkerboard procedure was used to optimize the coating antigen
and the antibody concentrations. For the ELISA, each well of a microtitre plate was coated with 100
μL coating antigen (DNSA-OVA or DNSHA-OVA) at the optimal dilution and then incubated
overnight at 4 ºC. The excess binding sites were blocked with 5% glycine for 3 h at 37 ºC. After
removal from the blocking solution, the optimal antibody dilution solution (50 μL) and DNSH
standard solution (50 μL) were added to the wells before the plate was incubated for 1 h at 37 ºC. After
3 washes with PBS with 0.05% Tween 20, horseradish peroxidase-labeled goat antirabbit IgG solution
(100 μL) was added to each well and the plate was incubated for 20 min at 37 ºC. The plate was
washed before 1% (w/v) tetramethylbenzidine (100 μL) was added. Finally, the plate was incubated
for 10 min at 37 ºC before 2 mol/L H2SO4 (50 μL) was added to stop the reaction. The plate was read
at 450 nm using an ELISA plate reader.
Relative absorbance was calculated using the formula B/B0, where B0 was the absorbance of the
well without DNSH and B was the absorbance of the well with DNSH. The standard curves were
constructed by plotting relative absorbance average values against the logarithm of DNSH
concentrations with three replicates per concentration. Sigmoidal curves were fitted to four-parameter
logistic equation and the standard deviations (n=3) were calculated, using Origin Program 7.5 software
packages.
The specificities of the ELISAs against several veterinary chemicals were determined and
calculated as the cross reaction rate (CR).
IC50 (DNSH )
CR (%) =
× 100
IC50 (test compound )
Preparation of samples
Chicken samples were purchased in a local retail outlet. The chicken was cut into small pieces. The
tested samples were respectively spiked with methanol solutions (10 μL) containing different DNSH
concentrations (0.1, 0.2, 0.8 and 2.0 nmol/g chicken). A sample without DNSH was used as a negative

Molecules 2008, 13
2247
control. The homogenized sample (1.0 g) was transferred into a glass tube and distilled water (0.9 mL)
and 1 mol/L HCl (0.1 mL) were added and mixed for 10 min using a vortex mixer. After centrifugation
for 2 min at 10,000 rpm, the supernatant phase was collected. The supernatant was mixed with ether (1
mL) for 60 s using a vortex mixer and then centrifuged for 2 min at 10,000 rpm. The water phase was
collected after centrifugation. The DNSH content of the water phase was evaporated to dryness at 50
ºC under nitrogen. Then, methanol (2 mL) was added, mixed for 10 min using a vortex mixer and
centrifuged for 2 min at 10,000 rpm. Finally, the methanol phase was evaporated to dryness at 50 ºC
under nitrogen. The obtained residue was redissolved in 0.5 mL of assay buffer mentioned above and
placed in an ultrasonic bath for 2 min, followed by mixing for 2 min with a vortex. DNSH content was
detected by the above-mentioned indirect ELISA method.
Acknowledgements
The work was supported by the Natural Science Foundation of Guangdong Province (06300421 and
06300423), the Headmaster’s Science Fund of High Schools (5100-k06133) and Public Bidding Items
Fund for a Great Breakthrough in Key Fields (2006A25005002) and the Research Fund for the
Doctoral Program of Higher Education (20050564011).
References and Notes
1.
2.
Commission Decision No. 2002/657/EC.
Verdon, E.; Couedor, P.; Sanders, P. Multi-residue monitoring for the simultaneous
determination of 5 nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoin, nifursol)
in poultry muscle tissue through the detection of their five major metabolites (AOZ, AMOZ,
SEM, AHD, DNSAH) by liquid chromatography coupled to electrospray tandem mass
spectrometry-In-house validation in line with Commission Decision 657/2002/EC. Anal. Chim.
Acta 2007, 586, 336-347.
3.
4.
Vahl, M. Analysis of nifursol residues in turkey and chicken meat using liquid chromatography-
tandem mass spectrometry. Food Addit. Contam. 2005, 22, 120-127.
Mulder, P. P. J.; Zuidema, T.; Keestra, N. G. M.; Kooij, P. J. F.; Elbers, I. J. W.; van Rhijn, J. A.
Determination of nifursol metabolites in poultry muscle and liver tissue. Development and
validation of a confirmatory method. Analyst 2005, 130, 763-771.
5.
6.
Nunes, G. S.; Toscano, I. A.; Barcelo, D. Analysis of pesticides in food and environmental
samples by enzyme-linked immunosorbent assays. Trends. Analyt. Chem. 1998, 17, 79-87.
Peterson, E. C.; Gunnell, M.; Che, Y. N.; Goforth, R. L.; Carroll, F. I.; Henry, R.; Liu, H. M.;
Owens, S. M. Using hapten design to discover therapeutic monoclonal antibodies for treating
methamphetamine abuse. J. Pharmacol. Exp. Ther. 2007, 322, 30-39.
7.
8.
Liu Y. H.; Jin M. J.; Gui W. J.; Cheng J. L.; Guo Y. R.; Zhu G. N. Hapten design and indirect
competitive immunoassay for parathion determination: Correlation with molecular modeling and
principal component analysis. Anal. Chim. Acta 2007, 591, 173-182.
Marvin H. G.; Bruce D. H. Hapten design for compound-selective antibodies: ELISAS for
environmentally deleterious small molecules. Anal. Chim. Acta 1998, 376, 83-91.

Molecules 2008, 13
9.
2248
Shen, Y. D.; Wang, Y.; Zhang, S. W.; Xiao, Z. L.; Sun, Y. M.; Bu, X. Z.; Gu, L. Q. Design and
efficient synthesis of novel haptens and complete antigens for the AOZ, a toxic metabolite of
furazolidone. Chin. Chem. Lett. 2007, 18, 1490-1492.
10. Gupta, G. L. New synthesis of salicylhydrazide and its nitro derivatives. Curr. Sci. 1964, 33,
616-17.
11. White, R. L. Jr.; Wessels, F. L.; Schwan, T. J.; Ellis, K. O. 1-[[[5-(Substituted phenyl)-2-
oxazolyl]methylene]amino]-2,4-imidazolidinediones, a new class of skeletal muscle relaxants. J.
Med. Chem. 1987, 30, 263-266.
12. El-Gendy, Z.; Morsy, J. M.; Allimony, H. A.; Abdel-Monem, W. R.; Abdel-Rahman, R. M.
Synthesis of heterobicyclic nitrogen systems bearing the 1,2,4-triazine moiety as anti-HIV and
anticancer drugs, part III. Pharmazie 2001, 56, 376-383.
13. Hong, X. Z.; Sun, M. Q. The Technology of Protein Linking. China Medical Technology Press:
Beijing, P. R. China, 1993; pp. 2-56.
14. Sashidhar, R. B.; Capoor, A. K.; Ramana, D. Quantitation of ε-amino group using amino acids as
reference standards by trinitrobenzenesulfonic acid. A simple spectrophotometric method for the
estimation of hapten to carrier protein ratio. J. Immunol. Method 1994, 167, 121-127.
15. Zhang Q.; Wang L. B.; Ahn K. C.; Sun Q.; Hu B. S.; Wang J.; Liu F. Q. Hapten heterology for a
specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus
insecticide fenthion. Anal. Chim. Acta 2007, 596, 303-311.
16. Wengatz I.; Stoutamire D.; Gee S. J.; Hammock, B. D. Development of an ELISA for the
detection of the pyrethroid insecticide fenpropathrin, J. Agric. Food Chem. 1998, 46, 2211-2221.
Sample Availability: Available from the authors.
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