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Hapten synthesis and development of an indirect competitive enzyme-linkedimmunosorbent assay for chlorpromazine in pork, chicken and swine liver

Yu-Dong Shen, Ben Xiao, Zhen-Lin Xu, Hong-Tao Lei, Hong Wang, Jin-Yi Yang and Yuan-Ming Sun*

Received 5th August 2011, Accepted 15th October 2011

DOI: 10.1039/c1ay05480e

The use of chlorpromazine (CPZ) as a sedative for livestock has been prohibited in the European Union

and many other countries. In this study, a new hapten 7-amino-chlorpromazine sulfoxide (hapten 1)

against CPZ was synthesized and coupled to ovalbumin (OVA) as an immunogen to produce

polyclonal antibody (PAb) specific for CPZ. An heterologous hapten 7-(4-carboxyl-phenylazo)-

chlorpromazine (hapten 2) was synthesized and coupled to bovine serum albumin (BSA) as coating

antigen to improve the assay sensitivity. The results showed that the hapten-heterologous systems

exhibited 20 times higher sensitivity than the hapten-homologous one. Based on the hapten-

heterologous system, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for CPZ

was developed, the IC50 value was 0.58 ng mL�1 and the limit of detection was 0.03 ng mL�1. The assay

showed no cross-reaction with the CPZ analogues. The average recoveries of CPZ from spiked samples

were estimated to range from 77.1% to 98.6%, with a coefficient of variation (CV) of less than 10.9%.

Linear regression analysis showed a good correlation between the CPZ concentrations obtained from

ELISA and HPLC analysis, which suggested that the ELISA is a convenient supplementary analytical

tool for monitoring CPZ.

Introduction

Chlorpromazine (CPZ) belongs to the group of phenothiazine

derivatives. It is widely used in human medicine in the therapy of

schizophrenia, organic psychoses and the manic phase of manic-

depressive illness.1–3 In veterinary medicine it was used as

a tranquillizer and as an antiemetic agent, causing calmness,

drowsiness and an indifference to surroundings, so it was

frequently employed to reduce stress during the transportation of

food producing animals to abattoirs.4 However, residues of CPZ

were found to have toxic effects, namely, cholestatic jaundice,

leukocytopenia, postural hypotension, extrapyramidal effects,

contact dermatitis and tardive dyskinesia as evaluated by the

European Agency for the Evaluation of Medicinal Products

(EMEA) and the Joint FAO/WHO Expert Committee on Food

Additives (JECFA) in 1991.5,6 The use of CPZ in food animals in

any way is totally prohibited due to their inclusion in Table 2

(prohibited substances) of the Commission Regulation (EC) No

17/97.7 The Ministry of Agriculture of the People’s Republic of

China also prohibited the addition of CPZ in feeding and

drinking water (No.176, 235).

Currently, a great variety of analytical methods are available

for the determination of CPZ in biological samples. These

Guangdong Provincial Key Laboratory of Food Quality and Safety, SouthChina Agricultural University, Guangzhou, 510642, China. E-mail:syd_tyx@163.com; ymsun@scau.edu.cn; Fax: +86 20 85288282; Tel:+86 20 85283925

This journal is ª The Royal Society of Chemistry 2011

methods include high performance liquid chromatography

(HPLC),8 liquid chromatography coupled with tandem mass

spectrometry (LC-MS),9 gas chromatography (GC),10 GC

coupled with MS (GC-MS),11 electrochemiluminescence

(ECL),12 chemiluminescence (CL),13,14 capillary electrophoresis

coupled with ECL (CE-ECL),15 spectrophotometry16 and

radioimmunoassay (RIA).17,18 However, instrumental methods

such as HPLC require complex and expensive instrumentation

that has to be managed by highly qualified personnel, and usually

involve extensive purification and often derivatization of the

target compounds; whilst RIA is not suitable for field testing and

requires the use of radioisotopes, making it predominately

a research tool.19 Mounsey et al.20 developed a polyclonal anti-

body-based fluoroimmunoassay for the determination of sulf-

oxide metabolites of commonly used phenothiazine and

thioxanthine neuroleptics. However, the assay showed low cross-

reactivity (1.4%) for CPZ.

Enzyme-linked immunosorbent assay (ELISA) has been

extensively used for the screening of veterinary drug residues in

food and animal tissues due to its sensitivity, specificity, rapidity

and simplicity.21 To the best of our knowledge, only one work

regarding the development of ELISA for CPZ has been repor-

ted.22 In this study, we used a new strategy to synthesize an

immunizing hapten 7-amino-chlorpromazine sulfoxide (hapten

1, Fig. 1) which retained the geometric and electronic properties

of CPZ. A new heterologous coating hapten 7-(4-carboxyl-phe-

nylazo)-chlorpromazine (hapten 2, Fig. 1) was also synthesized

Anal. Methods, 2011, 3, 2797–2803 | 2797

Fig. 1 Synthesis route of chlorpromazine haptens.

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and used to study the effect of heterologous hapten on ELISA

sensitivity. Polyclonal antibody was produced and used to

develop an indirect competitive ELISA (icELISA) with good

sensitivity and high specificity for CPZ. The ELISA was applied

in the determination of CPZ in pork, chicken and swine liver

samples and the results were further confirmed by HPLC.

Experiments

Reagents and chemicals

CPZ standard was obtained from International Laboratory USA

(San Bruno, USA). CPZ analogues acepromazine, prom-

ethazine, perphenazine, fluphenazine, 2-chlorophenothiazine

and azaperol were obtained from Guangzhou Institute for Drug

Control (Guangzhou, China). Bovine serum albumin (BSA),

ovalbumin (OVA) and goat anti-rabbit IgG–horseradish peroxi-

dase (HRP) conjugate were purchased from Wuhan Boster

Biological Technology Co., Ltd. (Wuhan, China). 3,30,5,50-tet-ramethylbenzidine (TMB), N,N-dicyclohexylcarbodiimide

(DCC), N-hydroxysuccinimide (NHS), Freund’s complete (cFA)

and in complete adjuvants (iFA) were purchased from Sigma

(St. Louis, MO, USA). Hydrochloric acid, glutaraldehyde,

sodium nitrite, Tween-20 and methanol were obtained from

Guangzhou Chemical Reagent Co., Ltd. (Guangzhou, China).

Silica gel GF 254 TLC plate (0.25 mm thickness, 20 � 20) was

obtained from Qingdao Haiyang Chemical Co., Ltd. (Qingdao,

China). Polystyrene ELISA plates were obtained from Xiamen

Yunpeng Biotech Co., Ltd. (Xiamen, China).

The buffers used for ELISA were as follows: 50 mM carbonate

buffer (pH 9.6) for coating plates, 10 mM PBST solution phos-

phate buffer saline (PBS, pH 7.4, containing 0.1% Tween-20) was

used for washing plates and sample neutralization, 0.1 M citrate

and sodium phosphate buffer (pH 5.4) for substrate buffer, and

2 M H2SO4 as the stopping reagent. TMB solution was prepared

by addition of 10 mL substrate buffer and 150 mL of 15 mg mL�1

TMB in dimethylformamide (DMF) and 2.5 mL of 6% (w/v)

H2O2. The water used in all the studies was ultrapure water

(18.2 MX cm) obtained from a Millipore Milli-Qultrapure water

system (Millipore, USA).

Instrumentation

ELISA plates were processed using aMultiskanMK2microplate

washer (Thermo Labsystems, America). Absorbance was

measured at a wavelength of 450 nm using a Multiskan MK3

2798 | Anal. Methods, 2011, 3, 2797–2803

microplatereader (Thermo Labsystems, USA). Ultraviolet spec-

trometry (UV) was recorded on a UV-3010 spectrophotometer

(Hitachi, Japan). HPLC analyses were performed using a Waters

1525 Binary HPLC Pump with Waters 2998 Photodiode Array

Detector (Waters, America). Mass spectrometry (MS) analyses

were performed using a Finnigan LCQDECA mass spectrometer

(Thermo, USA). Nuclear magnetic resonance (NMR) spectra

were obtained with the DRX-600 NMR spectrometers (Bruker,

Germany–Switzerland).

Hapten synthesis

Synthesis of 7-amino-chlorpromazine sulfoxide (hapten 1).

Freshly prepared acetyl nitrate (1.8 mL, prepared from 0.4 mL of

70% HNO3 and 1.4 mL of Ac2O at 0 �C) was added dropwise to

a solution of the appropriate CPZ hydrochloride (510 mg,

1.6mmol) in 2mLofAc2O at 0 �C.Themixturewas stirred at 0 �Cfor 1 h and then poured into ice-water. The solution was adjusted

to pH 8 and extracted three times with ethyl acetate. The organic

layer was washed with saturated brine and evaporated to dryness

under vacuum. The desired compound (CPZSO-NO2) was puri-

fied by column chromatography (chloroform : methanol ¼3 : 1). To a solution of CPZSO-NO2 (380 mg, 1 mmol) in

15 mL EtOH was sequentially added iron powder (336 mg,

5 mmol) and NH4Cl (336 mg, 5 mmol) in 10 mL H2O. The

mixture was refluxed for 3h, and then cooled to ambient

temperature and filtered. The filtrate was concentrated under

reduced pressure, diluted with water (5 mL) and a saturated

aqueous solution of sodium hydrogen carbonate (5 mL),

extracted with ethyl acetate (3 � 10 mL), dried over Na2SO4,

evaporated under reduced pressure and the resulting solid

product (hapten 1) was recrystallized from ethanol. MS (ESI

positive) m/z: 350.3 [M + H]+; 1H NMR (600MHz, d4-MeOH):

d 2.02–2.03 (m, 2H, CH2C), 2.25 (s, 6H, CH3), 2.44–2.47 (m, 2H,

CH2N), 4.29–4.31 (t, 2H, J ¼ 7.5 Hz, NCH2), 7.12–7.14 (dd, 1H,

J ¼ 2.82 Hz, J ¼ 3.0 Hz, CHar), 7.17–7.19 (dd, 1H, J ¼ 1.86 Hz,

J ¼ 8.28 Hz, CHar), 7.21–7.22 (d, 1H, J ¼ 2.7 Hz, CHar), 7.47–

7.49 (d, 1H, J ¼ 9.06 Hz, CHar), 7.62–7.63 (d, 1H, J ¼ 1.86 Hz,

CHar), 7.84–7.85 (d, 1H, J ¼ 8.34 Hz, CHar).

Synthesis of 7-(4-carboxyl-phenylazo)-chlorpromazine (hapten

2). p-Aminobenzoic acid (350 mg, 2.4 mmol) was dissolved in

3 mL of water and 0.7 mL of concentrated hydrochloric acid.

Sodium nitrite (173 mg, 2.5 mmol) dissolved in 1 mL water was

added dropwise and stirred for 30 min at 0 �C. CPZ

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hydrochloride (2.5 mmol) was dissolved in distilled water and

added gradually to the diazonium salt of p-aminobenzoic acid

with continuous stirring. The reaction was allowed to continue

overnight at 4 �C with constant stirring. The solution was then

adjusted to pH 5.6 with 0.5 M NaOH and extracted three times

with 30 mL of methylene chloride. The organic layer was washed

twice with water and evaporated to dryness under vacuum. The

product was purified on silica gel GF tlc plates developed in

methanol : chloroform : NH4OH (20 : 80 : 5), hapten 2 had an

Rf value of 0.27 while that of CPZ was 0.95, which was consistent

with the values reported in ref. 18.

Preparation of hapten–protein conjugates

Hapten 1 was coupled to OVA as an immunogen (conjugate 1,

Fig. 2) and coupled with BSA as a coating antigen (conjugate 2,

Fig. 2) using the glutaraldehyde method. Briefly, about 2 mL of

methanol containing 35 mg (100 mmol) of hapten 1 was added

dropwise into 4 mL of carbonate buffer containing 136 mg BSA

or 96 mg OVA. Then 100 mL of 25% glutaraldehyde diluted in

1 mL carbonate buffer was added dropwise to the mixture. The

solution was stirred for 4 h at room temperature and then dia-

lyzed against 10 mM PBS (pH 7.4) to obtain the immunogen and

coating antigen.

Hapten 2 was coupled with BSA using the active ester method

to be used as a coating antigen (conjugate 3, Fig. 2). 46.7 mg

(100 mmol) of hapten 2, NHS 17.0 mg (150 mmol) and DCC

31.0 mg (150 mmol) were dissolved in 1 mL of DMF. The acti-

vation reaction was carried out at 4 �C overnight with stirring.

The white dicyclohexylurea precipitate was removed from solu-

tion by centrifugation. The supernatant was added dropwise to

BSA (136 mg) in 10 mL PBS (10 mM, pH 7.4). The conjugate

mixture was stirred at 4 �C for 12 h and then dialyzed against

10 mM PBS (pH 7.4) for three days.

Production of polyclonal antibodies

Two New Zealand female rabbits (10 weeks, 1.5–2.0 kg) were

immunized 5 times using conjugate 1 at intervals of 21 days, at

the GuangdongMedical Laboratory Animal Center. For the first

Fig. 2 Synthesis route and structure

This journal is ª The Royal Society of Chemistry 2011

immunizations, each rabbit was immunized subcutaneously with

1 mg of immunogen, emulsified in Freund’s complete adjuvant

and the following four immunizations were administered using

Freund’s incomplete adjuvant. Eight days after the third

immunisation, blood was obtained for titer determination by

indirect ELISA. When the immunization finished, whole blood

was collected and allowed to coagulate overnight at 4 �C. Thenthe serum was divided into aliquots (1 mL) and stored at �20 �Cuntil use.

The icELISA procedures

For icELISA, 100 mL of coating antigen diluted with coating

buffer (pH 9.6) at the optimal dilution was piped into a microtitre

plate and incubated at 4 �C overnight. The excess binding sites

were blocked with 5% skimmed milk powder in PBS buffer for

3 h at 37 �C. Plates were washed 3 times with 250 mL well�1 of

PBST to remove the blocking solution, and then 50 mL antibody

diluted with PBS and 50 mL CPZ or its analogues were added to

each well. Unbound compounds were removed by washing five

times with wash buffer. After incubation for 1 h at 37 �C, 100 mLof HRP-conjugated goat anti-rabbit IgG solution diluted with

PBST were added to each well for 45 min at 37 �C then washed

five times with PBST. 100 mL of substrate solution were then

added to each well and the enzymatic reaction was stopped after

15 min incubation at 37 �C by addition of 50 mL well�1 of

stopping solution. Absorbance values were measured at 450 nm

using the ELISA plate reader. Competitive curves were obtained

by plotting relative absorbance of B/B0 against the logarithm of

the analyte concentration. Sigmoid competitive curves were fitted

to a four-parameter logistic equation by means of Origin 7.5

software.

Cross-reactivity

Cross reactivity (CR) was tested to determine the specificity of

the antibody. Six structurally related compounds were selected

and evaluated (Fig. 3). Standard solutions of testing compounds

were analyzed by the ELISA procedures described above. CR

s of chlorpromazine conjugates.

Anal. Methods, 2011, 3, 2797–2803 | 2799

Fig. 3 Structures of the compounds used in the determination of cross reactivity.

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was expressed as the ratio of IC50 values for CPZ to that for

analogues, e.g. CR (%) ¼ (IC50 CPZ/IC50 analogues) � 100%.

Preparation of food samples

The samples including pork, chicken and swine liver were

purchased from a local supermarket. Five gram of milled sample

was weighed into a polythene tube, each sample was spiked with

CPZ at final concentrations of 20, 60 and 180 ng g�1, and then

15 mL of acetonitrile was added and shaken vigorously for 15

min. Subsequently, 15 mL acetonitrile was added to the sample

and extracted again. The extraction solution was centrifuged at

room temperature for 10 min at 4000 � g. All the supernatant

was transferred and 15 mL n-hexane was added. After being

shaken vigorously for 10 min, the mixture was centrifuged for 10

min at 4000 � g. Then the subnatant was evaporated to dryness

at low pressure at 45 �C. The residue was then redissolved in

1 mL of methanol and the solution was filtered through a 0.22

mm membrane and assayed by HPLC. For ELISA, the residue

was redissolved in 10 mL of PBST and diluted 40 times in PBST

for ELISA analysis. In order to assess assay reproducibility, each

fortification level was tested in triplicate. The results obtained by

ELISA assay were compared with the data obtained by HPLC.

HPLC analysis of CPZ

A Waters high-performance liquid chromatograph (1525, USA)

equipped with aWaters 2998 tunable absorbance detector, which

was controlled by a wavelength switching programme, was used.

Standard solutions of CPZ at concentrations of 50, 100, 200, 500

and 2000 ng mL�1 in mobile phase were prepared in 10 mL

volumetric flasks. The solutions were filtered before injection.

The HPLC conditions were as following: the column (C18, 60 mm

� 4.6 mm, 5 mm) temperature was maintained at 25 �C using

aWaters temperature control module, the mobile phase was 80%

(v/v) methanol and 20% (v/v) 0.05 mol L�1 ammonium acetate

solution, the carrier flow rate was 1.0 mL min�1, absorbance was

measured using a single wavelength at 254 nm and the run time

was 4.008 min. In this study, peak area measurements for all

calculations were adopted.

2800 | Anal. Methods, 2011, 3, 2797–2803

Results and discussion

Design and synthesis of hapten/antigen

The key factor for generating antibodies allowing highly sensitive

and specific determination of small molecules is the design of the

hapten and the method used to link it to the carrier protein.23 The

basic requirement of hapten design is to maintain the original

molecular structure of the hapten and to ensure that the hapten is

exposed on the surface of carrier as much as possible.24 Liu

et al.22 reported that the side functional chain composed of N–

(CH2)3–N(CH3)2 is a very important structural feature for

generation of antibody for CPZ. Hubbard et al.17 suggested that

the 7-positions would be optimum for electrophilic substitution

of CPZ because of the participation of the lone pair on the

nitrogen and the chlorine generally deactivates an aromatic ring

to electrophilic attack. Therefore, in this study, hapten 1 (Fig. 1)

was designed to derive from the 7-positions of CPZ which means

it retained the main geometric and electronic properties of the

analyte and the linking site was far from the functional groups.

Therefore, it might be appropriate as an immunogen. The plate

coating antigen also plays a key role in ELISA sensitivity.

Heterology in the coating conjugate often results in weaker

antibody affinity providing higher sensitivity to the analytes.23

Therefore, an heterologous coating hapten (hapten 2, Fig.1) was

synthesized by a diazotized reaction on the 7-positions of CPZ as

described by Kawashima et al.18

Hapten 1 was coupled to OVA as an immunogen, while both

hapten 1 and hapten 2 were coupled to BSA for coating antigens.

The synthesized conjugates demonstrated qualitative differences

between the carrier protein and conjugate in the UV-Vis spectra,

suggesting successful hapten conjugation to the carrier protein.

The hapten coupling ratios with the carrier proteins were 12, 16

and 19 for hapten 1-OVA, hapten 1-BSA, and hapten 2-BSA,

respectively.

Preparation of polyclonal antibodies

Antisera from two rabbits injected with hapten 1-OVA were

collected (named antiserum #1 and antiserum #2). The titers of

antisera were determined by indirect ELISA (using homologous

coating antigen), which was 1/64,000 and 1/16,000 for antiserum

#1 and antiserum #2, respectively. The IC50 values for CPZ

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based on antiserum #1 and antiserum #2 in icELISA (using

homologous coating antigen) were 11.23 and 21.97 ng mL�1,

respectively. Antiserum #1 showed higher titer and better

sensitivity and therefore was chosen for the following

experiments.

Development of icELISA

Two formats of ELISAs, homologous and heterologous, are

commonly used. In homologous formats the same hapten is used

for immunization and assay purposes, whereas in heterologous

formats the immunizing hapten and the competitor hapten

(ELISA hapten) differ in their molecular structure. Heterologous

formats can often result in antibodies having a higher affinity

towards the analyte in comparison to the coating antigen or

tracer hapten.24–28

In this study, both homologous and heterologous icELISA for

CPZ were studied. A set of experimental parameters including

coating antigen concentrations, antibody dilutions, secondary

antibody dilutions as well as reaction buffers were evaluated to

obtained the optimal assay performance. The ratio of the Amax/

IC50 was used to ensure the optimum conditions for the assays.29

The outcome from this systematic approach guided the final

assay conditions to have a coating antigen concentration of

0.091 mg mL�1, a dilution of antibody of 1 : 64,000 and a dilution

of HRP-IgG antibody of 1 : 7000 in heterologous format, have

a coating antigen concentration of 0.131 mg mL�1, a dilution of

antibody of 1 : 16,000 and a dilution of HRP-IgG antibody of

1 : 7000 in homologous format. PBS (pH 7.4) containing 0.1%

Tween-20 was the optimal buffer for the antibody–antigen

reaction.

Based on the optimum conditions, two typical standard curves

for CPZ based on homologous and heterologous formats were

obtained as shown in Fig. 4. The IC50 for CPZ in heterologous

format was found to be 0.58 ng mL�1 while that in the homo-

logous format was 11.23 ng mL�1. The results obtained indicate

that the hapten-heterologous systems were 20 times more sensi-

tive than the corresponding hapten-homologous ones, and

a longer aromatic spacer-arm of the coating antigen was

Fig. 4 Comparison of homologous and heterogeneous icELISA results.

Data represented in mean � SD (standard deviation) and n ¼ 3.

This journal is ª The Royal Society of Chemistry 2011

beneficial for enhancing the assay sensitivity. Thus the heterol-

ogous system was selected for cross-reactivity studies and further

assay development and optimization.

Calibration curve of the icELISA

An icELISA calibration curve for CPZ was established (Fig. 5)

under the above optimum assay conditions. Seven concentra-

tions of CPZ (0, 0.06, 0.18, 0.54, 1.62, 4.86 ng mL�1) were applied

to the ELISA system, the linear regression equation was y ¼�0.37552x + 0.408, with a correlation determination (R2) of

0.9982. The IC50 of the assay was 0.58 ng mL�1, the limit of

detection (LOD, IC10) was 0.03 ng mL�1 and the workable range

(IC20–IC80) was 0.10–3.51 ng mL�1.

Specificity of the antibody

The assay specificity was evaluated by obtaining competitive

curves for several CPZ structurally related compounds (Fig. 3).

The IC50 values were calculated and compared with the IC50

value of CPZ. Cross-reactivity (CR) values for each compound

are given in Table 1. As shown in Table 1, the antibody shows no

cross-reaction between CPZ and 2-chlorophenothiazine,

perphenazine, fluphenazine, perphenazine, azaperol, less than

0.1% CR between CPZ and acepromazine, promethazine. The

low cross-reaction between CPZ and other structurally related

compounds suggests that the antibody is highly specific for CPZ.

The results indicated that the N–(CH2)3–N(CH3)2 side chain and

the chlorine play an important role in the immune response.

Recoveries test

In order to evaluate the accuracy and reproducibility of the

developed ELISA, three different types of samples including

pork, chicken and swine liver were spiked with a standard CPZ

solution to final concentrations of 20, 60 and 180 ng g�1,

respectively. These samples were analyzed by the competitive

ELISA method established above. For the ELISA we diluted the

samples of the agricultural extracts twenty times in PBST to

Fig. 5 Standard inhibition curves of chlorpromazine in heterogeneous

system.

Anal. Methods, 2011, 3, 2797–2803 | 2801

Table 1 Cross-reactivity of compounds structurally related to chlor-promazine in heterogeneous formats

CompoundsIC50 (ngmL�1)

Cross-reactivity(%)

Chlorpromazine 0.58 100Acepromazine 951 0.06Promethazine 1093 0.05Perphenazine >5800 <0.01Fluphenazine >5800 <0.012-Chlorophenothiazine >5800 <0.01Azaperol >5800 <0.01

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eliminate the presumable matrix effect. As shown in Table 2, the

average recoveries ranged from 77.1% to 98.6%, with a coeffi-

cient of variation (CV) of less than 10.9%. These results are

considered to be satisfactory for the assay of residues.30

Comparison of ELISA and HPLC

The accuracy of the analysis was determined by the comparative

detection of fortified CPZ at different concentrations using icE-

LISA and HPLC. The calibration curves obtained for HPLC

yielded an R2 of 0.9996 for CPZ with a linear equation of y ¼143.11x � 4995.52. Between icELISA and HPLC, linear regres-

sion analysis yields the equation y ¼ 1.01355x + 0.74237 and

a correlation coefficient of 0.9929. From the above results, it can

be seen that the icELISA developed for analysis of CPZ meets all

the requirements for residue analysis and is suitable for the

quantitative detection of CPZ at trace levels in food samples.

Conclusions

In this study, an icELISA was developed for the quantitative

detection of CPZ. The polyclonal antibody against CPZ was

generated by immunizing with 7-amino-chlorpromazine sulf-

oxide as a hapten conjugated to OVA. Heterologous assay, using

a hapten possessing an aromatic spacer arm conjugated to BSA

for the coating antigen, showed 20 times higher sensitivity than

homologous assays. The competitive inhibition experiments

under the optimized assay conditions indicated an IC50 of 0.58 ng

mL�1, and a lower detection limit of 0.03 ng mL�1. The heter-

ologous assay shows almost no cross-reactivity to other pheno-

thiazine derivatives. Three spiked food samples were analyzed by

Table 2 Recoveries of spiked samples determined by ELISA and HPLC (n

SampleSpiked (ngg�1)

ELISA

Measured (ng g�1)Recovery(%)

Pork 20 18.3 91.560 50.1 83.4180 162.4 90.2

Swine liver 20 19.7 98.660 48.3 80.5180 152.3 84.9

Chicken 20 17.9 89.560 46.2 77.1180 150.8 83.8

2802 | Anal. Methods, 2011, 3, 2797–2803

ELISA and the results were compared with those obtained by

HPLC, the results showed good stability, recovery and accuracy.

Therefore, this highly specific and reliable icELISA is suitable for

the sensitive analysis of CPZ in food samples.

Acknowledgements

This work was financially supported by the Guangdong

Provincial Municipal Science and Technology Project

(2010A080403005), Science and Technology Planning Project of

Guangdong Province (2010A090200084, 2009B011300005) and

National Natural Science Foundation of China (30901005).

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¼ 3)

HPLC

CV (%)Measured (ngg�1)

Recovery(%) CV (%)

6.8 16.6 83.1 9.18.1 53.8 89.6 7.73.9 166.7 92.6 1.610.2 18.1 90.3 5.67.1 57.8 96.4 7.18.4 168.1 93.4 10.94.6 16.8 83.9 3.43.5 53.4 89.0 6.96.5 144.9 80.5 9.0

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