Development of an Enzyme-Linked Immunosorbent Assay Specific To

Analytical Biochemistry 405 (2010) 41–49

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Analytical Biochemistry

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Development of an enzyme-linked immunosorbent assay speciﬁc to Sudan red I

Ting Xu a, Ke Yi Wei a, Jia Wang a, Sergei A. Eremin b, Shang Zhong Liu c, Qing X. Li d,JiLia,* a College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China b Department of Chemical Enzymology, Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow 119991, Russia c Department of Applied Chemistry, China Agricultural University, Beijing 100193, China d Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA article info abstract

Article history: To obtain antibodies to develop an enzyme-linked immunosorbent assay (ELISA) for the analysis of Sudan Received 2 March 2010 red I, haptens were designed and synthesized via four different strategies: (i) attachment of a spacer at Received in revised form 13 May 2010 the para position of the benzene ring, (ii) attachment of a spacer at the naphthol part, (iii) attachment Accepted 27 May 2010 of a spacer at the hydroxyl group of the Sudan red I molecule, and (iv) use of a fragment of the target mol- Available online 1 June 2010 ecule. A total of 10 haptens were used to generate immunogens, coating antigens, and polyclonal anti-

bodies. One of the heterologous ELISAs developed exhibited an IC50 of 1.6 ng/ml, a limit of detection Keywords: (LOD) of 0.03 ng/ml, and a dynamic range between 0.1 and 14 ng/ml. The assay had 13% cross-reactivity Sudan red dye with Para red and negligible cross-reactivity with other structure-related compounds. This ELISA was Sudan red I Enzyme-linked immunosorbent assay much more speciﬁc than those published previously. This assay was used to determine Sudan red I res- Color additive idues in tomato sauce and chili powder samples after simple pretreatment. The results were validated by Food adulteration comparison with high-performance liquid chromatography (HPLC). The average recoveries of Sudan red I Food analysis by ELISA and HPLC were in ranges of 70–97% and 82–114%, respectively, indicating suitability of the developed ELISA for screening of Sudan red I in foods. Ó 2010 Elsevier Inc. All rights reserved.

Synthetic Sudan red dyes I, II, III, and IV (Fig. 1) are widely used matography (HPLC)1 coupled with different detection methods, as coloring agents in chemical industries such as oils, fats, plastics, including mass spectrometry [12–14], ultraviolet–visible (UV–VIS) waxes, petrol, shoes, printing inks, shoe and ﬂoor polishing, and detection [15], and diode array detection (DAD) [16]. There have spirit varnishing [1,2]. Besides the chemical industry, they have been only a few reports on the gas chromatography–mass spectrom- been used without authorization and illegally in the food industry etry (GC–MS) analysis of these compounds, including a recent report to enhance and maintain the appearance of food products such as [11] on the determination of Sudan dyes in eggs by silylation prior to in chili-, curry-, curcuma-, and palm oil-containing foodstuffs GC–MS analysis. Several molecularly imprinting techniques have [3–5]. Sudan red I (1-phenylazo-2-naphthol), one of the most also been proposed and proven to be promising in the trace analysis frequently used Sudan dyes, is considered to be a genotoxic carcin- of Sudan red dyes [17–19]. ogen [6] and is classiﬁed as a category 3 carcinogen by the Interna- Although liquid chromatography–mass spectrometry (LC–MS) tional Agency for Research on Cancer [7]. Its presence is prohibited is able to separate and quantify four Sudan dyes simultaneously in foodstuffs for any purpose at any level worldwide. with high sensitivity and good recovery [20,21], complex extrac- Unfortunately, a variety of foodstuffs contaminated with Sudan tion and cleanup steps need to be taken, especially in the analysis dyes (particularly Sudan red I) have been detected recently of high-fat samples. Moreover, the scope of the instrumental throughout Europe and Asia [8–11]. Because the illegal use of the methods is limited to the quantitative detection in the laboratory dyes has major economic consequences for worldwide food industries as well as an adverse impact on public health, a number of articles concerning the development of extraction and detection 1 Abbreviations used: HPLC, high-performance liquid chromatography; UV–VIS, methods for Sudan dyes in foodstuffs have been published. Most ultraviolet–visible; DAD, diode array detection; GC–MS, gas chromatography–mass of the analytical methods applied for Sudan red dye determination spectrometry; LC–MS, liquid chromatography–mass spectrometry; LOD, limit of detection; ELISA, enzyme-linked immunosorbent assay; DCC, N,N0-dicyclohexylcar- in foodstuff samples are based on high-performance liquid chro- bodiimide; NHS, N-hydroxysuccinimide; TMB, 3,30,5,50-tetramethylbenzidine; HRP, horseradish peroxidase; BSA, bovine serum albumin; OVA, ovalbumin; IgG, immunoglobulin G; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PBST, PBS plus 0.05% Tween 20;

* Corresponding author. Fax: +86 10 62732017. IC50, half-maximum inhibition concentration; AU, arbitrary units; CV, coefﬁcient of E-mail address: [email protected] (J. Li). variation.

OH OH

N N N N CH3

H3C

Sudan red I Sudan red II

OH OH H3C

N N N N N N N N

H3C

Sudan red III Sudan red IV

N N NO2

Para red

Fig. 1. Chemical structures of Sudan red dyes (I, II, II, and IV) and Para red. because of the expensive instruments. On the other hand, simple, Louis, MO, USA). 6-Hydroxy-2-naphthoic acid was obtained from sensitive, and low-cost immunoassay techniques for Sudan Energy Chemical (Shanghai, China). 40-Amino-biphenyl-4-carbox- red dyes have been developed recently for screening purpose ylic acid was obtained from Avra Synthesis (India). Tween 20, [22–26]. Because the used haptens were designed following the dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 4-amino- same strategy to bind different lengths of hydrocarbon linkers to benzoic acid, 4-aminophenylacetic acid, 3-(4-aminophenyl)propi- the benzene ring of Sudan red I, all of the developed immunoassays onic acid, 4-(4-aminophenyl)butyric acid, aniline, 1-amino-2- showed high sensitivity to Sudan red I (limit of detection [LOD] of naphthol, and 2-naphthol were purchased from Beijing Chemical 0.01–0.2 ng/ml) but showed different specificity to the other Sudan (Beijing, China). Sudan red (II, III, and IV), Sudan red G, Para red, red dyes. One of the described enzyme-linked immunosorbent as- and Sunset yellow used for testing cross-reactivity were generous says (ELISAs) presented very high cross-reactivities with Sudan red gifts from Beijing Entry–Exit Inspection and Quarantine Bureau III and Para red (Fig. 1) [24]. (China). This study aimed to develop a more specific ELISA for Sudan red ELISA was carried out on 96-well polystyrene microplates (Xia I. Thus, a number of new haptens were designed and synthesized Men, China). The result was read spectrophotometrically at a following strategies different from those published previously wavelength of 450 nm with a Labsystems Dragon Wellscan MK3 [22–26], including attachment of a spacer at the para position of microplate reader (Helsinki, Finland). the benzene ring, attachment of a spacer at the naphthol part, attachment of a spacer at the hydroxyl group of the Sudan red I Hapten synthesis molecule, and use of a fragment of the target molecule. In addition, the influence of different hapten structures on immunoassay sen- Four types of haptens used for immunization and coating anti- sitivity and specificity is studied. Finally, the determination of Su- gen and the synthetic routes for haptens S1–S8 are presented in dan red I in tomato sauce and chili powder samples is presented to Fig. 2. Haptens S1–S6 were synthesized by the same procedure discuss the developed ELISA validation protocol, including the sen- as that reported by Ju and coworkers [24] using corresponding sitivity, recovery, and reproducibility in comparison with HPLC. commercial start materials. Haptens S7 and S8 were synthesized following the Williamson ether synthesis procedure [27]. The following subsections describe the synthesis procedure and the iden- Materials and methods tification of haptens. Chemicals and instruments 4-[(2-Hydroxy naphthalen-1-yl) diazenyl] benzoic acid (hapten S1) Sudan red I, N,N0-dicyclohexylcarbodiimide (DCC), N-hydroxy- To a stirring solution of 1.37 g of 4-aminobenzoic acid in water succinimide (NHS), 3,30,5,50-tetramethylbenzidine (TMB), horserad- (50 ml) cooled in an icewater bath was added dropwise a solution ish peroxidase (HRP), bovine serum albumin (BSA), ovalbumin of 12 ml of concentrated hydrochloric acid, followed by adding (OVA), goat anti-rabbit immunoglobulin G (IgG)–HRP, ethyl bromo- 0.7 g of sodium nitrite dissolved in 5 ml of water. The mixture acetate, 5-bromovaleric acid ethyl ester, and complete and incom- was stirred for 10 min, and then 2-naphthol (1.44 g) dissolved in plete Freund’s adjuvants were purchased from Sigma–Aldrich (St. 30 ml of sodium hydroxide solution (10%, w/v) cooled with ELISA specific to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49 43

Haptens type A

+ NH OH OH 2 N2

HCl, NaNO2 N N (CH2)n COOH 0-5 oC

(CH2)n (CH )n HOOC HOOC 2

S1: n=0; S2: n=1; S3: n=2; S4: n=3

HCl, NaNO 2 + COOH N COOH H2N 2 0-5 oC

OH OH

N N COOH

Hapten type B + NH N OH 2 2 HO

HCl, NaNO2 HOOC N N 0-5 oC

COOH S6 Haptens type C

HOOC (CH )n O HO CH3CH2O (CH2)n O 2 Br(CH )nCOOCH CH 2 2 3 NaOH N N N N N N

K2CO3

S7: n=1; S8: n=4 Haptens type D OH OH

NH2

HOOC S9 S10

Fig. 2. Structures of the hapten derivatives (hapten types A, B, C, and D) of Sudan red I and the synthesis routes for haptens S1–S8.

icewater was added. After stirring for 30 min, the mixture was vac- 175.99 (COOH), 166.87, 146.81, 142.41, 132.87, 131.24, 131.24, uum filtered and washed with water to obtain the crude product 4- 130.34, 129.72, 129.37, 128.31, 128.25, 127.00, 125.66, 121.92, [(2-hydroxy naphthalen-1-yl) diazenyl] benzoic acid (hapten S1), 117.55, 117.55 (Ar). which was further purified by recrystallization in methanol. Yield: 60%. 1H nuclear magentic resonance (NMR) (DMSO) d: 15.89 (1H, s, 2-(4-[(2-Hydroxy naphthalen-1-yl)diazenyl]phenyl) acetic acid ArOH), 13.03 (1H, s, COOH), 8.47 (1H, d, J = 7.7 Hz, Ar), 8.06 (2H, d, (hapten S2) J = 6.9 Hz, Ar), 7.96 (1H, d, J = 9.6 Hz, Ar), 7.88 (2H, d, J = 6.9 Hz, Ar), Yield: 47%. 1H NMR (DMSO) d: 15.70 (1H, s, ArOH), 12.43 (1H, s, 7.75 (1H, d, J = 7.7 Hz, Ar), 7.61 (1H, t, J = 4.4 Hz, Ar), 7.50 (1H, t, COOH), 8.59 (1H, d, J = 8.3 Hz, Ar), 7.98 (1H, d, J = 9.4 Hz, Ar), 7.87- J = 4.4 Hz, Ar), 6.80 (1H, d, J = 9.6 Hz, Ar). 13C NMR (DMSO) d: 7.80 (3H, m, Ar), 7.63 (1H, t, J = 7.3 Hz, Ar), 7.50–7.44 (3H, m, Ar), 44 ELISA specific to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49

13 6.98 (1H, d, J = 9.3 Hz, Ar), 3.67 (2H, s, CH2). C NMR (DMSO) d: J = 8.4 Hz, Ar), 8.03 (1H, d, J = 9.3 Hz, Ar), 7.96 (3H, d, J = 9.6 Hz, 13 172.55 (COOH), 167.01, 144.51, 139.49, 135.75, 132.84, 130.98, Ar), 7.68–7.48 (6H, m, Ar), 4.93 (2H, s, OCH2). C NMR (DMSO) 130.98, 129.25, 129.11, 128.97, 127.97, 125.80, 123.58, 121.42, d: 170.36 (COOH), 153.07, 147.74, 135.82, 131.60, 131.17, 129.66,

119.34, 119.34 (Ar), 30.82 (CH2). 129.66, 129.13, 128.24, 128.23, 127.22, 124.87, 122.76, 122.51, 122.51, 116.29, 66.53 (OCH2). 3-(4-[(2-Hydroxy naphthalen-1-yl)diazenyl]phenyl) propanoic acid (hapten S3) 5-[1-(Phenyl diazenyl)naphthalen-2-yloxy] pentanoic acid (hapten S8) Yield: 50%. 1H NMR (DMSO) d: 15.64 (1H, s, ArOH), 12.14 (1H, s, Yield: 44%. 1H NMR (DMSO) d: 12.03 (1H, s, COOH), 8.30 (1H, d, COOH), 8.60 (1H, d, J = 8.3 Hz, Ar), 7.98 (1H, d, J = 9.3 Hz, Ar), 7.85– J = 8.5 Hz, Ar), 8.04 (1H, d, J = 9.1 Hz, Ar), 7.97–7.92 (3H, m, Ar), 7.81 (3H, m, Ar), 7.63 (1H, t, J = 9.0 Hz, Ar), 7.50–7.42 (3H, m, Ar), 7.66–7.46 (5H, m, Ar), 7.45 (1H, t, J = 5.8 Hz, Ar), 4.21 (2H, t,

7.00 (1H, d, J = 9.0 Hz, Ar), 2.91 (2H, t, J = 7.5 Hz CH2CH2COOH), J = 5.7 Hz, OCH2CH2), 2.27 (2H, t, J = 7.0 Hz, CH2CH2COOH), 1.70 13 13 2.60 (2H, t, J = 7.5 Hz CH2CH2COOH). C NMR (DMSO) d: 173.78 (4H, m, J = 5.1 Hz, OCH2CH2CH2CH2). C NMR (DMSO) d: 174.50 (COOH), 165.79, 144.44, 142.13, 139.09, 132.83, 129.82, 129.82, (COOH), 153.18, 148.02, 135.58, 131.42, 131.32, 131.32, 129.59, 129.15, 129.04, 128.93, 127.96, 125.67, 123.30, 121.39, 119.68, 128.76, 128.20, 128.00, 127.94, 124.58, 122.62, 122.30, 122.30,

119.68 (Ar), 35.14 (CH2CH2COOH), 30.22 (CH2CH2COOH). 116.61 (Ar), 69.59 (OCH2), 33.43 (CH2COOH), 28.50 (OCH2CH2CH2), 21.33 (OCH2CH2CH2). 4-(4-[(2-Hydroxy naphthalen-1-yl)diazenyl] phenyl) butanoic acid (hapten S4) Preparation of immunogen and coating antigen Yield: 55%. 1H NMR (DMSO) d: 15.65 (1H, s, ArOH), 12.10 (1H, s, COOH), 8.61 (1H, d, J = 8.2 Hz, Ar), 7.98 (1H, d, J = 9.3 Hz, Ar), 7.86– All of the haptens having carboxylic acid could be converted to 7.74 (3H, m, Ar), 7.47 (1H, t, J = 7.3 Hz, Ar), 7.39 (1H, d, J = 8.4 Hz, the succinimide esters that are active esters for coupling haptens to Ar), 7.27 (2H, d, J = 7.5 Hz, Ar), 7.01 (1H, d, J = 9.3 Hz, Ar), 2.68 carrier proteins [28,29]. Haptens S1, S3, S4, S6, and S8 were conju-

(2H, t, J = 7.6 Hz CH2CH2COOH), 2.26 (2H, t, J = 7.3 Hz CH2CH2CH2), gated to BSA for serving as immunogens. All of the haptens were 13 1.85 (2H, m, J = 7.4 Hz CH2CH2CH2). C NMR (DMSO) d: 174.34 conjugated to OVA for coating antigens. Hapten S9 was diazotized (COOH), 165.43, 144.44, 142.94, 138.97, 132.81, 129.86, 129.86, and conjugated to OVA according to the published method [30]. 129.13, 129.02, 128.91, 127.96, 126.22, 122.75, 121.37, 119.80, Conjugates were separated from uncoupled haptens by dialyz-

118.74, 108.78, 108.78 (Ar), 34.26(CH2CH2CH2COOH), 33.18 ing against 0.01 M phosphate-buffered saline (PBS, pH 7.4) at (CH2CH2CH2COOH), 26.28 (CH2CH2CH2COOH). 4 °C for 3 days in the dark with frequent changes of the dialysis solution. The conjugate was lyophilized and stored at 4 °C until 40-[(2-Hydroxy naphthalen-1-yl) diazenyl]biphenyl-4-carboxylic acid use. (hapten S5) Yield: 40%. 1H NMR (DMSO) d: 15.83 (1H, s, ArOH), 13.05 (1H, s, Immunization protocol COOH), 8.57 (1H, d, J = 8.1 Hz, Ar), 8.05 (1H, d, J = 8.4 Hz, Ar), 8.00– 7.87 (9H, m, Ar), 7.80 (1H, d, J = 8.0 Hz, Ar), 7.46 (1H, t, J = 8.0 Hz, The immunization was done according to the protocol reported Ar), 6.93 (1H, d, J = 9.4 Hz, Ar). 13C NMR (DMSO) d: 168.62 (COOH), previously [22]. Two female New Zealand white rabbits were 144.89, 140.96, 140.04, 139.35, 132.84, 130.13, 130.13, 129.60, immunized for each immunogen (BSA–S1, BSA–S3, BSA–S4, BSA– 129.26, 129.07, 128.81, 128.25, 128.25, 128.03, 126.08, 126.08, S6, and BSA–S8). Final antiserum was collected 5 months following 126.03, 126.03, 123.96, 121.51, 119.77, 119.77 (Ar). the first immunization. Antiserum was obtained by centrifugation, stored at 20 °C, and used without purification. 6-Hydroxy-5-(phenyl diazenyl)-2-naphthoic acid (hapten S6) Yield: 58%. 1H NMR (DMSO) d: 15.84 (1H, s, ArOH), 13.06 (1H, s, Screening of antisera and coating conjugates COOH), 8.64 (1H, d, J = 8.6 Hz, Ar), 8.44 (1H, s, Ar), 8.11 (2H, t, J = 9.1 Hz, Ar), 7.90 (2H, d, J = 7.0 Hz, Ar), 7.58 (2H, t, J = 7.8 Hz, Optimum concentrations of antisera and coating conjugates Ar), 7.44 (1H, t, J = 7.3 Hz, Ar), 7.03 (1H, d, J = 9.4 Hz, Ar). 13C were chosen to produce absorbance values of approximately 0.6– NMR (DMSO) d: 168.60 (COOH), 167.23, 145.33, 140.04, 135.81, 1.2 U in the absence of analyte by the checkerboard titration. For 130.97, 129.97, 129.97, 129.02, 128.85, 128.85, 127.86, 127.38, this purpose, the affinity of antisera to different coating antigens 124.67, 121.63, 119.57, 119.57 (Ar). was determined in a noncompetitive indirect ELISA format by measuring the binding of serial dilutions (from 1:1000 to 1:128,000) of (E)-2-[1-(Phenyl diazenyl)naphthalen-2-yloxy]acetic acid (hapten S7) each antiserum to microtiter plates coated with different concen- A solution of ethyl bromoacetate (1.48 g) was added, under trations (from 1 to 1000 ng/ml) of each coating conjugate. nitrogen protection, into a suspension of Sudan red I (1.05 g) and potassium carbonate (10 g) in acetone (100 ml). The mixture was Competitive indirect ELISA heated in a status of circumfluence reaction for 24 h and cooled down to room temperature. Potassium carbonate was eliminated The effects of all the possible combinations of antiserum and from the mixture by vacuum filtrating through Whatman number coating antigen on the sensitivity of ELISA were evaluated by a 1 filter paper, followed by removing solvent from the filtrate. The competitive indirect ELISA using Sudan red I as competitor analyte. remains were dissolved in 50 ml of methanol with 4% (w/v) so- The most sensitive assay was found in the combination of antise- dium hydroxide. This solution was kept in circumfluence reaction rum against BSA–S4 and coating antigen OVA–S1, and then a set for 2 h and cooled down at room temperature. After removal of of experimental parameters (pH, ionic strength, and solvent) was methanol, the residue was dissolved in 50 ml of water. When the studied sequentially to evaluate their effects on the assay perfor- solution was acidified to approximately pH 6.0 with 6 M HCl, red mance. The effect of pH was evaluated using different PBST (PBS precipitations were separated out from the solution. The precipita- plus 0.05% Tween 20) pH values ranging from 4.9 to 10.0. To esti- tions were collected and dried in the air. Recrystallization of the mate the influence of ionic strength, concentrations of NaCl rang- crude precipitations in ether gave the final product (E)-2-[1-(phe- ing from 0 to 1.09 M were tested. The effect of methanol nyl diazenyl)naphthalen-2-yloxy]acetic acid (hapten S7). Yield: percentages ranging from 0% to 40% on the assay performance 42%. 1H NMR (DMSO) d: 13.11 (1H, s, COOH), 8.25 (1H, d, was studied. ELISA specific to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49 45

The optimized competitive ELISA was developed as follows. All Results and discussion of the incubations were performed at room temperature except the incubation with coating antigens. Microtiter plates were incubated Hapten design and synthesis with OVA–S1 conjugate (100 ng/ml, 100 ll/well) in 0.05 M carbonate–bicarbonate buffer (pH 9.6) overnight at 4 °C. The following To obtain desirable antibodies recognizing small molecules, day, the coated plates were washed four times with PBST and were suitable haptens having a spacer arm (linker) need to be synthe- blocked by incubation with 1% OVA in PBS (200 ll/well) for 1 h. sized and conjugated to proteins to elicit the immune response After another washing step, 50 ll per well of serial dilutions of of the host animal. Because production of antibodies is time- and the Sudan red I in 10% methanol–PBST was added, followed by cost-consuming, the hapten chemistry should be well considered 50 ll per well of antiserum diluted 1:10,000 with PBST. After an- to develop the most sensitive and selective immunoassay. In the other washing step, 100 ll per well of HRP-conjugated goat anti- case of Sudan red I, four types of haptens were used as antigen rabbit IgG diluted 1:5000 with PBST was added to the plates and (Fig. 2). Hapten type A has a linker joined to the benzene ring incubated for 1 h. The plates were then washed again, and 100 ll (S1–S5). Hapten type B contains a carboxyl acid on the naphthol per well of TMB solution (400 ll of 0.6% TMB–DMSO and 100 ll part of Sudan red I (S6). Hapten type C has a linker attached to of 1% H2O2 diluted with 25 ml of citrate–acetate buffer, pH 5.5) the hydroxyl group (S7 and S8). Hapten type D contains a fragment was added to the plates and incubated for 10 min. The reaction of the target structure (S9 and S10). was stopped by the addition of 50 llof2MH2SO4, and absorbance Haptens S1 to S4 were synthesized by connecting n-(4-amin- was read at 450 nm. The half-maximum inhibition concentration ophehyl)-carboxylic acid with 2-naphthol through an azo bond (IC50) value, an expression of the assay sensitivity, and the LOD, de- and used to evaluate the effect of different lengths of linker on 0 fined as the IC10 value, were obtained from a four-parameter logis- immunoassay. Hapten S5 was obtained by reacting 4 -amino- tic equation. biphenyl-4-carboxylic acid with 2-naphthol. The hydrocarbon linker in S2, S3, and S4 was replaced by a benzoic structure in S5, Cross-reactivity study which was used to study the effect of heterology in linker structure on immunoassay. For hapten S6, the same strategy as above Specificity of the optimized assay was tested by measuring the was implemented by starting new reaction materials 6-hydroxy- cross-reactivity using a group of structurally related compounds, 2-acid and aniline, introducing a carboxylic acid on the naphthol including Sudan red dyes, Para red, and the metabolites of Sudan part of Sudan red I. Another strategy was to use the hydroxyl dyes. The cross-reactivity (CR) was calculated as follows: CR group to obtain an ether ended in a carboxylic acid. Due to the (%) = [IC50 (Sudan red I)/IC50 (interferent)] 100. acidity of hydroxyl group, it is easy to generate the corresponding alkoxide with potassium carbonate; the subsequent reaction Sample preparation with ethyl bromoacetate (or 5-bromovaleric acid ethyl ester), followed by ester hydrolysis, leads to the acid S7 (or S8) in a good Tomato sauce or chili powder sample (2.0 g) free from the con- yield (Fig. 2). The use of fragmentary haptens provided the best tamination of Sudan dyes was weighed into a 50-ml beaker. An coating antigens in other immunoassays [31,32]. 1-Amino-2- appropriate amount of Sudan red I dissolved in methanol was added naphthol and 6-hydroxy-2-naphthoic acid were also used as hap- into the sample to make the final concentration of Sudan red I at four tens S9 and S10, respectively, which contain the naphthol struc- levels (0.1, 0.5, 2.0, and 10 lg/g in tomato sauce or 0.2, 1.0, 5.0, and ture of Sudan red I and the functional groups capable of coupling 20 lg/g in chili powder). The fortified sample was homogenized and protein. kept at 4 °C overnight. Sudan red I was extracted according to the The objective of preparing 10 haptens with varying linker and method reported by Ma and coworkers [16] with slight modifica- attaching positions in this study was 2-fold. The first objective tions. Briefly, 20 ml of methanol was infused into the sample and was to obtain diverse antisera from a multitude of haptens for a the extraction was performed ultrasonically for 5 min, followed by wider option of selecting a proper antiserum, and the selected hap- centrifugation at 5000 rpm for 10 min. The supernatant was col- tens for immunizing were type A (S1, S3, and S4), type B (S6), and lected and evaporated to dryness under vacuum at 50 °C. The resi- type C (S8). The other one was to investigate the effect of heterol- due was reconstituted with 5 ml of PBST containing 10% methanol ogy between immunizing and coating antigen haptens on the affin- and was vortexed occasionally at room temperature for 30 s. After ity of antibodies to the haptens and sensitivity of ELISA. passing through a 0.45-lm cellulose acetate membrane filter (All- tech, Unterhaching, Germany), the filtrate was analyzed with ELISA. Each sample was extracted in triplicate, and each extract was deter- Screening of antisera mined in triplicate. The blank (unspiked) samples were extracted in the same manner and used as control. The antisera collected after each boost were subjected to titration by the homology indirect ELISA. All of the antisera showed rel- HPLC analysis atively constant high titers after the fifth immunization and no significant affinity for OVA alone (data not shown). After the final Samples were extracted as described above except that residues bleeding, a battery of 10 different coating antigens was evaluated, were reconstituted with 2 ml of methanol and filtered through a and the results of the screening of the 10 antisera against each of 0.45-lm nylon membrane prior to HPLC analysis. the coating antigens are presented in Table 1. It would be difficult Sudan red I was analyzed on an Agilent HPLC system (Agilent to draw solid conclusions from the results of this type of experi- Technologies, Palo Alto, CA, USA) equipped with an Agilent eclipse ment because the immune responses may present a large interin-

XDB-C18 column (4.6 150 mm, 5 lm). The injection volume was dividual variation among animals and the nature of the target 20 ll. The mobile phase consisted of acetonitrile and water con- compounds and carrier proteins may affect the final behavior of taining 0.1% formic acid at a volume ratio of 70:30. The flow rate the antibodies. However, the results of the experiment showed was 1.0 ml/min. The detection wavelength was set at 478 nm. that there are certain trends in the behavior of the antibodies. The retention time of Sudan red I was 10.5 min. The concentrations Haptens S1, S3, and S4 differ only in the length of the linker, and of Sudan red I were calculated by calibration with the peak areas of they have been proven to be accessible to produce specific external Sudan red I standard. antibody against Sudan red I [22–26]. When they were used for 46 ELISA specific to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49

Table 1 Antiserum titration results with different coating antigens.

Antiserum Coating antigen (OVA–hapten) Immunogen Rabbit S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 BSA–S1 I HHHHL MNNL N IIHHHHL L NNNN BSA–S3 I HHHHMHNNNN IIHHHHL HNNL N BSA–S4 I HHHHMHLLLL IIHHHHMHL NL N BSA–S6 I L MMML H NNNN IILLLMLHNNNN BSA–S8 I NLLLNNHHNN IINNNNNNHHNN

Note. The concentration of each coating antigen is 10 ng per well. L, M, and H are arbitrary units corresponding to the dilution factor applied to the antiserum (L < 1:8000; 1:8000 6 M 6 1:32,000; H > 1:32,000) to obtain a signal absorbance between 0.6 and 1.2. N indicates a signal absorbance of less than 0.2 when the antiserum dilution is 1:1000. immunization, high titers were observed in all of the homologous To address the question of the effect of heterology on the affin- formats as well as in linker length heterologous formats (Table 1), ity and specificity of the antibodies as well as the question as to the suggesting that the heterology in linker length is not important for optimum coating antigen for immunoassay, the combination of the antigen recognition by these antisera. Low or medium titers antiserum S4 with different coating antigens is particularly dis- were observed in the linker structure heterology (S5) and in most cussed here. The IC50 values are dependent on the coating antigen of the linker position heterologies; nevertheless, high titers used (Table 2). As can be seen in the linker length heterology (S4/ showed up in the antisera produced from S3 and S4 against coating S1–S3), the shorter the linker length, the better sensitivity ob- antigen S6 (Table 1). The antiserum from S6 with zero spacer arm tained. Heterology is commonly used to eliminate problems asso- length shows high titers in homologies and medium titers in some ciated with the strong affinity of the antibodies to the spacer arm heterologies (Table 1); therefore, masking the hapten determinant that leads to no or poor inhibition by the target compound [35]. groups by using a short spacer arm does not always happen. In Thus, lower analyte concentrations can compete with these re- agreement with our view are several reports that have described agents, which result in better assay sensitivity. Hapten S1 is more the development of an acceptable assay using immunizing haptens structurally different from the immunizing hapten mimicking S4 with zero spacer arm length [33,34]. The antisera produced with and, thus, increases the assay sensitivity more remarkably, with

S8, which lacks the hydroxyl group in the naphthalene ring, gave the lowest IC50 of 1.8 ng/ml (Table 2). Perhaps due to the contribu- high titers only in the homology and the length heterology (S8/ tion of the benzoic linker in S5 and the difference of link site in S6, S7)(Table 1). It is noteworthy that all antisera show low or no the afﬁnity of antiserum to these haptens is also decreased and the titers against the fragment haptens S9 and S10 (Table 1), indicating assay sensitivity is improved (Table 2). It is not a surprise that high that the antibodies could not recognize these fragments well. IC50 values were observed in the heterology of coating haptens S7 and S8 because of the low afﬁnity of antiserum to these two hap- Competitive ELISA tens. Although low IC50 is observed in the heterology of fragmentary haptens S9 and S10, the maximum signal (A0) is weak and All of the combinations of antisera and coating antigens that the concentrations of antiserum and coating antigen used are high showed good titration results were used to carry out competitive (data not shown). In the current study, the heterology of antiserum assays so as to determine the most sensitive assay for Sudan red against hapten S4 and coating antigen S1 was selected for further I, and the results are given in Table 2. As expected, the antisera immunoassay development due to both high maximum signal from different haptens showed different patterns of recognition (data not shown) and the lowest IC50 value (Table 2). to Sudan red I. The antiserum from hapten S8 showed high IC50 values in both homology and heterology ELISAs, whereas antisera Chemical effects on ELISA from haptens S1, S3, and S6 showed medium IC50 values. The low IC50 values were obtained mainly in the heterology ELISAs The effects of different variables on assay performance (A0 and relating to the antiserum raised against immunizing hapten S4 (Ta- IC50) were studied at room temperature by the selected heterology ble 2), suggesting that this hapten containing a 3-hydrocarbon lin- ELISA. First, the effect of pH on the assay was tested (Fig. 3). IC50 ker at the para position of the benzene ring is the closest mimic of values and A0 changed approximately between 1.6 and 9.6 ng/ml Sudan red I among the synthesized haptens. and between 0.55 and 1.13 AU (arbitrary units), respectively, in a

Table 2

IC50 values (ng/ml) of Sudan red I by ELISA with different combinations of antisera and coating antigens.

Antiserum against Coating antigen (OVA–hapten) different immunogensa S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 BSA–S1 14.9 13.7 15.2 8.4 12.3 22.5 NA NA 34.6 NA BSA–S3 8.8 12.8 17.5 18.2 11.2 10.9 NA NA NA NA BSA–S4 1.8 6.8 10.2 16.7 2.8 2.5 27.5 30.6 6.6 5.7 BSA–S6 45.2 14.7 14.2 8.8 58.5 25.6 NA NA NA NA BSA–S8 NA NA NA NA NA NA 155 276 NA NA

Note. NA, no analysis. a For each immunogen, only data from the antiserum having higher titer by the homologous ELISA are shown. ELISA speciﬁc to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49 47

12 1.4 16 2.5

10 1.2 12 2.0 8 1.0 (ng/ml) (ng/ml) (AU) (AU) o 50 50 6 8 1.5 o A A IC IC 0.8 4 4 1.0 0.6 2

0 0.4 0 0.5 4 5 6 7 8 9 10 11 010203040 pH Methanol (%) Fig. 3. Effects of pH on the ELISA of Sudan red I. The data are averages of three Fig. 5. Effects of methanol percentage on the ELISA of Sudan red I. The data are replicates. averages of three replicates. pH range from 4.9 to 10.0. There were no signiﬁcant effects of pH ranging from 5.9 to 8.0 in the buffer on the IC50 value. The best 100 combination of IC50/A0 (IC50 = 1.6 ng/ml/A0 = 1.06) was obtained at pH 7.4, so this pH was selected for further optimization assays. The effect of ionic strength on the assay performance is shown 80 in Fig. 4. Essentially, IC50 and A0 values decreased gradually as buffer salt concentration increased, as has been observed previously for other immunoassays [31,36], probably due to similar biochem- 60 ical interactions between analyte and antibody. In the current study, the concentration of NaCl in PBST selected as a compromise 40 between signal and IC50 was 0.137 M. Methanol is commonly used in immunoassays and has been reported to cause the least negative effects on the performance of of control Percentage 20 immunoassays. Therefore, under the selected conditions above, the effect of methanol concentration on ELISA was studied. As shown in Fig. 5, A and IC increased as the proportion of metha- 0 50 0 nol increased, probably because the solvent enhanced antibody 10-2 10-1 100 101 102 binding to the coating antigen. Methanol was tolerated up to 10% Concentration of Sudan red I (ng/ml) with the good A0 at 1.10 AU and the low IC50 value at 1.7 ng/ml. Even under 20% of methanol, the sensitivity of ELISA is still good, Fig. 6. Calibration curve of Sudan red I by indirect ELISA. Plates were coated with maintaining the IC50 value at an acceptable level (3.6 ng/ml). 10 ng of OVA–S1 per well, and the antiserum produced with BSA–S4 was diluted Finally, the optimized ELISA used the assay buffer with 10% 10,000-fold in PBST. The data are averages of four replicates. methanol in 0.137 M PBST (pH 7.4). Fig. 6 shows a typical dose–re-

sponse curve of Sudan red I. This heterologous assay had a linear

range (IC20–80) of 0.1–14 ng/ml in the buffer system and an IC50 8 1.8 value of 1.6 ng/ml. The LOD in the buffer was deﬁned as the IC10 value (0.03 ng/ml). This result is comparable to the results in pre- vious reports [22–26], with the LOD in the range of 0.01–0.2 ng/ml. 1.5 6 Cross-reactivity study 1.2 (ng/ml)

(AU) Assay speciﬁcity was evaluated using a set of compounds struc-

50 4 o turally related to Sudan red I, and the cross-reactivity data for each IC A 0.9 compound is given in Table 3. As noted, all of the tested Sudan red dyes did not show significant cross-reactivity with Sudan red I 2 (<0.3%). A previously reported homogeneous ELISA [22], based on 0.6 the polyclonal antisera and coating antigen from the same hapten S4, showed higher cross-reactivity with Sudan II, III, and IV (3.4%, 9.5%, and 1.9%, respectively). It is suggested that the interindividual 0 0.3 variation among host animals and the assay format may affect the 0.0 0.2 0.4 0.6 0.8 1.0 1.2 specificity of the antibodies. The highest interference came from Concentration of NaCl (M) Para red, another widely used dye in foodstuffs, showing a cross- Fig. 4. Effects of ionic strength on the ELISA of Sudan red I. The data are averages of reactivity of 12.8% (Table 3). This result can be explained by the three replicates. fact that Para red is structurally different from Sudan red I, with 48 ELISA specific to Sudan red I / T. Xu et al. / Anal. Biochem. 405 (2010) 41–49

Table 3 method (Table 4). The results were comparable to those of HPLC Cross-reactivity of the antiserum with compounds structurally with average recoveries of 82–114% and CVs of 5.4–12.5% (Table 4). related to Sudan red I. These results showed that the ELISA could be useful for the pri- Compound Cross-reactivity (%) mary semiquantitative screening of Sudan red I in food matrices. Sudan red I 100 Sudan red II 0.11 Conclusions Sudan red III 0.23 Sudan red IV 0.22 Sudan red G <0.1 To develop a sensitive and selective immunoassay for Sudan red Para red 12.8 I, four types of hapten (differing in the structure of linker) have 1-Amino-2-naphthol 0.14 been synthesized to generate polyclonal antibodies and coating Sunset yellow <0.01 antigens. The introduction of a 3-hydrocarbon spacer at the para 2-Naphthol <0.01 Aniline <0.01 position of the benzene ring in a near perfect molecular mimic of Sudan red I (S4) aids in the recognition of Sudan red I while the antiserum is generated. A heterologous coating hapten (S1) having zero spacer between the Sudan red I and carrier protein dramati- only an extra nitro in the para position of benzene (Fig. 1). A mono- cally influences the affinity of the antiserum for the analyte in clonal antibody produced from hapten S1 showed very high cross- the competitive assay and, consequently, results in the highest reactivity with Sudan red I (100%), Sudan red III (91.3%), and Para sensitivity assay. The IC and LOD values of this assay for Sudan red (115.7%) [24]. Among the other tested compounds, only 1-ami- 50 red I are 1.6 and 0.03 ng/ml, respectively. Among the tested com- no-2-naphthol, the main metabolite of Sudan dyes [37], showed a pounds, only Para red exhibited a moderate cross-reactivity little cross-reactivity (0.14%) with Sudan red I. However, this com- (12.8%) with Sudan red I. In addition, acceptable recoveries (70– pound was rarely detected in foodstuffs and would not represent 97%) were obtained when applying this assay to the fortified toma- real interference. Sunset yellow, 2-naphthol, and aniline showed to sauce and chili powder samples. negligible cross-reactivity with Sudan red I (<0.01%). These results confirm that the designed hapten S4 is valuable for eliciting high- Acknowledgment affinity antisera to Sudan red I suitable for its specific detection at low microgram per liter levels. This work was supported, in part, by Beijing Municipal Sciences and Technology Committee and Beijing Municipal Commission of Matrix effect and ELISA validation Education, China.

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