Development of a biomimetic enzyme-linked immunosorbent assay method for the determination of methimazole in urine sample

Article abstract of DOI:10.1002/jccs.201190007

A fast and direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method was developed for the determination of methimazole (MMZ) in urine sample based on a molecularly imprinted film as an artificial antibody. This is the first example to monitor methimazole with a direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method. The imprinted film was directly synthesized on the well surface of MaxiSorp polystyrene 96-well plate and characterized. The results showed that it exhibited an antibody-like binding ability, rapid adsorption speed, high stability, which was particularly advantageous and suitable for BELISA development. The BELISA method established in this paper had a higher selectivity for MMZ than for the structurally related compounds and the IC50 (calculated as the concentration giving 50% inhibition of color development) and the detection limit values under optimized experimental conditions were 70 ± 4 μg L^-1 and 0.9 ± 0.04 μg L^-1, respectively. The method was applied to the determination of MMZ in spiked urine sample with excellent recoveries ranging from 90% to 95%, and the imprinted film was able to be reused for 20 times without loss of sensitivity. The results obtained by BELISA correlated well with that obtained by the high performance liquid chromatography (HPLC) method.

Full text of DOI:10.1002/jccs.201190007

Journal of the Chinese Chemical Society, 2011, 58, 463-469
463
Development of a Biomimetic Enzyme-linked Immunosorbent Assay
Method for the Determination of Methimazole in Urine Sample
Jun-Ping Wang, Wei-Wei Tang, Guo-Zhen Fang, Ming-Fei Pan and Shuo Wang*
Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science
and Technology, 29 The Thirteenth Road, Tianjin Economy and Technology Development Area,
Tianjin 300457, P. R. China
Received January 14, 2011; Accepted April 7, 2011; Published Online April 22, 2011
A fast and direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method
was developed for the determination of methimazole (MMZ) in urine sample based on a molecularly im-
printed film as an artificial antibody. This is the first example to monitor methimazole with a direct com-
petitive biomimetic enzyme-linked immunosorbent assay (BELISA) method. The imprinted film was di-
rectly synthesized on the well surface of MaxiSorp polystyrene 96-well plate and characterized. The re-
sults showed that it exhibited an antibody-like binding ability, rapid adsorption speed, high stability,
which was particularly advantageous and suitable for BELISA development. The BELISA method estab-
lished in this paper had a higher selectivity for MMZ than for the structurally related compounds and the
IC50 (calculated as the concentration giving 50% inhibition of color development) and the detection limit
values under optimized experimental conditions were 70 4 mg L-1 and 0.9 0.04 mg L-1, respectively.
The method was applied to the determination of MMZ in spiked urine sample with excellent recoveries
ranging from 90% to 95%, and the imprinted film was able to be reused for 20 times without loss of sensi-
tivity. The results obtained by BELISA correlated well with that obtained by the high performance liquid
chromatography (HPLC) method.
Keywords: Methimazole; Molecularly imprinted film; BELISA; HPLC.
INTRODUCTION
MMZ (1-methyl-2-mercaptoimidazole, tapazole)
scribed for the effectively monitoring MMZ in kinds of
samples including urine,7-11 plasma,6 tissues9,12-13 and se-
rum.15 Flow injection analysis,15 electrochemical detec-
(Fig. 1) is a kind of antithyroid drugs. Its action is to slow
iodide integration into tyrosine and thus inhibits the pro-
duction of thyroid hormones. So it is widely used in medi-
cine for treatment of hyperthyroidism.1 For it can promote
the metabolism of protein, it has been applied illegally to
animals’ feed to obtain more lean meat. Although MMZ
has been used in the treatment of hyperthyroidism, the un-
controlled introduction of this and other thyreostats into the
human’s food chain could have serious health implica-
tions.2 MMZ may cause side effects such as the irritation of
skin, impaired taste, olfaction, allergies or pharyngitis with
fever, and in rare occasions, nephritis and liver cirrhosis.3-4
Consequently, the use of MMZ in animal production has
been prohibited in many countries including European Un-
ion.5
So far, various analytical methods in TLC,6 GC-
MS,7-8 HPLC-UV9-11 or MS12-13 and CE14-15 have been de-
Fig. 1. Structures of MMZ, 2-mercaptoimidazole and
N-methylthiourea.
* Corresponding author. Tel: (+86) 22-60601456; Fax: (+86) 22-60601332; E-mail: s.wang@tust.edu.cn

464 J. Chin. Chem. Soc., Vol. 58, No. 4, 2011
Wang et al.
tion16-18 and molecularly imprinted polymer (MIP)19 for
MMZ are also developed. Among all of these methods,
HPLC is the most commonly used method because it is sen-
sitive and selective and it could be provided at a low level
of MMZ, but it is expensive and not suitable for extensively
prevalence. It is very significant to develop a simple and in-
expensive analytical method to monitor the residual quan-
tity of MMZ in biological samples.
Tianjin Kermel Chemical Reagents Co., Ltd. Tianjin,
China). Dimethylformamide (DMF), N-dicyclohexylcar-
bodiimide (DCC), N-hydroxysuccinate (NHS) and other
organic chemicals used for hapten synthesis were pur-
chased from Fluka. Horseradish peroxidase (HRP) was
purchased from Boehringer-Mannheim (Germany). Re-
agent grade hydrogen peroxide and Tween-20 were pur-
chased from Sigma (Sigma-Aldrich, USA). Maxisorp poly-
styrene 96-well plates were obtained from Nunc (Roskilde,
Denmark). The thin layer chromatography (TLC) plate was
purchased from Merck (Darmstadt, Germany).
Enzyme-linked immunosorbent assay (ELISA) is
sensitive and selective, and it can run many analytes simul-
taneously.20 However, this method has many disadvantages
such as the high cost, difficulty of the acquisition of anti-
body production, and the need of laboratory animals.
Moreover, the production of antibodies for toxic sub-
stances or immunosuppressants is particularly difficult, be-
cause they have effects on metabolism and the immune sys-
tem.21 Some studies have attempted to design and synthe-
size artificial antibody to replace antibodies.
HPLC-grade solvents used to prepare the LC-mobile
phase were methanol purchased from Concord Co. (Tian-
jin, China) and doubly deionized water (DDW, 18.2 MW
cm-1) obtained from a Water Pro water system (Labconco
Corp., Kansas City, MO, USA).
Solutions
5 × PBS: Na2HPO4·12H2O: 344 g, NaH2PO4·2H2O:
44.85 g, NaCl: 225 g, H2O: 5 L, when used, diluted to 1 ×
PBS. PBS/T: PBS with 0.05% Tween-20, Substrate Solu-
tion: Color Reagent A (anhydrous sodium acetate: 8.2 g,
b-cyclodextrin: 2.5 g, citric acid: 3.16 g, H2O: 1 L, car-
bamide peroxide: 0.429 g), Color Reagent B (TMB: 0.05 g,
DMSO: 5 mL). stop solution: 1.25 mmol L-1 H2SO4.
Instrumentation
Molecular imprinting technology (MIT) has been
proved to be an efficient method to produce functionalized
materials which are used as antibodys to recognize the tem-
plate from closely related compounds. MIP is inherent sta-
bility, low cost and ease of preparation. The MIP as anti-
bodies for application in ELISA should be provided with
appropriate wetting properties, a large surface area, and
easily accessible binding sites. But if it is directly synthe-
sized on the well surface of a MaxiSorp polystyrene
96-well plate, the traditional MIP is inhomogeneous and
difficult to remove the template. Moreover it is hard to con-
trol the thickness of the MIP. The parameters affecting the
performance of this BELISA method were discussed in de-
tail. This developed method was applied to the analysis of
MMZ in the urine of pig, and the results were validated by
the HPLC method.
HPLC system consisted of two LC-10ATVP pumps
and a Shimadzu SPD-10AVP ultraviolet detector (Shimadzu,
Kyoto, Japan). All separations were achieved on an analyt-
ical Hypersil-ODS column (4.6 mm × 250 mm long,
Thermo) at a mobile flow rate of 1 mL min-1 at the room
temperature. The detection wavelength and injection vol-
ume were 254 nm and 20 mL, respectively. Class-VP soft-
ware was used to acquire and process spectral and chro-
matographic data. A Cary 50-Bio UV spectrophotometer
(Bio-Rad, USA) and an ultrasonicator were also employed
in the adsorption test (l = 254 nm) and the preparation of
HPLC mobile phase, respectively. Analysis of mass spectra
(MS) was carried out on ESI mode on a Survegor-Lcce Ad-
vantage Max 10 mass spectrometer (Thermofinnigan,
USA).
EXPERIMENTAL
Materials and Reagents
MMZ (99.7%) was purchased from Beijing Yanjing
Pharmaceutical Co., Ltd. (Beijing, China). And pure stan-
dard of N-methylthiourea and 2-mercaptoimidazole (Fig.
1) were purchased from Sigma-Aldrich. Other chemicals
used for the polymer synthesis were the solvent absolute al-
cohol (Concord Co., Tianjin, China) the functional mono-
mer MAA (99%, Tianjin Chemical Reagent Research Insti-
tute, Tianjin, China) and the cross-linker EGDMA (98%,
Sigma–Aldrich) and the free radical initiator AIBN (99%,
Immunoassay absorbance was read in dual-wave-
length mode (450-650 nm) with a Labsystems 96-well
plate reader (Helsinki, Finland).
Synthesis of the Molecularly Imprinted Film
The imprinted film was directly polymerized on the
96-well plate wells as follows: MMZ (0.25 g, 2.19 mmol)

A Biomimetic BELISA Method for Monitoring Methimazole
J. Chin. Chem. Soc., Vol. 58, No. 4, 2011 465
was dissolved in 10 mL ethanol which was used as porogen
in a 25 mL round-bottom flask, then added Functional
monomer MAA (0.745 mL, 8.78 mmol), the mixture was
sonicated for 15 min. After 15 min, the cross-linker
EGDMA (0.415 mL, 2.19 mmol) was added to the mixture,
sonicated for 20 min. At last the initiator AIBN (0.050 g,
0.304 mmol) was added to the mixture. When AIBN was
dissolved, 200 mL of the mixture was placed in the wells of
a 96-well plate. The plate was sealed and irradiated by UV
irradiation for 12 h. After polymerization ended, the
96-well plate was extracted with 300 mL of methanol/ace-
tic acid (3:1, v/v) for 24 h by an ultracted cleaner, the eluent
was replaced by new methanol/acetic acid every 6 h. At last
the plate was extracted by 300 mL of the methanol for 6 h to
be free of MMZ, which was verified by detection of the
methanol eluent using UV spectrophotometer. The plate
was dried at room temperature.
duced pressure. ESI-MS: [M-H] 171, Fig. 2 (C6H8O2N2S,
M 172).
Enzyme Conjugate Preparation
The process for the synthesis of the active ester was
as Fig. 3.22 To prepare the enzyme conjugates, 6.8 mg of
NHS (0.06 mmol) and 24.8 mg of DCC (0.12 mmol) were
added to the hapten of MMZ solution (10.15 mg of hapten
of MMZ dissolved in 2.76 mL of dry DMF), and the mix-
ture was stirred for 24 h protected form light at room tem-
perature. After centrifugation, the supernatant was added
slowly to 10 mg of HRP in 2 mL of 50 mmol L-1 K2HPO4 in
ice-bath. The reaction solution was gently mixed by mag-
netic stirring bar and then left at 4 ºC overnight. The en-
zyme conjugated solution was then dialyzed against PBS.
For comparison, the non-imprinted film was prepared
in the same manner in the absence of template molecule
MMZ.
Under the condition of the water temperature in the
ultracted cleaner below 50 ºC and methanol/acetic acid
(3:1, v/v), the 96-well plate didn’t melt.
Characterization of the Imprinted Film
To measure the adsorption capacity, the imprinted
and non-imprinted wells of the 96-well plate were equili-
brated with 200 mL of 5% methanol in PBS containing
MMZ at various concentrations (10-120 mg L-1). After the
96-well plates were shaken (300 r min-1) for 60 min at room
temperature. The supernatants were diluted and measured
for the unextracted MMZ by a UV spectrometer at 254 nm,
and the adsorption capacity (Q) was calculated.
Fig. 2. Mass spectrum of the hapten of MMZ.
Uptake kinetics of the imprinted films by 40 mg L-1
MMZ was evaluated. After shaking for 5, 10, 20, 40, 60,
and 80 min at room temperature, the final concentration of
supernatants was diluted and determined by UV spectrom-
etry at 254 nm.
Synthesis of the hapten of MMZ
Briefly, 5.708 g MMZ was dissolved in 20 mL of
freshly distilled tetrahydrofuran (THF), and then 1.875 g
NaH (MMZ:NaH = 1:1.5, mole ratio) was added in batches.
This step should be occurred in ice-bath. When there were
no bubbles, 7.5 g bromoacetic acid was added. Finally, the
resulting residue was dissolved in water, and the pH of the
soultion was adjusted to 2 by the addition of HCl. After ex-
traction by ethyl acetate (3 × 30 mL), all of the organic frac-
tions were combined and evaporated to dryness under re-
Fig. 3. Reaction scheme for the synthesis of MMZ
hapten.

466 J. Chin. Chem. Soc., Vol. 58, No. 4, 2011
Wang et al.
Accurately calculated the volume of enzyme conjugated,
the same volume of glycerine was added to the solution,
and stored at -20 ºC before use.
Sample Analysis and HPLC Validation
The samples were determined using a HPLC at a mo-
bile flow rate of 1.0 mL min-1 under isocratic conditions,
and a mixture of methanol/water (90:10, v/v) was used as
the mobile phase. Class-VP software was used to acquire
spectral and chromatographic data. The injected sample
size was 20 mL, and the UV detection wavelength was 254
nm. All of the analyses were carried out in triplicate.
Standard Solution Preparation
For the construction of the calibration curve, six stan-
dard solutions containing MMZ in 5% PBS within the
range of 0.6 mg L-1 to 60 mg L-1 were prepared freshly in
glass tubes. The stock solution (1000 mg L-1 in methanol)
of MMZ was diluted to 60 mg L-1, and the solution of
60000 mg L-1 (in 5% PBS) was sequentially diluted to give
6000, 600, 60, 6, and 0.6 mg L-1.
RESULTS AND DISCUSSION
Direct Competitive BELISA Procedure
Characterization of the Novel Imprinted Film
First, 100 mL of 5% PBS was added to the control and
200 mL blank wells, and 100 mL of standard solution or
sample extracts was applied to the allocated wells. Then
100 mL of enzyme conjugate was immediately added to
each well, except for the blank wells, and the mixture was
incubated for 1.0 h. Following the washing with PBS/T so-
lution five times, 150 mL of substrate solution was added to
each well. The reaction was stopped after 30 min by adding
50 mL 1.25 mol L-1 H2SO4, and absorbances were recorded.
Finally, the imprinted film was extracted with 300 mL of
methanol/acetic acid (3:1, v/v) for 3.0 h by an ultrasonic
cleaner, followed with 300 mL of methanol for 2.0 h, for
the next BELISA procedure.
The isothermal adsorptions of imprinted and non-im-
printed films are plotted in Fig. 4. It was shown that the
amounts of the template molecules extracted by the im-
printed or non-imprinted film increased with increasing
MMZ initial concentrations. And under the same experi-
mental conditions, the adsorption capacity of the imprinted
polymer at each concentration was higher than that of the
non-imprinted polymer. And the adsorption capacity of the
imprinted film (1.5 mg well-1) was more than 1.5-fold that
of the non-imprinted film (1.1 mg well-1) at 120 mg L-1
concentration.
The adsorption kinetics curve should not changed
with the initial MMZ concentration. We could see the ad-
sorption capability was grown sharply under the concentra-
tion of 40 and 60 mg L-1 in Fig. 4, so uptake kinetics of
MMZ by the imprinted film was examined at 40 mg L-1
concentration (Fig. 5). Results indicated that the prepared
imprinted film had fast uptake kinetics, 68% of binding
was obtained within a short period of 10 min, and the ad-
sorption equilibrium was almost reached within 40 min.
Preparation of Samples
The urine sample were filtered by 0.22 mm filters
without any other pretreatment and stored in the precleaned
glass bottles at 4 ºC prior to ELISA or HPLC analysis. To
check the accuracy of the developed method, the samples
for spiking were determined to be free of MMZ with
HPLC. Briefly, 50, 100, or 150 mL of the standard MMZ so-
lution (1 mg L-1) was added to 5% methanol in urine.
Selectivity Studies
Cross-reactivity (CR) studies were carried out by
measuring the competitive curves and selectivity proper-
ties for other chemically related compounds under the opti-
mized conditions. The N-methylthiourea and 2-mercapto-
imidazole were the competitors with similar structure and
characteristics. Cross-reactivity was calculated as the per-
centage between the IC50 value (calculated as the concen-
tration giving 50% inhibition of color development) for
MMZ and the IC50 value for the interfering compound with
the following equation:
CR% = {IC50(MMZ)/IC50(cross-reacting
compound)} × 100
Fig. 4. Adsorption isotherms of the imprinted and non-
imprinted films.

A Biomimetic BELISA Method for Monitoring Methimazole
J. Chin. Chem. Soc., Vol. 58, No. 4, 2011 467
The rapid adsorption kinetics of the imprinted film is an ob-
vious advantage for its application in the BELISA.
ity, and the sensitivity was reduced when the concentration
of methanol was more than 5%. Thus, 5% methanol PBS
solution was chosen as the preparing solvent for the routine
analysis of MMZ in urine samples.
Hapten Selection and Synthesis
As previous studies have shown, the spatial arrange-
ment, nature, and size of the enzyme conjugate must be tai-
lored to the template structure to achieve efficient competi-
tion for the MIP binding sites. The labeled conjugate show-
ing the best performance in a MIP-based assay was also the
one providing the highest sensitivity in an immunoassay
based on the same type of measurements.
As previously reported for the immunoassay, differ-
ent concentrations of immobilized antibodies in the 96-
well plate well may have a direct effect on the sensitivity of
the direct competitive ELISA.21 The effect of the imprinted
film thickness and polymerization time on the assay sensi-
tivity was also investigated in the present study. Results in-
dicated that when the plate was sealed and irradiated by UV
irradiation for 12 h, lower IC50 values (70 4 mg L-1) and
optimized competitive binding between MMZ and the HRP
conjugate were observed.
A significantly different binding between a HRP-la-
beled conjugate and HRP to the imprinted film clearly indi-
cated that hapten played an essential role in the specific
recognition of MIP in the BELISA. MMZ (Fig. 1) contains
one reactive sulfhydryl group, ideal for chemical modifica-
tion to produce a hemisuccinate derivative with a terminal
carboxyl group for conjugating to the enzyme.
Cross-Reactivity
The specificity of the developed BELISA was evalu-
ated in the presence of other structurally related com-
pounds. Cross-reactivity was calculated as the percentage
between the IC50 value for MMZ and the IC50 value for the
interfering compound, and the results are depicted in Table
1. The imprinted film had a higher selectivity for MMZ
than other related compounds, and different CR were ob-
tained with N-methylthiourea (1.1%), 2-mercaptoimida-
zole (1.3%). This may have resulted from the imprinting ef-
fect, the difference of the molecular interactions, and their
structures. During the preparation of the imprinted film, the
OH groups of MAA reacted with sulfhydryl groups of
MMZ and the polyaniline complex formed. After removal
of the MMZ, the imprinted cavities and specific binding
sites for the sulfhydryl groups in a predetermined orienta-
tion were generated.
The hapten was synthesized by reaction of the sulf-
hydryl group of MMZ with the bromoacetic acid (Fig. 3).
The carboxylic acid hapten was converted to the active es-
ter for the coupling of hapten to enzyme. The MMZ hapten
ester obtained was compatible with enzymes, making an
HRP-labeled conjugate possible.
Condition Optimization
The applicability of the developed BELISA method
using the imprinted film as an artificial antibody for the de-
termination of MMZ was evaluated. The preparing solvent
and thickness of the imprinted film were optimized to
achieve good sensitivity and precision. The solvent used in
the preparation of standard solutions and samples can af-
fect the BELISA. In order to investigate the influence on
assay performance, the methanol solutions at 5, 10, and
15% in PBS were tested for their effects by comparing the
standard curves. Methanol at 5% had higher assay sensitiv-
We also observed that the imprinted film had higher
selectivity for 2-mercaptoimidazole than for N-methylthio-
urea, as the structure of 2-mercaptoimidazole was more
closely related to the structure of MMZ than it (Fig. 1).
Hence, the structure and the binding sites of the template
molecules all play important roles in the recognition of the
Table 1. IC50 and cross-reactivity values for two competitors
with the developed BELISA method (mean S.D., n =
3)
Compounds
IC50 (µg L-1)
CR (%)
MMZ
70
4
100
1.1
1.3
N-methylthiourea
2-mercaptoimidazole
6500 24
5400 17
Fig. 5. Kinetic uptake plot of the imprinted films.

468 J. Chin. Chem. Soc., Vol. 58, No. 4, 2011
Wang et al.
polymer and the competition reaction of BELISA.
Table 2. Analysis results of spiked MMZ in urine samples by the
developed BELISA and HPLC methods (mean S.D., n
= 3)
Analytical Characterization
Spiked level (mg L-1)
HPLC (mg L-1)
BELISA (mg L-1)
The normalized competition curves obtained using
the imprinted film as the artificial antibody for MMZ stan-
dards at concentrations from 60000 mg L-1 to 0.6 mg L-1 in
5% PBS solution can be seen in Fig. 6. In the competitive
assay format, free MMZ effectively competed with the
HRP conjugate for the binding sites in the imprinted film
surface (76% inhibitor). Under optimized experimental
conditions, the IC50 of MMZ standard curves was 70 mg L-1,
and the limit of detection (LOD), calculated as the concen-
tration of standard solution causing 15% inhibition of color
development, was 0.9 mg L-1.
0.050
0.100
0.150
0.047 0.001
0.095 0.001
0.143 0.001
0.045 0.002
0.093 0.007
0.142 0.002
by comparative analysis of the spiked samples with HPLC
(Table 2), and no significant differences were observed
between the results obtained by both methods.
CONCLUSIONS
There have described were various analytical meth-
ods in TLC, GC-MS, HPLC-UV or MS, CE, flow injection
analysis and electrochemical detection for the effectively
monitoring MMZ in kinds of samples, but these methods
were too expensive or not suitable for extensively preva-
lence. Though the MIP has been used to monitor MMZ,
there was no report about using it as the artificial antibody
of ELISA. Now we developed a fast, direct competitive
BELISA method using the imprinted film of controlled
thickness as artificial antibody with the advantages of low-
cost, time-saving, ease of preparation compared with tradi-
tional antibody. This BELISA method exhibited excellent
performance in real sample analysis with a simple matrix
dilution step, which has potential for being applied in the
quantitative determination of MMZ. We also presented a
methodology for the application of MIP in routine immu-
noassays, although its sensitivity is lower. With the devel-
opment of MIT, the sensitivity and accuracy of MIP-based
immunoassays would be improved, and they can provide
an important analysis platform in the future.
Furthermore, the imprinted film had a reusability of
20 times of cycles, where the IC50 was 74 mg L-1 and IC15
was 0. 94 mg L-1.
Results indicated that there was little loss of sensitiv-
ity. Thus, the cost per analysis of the BELISA method was
drastically reduced, and it was really more suitable for
rapid detection.
Sample Analysis and Accuracy of the BELISA
In an attempt to evaluate the suitability and applica-
bility of the optimized assay for the measurement of real
samples, the urine sample spiked at 50, 100, and 150 mg L-1
were determined (with three replicates for each concentra-
tion). The analytical data are shown in Table 2. A good cor-
relation of results was observed with recoveries ranging
from 90% to 95%, indicating that there was little matrix ef-
fect in the analysis of sample using the developed BELISA
without any other sample treatment procedure except filtra-
tion. The accuracy of this BELISA method was validated
ACKNOWLEDGEMENTS
Mrs. Bing Liu is thanked for invaluable discussions
and suggestions. This work was supported by the Natural
Science Foundation of Tianjin (Project No. 09JCYBJC14300)
the Ministry of Science and Technology of China (Project
No. 2009BADB9B06 and 2009BADB9B03).
REFERENCES
1. Liu, J. K.; Yeo, H. C.; Overvik-Douki, E.; Hagen, T.;
Doniger, S. J.; Chu, D. W.; Brooks, G. A.; Ames, B. N. J.
Appl. Phys. 2000, 89, 21-24.
Fig. 6. MMZ BELISA standard curves using the im-
printed film as the antibody.
2. Martinez-Frias, M. L.; Cereijo, A.; Rodriguez-Pinilla, E.;

A Biomimetic BELISA Method for Monitoring Methimazole
J. Chin. Chem. Soc., Vol. 58, No. 4, 2011 469
Urioste, M. Lancet 1992, 339, 742-743.
14. Sun, J. Y.; Zheng, C. Y.; Xiao, X. L.; Niu, L.; You, T. Y.;
Wang, E. K. Electroanalysis 2005, 17, 1675-1680.
15. Wang, A.; Zhang, L.; Zhang, S.; Fang, Y. J. Pharm. Biomed.
Anal. 2000, 23, 429-436.
3. Edward, A. C.; Smith, C. M.; Reynard, A. M. Textbook of
Pharmacology; Saunders: Philadelphia, 1992; pp 652-656.
4. Genter, M. B. Biochem J. Mol. Toxicol. 1998, 12, 305-314.
5. European Community Directive 86/649 Off. J. Eur. Com-
mun. 1986, 36, Document Number L275.
16. Raby, C.; Lagorce, J. F.; Jambut-Absil, A. C.; Buxeraud, J.;
Catanzano, G. Endocrinology 1990, 126, 1683-1691.
17. Zhang, S.; Sun, W. L.; Zhang, W.; Qi, W. Y.; Jin, L. T.;
Yamamoto, K.; Tao, S. G.; Jin, J. Y. Anal. Chim. Acta 1999,
386, 21-30.
6. Brabander, H. F. De; Batjoens, P.; Van Hoof, J. J. Planar
Chromatogr. 1992, 5, 124-130.
7. Zou, Q. H.; Liu, Y.; Xie, M. X.; Han, J.; Zhang, L. Anal.
Chim. Acta 2005, 551, 184-191.
18. Aslanoglu, M.; Peker, N. J. Pharm. Biomed. Anal. 2003, 33,
1143-1147.
8. Zhang, L.; Liu, Y.; Xie, M. X.; Qiu, Y. M. J. Chromatogr. A
2005, 1074, 1-7.
19. Pan, M. F.; Wang, J. P.; Fang, G. Z.; Tang, W. W.; Wang, S. J.
Chromatogr., B 2010, 878, 1531-1538.
9. Hollosi, L.; Kettrup, A.; Schramm, K. W. J. Pharm. Biomed.
Anal. 2004, 36, 921-924.
20. Hage, D. S. Immunoassays Anal. Chem. 1995, 67, 455-462.
21. Urraca, J. L.; Moreno-Bondi, M. C.; Orellana, G.; Sellergren,
B.; Hall, A. J. Anal. Chem. 2007, 79, 4915-4923.
22. Li, Z. L.;Wang, S.; Lee, A. N.; Allan, R. D.; Kennedy, I.
R. Anal. Chim. Acta 2006, 569, 50-57.
10. Kusmierek, K.; Bald, E. Talanta 2007, 71, 2121-2125.
11. Zakrzewski, R. Technol. Biomed. Life Sci. 2008, 869, 67-74.
12. Wasch, K. De.; Brabander, H. F. B.; Impens, S.; Vandewiele,
M.; Courtheyn, D. J. Chromatogr., A 2001, 912, 311-317.
13. Cannavan, A.; Kennedy, D. G. Analyst 1997, 122, 963.