development of an ultra-sensitive chemiluminescence enzyme immunoassay for the determination of...

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Development of an Ultra-Sensitive ChemiluminescenceEnzyme Immunoassay for the Determination ofDiethylstilbestrol in SeafoodYan Zhang a , Hong Tao Lei a , Hong Wang a , Zhen Lin Xu a , Yu Dong Shen a , Yuan Ming Sun a

& Jin Yi Yang aa College of Food Science, South China Agriculture University, Guangdong Provincial KeyLaboratory of Food Quality and Safety, Laboratory of Quality and Safety Risk Assessment inAgricultural Products Preservation Ministry of Agriculture , GuangZhou 510642 , ChinaAccepted author version posted online: 22 May 2013.

To cite this article: Yan Zhang , Hong Tao Lei , Hong Wang , Zhen Lin Xu , Yu Dong Shen , Yuan Ming Sun & Jin Yi Yang (2013):Development of an Ultra-Sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol inSeafood, Analytical Letters, DOI:10.1080/00032719.2013.798794

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 1

Immunoassay

Development of an Ultra-Sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol in Seafood

Yan Zhang1, Hong Tao Lei1, Hong Wang1, Zhen Lin Xu1, Yu Dong Shen1, Yuan Ming

Sun1, Jin Yi Yang1

1College of Food Science, South China Agriculture University, Guangdong Provincial Key Laboratory of Food Quality and Safety, Laboratory of Quality and Safety Risk

Assessment in Agricultural Products Preservation Ministry of Agriculture, GuangZhou 510642, China

Tel.: +8613602496019. E-mail: [email protected]

Received: 04 February 2013 Accepted: 01 April 2013

Abstract

An ultra-sensitive indirect competitive chemiluminescence enzyme immunoassay was

developed for screening diethylstilbestrol in fish and shrimp samples. The concentration

of diethylstilbestrol that caused 50% inhibition of the binding enzyme marker (IC50) was

0.32 ng/mL and the limit of detection was 0.0068 ng/mL; the linear range was from 0.028

ng/mL to 3.60 ng/mL. The assay showed cross-reactivity of 7.1 % and 2.8 % with

dienestrol and hexoestrol, respectively, but negligible cross-reactivity with estradiol,

estrone, ethinyloestradiol, and progestin. The recovery from spiked fish and shrimp

samples varied from 68.5% to 92.5%, and the mean coefficients of variation within

groups and between groups were 6.2% and 8.0%, respectively. Our results indicated that

the assay is a simple, sensitive, specific, and accurate method for screening fish and

shrimp samples for diethylstilbestrol.

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KEYWORDS: Diethylstilbestrol; Chemiluminescence enzyme immunoassay (CLEIA);

Seafood

INTRODUCTION

Diethylstilbestrol (DES) is a synthetic non-steroidal estrogen, which was first synthesized

in 1938 in London. DES was widely used, both clinically and in the agricultural industry,

before the health risks associated with it became known. DES can produce the same

pharmacological and therapeutic effects as estradiol, a naturally occurring hormone used

to treat estrogen hypothyroidism and hormonal imbalance (Noller et al. 1974). In animals,

DES was widely used as a growth stimulant to improve feed conversion efficiency and

promote growth rates, resulting in increased protein metabolism and animal daily gain,

and reduced fat (Burroughs et al. 1955). However, there is evidence that DES has the

potential for mutagenic, teratogenic, and carcinogenic effects, which has raised

widespread concern (Nielsem et al. 2000). In the 1970s, the European Union (EU), the

United States, China, and several other countries studied the hazards of DES residues. As

a result, DES has been banned for use in food animals and as a veterinary drug. In China,

the allowed level for DES in seafood is 0.6 µg/kg. Under EU regulations, DES residue

levels must be less than 2 µg/kg. However, driven by powerful economic interests, illegal

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use of DES still exists (Burroughs et al. 1955; Dickson et al. 2003). Therefore, the

development of a simple, rapid, sensitive, and specific method to detect DES residues in

animal food products is desirable.

Analytical techniques used to determine DES include gas chromatography tandem mass

spectrometry (GC-MS) (Dickson et al. 2003) and liquid chromatography tandem mass

spectrometry (LC-MS) (Malone et al. 2010). The major disadvantages of LC-MS and

GC-MS are the complex sample clean-up and the high-cost of instruments and equipment.

In contrast, immunoassay methods for the determination of drugs are cost-effective,

simple, and inexpensive. These assays can identify more than one target and detect

positive samples from hundreds of samples in a single test.

The chemiluminescence immunoassay is a combination of sensitive chemiluminescence

detection and specific immunosorbent assay. The chemiluminescence enzyme

immunoassay (CLEIA), in which enzyme labels are detected by chemiluminescent (CL)

substrates, such as the luminol/peroxide/enhancer system for horseradish peroxidase

(HRP) or dioxetane-based substrates for alkaline phosphatase, is one of the most sensitive

immunoassay detection systems (Knopp et al. 2006; Magliulo et al. 2005). The CLEIA is

a rapid assay that has a large linear dynamic range, high sensitivity and specificity,

involves small sample volumes, and does not create any radioactive pollution (Zhao et al.

2006; Roda et al. 2000; Wu et al. 2007; Zhao et al. 2009; Fan et al.2009; Zheng et al.

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2011). CLEIA has been widely used in drug residue assays (Xu et al. 2006; Choi et al.

2010; Zhang et al. 2006), including applications in the environment (Zhang et al. 2007;

Xin et al. 2009; Long et al. 2009; Zhang et al. 2004), medicine (Lin et al. 2007; Zhou et

al. 2005; Wang et al. 2009; Xue et al. 2011; Ren et al. 2008), food (Kloth et al. 2009;

Yang et al. 2008; Lin et al. 2008), and other areas (Tudorache et al. 2006; Zhang et al.

2010; Roda et al. 2004). A rapid and sensitive CLEIA for the determination of fumonisin

B1 in food samples has been developed, in which the limit of detection (LOD) was 0.09

µg/L; this is an improvement in sensitivity of one to two orders of magnitude compared

with the enzyme-linked immunosorbent assay (ELISA) (Quan et al. 2006). Magliulo et al.

have developed a CLEIA for detecting aflatoxin M1 in milk; the LOD was 0.25 ng/kg,

and the limit of quantitation (LOQ) was 1 ng/kg, which is an improvement in sensitivity

of two orders of magnitude compared with the ELISA (Magliulo et al. 2005). In addition,

several reviews have reported an improvement in sensitivity of two to three orders of

magnitude using the CLEIA compared with using the ELISA (Yang et al. 2008; Xu et al.

2006).

We have developed a competitive indirect CLEIA based on a polyclonal-antibody and

horseradish peroxidase-labeled secondary antibody chemiluminescence system to detect

DES residues in seafood. This assay allows the rapid screening of DES residues.

MATERIALS AND METHODS

Apparatus

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Plates were washed in a Multiskan MK2 microplate washer (Thermo Scientific, Hudson,

USA) to flush unattached substances away. Chemiluminescent intensity was recorded

using a Wallac 1420 VICTOR3 multilabel counter (PerkinElmer, USA). Opaque high

binding 96-well plates were purchased from Shenzhen Jincanhua Industrial Co., Ltd.

(Shenzhen, China).

Reagents

Ovalbumin (OVA), bovine serum albumin (BSA), dicyclohexylcarbodiimide (DCC),

N-hydroxysuccinimide (NHS), DES, dienestrol (DIEN), hexoestrol (HEX), estradiol (E2),

estrone (E1), ethinyloestradiol (EST), progestin (P4), horseradish peroxidase (HRP), and

HRP-conjugated goat anti-rabbit IgG (secondary antibody) were obtained from

Sigma-Aldrich (St. Louis, USA). Methanol, acetone, and Tween-20 were obtained from

Tianjin Damao Chemical Reagent Co., Ltd. (Tianjin, China). The Super Signal West Pico

CL substrate (luminol/enhancer, A; stable peroxide buffer, B) was obtained from Pierce

Protein Research Products (Thermo Fisher Scientific Inc., Illinois, USA). The anti-DES

polyclonal antibody was prepared in our laboratory. All other reagents were of analytical

grade and obtained from a local chemical supplier (Yunhui Trade Co., Ltd., Guangzhou,

China).

Buffers

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The following buffers were used: (A) coating solution, carbonate buffer (100 mmol/L, pH

9.6); (B) blocking solution, 5% skim milk in PBST; phosphate buffered saline (PBS, 10

mmol/L, pH 7.4); PBST (PBS containing 0.05% Tween-20, pH 7.4); and (C) standard

solution matrix, Tris-HCl buffer (100 mmol/L, pH 8.0).

Preparation Of Hapten-Protein Conjugates

Hapten 1 was coupled to BSA, to synthesize immunogen 1 (hapten 1-BSA), via the active

ester method. Briefly, hapten 1(12 µmol), NHS (14.4 µmol), and DCC (14.4 µmol) were

dissolved in DMF (1000 µL). The mixture was stirred gently at 4°C overnight, and then

centrifuged at 10,956 × g for 5 min. The supernatant (900 µL) was added dropwise to

BSA (136 mg) in PBS (9 mL, pH 7.4). The mixture was stirred at 4 °C for 12 h and then

dialyzed against PBS (0.01 M, pH 7.4) at 4°C for 2 days to obtain immunogen 1.

Hapten 2 was coupled to OVA to be used as the plate coating antigen (hapten 1-OVA).

Briefly, hapten 2 (33.9 mg, 0.1 mmol) and isobutyl chloroformate (20 µL, 0.15 mmol)

were dissolved in DMF (15,000 µL). The mixture was stirred gently at 4°C for 0.5 h. The

reaction mixture was then added to carbonate buffer (4 mL, 1 mol/L, pH 9.6) containing

OVA (50 mg) and stirred gently at 4°C overnight. The resulting mixture was dialyzed

against physiological saline at 4°C for 2 days to give the plate coating antigen

Preparation Of Polyclonal Antibodies

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Antibodies were raised via an intramuscular injection of immunogen 1 given to two New

Zealand white rabbit for immunogen, weighing 1.5–2.0 kg. In brief, immunogen 1 (0.5

mg) was dissolved in saline and emulsified with an equal volume of Freund’s complete

adjuvant. The rabbit (housed at the Guangdong Medical Laboratory Animal Center) was

immunized four times using emulsified immunogen 1, at intervals of 28 days. Freund’s

incomplete adjuvant was used for the booster muscle injection. Blood was taken from the

rabbit on the eighth day after the fourth immunization. The obtained antiserum was

divided into aliquots (1 mL) and stored at -20°C until use. Negative serum was obtained

from an unimmunized rabbit.

Calibration Curves

Calibration curves were prepared for quantification. DES standard solution (1000 ng/mL)

was prepared from a 1 mg/mL stock solution in dry DMF and diluted with Tris-HCl to

provide a series of standards containing 8.1, 2.7, 0.9, 0.3, 0.1, 0.03, and 0 ng/mL of DES.

Immunoassay Procedure

Plates were coated with DES-OVA (1:16,000, 100 µL/well) in carbonate buffer overnight

at 37°C. The wells were washed two times with PBST and blocked with 5% skim milk in

PBST (120 µL/well) for 3 h at 37°C. After removing the liquid, the plates were dried at

37°C overnight. Individual DES standards or samples (50 µL/well) were added to the

wells followed by addition of the anti-DES antibody diluted (1:100,000) with Tris-HCl

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(50 µL/well). The wells were incubated for 30 min at 37 °C. After washing five times

with PBST, 100 µL of HRP-conjugated goat anti-rabbit IgG (secondary antibody) (1:8000)

was added and further incubated for 25 min at 37°C. After washing five times with PBST,

the CL substrate (50 µL of A and 50 µL of B) was added. The plate was shaken gently for

1 min at room temperature and the CL intensity was recorded (expressed as relative light

unit [RLU]). Competitive curves were obtained by plotting inhibition rate against the

logarithm of analyte concentration. Sigmoid curves were generated using OriginPro 8.0

software (OriginLab Corp., Northampton, MA, USA).

Sample Preparation

Blank samples of seafood (1 g) were homogenized, a DES standard solution and ethyl

acetate (5.0 mL) were added, and the mixture shaken strongly for 5 min. The samples

were centrifuged at 4000 ×g for 10 min and the homogenization process was repeated.

The supernatants were combined and dried under a steam of nitrogen (N2) at 45°C. The

residue was dissolved in Tris-HCl containing 5% methanol (1.0 mL) and n-hexane (2

mL). The sample was shaken for 2 min and then centrifuged at 4000 ×g for 5 min. Each

sample was analyzed 20 times.

Data Analysis

For each parameter, standards and samples were established and processed according to

the above immunoassay procedure. Sigmoid curves were generated using OriginPro 8.0

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software (OriginLab Corp., Northampton, MA, USA). Using the standard curves, mean

chemiluminescence intensity values were calculated as inhibition rates (B0-B/B0 × 100,

where B0 is the luminescent intensity of the control (Tris-HCl) and B is the luminescent

intensity of the samples; and RLUmax/IC50 (where IC50 = a 50% decrease in RLUmax). The

LOD is the smallest concentration of the analyte that produces a signal that can be

significantly distinguished from zero for a given sample matrix, with a stated degree of

confidence. There is a general consensus in favor of selecting the dose which inhibits

10% of the binding of the antibody with the enzyme tracer at 90% B/B0 (IC10). (Guo et al.

2007; Hennion et al. 1998; Xu et al. 2012). The equation is:

^p2 1 2 0y A (A A ) / (1 (x / x ) )

A1 = the asymptotic maximum; A2 = the asymptotic minimum; X0 = the x value at the

inflection point (corresponding to the analyte concentration that gives a 50% decrease in

RLUmax, i.e., when the value of B/B0 is 0.5); and P = the slope of curve at the inflexion

point (Zhu et al. 2012).

Evaluation Of Recovery

Diluted DES analytical standard solutions in methanol (1 µg/mL) were added to the

samples of seafood up to final concentrations of 10, 30 or 50 ng/mL. The samples were

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then subjected to ethyl acetate pretreatment as described above. The final solution, which

was determined 20 times, was used for CLEIA analysis.

RESULTS AND DISCUSSION

Optimization Of CLEIA

To improve the performance of the assay, and the sensitivity of the immunoreaction,

several experimental parameters, including the coating concentration, antibody dilution,

incubation time, competitive reaction time, dilution buffer and pH were investigated. For

each condition, standard curves for DES were established using the concentration of DES

and the RLU/RLU0 ratio.

RLUmax and IC50 values were acquired from the standard curves. The IC50 value and

RLUmax/IC50 ratio were used to evaluate the effect of different parameters on CLEIA

performance. Lower IC50 values and higher RLUmax/IC50 ratios indicated increased

sensitivity of the assay (Botchkareva et al. 2003; Pang et al. 2008).

Optimization Of Coating Concentration And Antibody Dilution Ratios

Dilution ratios of the coating concentration (1 mg/mL) and the antibody were

investigated using standard curves ranging from 12,000 to 20,000, and 60,000 to 120,000,

respectively (Figure 1). The IC50 value first decreased and then increased as the dilution

ratios of the coating concentration increased, and the reverse was observed for the

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RLUmax/IC50 ratio (Figure 1 a and b). The same trend was observed for the dilution ratio

of the primary antibody (Figure 1 c and d). The highest level of sensitivity was obtained

with coating concentration and antibody dilution ratios of 1:16,000 and 1:100,000,

respectively.

Optimization Of Reaction Times

The effect of varying competitive reaction times and horseradish peroxidase-labeled

secondary antibody reaction times was examined. Competitive reaction times of 20, 30,

40 and 50 min were investigated. The RLUmax/IC50 ratio reached a peak and then

stabilized after an incubation period of 30 min (Figure 2 a and b), indicating that an

incubation time of 30 min is sufficient for the antibody–antigen interaction to reach

equilibrium.

HRP-labeled secondary antibody reaction times of longer than 25 min caused a decrease

in the RLUmax/IC50 ratio (Figure 2 c and d), so a HRP-labeled secondary antibody

reaction time of 25 min was chosen.

Optimization Of The Dilution Buffer And Buffer Ph

Antigen–antibody binding is characterized by weak intermolecular bonds that can be

affected by the dilution buffer and pH value. The standard solutions of DES were

investigated in different buffers: PBS (10 mmol/L, pH 7.4), PBST (PBS containing

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0.05% Tween-20, pH 7.4), Tris-HCl buffer (100 mmol/L, pH 8.0), as well as deionized

water. The highest sensitivity and maximum RLUmax/IC50 value were obtained with the

Tris-HCl buffer (Figure 3 a and b).

Five pH values (7.2, 7.5, 7.8, 8.0 and 8.2) of Tris-HCl buffer were tested. The effect of

pH on the assay performance (IC50, RLUmax and RLUmax/IC50) is shown in Figure 3 c and

d. The IC50 value reached a minimum, and the RLUmax/IC50 ratio a maximum, at pH 8.0.

Analytical Parameters Of The Optimized Immunoassays

Dose-dependent inhibition rate curves obtained with the CLEIA using the optimized

conditions are presented in Figure 4. The LOQ, determined as the concentration causing

20–80% inhibition of maximal chemiluminescence intensity, was 0.028–3.60 ng/mL. The

LOD for DES was 0.0068 ng/mL, and the IC50 value was 0.32 ng/mL.

Cross-Reactivity

Specificity should be considered one of the most important factors for an immunoassay.

The specificity of the proposed anti-DES polyclonal antibody was evaluated by analyzing

the extent of cross-reactivity with six structurally-related compounds: DIEN, HEX, E2,

E1, EST and P4. The anti-DES polyclonal antibody showed some cross-reactivity with

DIEN and HEX (7.1% and 2.8%), but negligible cross-reactivity with other analogs.

(Table 1). This cross-reactivity may occur because the anti-DES antibody active site can

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recognize the two benzene rings in DIEN and HEX. Overall, the lack of cross-reactivity

indicates that the CLEIA method is suitable for the specific screening of DES.

Aquaculture Tissue Sample Analysis

Aquaculture tissue samples (negative for DES), including fish and shrimp, were spiked

with DES at six different concentrations (0.03, 0.1, 0.3, 0.9, 2.7, and 8.1 ng/mL) to assess

the precision of the assay. The samples were treated following the procedure described.

The mean value (± SD) and the coefficient of variation (CV) of the concentration of DES

in the different samples, determined from replicate analyses (n = 4) in the same run

(intra-assay) and in separate runs (inter-assay) (Even et al. 2007), are shown in Table 2.

The intra-assay precision values (measured as CV %) were all below 10%, and the

inter-assay values were all below 13%. These results indicate an acceptable degree of

parallelism and precision, when the assay is applied to real samples.

Recovery Values

To determine the sensitivity and reproducibility of the proposed CLEIA, recovery tests

from spiked samples were performed. Samples were spiked with DES at three

concentrations (10, 30 and 50 ng/mL) and analyzed using the CLEIA; the extraction

solution was determined 20 times for CLEIA analysis. Each sample was evaluated three

times to verify the repeatability. The results are shown in Table 3.

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The recovery values from seafood samples ranged from 68.5% to 92.5%, and the mean

recovery values, intra-assay and inter-assay, were 82.95% and 81.1%, respectively. The

CV values ranged from 4.1% to 10.6%, and the mean CV values, intra-assay and

inter-assay, were 6.2% and 8.0%, respectively. The reproducibility of this assay is

acceptable for use in screening.

Validation Assay

The CLEIA method for determination of DES was investigated compared with a standard

HPLC-MS method. Eight seafood samples were determined using the surrogate

calibration curve. Two determinations were performed for each sample, and the results

were compared with those obtained using the reference HPLC-MS method. Concordant

results were obtained with the two methods: four samples were positive for DES, and

four samples were scored as negative (Table 4). Good agreement (r2 = 0.9941) was

observed between the results obtained using the CLEIA and HPLC-MS, further

confirming the reliability of the CLEIA.

CONCLUSIONS

An ultra-sensitive and specific CLEIA was developed and applied to the determination of

DES residues in seafood. The sensitivity of the assay was improved through optimizing

the parameters of the competitive immunoreaction. The linear range was 0.028–3.60

ng/mL, the LOD was 0.0068 ng/mL, and the IC50 value was 0.32 ng/mL. The optimized

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CLEIA was 20–30 fold more sensitive than the ELISA (Wang et al. 2006; Deng et al.

2011; Sun et al. 2010). The CLEIA showed some cross-reactivity with DIEN (7.1%) and

HEX (2.8%), but negligible cross-reactivity with E2, EST, P4 or E1. The recovery values

for spiked seafood samples ranged from 68.5% to 92.5%, the CV values ranged from

4.1% to 10.6%, and the mean CV values, intra-assay and inter-assay, were 6.2% and

8.0%, respectively. Good agreement was obtained between the CLEIA and HPLC-MS

methods for screening DES. The CLEIA method developed should prove useful for the

real-time, large-scale screening of trace levels of DES residues in seafood.

REFERENCES

Botchkareva, A. E., S. A. Eremin., A. Montoya., J. J. Manclus., B. Mickova., P. Rauch., F.

Fini., and S. Girotti. 2003. Development of chemiluminescent ELISAs to DDT and its

metabolites in food and environmental samples. J. Immunol. Methods. 283: 45–47.

Burroughs, W., C. C. Culbertson., E. Cheng. W. H. Hale., and P. Homeyer. 1955. The

influence of oral administration of diethylstilbestrol to beef cattle. J. Anim Sci. 14:

1015–1024.

Choi, J. H., Y. T. Kim., and J. H. Lee. 2010. Rapid quantification of melamine in milk

using competitive 1,1′-oxalyldiimidazole chemiluminescent enzyme immunoassay.

Analyst. 135: 2445–2450.

Dow

nloa

ded

by [

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rsity

NY

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gham

ton]

at 0

2:09

24

May

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3

ACCEPTED MANUSCRIPT


Deng, S. L., P. Ling., and H. B. Liu. 2011. et al. Preparation and Identification of the

Specific Antibody against Diethylstibestrol. China Biotechnology. 31(6):106–110.

Dickson, L.C., J. D. MacNeil., J. Reid., and A.C. Fesser. 2003. Validation of screening

method for residues of diethylstilbestrol, dienestrol, hexestrol and zeranol in bovine urine

using immunoaffinity chromatography and gas chromatography/mass spectrometry.

J.AOAC Int. 84(4): 631–639.

Even, M. S., C. B. Sandusky., and N. D. Barnard. 2007. et al. Development of a novel

ELISA for human insulin using monoclonal antibodies produced in serum-free cell

culture medium. Clin. Bio. Chem. 40(1-2): 98–103.

Fan, A., Z. J. Cao., H. Li., M. Kai., and J. Z. Lu. 2009. Chemiluminescence platforms in

immunoassay and DNA analyses. Anal Sci. 25(5): 587–597.

Guo, Y. R., W. J. Gui., and S. T. Wang. 2007. Development and performance testing of

Chloramphenicol ELISA kit. J. CIFST. 7(6):118–123.

Hennion, M.C., and D. Barcelo. 1998. Strengths and limitations of immunoassays for

effective and efficient use for pesticide analysis in water samples: A review. Anal. Chim.

Acta. 362(1): 3–34.

Katrin, K., R. J. Maria., D. Andrea., D. Richard., M. Erwin., N. Reinhard., and S. Michael.

2009. A regenerable immunochip for the rapid determination of 13 different antibiotics in

raw milk. Analyst. 134: 1433–1439.

Knopp, D. 2006. Immunoassay development for environmental analysis. Anal. Bioanal.

Chem. 385: 425–427

Dow

nloa

ded

by [

Stat

e U

nive

rsity

NY

Bin

gham

ton]

at 0

2:09

24

May

201

3

ACCEPTED MANUSCRIPT


Lin, J. M., L. X. Zhao., and X. Wang. 2008. Chemiluminescene Immunoassay. Beijing:

Chemical Engineering Press., 4-6.

Long, F., H. C. Shi., and M. He. 2009. et al. Sensitive and rapid chemiluminescence

enzyme immunoassay for microcystin-LR in water samples. Anal. Chimica. Acta. 649:

123–127.

Magliulo, M., M. Mirasoli., P. Simoni., R. Lelli., O. Portanti., and A. Roda. 2005.

Development and validation of an ultrasensitive chemiluminescent enzyme immunoassay

for aflatoxin M1 in milk. J. Agric. Food Chem. 53(9):3300–3305.

Malone, E.M., C.T. Elliott., D.G. Kennedy., and L. Regan. 2010. Rapid confirmatory

method for the determination of sixteen synthetic growth promoters and bisphenol A in

bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem

mass spectrometry. J. Chromatogr. B. ,Analyt. Technol. Biomed Life Sci. 878(15-16):

1077–1084.

Nielsem, M., S. Bjrnsdottir., P. E. Hoyer., and A.G. Byskov. 2000. Ontogeny of oestrogen

receptor α in gonads and sex ducts of fetal and newborn mice. Reprod. Fertil. 118:

195–204.

Noller, K. L., and C. R. Fish. 1974. Diethylstibestrol usage: Its interesting past, important

present, and questionable future. Med Clin North Am. 58: 739–810.

Pang, G. F., F. Zheng., C. L.Fan, Y. Li., X. X. Ji., and M. L. Wang. 2008. Determination

of 450 pesticides and related chemical residues in drinking water–LC-MS-MS method.

Dow

nloa

ded

by [

Stat

e U

nive

rsity

NY

Bin

gham

ton]

at 0

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ACCEPTED MANUSCRIPT


Chinese National Standard (GB/T 23214–2008), Chinese Standard Press, Beijing, (in

Chinese).

Quan, Y., Y. Zhang., S. Wang., N. Lee., and I. R. Kennedy. 2006. A rapid and sensitive

chemiluminescence enzyme-linked immunosorbent assay for the determination of

fumonisin B1 in food samples. Anal. Chim. Acta. 580: 1–8.

Ren, S. Q., X. Wang., and B. J. Tang. 2008. Micro-plate chemiluminesence enzyme

immunoassay for clinincal determination of progesterone in Human Serum. Chinese J.

Anal. Chem. 36(6): 729–734.

Roda, A., P. Pasini., and M. Guardigli. 2000. et al. Bio- and chemiluminescence in

bioanalysis. Fresenius J. Anal. Chem. 366(6-7): 752–9.

Roda, A., P. Pasini., and M. Mirasoli. 2004. et al. Biotechnological applications of

bioluminescence and chemiluminescence. Trends in Biotechnol. 22(6): 294–303.

Sun, L., and X. M. Gao. 2010. Application of Enzyme Linked Immunosorbent Assay to

Determinate the Residueses of Diethylstilbestrol Residues in Meat and Meat Product.

Food Research and Development. 31(1): 122–125.

Tudorache, M., I.A. Zdrojewska., and J. Emneus. 2006. Evaluation of progesterone

content in saliva using magnetic particle-based immuno supported liquid membrane assay

(m-ISLMA). Biosens. Bioelectron. 22: 241–246.

Wang, W. J., J. Li., J. X. Zhao., and Z, Z. Guo. 2006. et al. Development of Monoclonal

Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol.

Chinese J. Chem. 24(12): 1758–1765.

Dow

nloa

ded

by [

Stat

e U

nive

rsity

NY

Bin

gham

ton]

at 0

2:09

24

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ACCEPTED MANUSCRIPT


Wang, W. J., S. C. Zhang., and C. H. Liu. 2009. et al. CE immunoassay with enhanced

chemiluminescence detection of erythropoietin using silica dioxide nanoparticles as

pseudo stationary phase. Electrophoresis. 30: 3092–3098.

Wijitar, D. C., S. Weena., J. M. Lin., C. Orawon., S. Lin., and X. T. Ying. 2007.

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay

for the determination of carcinoembryonic antigen. Anal. Bioanal. Chem. 387(6):

1965–1971.

Wu, J., Z. F. Fu., and F. Yan. 2007. et al. Biomedical and clinical applications of

immunoassays and immunosensors for tumor markers. Trends Anal.Chem. 26(7):

679–688.

Xin, T. B., X. Wang., J. Hui., S. X. Liang., and J. Lin. 2009. et al. Development of

magnetic particle-based chemiluminescence enzyme immunoassay for the detection of

17β-estradiol in environmental water, Appl. Biochem. Biotechnol. 158: 582–594.

Xu, C. L., C. F. Peng., and Y. B. Liu. 2006. et al. Chemiluminescence enzyme

immunoassay (CLEIA) for the determination of choramphenicol residues in aquatic

tissues. Luminescence. 21:126–128.

Xu, Z. L., W. J. Sun., and J. Y. Yang. 2012. et al. Development of a Solid-Phase

Extraction Coupling Chemiluminescent Enzyme Immunoassay for Determination of

Organophosphorus Pesticides in Environmental Water Samples. J. Agric. Food Chem.

60(9): 2069–2075.

Dow

nloa

ded

by [

Stat

e U

nive

rsity

NY

Bin

gham

ton]

at 0

2:09

24

May

201

3

ACCEPTED MANUSCRIPT


Xu, C.L., D.H. Yu., X.G. Chu., C.F. Peng., and Z.Y. Jin. 2006. A chemiluminescence

enzyme immunoassay (CLEIA) for the determination of 19-nortestosterone residues in

aquatic products. Anal. Lett. 39(4): 709–727.

Xue, P., Z. J. Zhang., and X. M. Zhang. 2011. Chemiluminescent Analysis of

Carcinoembryonic Antigen in Serum Samples. Chinese J. Anal.Chem. 39(1): 95–98.

Yang, M., Y. Kostov., H.A. Bruck., and A. Rasooly. 2008. et al. Carbon nanotubes with

enhanced chemiluminescence immunoassay for CCD-based detection of staphylococcal

enterotoxin B in food. Anal. Chem. 80(22):8532–8537.

Zhang, R. Q., N. Hizuru., S. Nobuaki., N. Koji., M. Takashi., and N. Kazumi. 2007. et al.

Sequential Injection Chemiluminescence Immunoassay for Nonionic Surfactants by

Using Magnetic Microbeads. Anal. Chim. Acta. 600: 105–113.

Zhang, H. M., W. T. Li., Z. H. Sheng., H. Y. Han., and Q. G. He. 2010. Ultrasensitive

detection of porcine circovirus type 2 using gold(III) enhanced chemiluminescence

immunoassay. Analyst. 135: 1680–1685.

Zhang, S., Z. Zhang., and W. Shi. 2006. et al. Development of a chemiluminescent

ELISA for determining chloramphenicol in chicken muscle. J. Agric. Food Chem. 54(16):

5718–5722.

Zhao, L. X., J. M. Lin., Z. J. Li., and X. T. Ying. 2006. Development of a highly

sensitive,second antibody format chemiluminescence enzyme immunoassay for the

determination of 17-estradiol in wastewater. J. Anal. Chem. 558: 290–295.

Dow

nloa

ded

by [

Stat

e U

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rsity

NY

Bin

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ton]

at 0

2:09

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Zhao, L.X., L. Sun., and X. G. Chu. 2009. Chemiluminescence immunoassay. Trends

Anal. Chem. 28(4): 404–415.

Zheng, G.J., F. Lu., Q. Li., and J. Zhen. 2011. et al. Magnetic Microparticle

Chemiluminescence Immunoassay Method for Determination of Estriol in Human Urine.

Chinese J.Anal.Chem. 39(1): 62–65.

Zhou, M., C. Guan., G. Chen., X. Xie., and S. Wu. 2005. Determination of theophylline

concentration in serum by chemiluminescent immunoassay. J Zhejiang Univ-Sci B. 6(12):

1148–1152.

Zhu, J. H., X. Q. Tao., and S. Y. Ding. 2012. et al. Micro-Plate Chemiluminescence

Enzyme Immunoassay for Determination of Zeranol in Bovine Milk and Urine. Anal. Lett.

45: 2538–2548.

Zhuang, H. S., M. C. Zhang., and J. Zhang. 2004. Research of chemiluminescence

immunoassay in Environmental detection. World Sci-Tech R&D. 26: 89–96.

Dow

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Table 1. Cross-reactivity of DES antisera with analogs

Compounds Constitutional

formula

IC50 (ng/mL) Cross-reactivity

(%)

Diethylstilbestrol

(DES)

0.41 100

Dienestrol (DIEN) 5.77 7.1

Hexoestrol (HEX) 14.64 2.8

Estradiol (E2)

＞4100 ＜0.01

Ethinyloestradiol

(EST)

＞4100 ＜0.01

Progestin (P4)

＞4100 ＜0.01

Estrone (E1)

＞4100 ＜0.01

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Table 2. Intra-assay and inter-assay precision at different concentration of DES

Intra-assay (n=4) Inter-assay (n=4)

DES

(ng/mL)

Mean ±SD CV (%) Mean ± SD CV (%)

8.1 8.05±0.338 4.2 8.00±0.440 5.5

2.7 2.70±0.124 4.6 2.60±0.148 5.7

0.9 0.80±0.038 4.8 0.90±0.064 7.1

0.3 0.25±0.015 5.9 0.30±0.024 8.0

0.1 0.09±0.007 8.0 0.10±0.010 10.3

0.03 0.03±0.003 9.1 0.04±0.005 12.4

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Table 3. Recoveries of DES from spiked fish and shrimp by CLEIA

Samples Spiked

level

(ng/mL)

intra-assay

(n=3)

inter-assay

(n=3)

Mean ±SD

(ng/mL)

Recovery

(%)

CV

(%)

Mean ±SD

(ng/mL)

Recovery

(%)

CV

(%)

Fish 10 7.55±0.39 75.5 5.2 6.85±0.73 68.5 10.6

30 25.17±1.96 83.9 7.8 22.65±1.04 75.5 4.6

50 40.95±2.64 81.9 6.4 44.60±1.83 89.2 4.1

Shrimp 10 7.35±0.68 73.5 9.3 7.49±0.48 74.9 6.4

30 27.12±1.66 90.4 6.1 26.04±1.88 86.8 7.2

50 46.25±3.15 92.5 6.8 45.85±2.28 91.7 5.0

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Table 4. Concentrations of DES from spiked fish and shrimp by CLEIA and HPLC-MS

Samples Concentration ( ng/mL )

CLEIA(n=4) HPLC-MS/MS(n=2)

Fish1 ＜LODa ＜LODb

Fish2 ＜LODa ＜LODb

Fish3 33.07±2.12 33.169±3.07

Fish4 35.14±2.87 35.610±4.18

Shrimp1 ＜LODa ＜LODb

Shrimp2 ＜LODa ＜LODb

Shrimp3 39.53±3.54 39.40±4.43

Shrimp4 39.12±4.12 39.37±5.90

LOD：Limit of detection.

aLOD is 0.0068ng/mL

bLOD is 0.041ng/mL

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Figure1. Effects of coating concentration (a, b) and antibody dilution (c, d) on the IC50

and RLUmax/IC50 ratio

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Figure2. Influence of competitive reaction time (a, b) and horseradish peroxidase-labeled

secondary antibody reaction time (c, d) on the IC50 and RLUmax/IC50 ratio

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Figure3. Effects of dilution buffer (a, b) and buffer pH (c, d) on the IC50 and

RLUmax/IC50 ratio

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Figure4. Normalized standard curve by CLEIA for DES under optimized conditions.

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Figure 5. The mechanism of the reaction used in CLEIA

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development of an ultra-sensitive chemiluminescence enzyme immunoassay for the determination of...

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