Determination of Amaranth in Beverage by IndirectCompetitive Enzyme-linked Immunosorbent Assay (ELISA)Based on Anti-amaranth Monoclonal Antibody

Bo Zhang & Daolin Du & Meng Meng & Sergei A. Eremin &

Victor B. Rybakov & Junhong Zhao & Yongmei Yin &

Rimo Xi

Received: 16 September 2013 /Accepted: 28 November 2013# Springer Science+Business Media New York 2013

Abstract Amaranth (E 123) is a member of azo dyes, and it isallowed to use in various foods. The acceptable maximumaddition of amaranth is strictly fixed because of its potentialrisk to physical health. The objective of this study was toprepare a specific anti-amaranth monoclonal antibody anddevelop an indirect competitive ELISA for amaranth quanti-fication analysis. The immunogen and the coating antigenwere designed by introducing a carboxyl group into amaranthfor the conjugation with carrier proteins. Based on the immu-nogen, the monoclonal antibody exhibits satisfactory perfor-mances and the proposed ELISA shows an IC50 of20.33 ng mL−1. The limit of detection is as low as

3.35 ng mL−1, and the linear standard curve of the methodranges from 3.0 to 243.0 ng mL−1. Additionally, the antibodyreflects minimal cross-reactivity (<1 %) with six related fooddyes (erythrosine, ponceau 4R, allura red, tartrazine, sunsetyellow FCF, and brilliant blue). The recoveries of amaranthspiked beverage samples are in the range of 85.8–100.7 %with low coefficient of variation values (<11.5 %). The datashows that the developed ELISA provides a simple, sensitive,specific, and accurate alternative for amaranth determinationand monitoring. Furthermore, it is the first time that icELISAof amaranth is developed based on monoclonal antibodies.

Keywords Amaranth . Azo dye .Monoclonal antibody .

ELISA

Introduction

Synthetic azo dyes are widely used for color enhancement ofpractically all types of produced foods because of their lowprice, high effectiveness, and excellent stability. Amaranth(sodium 3-hydroxy-4-((4 ′-sulfonatonaphthalen-1 ′-yl)diazenyl)naphthalene-2,7-disulfonate; E123), a class ofazo dyes, possesses great solubility in water and good stabilitytowards light and heat. It provides an easy and economicalway to improve the esthetic quality of foods. Toxicologystudies of amaranth began in the 20th century, and it wasconsidered potentially dangerous (Clode et al. 1987; Hashemet al. 2010; Holmberg 1978; Sarıkaya et al. 2012). The azoreduction product-aromatic amine derivatives, naphthionicacid, for example (Willes et al. 1980), are considered as thesource of the toxicity. So the using amaranth is restricted inmost countries even though its negative impact on the human

Bo Zhang and Daolin Du contributed equally to this work.

M. Meng :Y. Yin (*) :R. Xi (*)State Key Laboratory of Medicinal Chemical Biologyand College of Pharmacy, Nankai University, Tianjin 300071, Chinae-mail: yinyongmei@nankai.edu.cne-mail: xirimo@nankai.edu.cn

M. Meng :Y. Yin :R. XiTianjin Key Laboratory of Molecular Drug Research, NankaiUniversity, Tianjin 300071, China

B. Zhang :D. DuSchool of Environment, Jiangsu University, Zhenjiang 212013,China

B. ZhangTianjin Bioradar Biotechnology Co. Ltd., Tianjin 300457, China

S. A. Eremin :V. B. RybakovFaculty of Chemistry, M.V. Lomonosov Moscow State University,Moscow 119991, Russia

J. ZhaoTianjin Sungene Biotech Co., Ltd., Tianjin 300450, China

Food Anal. MethodsDOI 10.1007/s12161-013-9779-1

body has not been proven yet. In China, applying amaranth inthe food industry is constrained to frozen drinks, jam, sodas,beverage, cut-wine, integrated alcoholic beverages, jelly, andsyrup, and the maximum permissible amount of amaranth inthese foods ranges from 0.025 to 0.3 g kg−1.

Several literatures have described the analysis of amaranthand its separation from other food dyes. These include capil-lary electrophoresis (Berzas Nevado et al. 1999; Perez-Urquiza and Beltran 2000; Suzuki et al. 1994) and high-performance ion chromatography (Chen et al. 1998). High-performance liquid chromatography is also commonly used inamaranth detection, with the aid of liquid phase micro-extraction (Wu et al. 2013) or various detectors, includingdiode-array detector (DAD) (Culzoni et al. 2009; Miniotiet al. 2007; Stefania et al. 2013), UV-DAD (Alves et al.2008), and ion-pair liquid chromatography (LC) withphotodiode-array mass spectrometry (MS) (Fuh and Chia2002). With these methods, food dyes achieved great separa-tion and satisfactory quantitative detection. However, all ofthe existent methods require expensive apparatus as well ashighly skilled operators. The task of rapidly identifyingamaranth-contained foods in resource-constrained environ-ments remains a challenge.

The objective of this study was to develop a rapid andsensitive enzyme-linked immunosorbent assay (ELISA) foramaranth. Compared with the existed instrumental methods,ELISA exhibits the advantages of low cost, speediness, sim-ple, and convenience, which is especially fitting for field test.For this purpose, the immunogen of amaranth was firstlysynthesized. Its monoclonal antibody (MAbs) was also orig-inally prepared, and the immunoassay of amaranth was newlydeveloped in this study. Simultaneously, the performance ofthe assay was investigated including sensitivity, specificity,and recovery in beverage.

Materials and Methods

Chemicals, Apparatus, and Buffers

Amaranth, erythrosine, ponceau 4R, allura red, tartrazine,brilliant blue, bovine serum albumin (BSA), ovalbumin(OVA), Freund’s complete adjuvant (cFA), and Freund’s in-complete adjuvant (iFA) were supplied by Sigma-Aldrich (St.Louis, MO, USA). N -hydroxysuccinimide (NHS) was fromthe Academy of Military Medical Sciences (Military MedicalInstitute, Beijing, China). 3,3′,5,5′-tetramethylbenzidine(TMB) was purchased from Shanghai Sangon BiologicalEngineering Technology (Sangon Biotech Co., Ltd., Shang-hai, China). N ,N -dimethylformamide (DMF) and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC)were obtained from TCI (Tokyo Kasei Kogyo Co., Ltd.,Tokyo, Japan).

Samples were tested by microplate reader Model 680 fromBio-Rad Laboratories Headquarters (Bio-Rad, Hercules,USA). Polystyrene microtiter plates (96-well) were from JetBio-filtration Products, Co., Ltd. (Jet Biofil, Beijing, China).UV-Vis spectra of conjugates were scanned by SHIMADZUUV-1800 (SHIMADZU, Kyoto, Japan).

Ultra-pure deionize water was used for the preparation ofall buffers and reagents: (1) phosphate-buffered saline (PBS,pH 7.4): composed of 138.0 mM NaCl, 1.5 mM KH2PO4,7.0 mM Na2HPO4, and 2.7 mM KCl; (2) washing buffer(PBST): a PBS solution containing 0.05 % (v/v) of Tween20; (3) coating buffer: 0.05 M carbonate buffer (15.0 mMNa2CO3 and 35 mM NaHCO3, pH 9.6); (4) blocking buffer:PBS mixed with 1 % of OVA and 0.05 % (v/v) Tween 20; (5)substrate buffer: 400 mL of 0.6 % TMB-DMSO mixed with100 mL of 1 %H2O2 in the citrate-acetate buffer (pH 5.5); and(6) enzymatic stopping solution: 2.0 M hydrochloric acid.

Preparation of Immunogen and Coating Antigen

Before conjugation with the hapten, the carboxylic acidgroups of the carrier proteins (BSA and OVA) need to beconverted to primary amine groups with excess ofethylenediamine to obtain the cationic BSA (cBSA) andOVA (cOVA) (Kamps-Holtzapple et al. 1993; Lu et al.2006). The immunogen of amaranth was synthesized byEDC method with proper modification (Xue et al. 2012).Briefly, amaranth (10.0 mmol) was treated with thionyl chlo-ride (2.5 mL) in anhydrous DMF (100 μL) to produce sulfo-nyl chloride, firstly. Then, the resulted amaranth-sulfonylchloride (2.0 mmol) was allowed to react with glycine(2.0 mmol) for the introduction of carboxyl group, and theresultant was used as the hapten of amaranth. After that,7.0 mL of PBS containing 0.1-mmol amaranth hapten wasmixed with 5.0 mL of PBS containing 1.0-mmol EDC and0.5-mmol NHS, which were allowed to react for 2.0 h at roomtemperature. Then the mixture was added dropwise to10.0 mL of PBS containing 0.003-mmol cBSA. The conjuga-tion mixture was stirred at 4 °C overnight to get the immuno-gen of amaranth-cBSA. The coating antigen was prepared byconjugating amaranth with cOVA according to the same pro-tocol by utilizing 0.0028-mmol cOVA instead of cBSA. Fi-nally, the solution containing immunogen or coating antigenwas dialyzed against PBS, and the resultant was lyophilizedand stored in the refrigerator (−20 °C) for further use.

Production of Anti-amaranth Monoclonal Antibody

The animal experiments were performed in compliance withthe relevant laws and institutional guidelines and conductedwith the approval of Institutional Authority for LaboratoryAnimal Care. The procedure of monoclonal antibody genera-tion was modified based on the previous literature (Xu et al.

Food Anal. Methods

2010). Briefly, 80.0-μg amaranth-cBSA in cFA was intraperi-toneally injected into BALB/c mice for the first immunization.Three more immunizations were performed at 2-week intervalsusing the same immunogen in iFA. The immune response wasvalidated by detecting the titre of the antisera by ELISA. Afterthat, fusion and cloning technology was followed. Thesplenocytes from immunized mice were fused with SP2/0myeloma cells (7:1). The cells were screened by ELISA andcloned by limited dilution method to obtain stable hybridomacell lines secreting monoclonal antibodies. Then, the hybrid-oma cells were intraperitoneally injected to mice and asceticfluid containing anti-amaranth MAbs was obtained. Finally,acid-ammonium sulfate method was applied to purify theMAbs and the target MAbs were stored at −20 °C until use.

Preparation of horseradish peroxidase (HRP)-labeledAnti-amaranth MAbs

MAbs against amaranth were modified with HRP as describedbelow. Firstly, 2.0-mg HRP was dissolved in purified waterand 0.08 mL NaIO4 (0.1 M) was added by stirring, which wasallowed to react in dark for 20 min. The mixture was dialyzedby dialysis bag with 1 mM sodium acetate buffer (pH 4.4) at4 °C for 14 h and 0.01 M bicarbonate buffer (pH 9.5) at roomtemperature for 2 h, respectively. Simultaneously, 2.0-mganti-amaranth MAbs was dialyzed in 0.01 M bicarbonatebuffer (pH 9.5) for 2 h at room temperature. Then, thepurified HRP and MAbs solution were mixed sufficientlyand reacted at room temperature for 2 h in dark. After that,freshly prepared 0.04 mL NaBH4 (4.0 mg mL−1) was addeddropwise and the reaction was allowed to continue for 2more hours. Finally, the resultants were dialyzed by 0.05 MPBS (pH 7.4) for future use.

Optimization of Antibody Concentration and CoatingAntigenDilution

The checkerboard procedure was used to optimize the prop-er dilution of the coating antigen and the MAbs of amaranth.Each polystyrene microtiter well was coated with 100 μL ofcoating antigen, which was diluted at 1/500, 1/1000, 1/2000,1/3000, and 1/4000, respectively, and then the plates wereincubated at 37 °C for 2 h. The coated plates were washedfor three times using washing buffer and blocked with250 μL/well of blocking buffer at 4 °C overnight. Afterthree washes, MAbs-HRP at different dilutions were added(1/500, 1/1000, 1/1500, 1/2000, 100 μL/well) and it wasincubated at 37 °C for 0.5 h. After three washings, thesubstrate solution was added (100 μL/well) and it wasincubated for 15 min at 37 °C. Then, the stopping buffer(100 μL/well) was used to inhibit the enzymatic action andabsorbance at 450 nm was measured by an automatic mi-croplate reader.

Indirect Competitive ELISA (icELISA) Procedure

An icELISA protocol was developed for amaranth analysis.The procedure of the proposed icELISAwas mainly similar asdescribed above. Except that after removing blocking buffer,70 μL of competitors in diluted MAbs-HRP (30 μL) or blankbuffer were added to each well successively. After absorbancemeasurement at 450 nm, the inhibition ratio was expressed asfollows: % inhibition=% B /B0, where, B is the absorbance ofthe well containing competitor, andB0 is the absorbance of theblank well. The sensitivity of the method was indicated byIC50 and limit of detection (LOD) values, which were obtain-ed from the standard curve made by plotting absorbance at450 nm versus the logarithm of amaranth concentration.

Cross-reactivity Studies

The specificity of the MAbs against amaranth was evaluatedby cross-reactivity (CR) towards six common dyes includingerythrosine, ponceau 4R, allura red, tartrazine, sunset yellowFCF, and brilliant blue (Fig. 1). In the proposed icELISA,these compounds were used as the competitor, respectively.Accordingly, the resulting IC50 values were exploited to cal-culate the CR% values by the following equation:

CR% ¼ IC50; amaranth=IC50; dyes � 100%

Recovery of Amaranth in Beverage Samples

An amaranth-free carbonated drink samples were used to con-duct the recovery of amaranth for assessment of matrix effecton the accuracy and precision of the assay. The samples weretreated by the following method. To remove the gas of thebeverage, samples were boiled firstly. Then the samples werediluted for 50 times by 50 mM Tris-buffer (pH 7.4). For therecovery study, amaranth was spiked into the beverage to makethree levels at 0.2, 1.0, and 4.0 μg mL−1. The fortified sampleswere treated as described above and analyzed by the developedicELISA and the recovery data could be obtained. In addition,the performance of the proposed icELISA in 5 amaranth-contained beverage samples was compared with that of high-performance liquid chromatography (HPLC) methods.

Results and Discussion

Synthesis of Immunogen and Coating Antigen

In order to grant the immunogenicity to small molecular dye-amaranth, it must be conjugated with certain carrier protein.Generally, carboxylic, amino- or hydroxyl group is consideredas the ideal substitute that could react with carrier protein

Food Anal. Methods

Amaranth (E 123)

Ponceau 4R (E 124) Sunset Yellow FCF (E 110)

Tartrazine (E 102) Allura Red (E 129)

Brilliant Blue (E 133) Erythrosine (E 127)Fig. 1 Structures of amaranth (E123) and related dyes evaluated in this study

Food Anal. Methods

easily. As shown in Fig. 1, there is a naphthalene hydroxylgroup in the structure of amaranth; however, an azo-hydrazone tautomerism might happen between the hydroxyland the ortho-azo bond (Ball and Nicholls 1982, 1985;Jacques 1988; Kelemen et al. 1984). Additionally, the naph-thalene hydroxyl and azo bond are the representative struc-tures of azo dyes that most azo dyes shared. Thus, it wasdesigned to introduce a carboxylic group into amaranth bymodifying its naphthalene sulfonic group. The procedure ofhapten synthesis is shown in Fig. 2. The naphthalene sodiumsulfonate of amaranth was halogenated firstly, and glycinewas allowed to react with amaranth-sulfonyl chloride

subsequently to introduce a carboxyl group. Finally, the re-sulted carboxylic group modified amaranth was conjugated tocBSA, which was previously prepared by BSA treated withexcess of ethylenediamine (EDA) (Kamps-Holtzapple et al.1993). The coating antigen, amaranth-cOVA, was preparedwith cOVA by the same protocol.

The UV-Vis spectra of amaranth, cBSA, immunogen(amaranth-cBSA), and the physical mixture of cBSA andamaranth are shown in Fig. 3. Amaranth exhibits strongabsorbance peak at 520 nm, and the characteristic peak ofcBSA locates at 278 nm. However, the spectrum of amaranth-cBSA also shows the characteristic peak of amaranth at

Fig. 2 Synthetic routs of the immunogen (amaranth-cBSA conjugate)

300 400 500 600

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Abs

orba

nce

Wavelength(nm)

mixture of cBSA and amaranth (3.5mg mL-1-20µg mL-1)

amaranth-cBSA immunogen

amaranth

cBSA

Fig. 3 UV-Vis spectra ofamaranth (black), activatedbovine serum albumin (cBSA)(light gray), immunogen(amaranth-cBSA) (gray), andphysical mixture of cBSA andamaranth (dark gray)

Food Anal. Methods

520 nm. Dialysis was employed to purify the immunogen, andthe free amaranth could be cleared completely. Thus, theabsorbance peak at 520 nm is from the conjugated amaranth.However, the spectrum of the physical mixture of cBSA andamaranth is different to that of the immunogen (includingshapes and locations of the peaks), especially in the range of200–300 nm. It could be preliminarily concluded that ama-ranth was coupled into cBSA. The spectra-scanning results ofamaranth-cOVA are similar, and it is not cited here. However,supplementary evidence for the synthesis success would beprovided by characterization results of antibody.

The molar ratio of amaranth to cBSAwas simply estimatedby the UV-Vis spectrophotometric method (Zhang et al. 2012).The absorbance values of the hapten, carrier protein, and im-munogen were detected at the characteristic wavelength of the

hapten (λ=330 nm). Then, the molar absorbance coefficient (ε)values were calculated. Finally, the molar ratio of amaranth tocBSAwas directly calculated by the following formula:

hapten=protein molar ratio ¼ εimmunogen−εcBSA� �

=εamaranth

Based on the above method, the molar ratio of amaranth tocBSA is 10.5:1.

Characterization of Anti-amaranth Monoclonal Antibody

The performances of the anti-amaranth MAbs are character-ized by sensitivity and specificity. For the optimization study,the selected dilution for antibody is 1:1000 and that for coatingantigen is also found to be 1:1000. Based on the above

Fig. 4 Linear standard curve ofthe developed icELISA. Eachdata point represents the mean±SD of three determinations

Table 1 Comparison of various methods for amaranth analysis

Methods Instruments Sensitivity Food samples Reference

Proposed method ELISA microplate reader LOD=3.35 ng mL−1 Beverage Proposed method

Capillary zone electrophoresis P/ACE System 5500 with a diodearray detector.

LOD=1.1 μg mL−1

LOQ=3.6 μg mL−1Beverage, syrups (Perez-Urquiza

and Beltran 2000)

Capillary zone electrophoresis Capillary electrophoresis witha diode-array UV/Vis detector

LOD=0.61 μg mL−1

LOQ=2.01 μg mL−1Beverages, icelolly, syrups

(Berzas Nevadoet al. 1999)

HPLC Waters HPLC LOD=0.017 ng mL−1

LOQ=0.05 ng mL−1Fruit flavored drink (Wu et al. 2013)

HPLC-DAD Thermo Finnigan Spectra System LOD=10.2 ng mL−1 – (Minioti et al. 2007)

HPLC-DAD Agilent 1100 Series LOD=0.1 μg mL−1

LOQ=0.32 μg mL−1Solid juice powder (Alves et al. 2008)

Ion-pair liquid chromatography withphotodiode array and Electro-spraymass spectrometry

HP1050 gradient pump, HP1100photodiode array detector,HP-5989B mass spectrometer

LOD=0.01 μg mL−1 – (Fuh and Chia 2002)

Food Anal. Methods

optimized condition, the linear range of the proposed ELISAis 3.0–243.0 ng mL−1. The B /B0 values exhibit good linearity(B /B0=−0.383lgC+1.013, R2=0.99) with the logarithm ofamaranth concentration in this range (n =3). The standardcurve is generated as shown in Fig. 4. Accordingly, IC50

corresponding to the amaranth concentration that exhibited50 % inhibition of antibody to the coating antigen is20.33 ng mL−1 and the LOD (IC20) is 3.35 ng mL−1. Theresults of other studies are shown in Table 1 for comparison.

Specificity of the antibody is reflected by the cross-reactivity against six related food dyes including erythrosine,ponceau 4R, allura red, tartrazine, sunset yellow FCF, andbrilliant blue. As shown in Table 2, the CR value of ponceau4R is less than 1 % and that of other selected dyes is all lowerthan 0.1 %, which indicates excellent specificity of the anti-body towards all selected dyes. Among the studied dyes,ponceau 4R and amaranth are a couple of isomeric com-pounds (Fig. 1). However, the CR value of anti-amaranthMAbs against ponceau 4R is extraordinarily low (<1 %). Itmight be that the sodium sulfonate of amaranth was modifiedat position C4′when amaranth reacted with the carrier protein.Thus, the different substituent of disulfonate between ama-ranth (C2, C7) and ponceau 4R (C5, C7) results in the signif-icant difference in CR data. If cBSA reacted with amaranth atposition C2 or C7, the CR results of amaranth and ponceau 4Rwould not differ significantly. If so, the substitutes that anti-bodies recognize are absolutely identical between amaranthand ponceau 4R, which is contradicted by the CR data. There-fore, the specificity performance of anti-amaranth MAbs il-lustrates that the immunogen synthesis is successful, and itcould also evidence that the structure of immunogen is thesame as shown in Fig. 2. However, that is initiatory conjectureand further researches should be designed if a confirmativeconclusion is expected.

ELISA Performances in Beverage Samples

The developed icELISA was applied to detect the recoveryof amaranth in beverage. In the preliminary study, the LODof the method in beverage was determined accordingly,which was compared with that of PBS buffer. With the

Table 2 Cross-reactivities of related food dyes

Analytes IC50 (ng mL−1) Cross-reactivity (%)

Amaranth 20.33 100

Ponceau 4R 2336.78 0.87

Erythrosine 33883.33 0.06

Allura red 50825.02 0.04

Tartrazine 40660.17 0.05

Sunset yellow FCF 67766.67 0.03

Brilliant blue 101650.00 0.02

Table 3 Recovery of amaranth in beverage samples by the developedicELISA

Spiked level(ng mL−1)

n Found level(ng mL−1)

Recovery (%) CV (%)

200 3 171.8±16.5 85.9 9.6

1000 3 858.0±73.7 85.8 8.6

4000 3 4028.0±463.2 100.7 11.5

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

icE

LIS

A (

µg m

L-1)

HPLC (µg mL-1)

Fig. 5 Determination ofamaranth in beverage samplesusing HPLC and the developedicELISA for comparison study.The experimental points reportedare the average values of threeexperiments

Food Anal. Methods

previously mentioned preparation method for the samples,the LOD in beverage is as low as that of the buffer and thedetection range is also comparable when the beverage sam-ples were diluted for 50 times before determination. Basedon these results, the beverage samples were spiked at finallevels as 4.0, 20.0, and 80.0 ng mL−1. The recoveries for theabove three spiked levels are 85.9, 85.8, and 100.7 %,respectively with the coefficient of variation (CV) valuesof 9.6, 8.6, and 11.5 % (Table 3).

HPLC method was selected to perform the comparisonstudy. Four amaranth-contained beverage samples from dif-ferent sources in the local markets were detected in triplicateby HPLC and the developed icELISA, respectively. The re-sults of these two methods exhibit a linear relationship(Fig. 5). The linear equation is y =0.888x+0.0183, with acorrelation coefficient (r) of 0.999.

Conclusions

In summary, anti-amaranth MAbs with satisfactory perfor-mances were prepared by the well-designed immunogen forthe first time. Based on the monoclonal antibody, an icELISAwas developed for amaranth. The proposed method exhibitedan IC50 of 20.33 ng mL−1, and the LOD of detection was aslow as 3.35 ng mL−1. Furthermore, the cross-reactivities of sixrelated food dyes were all lower than 1 %. In beveragesamples, the recovery results of three different spiked levelsranged from 85.8 to 100.7%with low CV values (<11.5 %). Itcan be concluded that the ELISA explored in this study issensitive, specific and accurate, which provides an alternativefor rapid and simple assay for amaranth.

Acknowledgments The authors gratefully acknowledge the financialsupport of the National Natural Science Foundation (No. 81173017, No.31101277), Tianjin Science and Technology Program (No.11ZCGHHZ01200, No. 12ZXCXSY08400, 11ZXCXSY04500), andTianjin Research Program of Application Foundation and AdvancedTechnology (No. 12JCQNJC08900, 13JCZDJC29700).

Conflict of Interest Bo Zhang has no conflict of interest.Daolin Du has no conflict of interest.Meng Meng has no conflict of interest.Sergei A. Eremin has no conflict of interest.Victor B. Rybakov has no conflict of interest.Junhong Zhao has no conflict of interest.Yongmei Yin has no conflict of interest.Rimo Xi has no conflict of interest.

Ethical Standards—Informed consent This article does not containany studies with human subjects.

Ethical Standards—Animal rights The studies with animals wereperformed following all institutional and national guidelines for the careand use of laboratory animals, which were also mentioned in theMethodssection.

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