Food Chemistry 158 (2014) 310–314

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Development of ultrasensitive direct chemiluminescent enzymeimmunoassay for determination of aflatoxin M1 in milk

http://dx.doi.org/10.1016/j.foodchem.2014.02.1280308-8146/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: AFM1, aflatoxin M1; HRP, horseradish peroxidase; ELISA,enzyme-linked immunosorbent assay; CL, chemiluminescence; SPTZ, 3-(100-phe-nothiazinyl)-propane-1-sulfonate; MORP, 4-morpholinopyridine; ECR, enhancedchemuliminescence reaction; RLU, relative luminescence units; CV, coefficient ofvariation.⇑ Corresponding author. Address: School of Biomedical Sciences, Chung Shan

Medical University, No. 110, Sec. 1, Chien Kuo N. Road, Taichung, Taiwan. Tel.: +8864 24730022x11816; fax: +886 4 23248187.

E-mail address: fengyu@csmu.edu.tw (F.-Y. Yu).

Marina M. Vdovenko a, Chuan-Chen Lu b, Feng-Yih Yu b,c,⇑, Ivan Yu. Sakharov a

a Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russiab School of Biomedical Sciences, Chung Shan Medical University, Taichung 402, Taiwanc Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan

a r t i c l e i n f o

Article history:Received 18 January 2013Received in revised form 3 September 2013Accepted 23 February 2014Available online 6 March 2014

Keywords:Aflatoxin M1MilkEnzyme immunoassayChemiluminescencePeroxidaseEnhancement

a b s t r a c t

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detectingaflatoxin M1 (AFM1) was developed. To improve the sensitivity of the assay, a mixture of 3-(100-pheno-thiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used to enhance perox-idase-induced CL. The concentrations of the coating anti-AFM1 antibody and the conjugate of AFB1 withhorseradish peroxidase the conditions of the chemiluminescent assay were varied to optimise the condi-tion of the chemiluminescent assay. The lower detection limit values and dynamic working range of CL-ELISA of AFM1 were 0.001 ng mL�1 and 0.002–0.0075 ng mL�1, respectively. A 20-fold dilution of milksamples prevented a matrix effect of the milk and allowed measurement of AFM1 at concentrationsbelow than the maximum acceptable limit. Values of recovery within and between assays were 81.5–117.6% and 86–110.6%, respectively. The results of using the developed CL-ELISA to analyse samples ofsix brands of milk that were purchased in Taiwan revealed that AFM1 was absent from all studiedsamples.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Aflatoxins are highly toxic, mutagenic, carcinogenic, and terato-genic compounds (Betina, 1989; Krska et al., 2008). Aflatoxin M1(AFM1) is a major hydroxylated metabolite of aflatoxin B1(AFB1), which exhibits the highest toxicity among all aflatoxins.AFM1 is found in the milk and blood of animals that ingest AFB-containing feed (Battacone, Nudda, Palomba, Mazzette, & Pulina,2009; Sabino, Purchio, & Milanez, 1995). The consumption of milkand milk products by humans, particularly by children is quite highresulting in a potential a risk of exposure to AFM1. Evidence of haz-ardous human exposure to AFM1 through dairy products has beenreported (Galvano et al., 2001; Govaris, Roussi, Koidis, & Botsoglou,2002; Polan, Hayes, & Campbell, 1974; van Egmond, 1989).

According to the United States Food and Drug Administration(US-FDA), the concentration of AFM1 in milk should not exceed0.5 ng mL�1 (Wood, 1992). More stringent restrictions of the levelof AFM1 in milk for adult consumption have been set by the Euro-pean Union (0.05 ng mL�1) (Commission regulation, 2004). Inbaby-food products this level should not exceed 0.025 ng mL�1.AFM1 is frequently present in commercial milk samples and dairyproducts, and various milk samples have been found to containAFM1 levels greater than the maximum acceptable limit. In Indiaalmost 99% of contaminated milk samples contained more thanthe maximum legal level set by EU regulations and 9% of samplesexceeded the limit set by the USFDA (Rastogi, Dwivedi, Khanna, &Das, 2004).

Analytical methods that combine simplicity, a high detectionsensitivity and a high analytical throughput are required for theeffective screening and monitoring of AFM1 in foodstuffs at pptlevels. High-performance liquid chromatography (HPLC) with afluorescent detector and enzyme-linked immunosorbent assay(ELISA) are generally used in routine analysis (Shephard et al.,2012; Stroka & Anklam, 2002; van Egmond, 2004). Notably, HPLCis a complex and time-consuming method to imolement and it re-quires costly and bulky instrumentation. ELISA has none of theseshortcomings. ELISA is widely accepted as the ‘‘gold standard’’

M.M. Vdovenko et al. / Food Chemistry 158 (2014) 310–314 311

screening method (Ediage, Di Mavungu, Goryacheva, van Peteg-hem, & De Saeger, 2012).

Among practical of ELISA formats, the most sensitive is an assayin which the enzyme activity of peroxidase-labelled immunorea-gents is determined by an enhanced chemiluminescent (CL) reac-tion (ECR) (Fan, Cao, Li, Kai, & Lu, 2009; Marquette & Blum, 2009;Roda & Guardigli, 2012). This reaction principle is based on the per-oxidase-catalysed oxidation of luminol by hydrogen peroxide inthe presence of enhancers. Some CL-ELISAs for the determinationof the concentration AFM1 in milk have already been reported(Kanungo, Pal, & Bhand, 2011; Magliulo et al., 2005).

Recently, a mixture of 3-(100-phenothiazinyl)-propane-1-sulfo-nate (SPTZ) in combination with 4-morpholinopyridine (MORPH)has been demonstrated to be the most efficient mixture of enhanc-ers for peroxidise-induced CL (Marzocchi et al., 2008; Vdovenko,Della Ciana, & Sakharov, 2009; Vdovenko, Demiyanova, Chemleva,& Sakharov, 2012). Under optimised conditions, the ratio of the CLintensity generated in horseradish peroxidase (HRP)-induced ECRto its background value was higher than 140,000 (Vdovenkoet al., 2012). The application of these enhancers provides perspec-tives on the construction of sensitive CL-ELISA kits. The presentwork describes the ultrasensitive direct competitive CL-ELISA fordetermining the concentration of AFM1. To increase the sensitivityof the assay, the CL method was utilised to determine HRP activityusing SPTZ and MORPH as enhancers. The developed CL-ELISA wassuccessfully applied to determine the concentration of AFM1 inmilk samples.

2. Material and methods

2.1. Chemicals and reagents

Aflatoxin M1 (AFM1) and aflatoxin B1 (AFB1) were purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). AFM1 analyticalstandard solution (10 lg/ml), Certified Reference Material – fromSupelco (Bellefonte, PA, USA). Horseradish peroxidase (HRP, RZ3.0) was purchased from Roche (Mannheim, Germany) and usedwithout further purification. Sodium 3-(100-phenothiazinyl)-pro-pane-1-sulfonate (SPTZ) was prepared as described by (Marzocchiet al., 2008). Luminol, Tween 20, Tris, O-(carboxymethyl)hydroxyl-amine hemihydrochloride (CMO) and 4-morpholinopyridine(MORP) were from Aldrich (St. Louis, MO, USA). Black polystyreneplates (high protein binding) were obtained from Nunc (Roskilde,Denmark). 1-Ethyl-3-(30-dime-thylaminopropyl) carbodiimide(EDC), N-hydroxysuccinimide (NHS), and bovine serum albumin(BSA) from Sigma Chemical Co. (St. Louis, MO, USA), N-dimethyl-formamide (DMF) and H2O2 (30%) were from J. T. Baker (Phillips-burg, NJ, USA). The concentration of H2O2 was estimated bymeasuring the absorbance using e240 = 43.6 (Kulmacz, 1986).

The polyclonal antibodies specific to AFM1 (anti-AFM1-pAb)were produced by subcutaneous immunisation of rabbits with aAFM1-CMO-BSA conjugate as described by Wang, Liu, Hsu, andYu (2011). The purification of anti-AFM1-pAb was carried as fol-lows: at first step saturated (NH4)2SO4 was added to the rabbitantiserum to a 35% saturation. After incubation for 1 h and centri-fugation (8000 rpm for 30 min) the precipitated proteins were dis-carded, and the additional quantity of (NH4)2SO4 solution wasadded to a 50% saturation. The precipitated anti-AFM1-pAb wascentrifuged and then redissolved in distilled water. The volumeof the used water was equal to half of the original volume of anti-serum. The anti-AFM1-pAb was dialyzed against 2 L of 0.01 Mphosphate buffer with 0.15 M NaCl, pH 7.5 (PBS) for 72 h at 4 �Cwith two changes of the buffer. Finally, PBS was added to the ob-tained anti-AFM1-pAb up to the original antiserum volume. Theantibody sample was stored at �20 �C or lyophilized for future use.

2.2. Preparation of aflatoxin B1-CMO

The synthesis of AFB1-CMO was carried out as described byChu, Hsia, and Sun (1977). For this, 10 mg AFB1 and 15 mg CMOwere dissolved in a mixture containing 1.0 mL pyridine, 4.0 mLmethanol and 1.0 mL water. After the reaction completion the mix-ture was gently refluxed for 2.5 h with continuous magnetic stir-ring, it kept at room temperature overnight. Using a rotaryevaporator the reaction mixture was concentrated up to �1 mL.To purify AFB1-CMO a thin-liquid chromatography (TLC) was car-ried out on silica gel plates using chloroform:methanol (9:1) in1.5% acetic acid as an eluent. Localization of AFB1-CMO spot onthe plate was detected under UV light (365 nm). Then, AFB1-CMO was removed from the TLC plate and dissolved in chloroform.Finally, AFB1-CMO was dried in the open air.

2.3. Synthesis of aflatoxin B1-HRP conjugate

AFB1 was conjugated with HRP by a carbodiimide method asfollows: 1.0 mg of EDC freshly dissolved in 0.01 mL of DMF and0.8 mg of NHS in 0.01 ml DMF were added to 0.1 mL of AFB1-CMO solution (0.25 mg mL�1 of DMF). The mixture was kept atroom temperature for 2 h with continuous stirring. Then, 1.5 mgof HRP in 1.0 mL of 0.1 M NaHCO3, pH 8.3 was added to theAFB1-CMO solution dropwise, and the reaction solution was keptat room temperature for the next 2 h with stirring. The obtainedconjugate was dialyzed against 2 L of PBS for 72 h with twochanges of the buffer and stored at �20 �C or lyophilized for futureuse.

2.4. Determination of AFM1 by CL-ELISA

CL-ELISA was carried out using 96-wells black polystyreneplates (MaxiSorp, Nunc, Roskilde, Denmark). The plates werecoated by adding into each well 100 lL of anti-AFM1-pAb (dilution1:40,000) dissolved in PBS, and incubated at 4 �C overnight. Theplate was then washed using PBS with 0.05% Tween 20 (PBST) fourtimes (ELx 50 ELISA washer from Bio-Tek instruments, USA) andblocked by adding 170 lL of PBS containing 0.1% BSA for 30 minat 37 �C. The plate was washed four times with PBST. Subsequently,50 lL of AFB1-HRP (dilution 1:20,000) in 10 mM PBS, pH 7.4 and50 lL of AFM1 (0.00002–0.2 ng mL�1) or milk sample in dilution1/10 were added to each well. The competitive step of the assayproceeded for 1 h at 37 �C. The plates were washed again as de-scribed above. Finally, 100 lL of freshly prepared substrate solu-tion (80 mM Tris, pH 8.3, containing 0.17 mM luminol, 2.1 mMSPTZ, 8.75 mM MORP, and 1.75 mM H2O2) (Vdovenko et al.,2012) were added to each well and stirred. Chemiluminescenceintensity was monitored after incubation for 5 min at room tem-perature on a Vmax automatic ELISA reader (FlexStation 3, Molec-ular Devices).

2.5. Preparation of spiked samples

Milk samples were purchased from Taiwanese stores. Each sam-ple (10 ml) was centrifuged twice at 4 �C at 19,500 g for 5 min.Prior to ELISA the free-fat milk was diluted 10 times using PBS.

2.6. Data analysis

Standards and samples were run in triplicates, and the meanvalues were processed. Standard curves were obtained by plottingthe light intensity against the logarithm of the analyte concentra-tion and fitted to a four-parameter logistic equation using theOrigin 6.0 Professional software (OriginLab Corp., United States):

312 M.M. Vdovenko et al. / Food Chemistry 158 (2014) 310–314

Y ¼ fðA� DÞ � ð1þ ðx=CÞBÞg þ D;

where A is the asymptotic maximum (intensity in the absence of ananalyte, Imax), B is the curve slope at the inflection point, C is the xvalue at the inflection point, and D is the asymptotic minimum (Imin,background signal).

3. Results and discussion

3.1. Optimal concentrations of coating antibody and conjugate AFB1-HRP

An assay for determining the concentration of AFM1 in milkwas developed on the basis of a competitive CL-ELISA. A schemeof the direct CL-ELISA was presented in Fig. 1. The polyclonalanti-AFM1 antibody that was used in this work was produced byimmunising a rabbit with the AFM1-BSA conjugate. As reportedpreviously (Wang et al., 2011), an affinity of this antibody towardsAFM1 is similar to that toward AFB1. Accordingly, in the develop-ment of the assay, the AFB1-HRP conjugate was used instead of theAFM1-HRP conjugate to reduce cost without worsening the analyt-ical characteristics of the assay, as AFB1 is significantly less expen-sive than AFM1. The ability of the anti-AFM1 antibody to react withAFB1 is not a drawback of the method, as its purpose is to estimatethe AFM1 content in milk samples that do not contain AFB1 as re-ported previously (Battacone et al., 2009; Sabino et al., 1995).

The sensitivity of competitive ELISA depends on the concentra-tions of the capture antibody and the enzyme-labelled antigen.Therefore, a set of calibration curves for determining the concen-trations of AFM1 were obtained by varying the concentrations ofpolyclonal antibodies and HRP conjugates. All calibration curveswere of a form that was typical of competitive ELISA (data notshown). The values of IC10, IC50, dynamic working range (IC20–IC80) as well as background (Imin) were selected as the parametersused in estimating the efficiency of the assay.

As seen in Table 1, the values of background were low for allused combinations of concentrations of anti-AFM1-pAb andAFB1-HRP. When many diluted solutions were used (combination5) the analytical parameters were not calculated because of highCV values. A comparison with the other combinations revealed thatthe most sensitive CL-ELISAs were obtained using combinations 1and 2. For subsequent work, combination 2 (1:40,000/1:40,000)was chosen as optimal, because it was associated with the con-sumption of half as much AFB1-HRP consumption as other combi-nations. The lower detection limit value (LDL) equal to IC10, IC50,and the working (linear) range (IC20–IC80) of the developed methodwere 0.001, 0.035 and 0.002–0.0075 ng mL�1, respectively. Thecoefficient of variation (CV) for determining AFM1 concentrationswithin the working range of the assay was 4–13% (n = 6).

The characteristics of the developed assay, in which the enzymeactivity was measured using ECR with SPTZ/MORPH as enhancers,were compared with those of colorimetric ELISA (COL-ELISA) of

Fig. 1. Scheme of direct competitive CL-EL

AFM1 as described previously (Wang et al., 2011). Both assayswere developed using the same immunochemical reagents, butthe principles of detection of HRP activity differed. As shown inTable 2, the obtained results demonstrated that the LDL of CL-ELI-SA is 2 times lower than that of the COL-ELISA. The additionaladvantage of the CL-ELISA is its broader working range. Also, thereplacement of the colorimetric method of measuring HRP activitywith the chemiluminescent method shortened the time of suchmeasurement and also reduced 3-folds of consumption of anti-AFM1 antibody.

3.2. Analysis of spiked and real samples

To demonstrate the practicality of the proposed CL-ELISA, AFM1concentration in spiked milk samples was measured. Various levelsof AFM1 (0.002–0.006 ng mL�1) were added to an AFM1–free milksample which was diluted to varying degrees using PBS; then, theAFM1 concentration in the obtained solutions was measured bythe CL-ELISA. When the dilution of the spiked sample was 1:4,the recovery and coefficient of variation (CV) were in range 123–180% and 9.4–35%, respectively. The obtained results indicated thatunder the assay conditions, the milk exhibited a strong matrixeffect.

One of the approaches that is widely used to prevent the matrixeffect is the dilution of test samples. Accordingly, prior to the CL-ELISA, spiked milk sample was diluted by a factor of 10 and 20times. The sample with 10-fold dilution yielded unsatisfactoryrecovery and CV (Table 3), whereas the 20-fold dilution yieldedrecovery values of 92–100%.

Analysis of 3 spiked milk samples with 20-fold dilution (Table 4)by the assay showed recovery values in the range of 82–118% andCVs (n = 4) that did not exceed 11%. Also, the values of recovery be-tween assays obtained at the performance of the assay day by day(n = 4) were in range of 86–110.6% with CVs less than 7.7%. There-fore, the matrix effect of milk was prevented by 20-fold dilution ofthe milk sample.

Based on the fact that in the CL-ELISA the dilution of milk sam-ples should be 1:20, a minimum concentration of AFM1 that maybe measured in real milk samples was calculated. The lowest valueof the working range of the assay in buffered solution(0.002 ng mL�1) should be multiplied by the dilution factor (20)yielding 0.04 ng mL�1 which was lower than the maximum accept-able limits that are set in both the USA and the European Union.The obtained results demonstrate that the sensitivity and precisionof the developed CL-ELISA were suitable for quantifying AFM1 inmilk samples.

The developed assay was used to analyse 6 samples of milk thatpurchased in stores in Taiwan. Our results demonstrated thatAFM1 was absent from all studied samples. This finding suggeststhat milk products that are sold in Taiwan are generally safe forconsumers.

ISA for determination of aflatoxin M1.

Table 1Optimization of experimental conditions in competitive step of CL-ELISA for determination of AFM1 in buffered solutions.

No. Anti-AFM1-pAb /AFB1-HRP Background (Imin) IC10 (ng mL�1) IC50 (ng mL�1) IC20–IC80 (ng mL�1)

1 1:40,000/1:20,000 30 0.001 0.005 0.002–0.00952 1:40,000/1:40,000 30 0.001 0.0035 0.002–0.00753 1:40,000/1:60,000 20 0.002 0.007 0.004–0.0154 1:20,000/1:40,000 20 0.003 0.009 0.008–0.0705 1:60,000/1:40,000 20 ND ND ND

ND–not determined.

Table 2Comparison of analytical parameters of ELISAs with colorimetric and chemiluminescent detections used for the determination of AFM1.

CL-ELISA COL-ELISA

IC10, ng mL�1 0.001 0.002IC50, ng mL�1 0.0035 0.014IC20–IC80, ng mL�1 0.002–0.0075 0.006–0.06Time of substrate reaction, min 5 10Concentration of anti-AFM1-pAb used in the coating step, lg/ml 0.34 1

Table 3Recovery and CVs of AFM1 from spiked milk samples.

[Spiked AFM1] (ng mL�1) Dilutions of milk sample

1/4 (n = 3) 1/10 (n = 3) 1/20 (n = 3)

Recovery (%) CV (%) Recovery (%) CV (%) Recovery (%) CV (%)

0.002 160 ± 15 9.4 139 ± 13 9.4 100 ± 1 1.00.004 180 ± 29 16.1 170 ± 27 15.9 96 ± 1 1.00.006 123 ± 43 35.0 143 ± 50 35.0 92 ± 3 3.3

Table 4Analysis of milk samples spiked with 3 different concentrations of AFM1 within the working range by CL-ELISA.

Milk sample Expected (ng mL�1) Within assayb (n = 4) Between assayc (n = 4)

Founda (ng mL�1) CV (%) Recovery (%) Founda (ng mL�1) CV (%) Recovery (%)

1 0.04 0.04 ± 0.0004;0.039 ± 0.004;

0.038 ± 0.003;0.043 ± 0.002

0.5–9.6 95.2–107.9 0.040 ± 0.002 5.5 100.2 ± 5.5

0.08 0.077 ± 0.0003;0.08 ± 0.002;

0.071 ± 0.006;0.073 ± 0.003

0.4–8.0 89.0–99.9 0.075 ± 0.004 5.3 93.9 ± 5.0

0.12 0.11 ± 0.003;0.098 ± 0.008;

0.105 ± 0.01;0.10 ± 0.003

2.8–11 81.5–92 0.103 ± 0.006 5.4 86.0 ± 4.6

2 0.04 0.04 ± 0.002;0.044 ± 0.004;

0.038 ± 0.003;0.044 ± 0.003

4.7–8.3 94.4–110.3 0.041 ± 0.003 7.7 103.4 ± 8.0

0.08 0.083 ± 0.004;0.083 ± 0.006;

0.094 ± 0.009;0.094 ± 0.006

4.2–9.7 103.3–117.6 0.089 ± 0.007 7.3 110.6 ± 8.1

0.12 0.12 ± 0.01;0.134 ± 0.013;

0.118 ± 0.006;0.114 ± 0.007

4.9–9.8 94.6–111.6 0.121 ± 0.009 7.3 101.0 ± 7.4

0.04 0.039 ± 0.003;0.044 ± 0.0002;

0.038 ± 0.004;0.039 ± 0.002

0.5–9.7 94.2–110.4 0.04 ± 0.003 7.2 99.8 ± 7.2

3 0.08 0.085 ± 0.008;0.083 ± 0.001;

0.084 ± 0.007;0.077 ± 0.005

1.6–8.8 95.8–105.8 0.08 ± 0.004 4.4 102.3 ± 4.5

0.12 0.116 ± 0.007;0.109 ± 0.006;

0.123 ± 0.01;0.113 ± 0.006

5.1–8.4 90.6–102.9 0.115 ± 0.006 5.4 96.0 ± 5.2

a The report data are the mean ± SD.b The assays are carried out in four replicates on the same day.c The assays are carried out in four different days.

M.M. Vdovenko et al. / Food Chemistry 158 (2014) 310–314 313

4. Conclusion

This work developed ultra-sensitive CL-ELISA for determiningthe concentration of AFM1 in a buffer solution. The high sensitivityof the assay was achieved using the chemiluminescent method formeasuring HRP activity in the presence of SPTZ and MORPH(enhancers). The values of LDL and the dynamic working range ofthe CL-ELISA of AFM1 were 0.001 ng mL�1 and 0.002–0.0075 ng mL�1, respectively. Twenty-fold dilution of the milk

samples completely prevented the matrix effect. The values ofrecovery within and between assays were 81.5–117.6% and86–110.6%, respectively. Therefore, the developed CL-ELISA is avaluable tool for the routine quality control of milk.

Acknowledgements

The authors thank the Russian Foundation for Basic Research(11-04-92005-NNS_a) and the National Science Council of the

314 M.M. Vdovenko et al. / Food Chemistry 158 (2014) 310–314

Republic of China (Taiwan) for Taiwan-Russia Cooperation GrantNSC-100-2923-B-040-001-MY2 for financial support.

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