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Development of a chemiluminescent enzyme immunoassay for thedetermination of dexamethasone in milk

Marina M. Vdovenko,a Anastasia V. Gribas,b Alexandra V. Vylegzhaninac and Ivan Yu. Sakharov*a

Received 19th March 2012, Accepted 22nd May 2012

DOI: 10.1039/c2ay25278c

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the

determination of dexamethasone (DEX) was developed using soybean peroxidase (SbP) as an enzyme

label. A mixture of 3-(100-phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine

(MORPH) was used as an enhancer of SbP-induced chemiluminescence. Varying the concentrations of

the capture antigen (DEX-ovalbumin) and specific anti-DEX antibody, the conditions of the assay

were optimized. The values of IC10, IC50 and working range (IC20–IC80) of the CL-ELISA of DEXwere

0.02, 0.9, 0.08–9.3 ng mL�1, respectively. It was shown that a pretreatment of cow milk samples by

centrifugation and 25%methanol prevented the matrix effect of whole milk. The coefficient of variation

(CV) and recovery values from the spiked milk samples estimated by the developed CL-ELISA were in

the range of 2.2 to 9.9% and 82 to 142%, respectively.

Introduction

Synthetic glucocorticoids including dexamethasone (DEX)

(Fig. 1) are extensively used as therapeutic agents in veterinary

practice for the treatment of metabolic diseases and inflamma-

tory disorders in farm animals.1–3 Glucocorticoids are also

utilized illegally as growth promoters, thus allowing an increase

in production of animal origin with minimisation of the associ-

ated expenses.4–7 The high pharmacological activity of most

Fig. 1 Chemical structure of dexamethasone.

aDepartment of Chemistry, Lomonosov Moscow State University,Moscow, 119991, Russia. E-mail: sakharovivan@gmail.com; Fax: +7495 9395417; Tel: +7 495 9393407bG.V. Plekhanov Russian Economic University, 115998 Moscow, RussiacThe All-Russian State Centre for Quality and Standardization ofVeterinary Drugs and Feed, 123022 Moscow, Russia

2550 | Anal. Methods, 2012, 4, 2550–2554

synthetic corticosteroids makes the residues of these molecules

potentially dangerous for meat consumers due to the risk of toxic

effects on humans.8 Recently, it was demonstrated that long term

administration of DEX in low doses induced thymus atrophy in

beef cattle.9

As the use of growth promoters has been banned in all

European Community countries since 1986,10 the DEX content

in food products is regulated by some governmental organiza-

tions. Thus, the European Commission set the maximum residue

limits (MRLs) for DEX contamination equal to 0.75 mg kg�1 in

muscle and kidney, 2 mg kg�1 in liver and 0.3 mg kg�1 in milk.11 In

Russia MRLs are 0.5 mg kg�1 in muscle and kidney, 2.5 mg kg�1

in liver and 0.3 mg kg�1 in milk.12

To minimize the risk of human exposure to DEX and to

control the content of DEX in food and feed samples some

analytical techniques have been developed. The European

Pharmacopoeia13 uses ultraviolet-visible spectrophotometry and

high-performance liquid chromatography (HPLC) techniques as

standard methods to detect DEX. The U.S. Pharmacopeia14 and

Japanese Pharmacopoeia15 also use HPLC for the determination

of DEX. The problems encountered by using such methods

include a large susceptibility for interference in spectroscopic

analysis,16 a need for derivatization or time-consuming extrac-

tion procedures in chromatographic procedures,17 and addition

of organic solvents that contribute to reducing the lifetime of the

columns and equipment,18 making this technique unfeasible for

routine analysis. The additional disadvantages of HPLC are the

need for expensive equipment and highly trained personnel.

Among several electroanalytical techniques employed in

studies with DEX, square-wave adsorptive voltammetry can be

highlighted. This technique involves no cleanup procedures.

Unfortunately, it has a relatively high detection limit.19 A more

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promising technique is enzyme immunoassay due to the

following advantages: high specificity and sensitivity, possibility

to analyze a large number of samples, ease of automation and

the lack of requirement for time-consuming procedures and

sophisticated equipment compared with chromatographic

methods.

It is well known that colorimetric, fluorometric and chemi-

luminescent methods are used to detect peroxidase activity in

enzyme immunoassays. The chemiluminescent method has the

highest sensitivity. It is based on peroxidase-catalyzed oxida-

tion of luminol in the presence of enhancers, where light is

formed as the result of the enhanced chemiluminescence reac-

tion (ECR). As reported by Vdovenko et al.,20 the highest CL

intensity was obtained in ECR catalyzed by anionic soybean

peroxidase (SbP) using 3-(100-phenothiazinyl)-propane-1-sulfo-nate (SPTZ) in combination with 4-morpholinopyridine

(MORPH) as enhancers. Advantages of the employment of the

SbP/SPTZ/MORPH system have been demonstrated in the

development of ultrasensitive ELISAs for the determination of

human thyroglobulin and ochratoxin A.21,22 As well as the high

sensitivity of SbP in ECR, this commercially available peroxi-

dase also showed a unique stability.23,24 These characteristics of

SbP show good potential for the wide application of this

enzyme in CL-ELISA.

In the present work we describe an indirect competitive

enzyme-linked immunosorbent assay (ELISA) for the determi-

nation of DEX. To increase the sensitivity of the assay, for the

determination of peroxidase activity we applied ECR based on

the ultrasensitive SbP/SPTZ/MORPH system. The developed

CL-ELISA was successfully applied for the determination of

DEX in whole milk samples.

Materials and methods

Materials

Dexamethasone (DEX) was purchased from Sigma-Aldrich

(USA). A standard solution of DEX was prepared as 1 mg mL�1

in absolute ethanol. Soybean peroxidase (SbP, RZ 2.8) was

purchased from Bio-Research Products Ltd. (USA) and used

without further purification. Sodium 3-(100-phenothiazinyl)-propane-1-sulfonate (SPTZ) was prepared as described by

Marzocchi et al.25 Luminol, Tween 20, Tris, casein (sodium salt)

were obtained from Sigma (USA); 4-morpholinopyridine

(MORPH) was from Aldrich (USA); and H2O2 (30%) were from

ChimMed (Russia). Ethanol and methanol were obtained from

Merck (Darmstadt, Germany). Black polystyrene plates (high

protein binding) were obtained from Nunc (Denmark).

The concentration of H2O2 was estimated by measuring the

absorbance using 3240 ¼ 43.6 L mol�1 cm�1.26

Dexamethasone conjugated with ovalbumin (DEX-OVA) was

synthesized as described previously.27 The polyclonal antibodies

specific to DEX (anti-DEX-Ab) were produced by subcutaneous

immunization of rabbits by a conjugate of DEX and bovine

serum albumin. Anti-DEX antiserum was stored in 50% glycerol

at �20 �C.DEX-free cow’s milk samples were obtained from local dairies

and analyzed on the same day.

This journal is ª The Royal Society of Chemistry 2012

Conjugation of sheep anti-rabbit IgG antibody with SbP

The sheep anti-rabbit IgG antibody (SAR) was conjugated with

SbP by a periodate method as described by Sakharov et al.28 For

this, 0.2 mg of SbP was dissolved in 0.2 mL of distilled water and

supplemented with sodium periodate to a final concentration of

32 mM. Periodate oxidation of peroxidase was performed at

room temperature for 20 min in darkness. To remove the excess

oxidant and its degradation products the dialysis of the reaction

mixture was carried out against 1 mM sodium acetate buffer, pH

4.3 overnight at 4 �C. The next day the obtained peroxidase

solution was supplemented with 120 mL of SAR (10 mg mL�1)

and incubated at room temperature for 2 hours with constant

stirring. To reduce the formed Schiff bases, 20 mL of freshly

prepared NaBH4 solution (4 mg mL�1) was added. The reaction

was carried out for 1 hour at room temperature. The conjugate

SAR–SbP was extensively dialyzed against 10 mM phosphate

buffer with 0.15 M NaCl (PBS), pH 7.4 overnight and stored in

50% glycerol at �20 �C.

Determination of DEX by CL-ELISA

CL-ELISA was carried out using 96-well black polystyrene

plates (MaxiSorp, Nunc, Denmark). The plates were coated by

adding into each well 100 mL of DEX-OVA (dilution 1 : 8000)

dissolved in 50 mM carbonate buffer, pH 9.6, and incubated at

4 �C overnight. The plate was then washed using PBS with 0.05%

Tween 20 (PBST) four times and blocked by adding 100 mL of

PBS containing 0.1% casein for 60 min at 37 �C. The plate was

washed four times with PBST. Subsequently, 50 mL of DEX

(0.0004 to 4000 ng mL�1) and 50 mL of anti-DEX antiserum

solution (dilution 1 : 8000) were added to each well. Anti-DEX

antiserumwas dissolved in PBS or PBS with 0.2% casein and 25%

methanol, while DEX was dissolved in PBS containing 0.2%

casein and 25% methanol or in milk samples. The competitive

step of the assay proceeded for 1 h at 37 �C. The plates were

washed again as described above. Then, 100 mL of the conjugate

SAR–SbP (dilution 1 : 8000) dissolved in PBST with 0.1% casein

was added to each well. The plates were incubated for 1 h at

37 �C and then washed with PBST four times. Finally, 100 mL of

freshly prepared substrate solution (50 mM Tris–HCl buffer, pH

8.3 with 0.75 mM luminol, 1 mM SPTZ, 1 mM MORPH and

0.5 mM hydrogen peroxide20) were added to each well and stir-

red. Chemiluminescence intensity was monitored at room

temperature on a luminescence reader Zenyth 1100&3100

(Anthos Labtec Instruments GmbH, Austria). Integration time

was 1.0 s; and the interval and intensity of stirring prior to CL

reading were 5 s and medium, respectively.

Pretreatment of milk samples

Preparation of fat-free milk. The milk samples were centri-

fuged at 970 � g for 15 min at 4 �C to remove fat; the upper

creamy layer was completely discarded.

Pretreatment using Carrez method. 250 mL of 0.36 M

K4Fe(CN)6$3H2O and 250 mL of 1.04 M ZnSO4$7H2O were

added successively at 20 min intervals to 5 mL of fat-free milk.

The obtained mixture was centrifuged at 2660 � g for 10 min at

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15 �C to precipitate proteins. The obtained supernatant was used

in CL-ELISA.

Pretreatment using trichloroacetic acid. Trichloroacetic acid

was added to the fat-free milk to a final concentration of 1.7% at

room temperature. The milk sample was centrifuged at 2660 � g

for 10 min at 15 �C after 10 min incubation with shaking. The

obtained supernatant was used in the CL-ELISA.

Pretreatment using methanol. Methanol was added to the

defatted milk sample to 25% concentration and left for 20 min

stirring at room temperature. The precipitated proteins were

discarded by centrifugation at 2660 � g for 10 min at 15 �C. Theprotein-depleted milk samples were used in the analysis.

Preparation of spiked milk samples

Whole cow milk samples were mixed with a stock solution of

DEX (1 mg mL�1 in ethanol). Using each milk sample, the spiked

solutions with DEX concentrations equal to 2, 8 and 16 ng mL�1

were prepared. Prior to the analysis the spiked milk samples were

centrifuged and treated with 25% methanol as described above.

Data analysis

Standards and samples were run in triplicate, and the mean

values were processed. Standard curves were obtained by plot-

ting the light intensity against the logarithm of the analyte

concentration and fitted to a four-parameter logistic equation

using the Origin 6.0 Professional software (OriginLab Corp.,

United States):

Y ¼ {(A � D) O (1 + (x/C)B) + D}

where A is the asymptotic maximum (intensity in the absence of

an analyte, Amax), B is the curve slope at the inflection point, C is

the x value at the inflection point, and D is the asymptotic

minimum (Amin, background signal).

Results and discussion

Choice of the optimal concentrations of coating antigen and

specific antibody

While performing an indirect competitive ELISA (Fig. 2), the

sensitivity of the assay depends upon the concentration of

coating antigen and the specific antibody. Accordingly, varying

the concentrations of DEX-OVA (coating antigen) and anti-

Fig. 2 Scheme of indirect competitive CL-ELISA for determination of

dexamethasone used in this work.

2552 | Anal. Methods, 2012, 4, 2550–2554

DEX-Ab (specific antibody), a set of calibration curves for DEX

determination was obtained. All curves had a form typical of a

competitive ELISA (data not shown). The values of IC10, IC50,

working range (IC20–IC80), the highest analytical signal (Amax)

and the ratio of Amax to Amin (Amax/Amin) were selected as the

parameters used for estimation of the assay efficiency.

As seen in Table 1, the values of parameters such as IC10, IC50

and IC20–IC80 were similar to all used combinations of concen-

trations of DEX-OVA and anti-DEX-Ab. The exception was the

combination of DEX-OVA and anti-DEX-Ab with dilutions of

1 : 16 000 and 1 : 16 000, respectively, where the values of the

used parameters were higher. Thus, choosing the optimum

concentrations of the coating antigen and the specific antibody

we focused on such parameters as Amax and Amax/Amin. The

highest Amax/Amin value, and, hence, the maximum sensitivity of

the assay, was obtained for the combination of DEX-OVA and

anti-DEX-Ab with dilutions of 1 : 8000 and 1 : 16 000, respec-

tively. Moreover, in this case the Amax was also high. Therefore,

the solutions of DEX-OVA and anti-DEX-Ab with dilutions of

1 : 8000 and 1 : 16 000, respectively were selected as optimal

ones. Under these conditions the values of IC10, IC50 and IC20–

IC80 were 0.02, 0.5 and 0.08–3.4 ng mL�1, respectively (Fig. 3,

curve 1). Thus, the developed DEX CL-ELISA had a low

detection limit and high sensitivity.

The analytical characteristics mentioned above were obtained

when the competitive reaction was carried out in the presence of

Tween 20. The use of detergents such as Tween 20 and Triton

X100 in ELISA, which are applied to prevent nonspecific inter-

actions, is a traditional practice in immunoassays.29,30 However,

if the competitive reaction in the DEX CL-ELISA was carried

out in buffer, which did not contain Tween 20, the values of IC10,

IC50 and IC20–IC80 were 0.02, 0.9, 0.08–9.3 ng mL�1 (Fig. 3,

curve 2), respectively. The effect of Tween 20 on analytical

parameters of competitive ELISA has also been reported previ-

ously.27,31 Therefore, a depletion of Tween 20 from the buffer

solution used in the competitive step of the assay allowed to

expand the working range, preserving the detection limit value.

Comparison of the detection limits of the developed assay

(0.02 ng mL�1) and ELISAs reported previously (0.15 ng

mL�1(ref. 32) and 3.1 ng mL�1(ref. 33)) showed that of the use of

the chemiluminescent detection based on SbP–SPTZ–MORPH

allowed for a significant increase in the sensitivity of ELISA for

determination of DEX.

Pretreatment of milk samples

To measure DEX concentration in cow milk we adapted the

assay developed for determination of the analyte in buffer

samples. Whole milk is a colloidal system of complex composi-

tion and consists of many hydrophilic and hydrophobic

compounds. It is well known from the literature that analytes can

not be determined in samples of whole milk due to its high matrix

effect.34,35 The results of our measurement of DEX in whole milk

by CL-ELISA confirmed this (data not shown).

One of the interfering factors for the determination of analytes

in milk by enzyme immunoassay is milk fat.36–38 Comparison of

curves 2 and 3 (Fig. 3) showed that a removal of fat from whole

milk samples by centrifugation did not prevent the matrix effect.

This journal is ª The Royal Society of Chemistry 2012

Table 1 Determination of the optimal concentrations of coating antigen (DEX-OVA) and specific anti-DEX-Ab for determination of DEX by CL-ELISAa

DEX-OVA, dilutionb Anti-DEX-Ab, dilutionb Amax Amax : Amin

IC10,ng mL�1 IC50, ng mL�1

IC20–IC80,ng mL�1

1 : 8000 1 : 16 000 94 000 128 0.02 0.5 0.08–3.41 : 16 000 1 : 4000 221 100 67 0.035 0.5 0.09–2.8

1 : 8000 127 460 80 0.03 0.5 0.08–2.91 : 16 000 68 400 119 0.045 0.9 0.13–6.7

1 : 32 000 1 : 8000 76 400 80 0.02 0.5 0.07–3.4

a In the competitive step the reaction solution contained 50 mL of anti-DEX-Ab in PBS and 50 mL of DEX in PBS with 0.1% Tween 20, 0.2% casein and25% methanol. b The values corresponded to the dilutions of DEX-OVA and anti-DEX-Ab in the reaction solution.

Fig. 3 Determination of dexamethasone in milk samples by CL-ELISA.

In the competitive step the reaction solution contained (1) 50 mL of anti-

DEX-Ab in PBS and 50 mL of DEX in PBS with 0.1% Tween 20, 0.2%

casein and 25%methanol, (2) 50 mL of anti-DEX-Ab in PBS and 50 mL of

DEX in PBS with 0.2% casein and 25%methanol, (3) 50 mL of anti-DEX-

Ab in PBS with 0.2% casein and 25% methanol and 50 mL of DEX in

defatted milk, (4) 50 mL of anti-DEX-Ab in PBS with 0.2% casein and 50

mL of DEX in defatted and protein-depleted milk sample containing 25%

methanol. The dilution of anti-DEX-Ab in each well was 1 : 16 000.

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This led us to the development of a more sophisticated method

for milk sample pretreatment.

We applied three different reagents to precipitate proteins in

defatted milk: Carrez solution, trichloroacetic acid and meth-

anol. The pretreatment of milk samples using these reagents has

been reported previously.31,39,40 To choose the most appropriate

Table 2 Recovery of DEX from spiked milk samples using CL-ELISA

Milk samples

Concentration of DEX in milk samples

Spiked, ngmL�1 Measured by CL-ELI

No. 1 2.0 2.84 � 0.088.0 6.56 � 0.6516.0 20.52 � 0.64

No. 2 2.0 2.75 � 0.068.0 7.16 � 0.4816.0 20.20 � 1.12

a These data are the mean � SD.

This journal is ª The Royal Society of Chemistry 2012

pretreatment method, milk samples with different concentrations

of DEXwere defatted and then treated by the denaturants. In the

obtained samples the DEX concentration was estimated by the

CL-ELISA. Unfortunately, the calibration curves obtained for

the samples treated by Carrez solution and trichloroacetic acid

had a low precision (CVs were 20% and 50%, respectively) and,

hence, these reagents were not used in further work. The reasons

for this fact are not yet clear. It is possible that trichloroacetic

acid and components of Carrez solution remaining in milk

samples after pretreatment can interact strongly with the coating

antigen and its complex with anti-DEX antibody and are not

removed during washing steps. In turn, these compounds can

affect the oxidation of luminol and the formation of light. High

sensitivity of luminol oxidation to the addition of different

compounds is a well known fact.41–43

Contrary to Carrez solution and trichloroacetic acid, while

using methanol (25% v/v) to precipitate milk proteins, the

precision of the DEX concentrations values measured by CL-

ELISA was good (CVs were less than 14%). Moreover, as seen in

Fig. 3 (curves 2 and 4), the calibration curves obtained for DEX

in the buffer without Tween 20 and in the defatted milk treated

with methanol were similar. Therefore, the pretreatment of

whole milk samples by centrifugation and 25% methanol

prevents the matrix effect of cow milk on the performance of

CL-ELISA.

Analysis of spiked milk samples with CL-ELISA

The ability to test real samples using the developed CL-ELISA

was estimated using DEX-spiked whole milk samples. The

concentrations of DEX in the spiked samples were 2, 8 and 16 ng

Recovery, % CV, %SAa, ng mL�1 (n ¼ 3)

142.0 � 4.0 2.882.0 � 8.1 9.9128.3 � 4.0 3.1137.5 � 3.0 2.289.5 � 6.0 6.7126.3 � 7.0 5.5

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mL�1 (1.9, 7.6 and 15.2 mg kg�1). The results of the determination

of DEX concentration in milk samples are summarized in Table

2. The coefficient of variation (CV) and recovery values were in

the range of 2.2 to 9.9% and 82 to 142%, respectively. These

results allowed us to conclude that the developed CL-ELISA

permits the correct measurement of DEX in whole milk samples.

Conclusions

In this work we have developed a sensitive CL-ELISA for the

determination of DEX in buffer solution. The high sensitivity

of the assay was achieved by using a chemiluminescence

method to measure the enzyme activity of SbP in the presence

of SPTZ and MORPH (enhancers). Analytical parameters such

as IC10, IC50 and IC20–IC80 for the calibration curve of DEX

determined by CL-ELISA were 0.02, 0.9, 0.08–9.3 ng mL�1,

respectively. A combination of defatting of milk and precipi-

tation of proteins by methanol allowed prevention of the

matrix effect and, hence, to correctly evaluate DEX content in

whole milk samples by the developed assay. Therefore, the

developed CL-ELISA could provide a valuable tool for sensi-

tive determination of DEX in milk.

Acknowledgements

The authors thank the Russian Foundation for Basic Research

for financial support (11-04-92005-NNS_a) and Dr O.S. Soko-

lova (Biology Faculty, MSU, Russia) for providing the

luminometer.

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