A sensitive competitive enzyme immunoassay for detection of erythrosine in foodstuffs

Food Control 47 (2015) 472e477

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A sensitive competitive enzyme immunoassay for detection of erythrosine in foodstuffs Zhihuan Xu a, b, 1, Lei Zheng a, b, 1, Yongmei Yin a, b, Jing Wang a, b, Peng Wang a, b, Linlin Ren a, b, Sergei A. Eremin c, Xiaodan He d, Meng Meng a, b, *, Rimo Xi a, b, * a

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia d Tianjin Sungene Biotech Co., Ltd., Tianjin 300450, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2013 Received in revised form 18 July 2014 Accepted 19 July 2014 Available online 28 July 2014

An enzyme immunoassay for erythrosine (Ery) in food products was developed in this study. Anti-Ery polyclonal antibody was obtained by immunizing rabbits with Ery-cationized BSA (cBSA) conjugates. Coupling ratios of erythrosine to carrier proteins were measured to be 16.6:1 in immunogen and 13.5:1 in coating antigen. The developed method showed high sensitivity with the IC50 value up to 29.1 ± 6.79 ng mL
1. In food samples (healthy energy drink, breezer, grape juice, coca-cola sugar, fermented bean curd and tomato paste), the limit of detection (LOD) values of the developed method ranged from 2.2 ng mL
1 to 8.3 ng mL
1. The recoveries ranged from 86.3% to 115.5%. Intra-assay and inter-assay variation were lower than 9.6% and 10.7% respectively. The method could specifically recognize Ery among commonly used food colors, and the results obtained with the proposed immunoassay were well related with that of the reference high performance liquid chromatography (R2 > 0.9999). Therefore, the proposed method could be selectively used for rapid screening Ery in the mentioned foodstuffs. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Erythrosine Polyclonal antibody Enzyme immunoassay Liquid chromatography Foods

1. Introduction The wide use of synthetic colors as food additives has increasingly drawn attention in recent years. The emergence of related accidents has also attracted high concern by customers. Synthetic colors usually include the compounds with azo bond, benzene and xanthene. Although most food dyes are expected to be safe if their dosages are strictly limited, some studies have indicated carcinogenicity and toxicity related to commonly used food dyes (Combes & Haveland-Smith, 1982). Erythrosine (Ery, C20H6I4Na2O5), a kind of xanthene colors, is often added into processed food products and beverages. Excessive Ery can cause negative impact on human body. It has been found that Ery inhibits dopamine uptake in rat caudate synaptosomes “uncompetitively” (Lafferman & Silbergeld, 1979) and decreases

* Corresponding authors. State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China. Tel.: þ86 (22) 23499986; fax: þ86 (22) 23507760. E-mail addresses: [email protected] (M. Meng), [email protected] (R. Xi). 1 The first two authors contributed equally to this work. http://dx.doi.org/10.1016/j.foodcont.2014.07.033 0956-7135/© 2014 Elsevier Ltd. All rights reserved.

nonsaturable binding of dopamine to the synaptosome membrane (Logan & Swanson, 1979). Based on the findings, many countries have already issued specific directions for its use. In China, the maximal usage of the Ery is limited to 0.05 g kg
1 in drinks and certain types of food products. Given the potentially serious risk, accurate determination of synthetic colors is very important. Presently, analytical methods for the detection of Ery include high performance liquid chromatography (Kirschbaum, Krause, Pfalzgraf, & Brückner, 2003; Miao et al., 2012), capillary electrophoresis (Chou, Lin, Cheng, & Hwang, 2002; bel, Kra senský, Kuo, Huang, & Hsieh, 1998; Ryvolov a, T aborský, Vra , & Preisler, 2007), thin layer chromatography (Baranowska, Zydron & Szczepanik, 2004), micellar electrokinetic chromatography (Brumley, Brownrigg, & Grange, 1994), spectrophotometry (Nevado, Flores, & Llerena, 1994) and stripping voltammetry (Alghamdi, 2005). Although these methods have served as important measurements for the evaluation of Ery, the drawbacks such as high cost, time-consuming operation, and complicated pretreatment cannot be neglected. In this work, an enzyme-linked immunosorbent assay (ELISA) was developed as an alternative method for sensitive determination of Ery because of its benefit in high throughput screening. The

Z. Xu et al. / Food Control 47 (2015) 472e477

ELISA protocol was developed based on the polyclonal antibody obtained by immunization procedure. Compared with instrumental assays, the developed immunoassay can be carried out without highly-skilled operation procedure or complicated pretreatment of food samples, and thus could serve as a reliable screening method. 2. Materials and methods 2.1. Materials and instruments Erythrosine and related food dyes (Rhodamine B, Orange II, Sunset Yellow FCF, Tartrazine, Quinoline Yellow, New Coccine, Amaranth, Chromotrope FB), ovalbumin (OVA), bovine serum albumin (BSA), Freund's complete adjuvant (cFA), Freund's incomplete adjuvant (iFA), N,N0 -carbonyldiimidazole (CDI), N,Ndimethylformamide (DMF), trinitrobenzene sulfonic acid (TNBS), 1(3-dimethylaminopropyl)-ethylcarbodiimide hydrochloride (EDC$HCl) and sodium dodecyl sulfate (SDS) were supplied by SigmaeAldrich (St. Louis, MO, USA). Goat anti-rabbit IgG labeled with horseradish peroxidase (HRP), 3,30 ,5,50 -tetramethylbenzidine (TMB), urea hydrogen peroxide and ammonium sulfate were from Sangon Biotech Co. (Shanghai, China). Crocein Orange G was purchased from TCI (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan). The buffers used in this research were as follows: (1) coating buffer: 50 mmol L
1 carbonate buffer (15 mmol L
1 Na2CO3 and 35 mmol L
1 NaHCO3, pH9.6); (2) PBS solution: sodium phosphatebuffered saline (0.138 mol L
1 NaCl, 2.7 mmol L
1 KCl, 1.5 mmol L
1 KH2PO4, and 7 mmol L
1 Na2HPO4, pH7.4); (3) blocking buffer: 10 g L
1 OVA in PBS containing 0.05% (v/v) Tween20; (4) washing buffer (PBST): PBS buffer with addition of 0.05% (v/v) Tween20; (5) substrate buffer: TMB solution (1 mmol L
1 TMB mixed with an equal volume of 1 mmol L
1 urea hydrogen peroxide citrate buffer, pH5.5); (6) stopping solution: 2 mol L
1 hydrochloric acid; (7) borate buffer: 0.01 mol L
1 Na2B4O7$10H2O, pH9.2. ELISA plates FEF-100-096 were purchased from JET Bio-filtration Products Co. (Guangzhou, China). ELISA signal at 450 nm was read by a Model 680 microplate reader (Bio-Rad Laboratories, Beijing, China). UV spectra were collected on a U-4100 spectrophotometer (Hitachi Co., Kyoto, Japan). Protein products were purified using dialysis bag (MWCO ¼ 14,000) from Solarbio (Beijing, China). Hapteneprotein conjugates were lyophilized using an FD-1 freezedrier (Boyikang Technology Corporation, Beijing, China). Centrifugation was carried out in a Biofuge Stratos refrigerated centrifuge (Heraeus, Shanghai, China). 2.2. Preparation of immunogen and coating antigen for Ery Before coupling with carrier proteins, Ery (20.5 mg) was dissolved in 2 mL of HCl (0.02 mol L
1) for acidizing treatment. The reaction mixture was stirred slowly for 6 h at room temperature in the dark and subsequently centrifuged at 6000g for 5 min. After washing twice with distilled water, the precipitation was redissolved into 3 mL of DMF. In order to increase the conjugation efficiency of Ery to carrier proteins, the carboxylic acid groups in carrier proteins were converted into primary amine groups with excess of ethylenediamine (Liu et al., 2007) to obtain cationized BSA (cBSA) or OVA (cOVA). Ethylenediamine (15 mL) was added dropwise to 30 mL of PBS at 4  C and then 12 mol L
1 HCl was slowly added to obtain the product at pH7.4. Afterward, BSA (1 g) and EDC (0.639 g) were dissolved into the mixture solution with continuous stirring and then incubated for 4 h at room temperature. The final reaction solution was dialyzed against PBS for 3 days and distilled water for another 3 days. White solid was obtained by lyophilizing and stored at
20  C until use.

473

The immunogen cBSAeerythrosine (cBSAeEry) and coating antigen cOVAeerythrosine (cOVAeEry) were prepared via a N,N0 carbonyldiimidazole (CDI) method showed in Fig. 1 (Rao, Wang, Cessac, & Moore, 1998). First, 15.9 mg CDI was dissolved into 1 mL of DMF and then added to acidized-Ery solution (10.3 mg mL
1). The mixture was incubated for 24 h at room temperature in dark. Then, 47.6 mg cBSA was dissolved in 6 mL of borate buffer (0.01 M, pH9.2) and added dropwise to the mixture. The reaction mixture was kept for 24 h at room temperature. After that, the final reaction solution was dialyzed under stirring against PBS for 3 days and distilled water for another 3 days. The solution was lyophilized to obtain the cBSAeEry conjugate. The cOVAeEry was prepared according to the similar method. Trinitrobenzene sulfonic acid assay was performed to determine the conjugation percentage of hapten to carrier protein using TNBS reagent (Sreenath & Venkatesh, 2007). To 1 mL of BSA solutions (50e200 ng mL
1), 1 mL of 4% NaHCO3 (pH8.5) and 1 mL of 0.01% freshly prepared TNBS solutions were added. The reaction mixture was incubated for 2 h at 42  C followed by the addition of 10% SDS (1 mL) and 1 mol L
1 HCl (0.5 mL), and then the absorbance at 335 nm was recorded. A negative control was performed at the same time for blank corrections. After the standard curve of BSA was established, 100 ng mL
1 cBSA, cOVA, cBSAeEry, cOVAeEry were investigated by the same procedure to obtain conjugation percentage. The ratio was evaluated based on the decrease in the absorbance compared with that of an identical concentration of standard BSA. 2.3. Immunization and polyclonal antibody preparation To raise polyclonal antibodies, two male New Zealand white rabbits weighing approximately 2 kg were subcutaneously immunized with the cBSAeEry immunogen at the back. Before the immunization, 1 mL of blood was taken from each rabbit to get the negative serum. For the first immunization, 0.5 mg of immunogen in 0.5 mL of physiological saline was emulsified with equal volume of Freund's complete adjuvant. The prepared immunogen was then injected via the multiple-site injection method. For booster immunizations, the amount of immunogen was decreased to 0.25 mg and iFA was used to replace cFA. Booster immunizations were given four times at 15 days intervals. The fifth immunization was administered using 0.5 mL of physiological saline containing 0.25 mg immunogen. Seven days after the last immunization, the rabbits were exsanguinated by heart puncture after anaesthetized by diethyl ether to collect blood samples. The blood samples were left to coagulate at room temperature for 2 h and then overnight at 4  C. The antiserum was isolated by centrifugation at 8000g for 10 min and stored at
20  C with 0.02% sodium azide (w/w). 2.4. The purification and quantification of the polyclonal antibodies Owing to different solubilities of proteins in saline solution, purification of the polyclonal antibodies was conducted by precipitation with saturated ammonium sulfate (SAS) (Wu & Han, 2010). Firstly, the SAS solution was prepared by dissolving ammonium sulfate (400 g) into 500 mL of distilled water at 70e80  C. The solution cooled down and crystallized out ammonium sulfate crystals at room temperature and the supernatant was SAS solution. Then the antibodies were purified stepwise with 50%, 33%, and 33% (v/v) of SAS to remove impurities. The final antibodies precipitation was redissolved in physiological saline for dialysis and further determination. The spectrophotometer was employed to quantify the antibodies. The concentration was calculated with LowryeKalckaras formula as follows:

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Z. Xu et al. / Food Control 47 (2015) 472e477

Fig. 1. Synthesis of immunogen (cBSAeEry).

  Protein concentration mg mL
1 ¼ ð1:45A280
0:74A260 Þ  Dilution ratio of pAb: A280 and A260 represent the absorption of pAb at 280 nm and 260 nm respectively. 2.5. Optimization of the immunoassay To optimize the concentrations of coating antigen and the antibody, the checkerboard procedure was performed in a 96-well microplate. The plate was coated with coating antigen dissolved in coating buffer (pH9.6) from 0.2 mg mL
1 to 10 mg mL
1 (100 mL per well). The plate was incubated at 37  C for 2 h and then the excess coating antigen was removed with washing buffer (300 mL per well, three times). Then blocking buffer was added into the plate (250 mL per well) which was subsequently incubated at 37  C for 2 h. After washing, antibody diluted in PBS (1:1000e1:80,000, 100 mL per well) was added and kept at 37  C for 30 min. The wells were washed three times again, and then goat anti-rabbit IgG-HRP (1:20,000, 100 mL per well) was added into each well followed by incubation at 37  C for 30 min. 100 mL of TMB substrate solution was added after washing with PBST. After incubation for another 10 min, enzymatic color development was immediately stopped by stopping solution (100 mL per well). The absorbance values at 450 nm were read using microplate reader. 2.6. Development of an indirect competitive ELISA (icELISA) The sensitivity of the antiserum was evaluated by the icELISA protocol under the optimum condition. The procedures for the icELISA were the same as before except that after removal of blocking buffer, 50 mL of analyte diluent was added followed by the addition of 50 mL diluted antibody. The Ery reference substances with the concentration from 1  105 ng mL
1 to 0.01 ng mL
1 were served as the competitors. The inhibition curve was obtained by plotting inhibition ratio B/B0 versus the logarithm of competitor concentration. The B signifies the absorbance value of the well containing competitors and the B0 signifies the absorbance value with PBS instead of competitors which served as blank control. The IC50 value was adopted to represent the concentration of competitor that leads to 50% decrease of the maximum signal. The value was taken as the parameter for the evaluation of sensitivity. To determine the specificity of the assay, cross-reactions were performed with functionally and structurally similar analogs by the icELISA method. The studied compounds included Rhodamine B, Orange II, Sunset Yellow FCF, Tartrazine, Quinoline Yellow, New Coccine, Amaranth, Chromotrope FB and Crocein Orange G. Crossreactivity (CR%) values that represent the specificity were calculated as follows:

100, 300 ng mL
1) were selected according to the linear range of the standard calibration curve. The experiments were repeated five times a day and carried out for three days. 2.7. Analysis of Ery in foods by icELISA Four kinds of commercial food products in which Ery are commonly added were purchased from local supermarkets and tested. These foodstuffs include drinks (healthy energy drink, breezer and grape juice), coca-cola sugar, fermented bean cured and tomato paste. Before used for analysis, samples need to be treated as follows: Drinks: Breezer was boiled to remove CO2 and coca-cola sugar (5 g) was dissolved in 10 mL of distilled water. Grape juice (10 mL) was centrifuged at 10,000g for 10 min and then filtered. The matrices were then diluted before analysis. Fermented bean curd: 2.5 g of sample was mixed with 10 mL of PBS and centrifuged at 10,000g for 10 min and then filtered. Tomato paste: 5 g of sample was mixed with 10 mL of PBS and centrifuged at 10,000g for 15 min. The supernatant was mixed with 5 mL of hexane followed by centrifugation under the same condition as above. The aqueous phase was stored for future use. Sugar: coca-cola sugar (5 g) was dissolved in 10 mL of distilled water and then diluted for further analysis. IC50 values of Ery in each prepared sample were compared with that in PBS to obtain the optimum dilution. The limit of detection (LOD) of each matrix was evaluated by the icELISA and defined as the average concentration determined in 20 non-spiked samples plus three times of the standard deviation (SD). Recoveries and coefficients of variation (CVs) were determined by the performance of the icELISA in different matrices spiked with Ery at levels of 10, 100, 300 ng mL
1. 2.8. Comparison with HPLC analysis The confirmatory HPLC was performed on an Ultimate 3000 liquid chromatography system (Dionex, Sunnyvale, CA, USA) with Inert Sustain C18 column (250  4.6 mm i.d., 5 mm, Shimadzu-GL, Shanghai, China). The detection procedure was carried out with a methanol:ammonium acetate (0.02 mol L
1) mobile phase gradient changing from 70:30 (v/v) to 80:20 (v/v) in 10 min and then 70% methanol in next 5 min with a flow rate of 1 mL min
1. The signals of diluted Ery solutions ranging from 10 mg mL
1 to 400 mg mL
1 were collected at the wavelength of 254 nm. The standard curve was constructed by plotting the peak area versus Ery concentration. 100, 200, 300 mg mL
1 were chosen to investigate the accuracy and precision of the analytical manner.

CRð%Þ ¼ ðIC50 of Ery=IC50 of cross
reacting compoundÞ  100%:

2.9. Statistical analysis

Inter- and intra-assay variations were adopted to evaluate the accuracy and precision of the assay. Three Ery concentrations (10,

All the experiments were performed at least three times, and the data were expressed as the mean ± standard deviation (S.D.).

Z. Xu et al. / Food Control 47 (2015) 472e477

1. Ery (526 nm) 2. cBSA-Ery (542 nm) 3. cBSA (278 nm)

4.0 3.5

0.9 0.8 0.7

3.0

0.6

2.5

B/BO

UV absorbance

475

1

2.0

0.5 0.4 0.3

1.5

2

0.2

1.0

3

0.5

0.1 0.01

0.0

0.1

1

10

100

1000

10000

-1

Ery(ng mL ) 300

400

500

600

Wavelengh(nm) Fig. 2. UV spectra of: (1) Ery, (2) immunogen (cBSAeEry), (3) cBSA.

The inhibition curve was generated by plotting inhibition ratio (B/ B0) versus the logarithm value of Ery concentration by OriginPro 8.5 (OriginLab®, MA, USA).

Fig. 3. Inhibition curve of Ery determination by the icELISA method (n ¼ 4).

cOVAeEry could be calculated. As was shown in Table 1, TNBS reactive amino groups in carrier proteins increased after modification by ethylenediamine, and conjugation ratios were high with 16.6:1 for cBSAeEry and 13.5:1 for cOVAeEry. 3.2. Characterization of polyclonal antibodies

3.1. Characterization of immunogen and coating antigen for Ery Ery belongs to small molecule and needs to be covalently coupled with carrier proteins to enhance its immunogenicity. Therefore, appropriate synthesis of immunogen is significant for development of antibody. As illustrated in Fig. 1, the carboxylic groups in hapten were applied to link with the carrier protein (cBSA or cOVA). To characterize the conjugation, the UV spectra of Ery, cBSA, cBSAeEry, cOVAeEry were collected. As showed in Fig. 2, the UV profile of cBSA possessed the maximal absorption peak at 278 nm while cBSAeEry showed UV absorption peak at 542 nm. This difference was probably owing to the Ery coupling which has absorption peak at 526 nm. In addition, both immunogen and coating antigen remained pink after purification. These results indicated the successful conjugation between hapten and carrier proteins. For further research, the conjugation ratio was determined by trinitrobenzene sulfonic acid assay. The assay was based on the existence of TNBS reactive amino groups on the carrier protein (Sreenath & Venkatesh, 2007). It is known that BSA and OVA have 59 and 20 TNBS reactive amino groups respectively (Hermanson, 2008). A standard curve (y ¼ 0.00152x
0.03246) was obtained and showed good linear relationship of BSA concentration with its absorbance at 335 nm (R2 > 0.9886). Using this calibration curve, the amount of reactive amino groups on cBSA, cOVA, cBSAeEry and

Table 1 Coupling ratios of immunogen (cBSAeEry) and coating antigen (cOVAeEry). Sample

Amino groups

Conjugation ratio

BSA cBSA cBSAeEry OVA cOVA cOVAeEry

59.0 90.5 75.5 20.0 53.2 46.0

e e 16.6:1 e e 13.5:1

According to the results of purification and quantification, the concentration of the polyclonal antibodies was figured out to be 11.7 mg mL
1. The features including titer, sensitivity, specificity, stability and repeatability were used to characterize the antibodies. From the results of the checkerboard protocol, the optimum concentrations of coating antigen and antibody were 2 mg mL
1 and 1:20,000 respectively. The indirect competitive ELISA was performed accordingly and the titer of the obtained antibody was up to 320,000, which was defined as the reciprocal of the dilution that results in an absorbance value twice the background. The sensitivity of the developed assay was featured by the IC50 value from inhibition curve. The representative inhibition curve for Ery was shown in Fig. 3, which indicated IC50 value was 29.1 ± 6.79 ng mL
1. The representative standard curve (R2 > 0.999) described in Fig. 4 showed good linear relationship between B/B0

0.9 0.8 0.7

B/BO

3. Results and discussion

0.6 0.5 0.4 0.3 0.2 10

100 -1

Ery(ng mL ) Fig. 4. Linear standard curve of the developed icELISA method (n ¼ 4).

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Z. Xu et al. / Food Control 47 (2015) 472e477

Intra-assay CV (%)

Inter-assay CV (%)

9.3 1.7 2.2 9.6 15.9 2.6 5.3 5.1 2.8

13.4

five times a day and repeated in three different days to determine the intra-assay and inter-assay variation. As indicated in Table 2, the intra-assay coefficient of variation (CV) varied from 1.7% to 15.9% in three days, and the inter-assay CV was less than 13.4%. The results indicated that the developed immunoassay possessed good stability and repeatability.

10.2

3.3. Analysis of Ery in foods by icELISA

Table 2 Accuracy and precision evaluation of Ery determination in PBS system by the icELISA method. Added amount (ng mL
1)

Day

Measured amount (ng mL
1)

10

1 2 3 1 2 3 1 2 3

10.3 7.9 10.3 113.8 113.8 111.2 307.7 309.3 309.4

100

300

± ± ± ± ± ± ± ± ±

1.0 0.1 0.2 10.9 18.1 2.9 16.3 15.7 8.5

4.2

Table 3 Dilution factors of the food samples investigated in the study for the icELISA. Matrix

Dilution factors

IC50 (ng mL
1)

PBS buffer Healthy energy drink Breezer

e 1:20 1:40 1:100 1:10 1:20 1:50 1:5 1:10 1:20

29.1 ± 6.79 93.0 59.3 50.6 38.2 60.8 70.5 277.4 76.9 23.8

Grape juice

Matrix

Dilution factors

IC50 (ng mL
1)

Fermented bean curd

1:5 1:10 1:20 1:5 1:10 1:20 1:5 1:10 1:20

56.8 37.2 25.3 85.6 25.7 25.1 132.4 182.9 41.7

Coca-cola sugar

Tomato paste

The dilution factors in bold are optimal and chosen for future experiments.

value (y) and the logarithmic concentration of Ery (x) from 5.0 ng mL
1 to 300 ng mL
1. Compared with the monoclonal antibody of Ery we produced by carbodiimide method (IC50 ¼ 680 ng mL
1) (Zhang, Du, Yin, & Zheng, 2014), the polyclonal antibody in this work is more sensitive. The specificity of the antibody was evaluated by the performance of cross-reaction with the selected analogs by icELISA. Structural analog (Rhodamine B) and functional analogs (Orange II, Sunset Yellow FCF, Tartrazine, Quinoline Yellow, New Coccine, Amaranth, Chromotrope FB and Crocein Orange G) were tested and little cross-reaction (<0.1%) was observed. To assess the accuracy and precision of the icELISA method, a series of Ery diluents (10, 100, 300 ng mL
1) in PBS was detected

Matrix interference is an important factor that cannot to be neglected (Wu, Chang, Ding, & He, 2008). Four kinds of food samples (drinks, coca-cola sugar, fermented bean curd and tomato paste) were selected to determine the matrix interference on the application of the method. For drinks group, healthy energy drink, breezer and grape juice were tested. The matrices were subjected to series of dilution after pretreatment to obtain the most similar IC50 values with that in PBS system. The dilution ratios and corresponding IC50 values in each group were showed in Table 3, and it indicated that 1:100 for healthy energy drink, 1:10 for breezer, 1:20 for grape juice, 1:10 for coca-cola sugar, 1:20 for fermented bean curd and 1:20 for tomato paste would be the appropriate dilutions for further analysis. Based on the LOD results (7.2 ng mL
1 for healthy energy drink, 8.3 ng mL
1 for breezer, 5.2 ng mL
1 for grape juice, 7.9 ng mL
1 for coca-cola sugar, fermented bean 6.0 ng mL
1 for curd, 5.7 ng mL
1 for tomato paste), the matrices were finally fortified with three different concentrations (10, 100, 300 ng mL
1) and the proposed icELISA was carried out five times a day and three days in a row. Ery content in the sample was determined by comparing the measured absorbance with the calibration curve. As showed in Table 4, the analytical recoveries were 86.3e115.5% with intra-assay variation less than 9.6% and inter-assay variation less than 10.7%. The results made the analytical method a reliable screening manner in the real samples. 3.4. Comparison with HPLC analysis The performance of the HPLC was presented in Fig. 5. The standard curve showed in the inset indicated excellent linearity from 10 mg mL
1 to 400 mg mL
1 with the R2 up to 0.9999. The LOD value for the HPLC method was measured to be 330 ng mL
1, which demonstrated that the proposed icELISA method is more sensitive. The accuracy experiment conducted three times at three

Table 4 Results of recovery and coefficient of variation for Ery determination from food samples by the icELISA method. Sample

Spiked (ng mL
1)

Inter-assay Detected (ng mL
1)

Healthy energy drink

Breezer

Coca-cola sugar

Fermented bean curd

Grape juice

Tomato paste

10 100 300 10 100 300 10 100 300 10 100 300 10 100 300 10 100 300

11.2 102.8 310.2 10.8 104.1 299.1 10.5 96.2 302.6 10.8 102.1 300.4 10.0 105.7 306.9 10.6 100.6 296.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.1 8.4 15.0 0.9 7.3 11.6 1.0 9.2 18.6 1.2 7.3 14.6 0.7 4.5 9.5 0.9 4.6 9.4

Intra-assay Recovery (%)

CV (%)

Detected (ng mL
1)

112.4 102.8 103.4 107.7 104.1 99.7 104.9 96.2 100.9 108.0 102.1 100.1 100.0 105.7 102.3 105.7 100.6 99.0

9.6 8.2 4.8 8.6 7.0 3.9 9.4 9.6 6.2 10.7 7.2 4.9 6.8 4.2 3.1 8.1 4.6 3.2

10.6 106.6 308.7 11.6 105.7 293.3 9.7 86.3 301.7 10.1 106.0 301.9 10.3 104.9 306.2 10.2 98.8 296.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 8.3 22.7 1.0 4.5 8.7 0.9 4.6 18.0 0.8 7.1 9.1 0.4 5.1 12.0 0.5 4.8 11.6

Recovery (%)

CV (%)

105.8 106.6 102.3 115.5 105.7 97.8 97.4 86.3 100.6 100.9 106.0 100.7 102.6 104.9 102.1 102.2 98.8 98.7

8.4 7.8 7.3 8.6 4.3 3.0 9.6 5.3 6.0 7.5 6.7 3.0 3.8 4.9 3.9 4.8 4.9 3.9

Z. Xu et al. / Food Control 47 (2015) 472e477

477

Fig. 5. Confirmatory HPLC chromatogram of Ery and linear standard curve of the HPLC method (inset).

Table 5 Detection of Ery by icELISA and HPLC method (n ¼ 3). Added amount (mg mL
1)

icELISA Measured amount (mg mL
1)

Recovery (%)

HPLC Measured amount (mg mL
1)

Recovery (%)

100 200 300

107.4 ± 8.1 203.6 ± 2.2 306.9 ± 16.7

107.4 101.8 102.3

102.5 ± 0.4 204.2 ± 4.2 303.4 ± 2.9

102.5 102.1 101.1

concentrations (100, 200 and 300 mg mL
1) in Table 5 also showed consistent results with that of the icELISA. 4. Conclusions A sensitive enzyme immunoassay depending on polyclonal antibody has been developed in this work. The method demonstrated good accuracy and repeatability in four types of foods (drinks, coca-cola sugar, fermented bean cured and tomato paste). Although the immunoassay may be susceptible to matrix interference, the effect could easily be avoided by appropriate sample dilution for the proposed assay. All the features and the correspondence with the reference HPLC method contributed to demonstrate that the immunoassay could be used as the rapid and reliable method for the screening of Ery in mentioned food samples. Acknowledgments This work was supported by the financial support of National Natural Science Foundation of China (Nos. 81173017, 31101277), the National High-Tech Research and Development Program of China (863 Program) (No. 2014AA022303), Tianjin Science and Technology Program (Nos. 11ZCGHHZ01200, 12ZXCXSY08400, 12JCQNJC08900), and Tianjin Research Program of Application Foundation and Advanced Technology (13JCZDJC29700). References Alghamdi, A. H. (2005). Determination of Allura Red in some food samples by adsorptive stripping voltammetry. Journal of AOAC International, 88, 1387e1393.

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