Food Chemistry 125 (2011) 1359–1364

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Food Chemistry

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

Analytical Methods

An ultra-sensitive monoclonal antibody-based competitive enzymeimmunoassay for aflatoxin M1 in milk and infant milk products

Di Guan a,b,c,1, Peiwu Li a,b,c,⇑,1, Qi Zhang b,c,⇑, Wen Zhang a,b, Daohong Zhang a,b, Jun Jiang a,b

a Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Chinab Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Wuhan 430062, Chinac Quality Inspection and Test Center for Oilseeds Products MOA PRC, Wuhan 430062, China

a r t i c l e i n f o

Article history:Received 31 December 2009Received in revised form 30 September2010Accepted 3 October 2010

Keywords:Aflatoxin M1

Monoclonal antibodyEnzyme-linked immunosorbent assayMilk

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.006

⇑ Corresponding authors. Address: Oil Crops Researcof Agricultural Sciences, Wuhan 430062, China. Tel.: +86812862 (P. Li). Key Laboratory of Oil Crop Biology oWuhan 430062, China (Q. Zhang).

E-mail address: peiwuli@oilcrops.cn (P. Li).1 Both of Di Guan and Peiwu Li ranked as first autho

a b s t r a c t

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M1 (AFM1), named as 2C9, wasselected by semi-solid HAT medium. It exhibited high affinity for AFM1 of 1.74 � 109 L/mol and no cross-reactivity to aflatoxin B1, B2, G1 and G2. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM1 in milk and infant milk products. Assays wereperformed in the AFM1-BSA coated (0.0625 lg/mL) ELISA format in which the antibody was diluted1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assayperformance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-basedcereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery rangedfrom 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data werecompared with a reference high-performance liquid chromatography method.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction Several methods have been described for the determination of

Aflatoxins are highly toxic and carcinogenic compounds, whichare a group of structurally related toxic metabolites produced byAspergillus flavus, Aspergillus parasticus and Aspergillus nomius (Crep-py, 2002; Sweeney & Dobson, 1998). Aflatoxins frequently contam-inate a wide range of foods and animal feedstuffs. Aflatoxin B1 (AFB1)is the most toxic. And World Health Organization classifies AFB1 as ahuman carcinogen and proposes no safe dose (Anklam, Stroka, &Boenke, 2002). AFM1 is the hydroxylated metabolite of AFB1. Lactat-ing animals that ingest feedstuffs contaminated with AFB1 excreteAFM1 into milk (Polan, Hayes, & Campbell, 1974), and subsequentlyit can be found in a large variety of milk products. Although AFM1 isless carcinogenic and mutagenic than AFB1 (Neal, Eaton, Judah, &Verma, 1998), it is also a health danger and has been classified asGroup 1 carcinogen (IARC, 2002). As milk and milk products arewidely consumed by infants and children who are more susceptibleto adverse effect of mycotoxins, the presence of AFM1 in milk repre-sents a worldwide concern. The European Union limit for AFM1 is0.050 lg/L in milk and 0.025 lg/kg in infant formulas (Byrne,2004). However, in China and United States, the regulations keepAFM1 levels below 0.5 lg/L.

ll rights reserved.

h Institute, Chinese Academy86 27 86812943; fax: +86 27f the Ministry of Agriculture,

rs.

AFM1, including the fluorescence detection after immunoaffinityclean-up (Chiavaro, Cacchioli, Berni, & Spotti, 2005), high perfor-mance liquid chromatography with fluorescent detector (Beebe &Takahashi, 1980; Dragacci, Grosso, & Gilbert, 2001) or mass spec-trometry (Cavaliere, Foglia, Pastorini, Samperi, & Laganà, 2006).These techniques have high sensitivity and accuracy, but requireextensive sample preparation, expensive equipment and well-trained personnel. Recently, ELISA methods have also been de-scribed and were mainly used in routine analysis (Anfossi et al.,2008; Pei, Zhang, Eremin, & Lee, 2009; Thirumala-Devi et al.,2002). These methods have been shown to be simple, the portabil-ity of the equipment, hand-holding validation, and reliable for theanalysis of a large number of samples. Considering the hugerequirement of antibodies, we here prepared a sensitive and spe-cific Mab against AFM1, which has benefits of uniform, constantproperties and unlimited production (Li, Zhang, & Zhang, 2009).With these Mabs, we developed, optimised and validated anultra-sensitive competitive immunoassay for AFM1 in milk andinfant milk products.

2. Materials and methods

2.1. Chemicals and instruments

Aflatoxin B1, B2, G1, G2 and M1 standard solution, aflatoxinM1-BSA conjugate (4 mol aflatoxin M1 per mol BSA), goatanti-mouse immunoglobulin horseradish peroxidase (IgG-HRP),

1360 D. Guan et al. / Food Chemistry 125 (2011) 1359–1364

mouse monoclonal antibody ISO2-1 kits, OVA (agrose gel electro-phoresis grade), methyl cellulose, complete and incompleteFreund’s adjuvants, 3,30,5,50-tetramethyl benzidine (TMB), hypo-xanthine–aminopterin–thymidine (HAT), hypoxanthine-thymidine(HT), and polyethylene glycol 1450 (PEG 1450, 50%) were pur-chased from Sigma–Aldrich (St. Louis, MO). RPMI-1640 mediumwith l-glutamine and HEPES (free acid, 283.3 g/L) were obtainedfrom HyClone. Fetal bovine serum, penicillin (+10,000 U/mL) andstreptomycin (+10,000 lg/mL) were from Gibco. Hybridoma Fu-sion and Cloning Supplement (HFCS) was obtained from Roche(Switzerland). Unless otherwise stated, all other inorganic chemi-cals and organic solvents were of analytical reagent grade or better.Water was obtained from a MilliQ purification system (Millipore).Female Balb/c mice were purchased from Centers for Disease Con-trol and Prevention of Hubei province.

The absorbance at 450 nm was detected by using a SpectraMaxM2e microplate reader (Molecular Devices, USA). Cell cultureplates (6, 24 and 96 wells) were from Iwaki, Japan. Polystyrene96-well microtiter plates were from Costar (Corning, Massachu-setts, USA). HPLC series (Agilent 1200) were consisted of a fluores-cence detector, a ODS-3 column (3 lm particle size, 150 � 4.6 mmI.D.), for which samples were cleaned up by immunoaffinity col-umns supplied by Beijing Chinainvent Instrument Tech. Co. Ltd.(Beijing, PRC).

2.2. Competitive ELISA

Plates were coated at 37 �C with 100 lL/well of the appropriateAFM1-BSA concentration in 0.05 M carbonate–bicarbonate buffer(pH 9.6). After incubation for 2 h, plates were washed three timeswith PBST (PBS with Tween-20: 8 g/L NaCl, 1.15 g/L Na2HPO4,0.2 g/L KH2PO4, 0.2 g/L KCl, and 0.05% Tween-20, v/v) and thenblocking with 1.5% OVA in PBST (200 lL/well) for 1 h at 37 �C. Afteranother washing step, 50 lL/well of MAb diluted in PBS and 50 lL/well of analyte solution were added, and incubated for 1 h. Follow-ing a washing step, goat anti-mouse HRP conjugate (1:5000 inPBST, 100 lL/well) was added and incubated for 1 h at 37 �C. Theplates were washed again, and 100 lL/well of TMB solution(3.3 lL of 30% H2O2, 400 lL of 0.6% TMB in DMSO per 25 mL of ace-tate buffer, pH 5.5) was added. The color development was stoppedafter 15 min with 2 M H2SO4 (50 lL/well). The absorbance wasmeasured at 450 nm. Sigmoidal curves were fitted to a logisticequation (Raab, 1983) from which IC50 values (concentration atwhich binding of the antibody to the coating antigen is inhibitedby 50%) were determined.

2.3. Non competitive ELISA

A non competitive ELISA was used to determine the antibody ti-ters of mouse sera or cell culture supernatants. The procedureswere identical with that of competitive ELISA except for the detec-tion of the antigen, AFM1 and its related competitors.

2.4. Preparation and characterisation of monoclonal antibodies

2.4.1. ImmunisationThree six-week-old female Balb/c mice were subcutaneously

immunised with the immunogen at approximately three-weekintervals. The immunogen used was AFM1-BSA conjugate (50 lg)dissolved in 0.2 mL of sterilised 0.85% NaCl solution and emulsifiedwith an equal volume of Freund’s adjuvant. The initial injectionwas given using Freund’s complete adjuvant and the rest twoinjections were given with Freund’s incomplete adjuvant. Themouse with the highest titer of antiserum was given booster injec-tion three days before cell fusion. No adjuvant was used for thebooster injection.

2.4.2. Monitoring antiserum titers by two-step procedureAntiserum was collected from the tail vein of each mouse. BSA

was added to eliminate the effect of anti-BSA in serum. The opti-mum concentration of BSA was chosen by non competitive ELISAwith 1 lg/mL BSA coated. Then, the anti-AFM1 titers were checkedby non competitive ELISA as described in Section 2.3.

2.4.3. Cell fusion and selectionThis protocol was modified from Davis, Pennington, Kubler, and

Conscience (1982). Briefly, the mouse with the highest antibody ti-ter was sacrificed, spleen was removed aseptically and splenocyteswere fused with SP2/0 myeloma cells using 50% (v/v) PEG1450.Then the fused cells were mixed with selective semi-solid media(RPMI 1640medium supplemented with 20% (v/v) foetal bovineserum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 1% (v/v) HEPES, 1% (w/v) methyl cellulose, 2% (v/v) HFCS, HAT), and pla-ted to 6-well plate (1.5 mL/well). After two weeks, lots of whitedots visible to the naked eyes were removed into 96-well plates,respectively.

Supernatants from 96-well plates were checked by two-stepscreening procedure. First of all, supernatants were determinedby non competitive ELISA. Then, the positive well was checkedby competitive ELISA using AFM1 as competitor.

2.4.4. Antibody production and purificationThe antibodies were prepared on a large scale as ascetic fluid, by

inoculating the hybridoma cells into pristine-treated BALB/c mice.The IgG fractions were prepared by ammonium sulfate precipita-tion followed by the protein A column (Kobayashi, Oiwa, Kubota,Sakoda, & Goto, 2000).

2.5. Characterisation of antibodies

The isotypes of the Mabs were performed with the isotyping kitaccording to the protocol provided by the manufacturer. To evalu-ate the cross-reactivity of the antibodies, tests were made usingaflatoxin M1, B1, B2, G1 and G2. The protocol used was competitiveELISA as described above. These data were converted to a plot ofantibody inhibition, expressed as B/B0%, where B was the absor-bance at each concentration of analyte and B0 was the absorbancein the absence of analyte. CR for different aflatoxins was deter-mined by comparing the IC50 values of analytes and calculatedas: CR (%) = [IC50 (AFM1)/IC50 (analyte)] � 100 (Kolosova, Shim,Yang, Eremin, & Chung, 2006). The affinities of the antibodies weredetermined by indirect non-competitive ELISA (Beatty, Beatty, &Vlahos, 1987).

2.6. Optimisation of a Competitive ELISA

With the checkerboard procedure, the appropriate concentra-tions of coating antigen were prepared by serial dilutions from0.5 to 0.0625 lg/mL of AFM1-BSA with a dilution factor of 2 inthe carbonate-bicarbonate buffer and primary antibody (seriallydiluted purified monoclonal antibodies with 2-fold dilution from1:2500 to 1:30,000 in the PBS). The optimum reagent concentra-tions were defined as those, which give maximum absorbancearound 1.0 in the absence of analyte with minimum reagent ex-penses (Zeng et al., 2007).

Assay optimisation was performed using AFM1 as the competi-tor analyte. A set of experimental parameters (ionic strength, pH,and blocking reagent) were studied sequentially to improve thesensitivity of the immunoassay. The main criterion used to evalu-ate immunoassay performance was IC50. To determine the effect ofsalt concentration on the assay performance, PBS at 0, 10, 20, 40,80, and 160 mM with constant pH of 7.0 was tested. The effect ofpH was evaluated using different PBS solutions, ranging from pH

D. Guan et al. / Food Chemistry 125 (2011) 1359–1364 1361

5.0 to 9.0. Finally, the influence of blocking reagent (1.5% gelatin,1.5% BSA, and 1.5% OVA) was investigated.

2.7. Analysis of samples

Samples were obtained from local markets and supermarketsand analysed before their expiration dates. Infant formula andmilk-based cereal weaning food (10 g) were suspended in 100 mLof warm deionised water. Subsequently, these samples as well asliquid milk samples were centrifuged at 3500g for 10 min at 4 �C.The upper fat layer was completely removed, and the aqueouslayer was directly used for the analysis.

The quantitative analysis of AFM1 in samples was performed byindirect competitive ELISA. Standard curves were obtained usingAFM1 standard solutions prepared in deionised water or in AFM1-free milk samples extract.

The matrix effect, the recovery percentage and the limit ofdetection were assayed using blank samples.

To evaluate the accuracy and to validate the method, a compar-ative study using both the developed competitive ELISA and a HPLCreference procedure (Wang, Zhang, Zhang, & Shao, 2003) was per-formed. A HPLC system equipped with a 150 � 4.6 mm, particlesize 3 lm, ODS-3 column was used. The mobile phase consistedof acetonitrile and water at a volume ratio of 33:67, delivered tothe column at a rate of 1 mL/min. Detection was made by a spec-

Fig. 1. Influence of different of factors, coating antigen and antibodies ratio (a), pH (b), ionmeans of three independent experiments.

trofluorometer, the excitation and emission wavelengths beingset at 365 and 435 nm, respectively.

3. Results and discussion

3.1. Monitoring antiserum titers

Since AFM1 is low molecular weight and devoid of antigenicity,it must be coupled to protein carrier in order to elicit an immuneresponse. Antibodies produced against a hapten-carrier antigenresult in antibodies against for the hapten, the carrier and alsofor various mixtures of these molecules (Heussner, Moeller, Day,Dietrich & O’Brien, 2007). So the carrier protein used for monitor-ing and screening is different from the immunisation. But commer-cial AFM1-carrier is only AFM1-BSA, we have to prepare AFM1

conjugating with other carrier proteins or develop other methods.Preparation of AFM1-protein conjugates is high cost (AFM1, RMB¥0.1 million per mg) and harmful to the operators. Therefore wedeveloped some other methods to solve this problem.

Two-step procedure was applied to monitor antiserum titers.Mice were immunised with AFM1-BSA, so BSA was added in serumat first to eliminate the effect of anti-BSA. With 20 mg/mL BSA, theabsorbance was decreased to a level similar to blank control value.Therefore the antiserum containing 20 mg/mL BSA was chosen.Then, titers of anti-AFM1 were checked by non competitive ELISA.

ic strength (c) and blocking reagents (d) on the performance of assay. Results are the

Fig. 2. Standard curve for AFM1. Each point represents the mean ± SD from fivedeterminations in competitive ELISA.

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Among the three mice, the titer of 2# mouse was higher than theothers. So the 2# mouse should be used for the cell fusion.

3.2. Cell fusion and selection

Selective semi-solid media used in this paper was modifiedfrom Davis et al. (1982). HFCS added in this media optimises cellgrowth of freshly fused hybridomas. It does not need the prepara-tion of feeder cells. Hybridoma colonies we obtained from thismedium are monoclonal from the start. It avoids the step of reclon-ing and saves much time. 14 to 21 days after cell fusion, 658 iso-lated colonies visible to the naked eye were removed into 96-well plates. The well that had high cell density was tested bynon competitive ELISA. And then, 43 positive hybridomas werescreened out by competitive ELISA. Finally 7 stable clones were ob-tained. One is described in this paper. We will refer to the antibodyproduced by this clone as 2C9.

3.3. Characterisation of antibodies

The isotype of 2C9 was found to be IgG2a. The cross-reactivityof 2C9 to aflatoxins was determined by competitive ELISA. The re-sults demonstrate that the antibodies did not react with aflatoxinB1, B2, G1 and G2, which showed highly specific for AFM1. Theaffinity constant for 2C9 was determined to be 1.74 � 109 L/molby non-competitive ELISA.

Table 1Recovery analysis of AFM1 by competitive ELISA.

Matrix Expect

Milk Within assayb (n = 6) 1560

240Between assayc (n = 6) 15

60240

Milk-based cereal weaning food Within assayb (n = 6) 1560

240Between assayc (n = 6) 15

60240

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

3.4. Optimisation of a competitive ELISA

The optimal coating antigen and antibodies ratio were checkedby checkerboard procedure and competitive ELISA. A satisfyingcompromise between the lowest limit of detection and the mini-mum reagent expenses was obtained by using a 0.0625 lg/mLAFM1-BSA and a 1:10,000 dilution of the antibody (Fig. 1a).

To identify the effect, originating from different physicochemi-cal factors, on ELISA and optimise assay performance, pH, buffer io-nic strength and blocking solution were investigated (Fig. 1).

The value of pH is one of the key factors influencing the charac-teristics of assay. From Fig. 1b, we can see that acid solution resultin lower IC50 value. But it was a little influence at pH 5.0. Therefore,the value of pH 6.0 was selected as the optimum for the assay,based on the favourable IC50 value. Ionic strength influenced ELISAperformance (Fig. 1c). And optimum concentration selected was0.02 M, which led to the lowest IC50 value. The effect of blockingreagents, gelatin, BSA and OVA were tested, because they are usu-ally used in ELISA system to eliminate non-specific binding. Finally,1.5% OVA with the lowest IC50 value was selected (Fig. 1d).

Through studies of several factors, the main parameters of ELISAprocedure were determined: the blocking reagent was 1.5% OVA,pH was 6.0, and ionic strength was 0.02 M.

3.5. Matrix effect, recovery and real sample analysis

To evaluate milk matrix effect, AFM1 calibration curves pro-duced in either deionised water or AFM1-free milk samples extractwere compared. A significant matrix effect was observed by com-paring calibration curves. The calibration curves in milk andmilk-based cereal weaning food were influenced by the new envi-ronment. So we used milk matrix and milk-based cereal weaningfood matrix to prepare calibration curves, respectively (Fig. 2).The IC50 value was 67 ng/L for milk and 80 ng/L for milk-based cer-eal weaning food. The limit of detection, defined as the concentra-tion corresponding to 90% of B/B0, was 3 ng/L for milk and 6 ng/Lfor milk-based cereal weaning food.

Recoveries were assessed (Table 1) by spiking with AFM1 blanksamples. The precision of the method was determined by analysingreplicates of AFM1 fortified milk samples. The assays were carriedout in six replicates on the same day for the within assay precisionevaluation and in six different days for the between assay precisionevaluation. The values of the mean, SD, and RSD were calculated ateach theoretical concentration level and are summarised in Table2. It can be concluded that the use of Mab 2C9 in an optimisedcompetitive ELISA format to analyse AFM1 in milk products dem-onstrated reliable reproducibility.

ed (ng/L) Founda (ng/L) Recoverya (%) RSD (%)

15.8 ± 1.1 105.3 ± 7.3 7.057.6 ± 2.7 96.0 ± 4.5 4.7

233.7 ± 5.9 97.4 ± 2.5 2.516.4 ± 1.3 109.3 ± 8.7 9.456.0 ± 4.5 93.3 ± 7.5 8.0

234.2 ± 10.0 97.6 ± 4.2 4.314.1 ± 1.0 94.1 ± 7.0 7.461.3 ± 3.9 102.2 ± 6.6 6.4

235.6 ± 6.9 98.2 ± 2.9 2.913.8 ± 1.3 91.9 ± 8.8 9.656.3 ± 4.2 93.8 ± 7.0 7.4

236.2 ± 9.0 98.4 ± 3.8 3.8

Table 2Comparison of results obtained using competitive ELISA and reference HPLC method.

Number Sample HPLC ng/L ELISA ng/L

1 Milk 63.4 57.42 284 225.53 37.2 26.34 NDa ND5 68.9 62.16 111.8 81.07 43.4 36.28 52.4 45.49 31.7 28.9

10 262.4 197.811 252.3 214.112 12.1 8.413 ND 4.214 25.4 19.415 301.2 278.716 Milk powder ND ND17 19.1 12.018 20.5 19.919 56.2 48.420 ND ND21 253.4 223.922 106.7 89.923 24.4 21.824 18.2 13.125 Milk based cereal weaning food ND ND26 ND ND27 ND 9.528 21.6 15.829 ND ND30 ND ND

a ND: not detected.

Fig. 3. Correlation of results obtained by both competitive ELISA and referenceHPLC method on milk samples. The linear regression analysis yielded a goodcorrelation between methods (y = 1.1825x + 0.7752, r2 = 0.9838, n = 30).

D. Guan et al. / Food Chemistry 125 (2011) 1359–1364 1363

A total of 30 samples were analysed with developed competi-tive ELISA method. For each sample, two determinations were per-formed and the results were compared with those obtained usingthe reference HPLC method.

Concordant results were obtained with the two methods (Table2): 23 samples were positive for AFM1, and 7 samples were scoredas negative. The linear regression analysis (Fig. 3) yielded a goodcorrelation between methods (y = 1.1825x + 0.7752, r2 = 0.9838,n = 30). Overall, the competitive immunoassay developed in thisstudy can be applied for the determination of AFM1 in milk and in-fant milk products, with accuracy and precision comparable withthose obtained with the reference method.

4. Conclusions

The goal of sensitive and specific monoclonal antibody againstAFM1 was achieved. It exhibited high affinity for AFM1 of1.74 � 109 L/mol and no cross-reactivity to aflatoxin B1, B2, G1

and G2. A competitive ELISA was developed for the detection ofAFM1 by determining the optimal AFM1-BSA coated antigen andMab (2C9) in a checkerboard fashion and competitive ELISA, whichwere defined to be 0.0625 lg/mL and dilution of 1:10,000, respec-tively. Considering the effect of factors such as pH, ionic strength,blocking solution and matrix on the performance of ELISA, theIC50 values of 67 ng/L for milk and 80 ng/L for milk-based cerealweaning food, the limit of detection of 3 and 6 ng/L for differentmilk matrix, inter-assay and intra-assay variations of less than10%, and the recoveries ranged from 91 to 110% were obtained.According to the analysis of milk products, an agreement resultwas obtained when the data were compared with a referencehigh-performance liquid chromatography method. Therefore, theproduced Mab 2C9 and the developed competitive ELISA couldprovide a valuable tool for sensitive determination of AFM1 in milkand infant milk products.

Acknowledgements

This work was supported by the Key Project of Ministry of Agri-culture (2010-G1, 2009-Z46) and Special Foundation of Presidentof the Chinese Agricultural Academy of Sciences.

References

Anfossi, L., Calderara, M., Baggiani, C., Giovannoli, C., Arletti, E., & Giraudi, G. (2008).Development and application of solvent-free extraction for the detection ofaflatoxin M1 in dairy products by enzyme immunoassay. Journal of Agriculturaland Food Chemistry, 56(6), 1852–1857.

Anklam, E., Stroka, J., & Boenke, A. (2002). Acceptance of analytical methods forimplementation of EU legislation with a focus on mycotoxins. Food Control,13(3), 173–183.

Beatty, J. D., Beatty, B. G., & Vlahos, W. G. (1987). Measurement of monoclonalantibody affinity by non-competitive enzyme immunoassay. Journal ofImmunological Methods, 100(1–2), 173–179.

Beebe, R., & Takahashi, D. (1980). Determination of aflatoxin M1 by high-pressureliquid chromatography using fluorescence detection. Journal of Agricultural andFood Chemistry, 28(2), 481–482.

Byrne, D. (2004). Amending Regulation (EC) No 466/2001 as regards aflatoxins andochratoxin A in foods for infants and young children. Official Journal of theEuropean Union. COMMISSION REGULATION (EC) No, vol. 683 (pp. 3–5).

Cavaliere, C., Foglia, P., Pastorini, E., Samperi, R., & Laganà, A. (2006). Liquidchromatography/tandem mass spectrometric confirmatory method fordetermining aflatoxin M1 in cow milk comparison between electrospray andatmospheric pressure photoionization sources. Journal of Chromatography A,1101(1–2), 69–78.

Chiavaro, E., Cacchioli, C., Berni, E., & Spotti, E. (2005). Immunoaffinity clean-up anddirect fluorescence measurement of aflatoxins B1 and M1 in pig liver:Comparison with high-performance liquid chromatography determination.Food Additives and Contaminants: Part A, 22(11), 1154–1161.

Creppy, E. (2002). Update of survey, regulation and toxic effects of mycotoxins inEurope. Toxicology Letters, 127(1–3), 19–28.

Davis, J. M., Pennington, J. E., Kubler, A. M., & Conscience, J. F. (1982). A simple,single-step technique for selecting and cloning hybridomas for the productionof monoclonal antibodies. Journal of Immunological Methods, 50(2), 161–171.

Dragacci, S., Grosso, F., & Gilbert, J. (2001). Immunoaffinity column cleanup withliquid chromatography for determination of aflatoxin M1 in liquid milk:Collaborative study. Journal of AOAC International, 84(2), 437–443.

Heussner, A. H., Moeller, I., Day, B. W., Dietrich, D. R., & O’Brien, E. (2007).Production and characterization of monoclonal antibodies against ochratoxin B.Food and Chemical Toxicology, 45(5), 827–833.

IARC (2002). Some mycotoxins, naphtalene and styrene. IARC monographs on theevaluation of carcinogenic risk to humans: Vol. 82. International Agency forResearch on Cancer. (pp. 171–300).

Kobayashi, N., Oiwa, H., Kubota, K., Sakoda, S., & Goto, J. (2000). Monoclonalantibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody: An approach to noncompetitive haptenimmunoassays based on anti-idiotype or anti-metatype antibodies. Journal ofImmunological Methods, 245(1–2), 95–108.

Kolosova, A. Y., Shim, W. B., Yang, Z. Y., Eremin, S. A., & Chung, D. H. (2006). Directcompetitive ELISA based on a monoclonal antibody for detection of aflatoxin B1.

1364 D. Guan et al. / Food Chemistry 125 (2011) 1359–1364

Stabilization of ELISA kit components and application to grain samples.Analytical and bioanalytical chemistry, 384(1), 286–294.

Li, P., Zhang, Q., & Zhang, W. (2009). Immunoassays for aflatoxins. Trends inAnalytical Chemistry, 28(9), 1115–1126.

Neal, G., Eaton, D., Judah, D., & Verma, A. (1998). Metabolism and toxicity ofaflatoxins M1 and B1 in human-derived in vitro systems. Toxicology and AppliedPharmacology, 151(1), 152–158.

Pei, S., Zhang, Y., Eremin, S., & Lee, W. (2009). Detection of aflatoxin M1 in milkproducts from China by ELISA using monoclonal antibodies. Food Control,20(12), 1080–1085.

Polan, C. E., Hayes, J. R., & Campbell, T. C. (1974). Consumption and fate of aflatoxinB1 by lactating cows. Journal of Agricultural and Food Chemistry, 22(4), 635–638.

Raab, G. (1983). Comparison of a logistic and a mass-action curve forradioimmunoassay data. Clinical Chemistry, 29(10), 1757–1761.

Sweeney, M., & Dobson, A. (1998). Mycotoxin production by Aspergillus, Fusariumand Penicillium species. International journal of food microbiology, 43(3),141–158.

Thirumala-Devi, K., Mayo, M., Hall, A., Craufurd, P., Wheeler, T., Waliyar, F., et al.(2002). Development and application of an indirect competitive enzyme-linkedimmunoassay for aflatoxin M1 in milk and milk-based confectionery. Journal ofAgricultural and Food Chemistry, 50(4), 933–937.

Wang, J., Zhang, P., Zhang, Y., Shao, M. (2003). Determination of aflatoxin M1

content in milk and milk powder-Cleanup by immunoaffinity chromatographyand determination by high-performance liquid chromatography andfluorometer. Standard of PR China, GB/T 18980-2003.

Zeng, K., Yang, T., Zhong, P., Zhou, S., Qu, L., He, J., et al. (2007). Development of anindirect competitive immunoassay for parathion in vegetables. Food Chemistry,102(4), 1076–1082.