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Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection
Talanta 77 (2008) 138–143
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Talanta journal homepage: www.elsevier.com/locate/talanta
Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection A. Radoi ∗ , M. Targa, B. Prieto-Simon, J.-L. Marty IMAGES, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France
a r t i c l e
i n f o
Article history: Received 4 March 2008 Received in revised form 22 May 2008 Accepted 29 May 2008 Available online 5 June 2008 Keywords: Superparamagnetic nanoparticles ELISA Aflatoxin M1 Milk
a b s t r a c t Five different clones of antibodies developed against the aflatoxin M1 were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM1 in milk, in a wide linear working range (4–250 ng/L). The IC50 value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM1 ), showing an excellent percentage of recovery, close to 100%. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxins are highly toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus on a variety of agricultural commodities [1] and [2]. These fungi grow under particular conditions of temperature and humidity on a great variety of food commodities and animal feed materials. Contamination, either before or after harvest, of corn, peanuts, cereal crops, figs, etc., is a common occurrence [3–5]. Aflatoxin M1 (AFM1 ) is the hydroxylated metabolite of aflatoxin B1 (AFB1 ). Mammals that ingest aflatoxin B1 contaminated diets excrete amounts of the principal 4-hydroxylated metabolite known as aflatoxin M1 into milk, and subsequently it can be found in a large variety of dairy products. The toxic and carcinogenic effects of AFM1 have been convincingly demonstrated in laboratory investigations [6]. The demonstrated toxic and carcinogenic effects of AFM1 recently lead WHO-IARC to change its classification from group 2 to group 1 [7]. AFM1 is relatively stable during pasteurisations, storage, and preparation of various dairy products [6] and [8] and therefore AFM1 contamination poses a significant threat to human health, especially to children, who are the major consumers of milk. European Community legislation limits the concentration of aflatoxin M1 , in milk and dried or processed milk products intended for adults, at 0.050 ppb (g/kg) [9] and at 0.025 ppb (g/kg) for milk
intended for infants or for baby-food production [10]. The official methods of sampling and analysis are regulated by the European Commission Directives [11]. A high performance liquid chromatography analysis with fluorimetric detection (HPLC-FD) coupled with a clean-up treatment by immunoaffinity columns (IC) is the reference method used for the determination of aflatoxins in milk [12]. This procedure is long and laborious and requires expensive equipment and well-trained personnel. Other methods for AFM1 determination have also been proposed. Of significant importance are thin-layer chromatography [13], fluorescence detection after immunoaffinity clean-up [14], liquid chromatography coupled to mass spectrometry [15], and immunoenzymatic assays. Immunochemical assays are rapid, simple, specific, sensitive and even in portable format, have become the most common quick methods for the routine analysis of mycotoxins in food and feed materials [16–20]. ELISA (Enzyme-Linked Immunosorbent Assay) is wellestablished as a high throughput assay with low sample volume requirements, and often has less sample clean-up procedures compared to conventional HPLC methods, and its standardisation for the application to milk sample analysis has been reported in International Standards Organisation guidelines [21]. 2. Experimental 2.1. Safety note
∗ Corresponding author. E-mail address: [email protected] (A. Radoi). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.05.048
Aflatoxins are highly carcinogenic and should be handled with extreme care. Aflatoxin contaminated labware should be
A. Radoi et al. / Talanta 77 (2008) 138–143
decontaminated with an aqueous solution of sodium hypochlorite (5%). 2.2. Materials and apparatus Polystyrene microtitre plates, MaxiSorpTM and PolySorpTM , were purchased from NUNCTM (Roskilde, Denmark). The ERM (European Reference Material) BD282 (zero level of AFM1 ) was purchased from the Institute for Reference Materials and Measurements (IRMM, Belgium). I’screen AFLA M1 MILK test kit was from Tecna S.r.l. (Trieste, Italy). Milk samples were obtained from local supermarkets. Aflatoxin M1 (AFM1 , A6428), aflatoxin M1 linked to bovine seric albumin (BSA-AFM1 , A6412), anti-rat goat IgG linked to alkaline phosphatase (rat AbII -AP, A8438), anti-mouse IgG linked to alkaline phosphatase (AbII -AP, A3562) or to horseradish peroxidase (AbII HRP, A4416), 3,3 ,5,5 -tetramethylbenzidine (TMB, T0440) liquid substrate for ELISA, bovine seric albumin (A9647) were purchased from Sigma (St. Louis, MO, USA). Alkaline phosphatase substrate (p-nitrophenol) was from Fluka Chemie (Buchs, Switzerland). Superparamagnetic nanoparticles (d = 300 nm) coated with affinity purified goat anti-mouse IgG (Bio-Adembeads Antibodies Goat anti-Mouse IgG) and Bio-Adembeads Protein G (uniform sized superparamagnetic nanoparticles conjugated with protein G) were from Ademtech S.A. (Pessac, France). Five different types of monoclonal antibodies against aflatoxin M1 were tested: clone 1C6 (Acris Antibodies GmbH, Hiddenhausen, Germany, 0.5 mg/mL), clones 3G11 and 6G4 (Soft Flow Biotech˝ Hungary, 1 mg/mL) and clones (confidential nology Ltd., Gödöllo, source) ATX9 (1.78 mg/mL) and ATX2 (0.93 mg/mL). All other reagents were from Sigma (St. Louis, MO, USA). A Multiskan EX (Thermo Life Sciences, Cergy-Pontoise, France) microplate photometer was utilised for colorimetric measurements. Adem-Mag 96 (adapted for 96-well microtitre plates) and Adem-Mag SV (single magnet position adapted for both 1.5 mL or 2 mL microtubes) were from Ademtech S.A. (Pessac, France). A horizontal shaker (IKA, Vibrax-VXR) was also utilised. 2.3. Samples preparation Milk samples (available on local markets) were centrifuged (10 min, 3000 × g, 10 ◦ C, Beckman centrifuge-model J2-21), and the skimmed milk was assayed. All the samples were also assayed using the I’screen AFLA M1 MILK test kit, following the recommendations indicated by the supplier. The ERM-BD282 milk powder was handled as recommended in the instructions for use, data provided with the certified material. For recovery studies, it was spiked before centrifugation (10 min, 3000 × g, 10 ◦ C) and recovery percentage was calculated. 2.4. Spectrophotometric ELISA 2.4.1. Indirect competitive ELISA (ic-ELISA) protocol In the indirect competitive ELISA (ic-ELISA) format, the BSAAFM1 was adsorbed onto the wells of the microtitre plate (MaxiSorpTM ) during the coating step, performed in 50 mM carbonate buffer (CB), pH 9.80. The coating volume was 100 L/well and the plate was incubated at 4 ◦ C, over night (ON). Then the incubated ELISA plate was covered with 150 L/well of 1% (w/v) BSA solution (blocking step) prepared in 15 mM phosphate buffer saline (PBS), pH 7.40, for 45 min, at room temperature (RT, 22 ◦ C). The competition was allowed to proceed, by adding inside the wells non-labelled aflatoxin M1 (90 L) and primary (AbI ) antiAFM1 antibody (10 L); solutions were prepared in PBS and the
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competition time was 1 h, at room temperature. Then a solution (100 L) prepared in PBS of secondary (AbII ) anti-IgG antibody was added and allowed to react (1 h), at RT. Finally, 100 L of substrate solution was added and the absorbance was read. Washing (3 × 200 L) was performed after each step, by using a solution of 0.05% (v/v) of Tween prepared in PBS (2 × 200 L) followed by only PBS (1 × 200 L). All the five antibodies (clones 1C6, 3G11, 6G4, ATX9 and ATX2) developed against AFM1 were assayed in the ic-ELISA format. The optimised conditions (coating concentrations and dilutions of primary antibody), for the three different clones, when using an alkaline phosphatase labelled secondary antibody (AbII -AP, 1/1000, v/v), were: 3G11 (1/1280, v/v) and BSA-AFM1 (25 ng/mL); 6G4 (1/1280, v/v) and BSA-AFM1 (25 ng/mL); ATX2 (1/1600, v/v) and BSA-AFM1 (12.5 ng/mL). A solution of para-nitrophenyl phosphate (p-NPP, 2 mg/mL) solubilised in 10% diethanolamine buffer (DEA, pH 9.80) was used as substrate. The absorbance was read after 30 min, using the 405 nm filter. When using as secondary antibody an anti-mouse IgG labelled with horseradish peroxidase (AbII -HRP, 1/2500, v/v) the dilutions of primary antibodies were kept constant as before (i.e. for clones 3G11 and 6G4 the dilution was 1/1280, v/v and for ATX2 clone it was 1/1600, v/v), only the coating concentration varied (25 ng/mL of BSA-AFM1 ). The AbII -HRP was allowed to bind the anti-AFM1 primary antibody during an incubation time of 30 min, and the absorbance was read also after 30 min. TMB liquid substrate for ELISA was used as chromogen, the absorbance being measured at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). 2.4.2. Direct competitive ELISA (dc-ELISA) protocol In the direct competitive ELISA (dc-ELISA) assay, the 96-well ELISA plate (MaxiSorpTM ) was coated with anti-AFM1 antibodies (clones G11, 6G4, and ATX2). The coating step (100 L/well, 4 ◦ C, ON) was performed in 50 mM carbonate buffer, pH 9.80. The blocking step (45 min) was performed at 22 ◦ C, using 150 L/well of 1% (w/v) BSA solution (prepared in 15 mM PBS, pH 7.40). The competition was allowed to proceed for 45 min and at RT, by adding inside the wells non-labelled aflatoxin M1 (90 L) and horseradish peroxidase labelled aflatoxin M1 (AFM1 -HRP, 10 L). Finally, 100 L of substrate solution was added and the absorbance was read 15 min after the TMB solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). Washing (3 × 200 L) was performed after each step, by using a solution of 0.05% (v/v) of Tween prepared in PBS (2 × 200 L) followed by only PBS (1 × 200 L). The optimised conditions (coating and dilution of labelled AFM1 ) for the three different clones were: 3G11 (0.4 g/mL) and AFM1 -HRP (1/180, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/180, v/v); ATX2 (0.5 g/mL) and AFM1 -HRP (1/120, v/v). 2.5. Direct competitive ELISA based on Bio-Adembeads Protein G This simplified version of dc-ELISA format relies on the use of uniform sized superparamagnetic nanoparticles conjugated with protein G as support for immobilizing the anti-AFM1 antibodies. No blocking step was necessary and as working buffer a solution at 0.05% (v/v) of Tween prepared in PBS was employed for all the steps:
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coating, competition and washing. Prior to use the nanoparticles were washed twice with working buffer (1400 L) for removing the Proclin 300. Briefly the optimised procedure was the following:
3G11 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); ATX2 (1.5 g/mL) and AFM1 -HRP (1/80, v/v). 2.7. Calibration curves for spectrophotometric ELISA
coating: 14 L of superparamagnetic nanoparticles conjugated with protein G were dispersed into 1400 L of antibody solution prepared in working buffer and allowed to react for 20 min; then the particles were collected using the Adem-Mag SV and washed twice with working buffer (1400 L) and finally resuspended in 1400 L; 30 L of this dispersion were added inside the wells and the buffer was removed, meanwhile the nanoparticles were collected onto the inner wall of the well by using the Adem-Mag 96; competition: 90 L of AFM1 and 10 L of AFM1 -HRP were allowed to compete (PolySorpTM microtitre plates were utilised) for the antibody binding sites, during 20 min; during coating and competition, an horizontal shaker (200 rpm) was employed. Washing steps were performed (300 L/well, 1 min) and when the different solutions were discarded from the wells, the superparamagnetic nanoparticles were collected by using the magnets available on the Adem-Mag 96. The TMB liquid substrate for ELISA was utilised (150 L/well) and the absorbance was read 15 min after the substrate solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). The optimised amount of antibody against AFM1 and the dilution of AFM1 -HRP used, for each of the three tested clones, is: 3G11 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); ATX2 (0.5 g/mL) and AFM1 -HRP (1/120, v/v). 2.6. Direct competitive ELISA based on Bio-Adembeads anti-Mouse IgG This simplified version of dc-ELISA format relies on the use of superparamagnetic nanoparticles coated with affinity purified goat anti-mouse IgG as support for antibody binding. The procedure is similar with the one previously described, when the Bio-Adembeads Protein G superparamagnetic particles were utilised. Prior to use the nanoparticles were washed twice with working buffer (1400 L) for removing the Proclin 300. The optimised procedure was the following: coating: 13 L of superparamagnetic nanoparticles coated with affinity purified goat anti-mouse IgG were dispersed into 1400 L of antibody solution prepared in working buffer and allowed to react for 20 min; then the particles were collected using the AdemMag SV and washed twice with working buffer (1400 L) and finally resuspended in 1400 L; 100 L of this dispersion were added inside the wells and the buffer was removed; competition: 90 L of AFM1 and 10 L of AFM1 -HRP were allowed to compete (PolySorpTM microtitre plates were utilised) for the antibody binding sites, during 20 min; during coating and competition, an horizontal shaker (200 rpm) was employed. Washing steps were performed as described above. The TMB liquid substrate for ELISA was utilised (150 L/well) and the absorbance was read 15 min after the substrate solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). The optimised amount of antibody against AFM1 and the dilution of AFM1 -HRP used, for each of the three tested clones, is:
Spectrophotometric ELISA standard curves were obtained using AFM1 standard solutions prepared in PBS or in BD282 reference material. Each experiment was performed in triplicate and the mean of each value was used for curve fitting. The calibration curves (absorbance at 650 or 405 nm vs. antigen concentration) were fitted using “non-linear four parameter logistic calibration plots” [22]. To allow the direct comparison of different calibration curves, absorbance values were converted into their corresponding test inhibition values (A/A0 , %) as follows: %
A A − Asat = × 100 A0 A0 − Asat
(1)
where A is the absorbance value of competitors, Asat and A0 are the absorbance values corresponding to the saturating and the non-competition antigen, respectively (as evaluated by the four parameters logistic function). The matrix effect, the recovery percentage and the limit of detection were assayed using blank samples prepared using the ERM-BD282 (zero level of AFM1 ) powder milk. Recovery was assessed by spiking blank milk samples with a known amount of AFM1 standard solution. The detection limit (LOD) was calculated as the concentration corresponding to 90% of A/A0 [23]. The midpoint value (IC50 ) was evaluated as the concentration of AFM1 at 50% A/A0 . The working range was evaluated as the toxin concentration that gives test inhibition values of 80 and 20% of A/A0 . The data obtained for each curve were plotted and fitted using a SigmaPlot software (SPSS), and a regression analysis on the linear portion of the sigmoidal curves was also performed. The slopes obtained from the regression analysis were used to evaluate the matrix effect and the recovery of the assay. 3. Results and discussion 3.1. Spectrophotometric ELISA Indirect and direct competitive ELISA formats were assayed to characterise analytically 5 clones of antibodies developed against AFM1 . This study was aimed also towards the possibility to reduce the time of analysis with respect to the classical ELISA competitive techniques. The indirect competitive format of ELISA was utilised to ascertain how many of the five antibodies (clones 1C6, 3G11, 6G4, ATX9 and ATX2) available in our laboratory were able to bind the aflatoxin M1 . Three of them (clones 3G11, 6G4 and ATX2) were able to recognise and compete for the free AFM1 during the competition step, meanwhile the other two clones were not able to recognise AFM1 (data not shown). Two types of secondary antibodies, labelled with alkaline phosphatase (Fig. 1) or horseradish peroxidase (Fig. 2), were used to trace the AFM1 , but no significant difference, with respect to linear working range or midpoint value, was achieved (Table 1), since the determining factor for this format is the coating step. In fact, maintaining constant the amount of one primary antibody (for example ATX2, 1/1600, v/v) and varying the coating concentrations (50, 25 and 12.5 ng/mL of BSA-AFM1 ), the midpoint value shifted from 430 to 120 and from 120 to 47 ng/L, respectively, when an AbII -AP was used. When using an AbII -HRP only the time analysis was shortened by 30 min, since it was possible to reduce the binding time with the primary antibody,
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Fig. 1. Indirect competitive ELISA; secondary antibody labelled with alkaline phosphatase; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 60 min competition time.
Fig. 2. Indirect competitive ELISA; secondary antibody labelled with horseradish peroxidase; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 60 min competition time. Table 1 Analytical parameters for spectrophotometric ELISA Clone
LWR (ng/L)
IC50 (ng/L)
LOD (ng/L)
ic-ELISA (AbII -AP)-165 min 3G11 15–250 6G4 15–500 ATX2 15–125
75 84 47
15 15 15
ic-ELISA (AbII -HRP)-135 min 3G11 15–250 6G4 8–500 ATX2 8–125
76 72 35
15 8 8
dc-ELISA-90 min 3G11 6G4 ATX2
36 40 36
12.5 12.5 12.5
12.5–80 12.5–80 12.5–100
dc-ELISA Bio-Adembeads Protein G-20 min 3G11 4–250 6G4 12–500 ATX2 30–500
15 50 150
4 12 30
dc-ELISA Bio-Adembeads anti-Mouse IgG-20 min 3G11 8–125 30 6G4 10–500 50 ATX2 10–250 50
8 10 10
LWR: linear working range; IC50 : midpoint value; LOD: limit of detection.
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Fig. 3. Direct competitive ELISA; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 45 min competition time.
presumably due to a better affinity of AbII -HRP towards the primary antibody. When the three clones (3G11, 6G4 and ATX2) were assayed in the direct competitive format (Fig. 3), all of them showed, more or less, an identical midpoint value (around 40 ng/L). The linear working range (LWR) was narrower then the one obtained in the ic-ELISA procedure but the limits of detection were comparable with the ones obtained for the ic-ELISA. However, the dc-ELISA format is more rapid then the ic-ELISA format (90 min against 165 or 135 min) therefore it was further optimised, by transferring the entire procedure (coating, competition, etc.) onto the superparamagnetic nanoparticles coated with protein G or anti-IgG. One of the main advantage of using nanoparticles resides in the short interaction time between the biological components and the protein G or anti-IgG coated nanoparticles. The whole process could be defined as “homogeneous”, since the interaction between the Ag and the Ab takes place at “nano” level. Moreover, being superparamagnetic, these nanoparticles can be easily separated from bulk solution, allowing also a versatile manipulation. Confronted with the ic- and dc-ELISA, the coating time was reduced to 20 min, instead of an over night incubation time (12–14 h). Moreover, the already incubated nanoparticles could be stored separately as ready to use nanobeads for performing just the competition step in a dc-ELISA. One time consuming factor was a priori overcame, since no blocking step was necessary. This was achieved by adding a non-ionic surfactant into the working buffer, as described before. Then, the competition time was even further reduced, when compared with an ic- or dc-ELISA, till 20 min (Figs. 4 and 5). When the competition curves performed by the aid of superparamagnetic nanoparticles were compared with the ones obtained from ordinary ic- and dc-ELISA, a different shape of these curves was evident, since the affinity of the tested clones towards the reactive coating biomaterial of the nanoparticles, protein G and anti-IgG, respectively, hardly depends on this factor. When a dcELISA based on Bio-Adembeads anti-Mouse IgG was performed, the behaviour of the clones 3G11, 6G4 and ATX2 was more or less identical, and similar with the one observed in the classical icand dc-ELISA. A better discrimination was further achieved when a dc-ELISA based on Bio-Adembeads Protein G was assayed. The clone 3G11 showed to be the best antibody, with the lowest IC50 (15 ng/L) and it was further used to assess the matrix effect, the recovery percentage, when ERM-BD282 powder milk was fortified with known amounts of AFM1 , and to test locally available milk samples.
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Fig. 4. Direct competitive ELISA based on Bio-Adembeads Protein G; coating (20 min) performed at room temperature; no blocking step; 20 min competition time.
Fig. 6. Competition curves performed in buffer and in certified reference material (BD282, zero level of AFM1 ); clone 3G11 immobilised on Bio-Adembeads Protein G was utilised to assess the matrix effect.
3.2. Matrix effect, recovery and real sample analysis Certified reference material (BD282, zero level of AFM1 ) was reconstituted as indicated in the certification report supplied by the IRMM, Belgium. After reconstitution and centrifugation, a calibration curve in matrix was performed and it was observed that the calibration curve in milk was influenced by the new environment (Fig. 6). The IC50 shifted almost 3 times (41 ng/L) then the midpoint value obtained in buffer (15 ng/L). The linear working range (4–250 ng/L) was slightly modified, being essentially the same as for the standard competition curve performed in PBS. This is very important since the maximum accepted level of AFM1 (50 ng/L) is well fitted into this linear working range. Recovery was assessed (Table 2) by spiking with aflatoxin M1 the BD282 reconstituted material. The fortified (30, 60 120 ng/L of AFM1 ) blank milk samples were interpolated from the calibration curve performed using reconstituted certified reference material. The precision was determined by calculating the relative standard deviation (%R.S.D.) for the replicate measurements and the accuracy (%R.E.) was calculated by assessing the agreement between measured and nominal concentration of the fortified samples.
Table 2 BD282, zero level of AFM1 certified reference material was employed; clone 3G11 immobilised on Bio-Adembeads Protein G was utilised to asses the recovery efficiency AFM1 added (ng/L)
AFM1 found (ng/L)
R.S.D. (%)
R.E. (%)
Recovery (%)
30 60 120
28 58 118
7 5 3
−7 −3 −2
93 97 98
%R.E. (relative error) = [(measured value − true value)/true value] × 100; %R.S.D. (relative standard deviation) = standard deviation/mean × 100; n = 6.
Six different brands of commercially available milk samples, three declared to be produced following the rules of biological agriculture and three as fresh milk, were assayed and for all of them the response was negative, since the absorbance values were at the same level as for the “no competition” point of the standard calibration curve. The obtained results were confronted with the response obtained by using a commercial available kit for aflatoxin M1 detection (AFLA M1 MILK), and again a negative response was obtained. 4. Conclusions
Fig. 5. Direct competitive ELISA based on Bio-Adembeads anti-Mouse IgG; coating (20 min) performed at room temperature; no blocking step; 20 min competition time.
In this work, five different antibodies developed against the aflatoxin M1 were investigated by using different ELISA schemes based on classical indirect and direct competitive formats, and on the use of superparamagnetic nanoparticles. From all the five investigated antibodies, the most powerful, i.e. showing the best IC50 value and limit of detection, was the clone 3G11. Superparamagnetic nanoparticles conjugated with protein G and anti-mouse IgG were utilised to further reduce the coating (till 20 min, instead of 12–14 h necessary for the over night coating procedure) and the competition time. Also, one time consuming factor was a priori minimised, since no blocking step was necessary. Being superparamagnetic, these nanoparticles are easily separated from the bulk solution, allowing also a versatile manipulation. The use of superparamagnetic nanoparticles allowed us to demonstrate that the classical ELISA procedures, which sometimes are time consuming, could be further improved by decreasing the coating and competition time and by eliminating steps that cannot be neglected, like the blocking step. The feasibility of this dc-ELISA based on the use of pre-coated (protein G or anti-IgG) nanoparticles was confirmed by performing
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competition curves in milk reference material, showing that the matrix effect is not greatly affecting neither the linear working range, neither the recovery rate nor the midpoint value. In conclusion, we have demonstrated that a competitive immunoassay for AFM1 based on the use of superparamagnetic nanoparticles is reliable, easy to perform and time efficient. Acknowledgement This work was supported by a financial contribution from the programme EraSME Food for Better Human Health (project FASTDETECT). References [1] W.F. Busby Jr., G.N. Wogan, Food-Borne Infections and Intoxications, second ed., Academic Press, New York, 1979, pp. 519–610. [2] W.H. Butler, Mycotoxins, Elsevier Scientific Publishing, New York, 1974, pp 1–28. [3] J.W.O. Ellis, P. Smith, B.K. Simpson, Food Sci. Nutr. 30 (1991) 403. [4] J.P. Jouany, Anim. Res. 51 (2002) 81. [5] D. Miller, J. Stor. Prod. Res. 30 (1994) 1. [6] L. Stoloff, J. Food Prot. 43 (1989) 226. [7] IARC, International Agency for Research on Cancer, Monograph on the Evaluation of Carcinogenic Risk to Humans, vol. 82, World Health Organisation, Lyon, France, 2002, p. 171. [8] R.D. Stubblefield, G.M. Shannon, J. Assoc. Off. Anal. Chem. 57 (1974) 847. [9] European Commission Regulation, No. 466/2001/EC of 8 March 2001, Setting Maximum Levels for Certain Contaminants in Foodstuffs, Official Journal of European Communities L077, 2001, pp. 1–13.
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[10] European Commission Regulation, No. 683/2004/EC of 13 April 2004, Amending Regulation (EC) No. 466/2001 as Regards Aflatoxins and Ochratoxin A in Foods for Infants and Young Children, Official Journal of European Communities L106, 2004, pp. 3–5. [11] European Commission Directive, 2002/26/EC of 13 March 2002 Laying Down the Sampling Methods and the Methods of Analysis For The Official Levels of Ochratoxin A in Foodstuffs, Official Journal of European Communities L075, 2002, pp. 38–43. [12] ISO, Milk and milk powder. Determination of aflatoxin M1 content, in: Clean-up by Immunoaffinity Chromatography and Determination by High-Performance Liquid Chromatography. Standard 14501, International Standards Organisation, Geneva, Switzerland, 1998. [13] F. Grosso, J.M. Fremy, S. Bevis, S. Dragacci, Food Addit. Contam. 21 (2004) 348. [14] E. Chiavaro, C. Cavicchioli, E. Berni, E. Spotti, Food Addit. Contam. 22 (2005) 1154. [15] C. Cavaliere, P. Foglia, E. Pastorini, R. Samperi, A. Lagana, J. Chromatogr. A 1101 (2006) 69. [16] M. Magliulo, M. Mirasoli, P. Simoni, R. Lelli, O. Portanti, A. Roda, J. Agric. Food Chem. 53 (2005) 3300. [17] L. Micheli, R. Greco, M. Badea, D. Moscone, G. Palleschi, Biosens. Bioelectron. 21 (2005) 588. [18] M.L. Rodriguez Velasco, M.M. Calonge Delso, D. Ordonez Escudero, Food Addit. Contam. 20 (2003) 276. [19] J. Stroka, E. Anklam, Trends Anal. Chem. 21 (2002) 90. [20] K. Thirumala-Devi, M.A. Mayo, A.J. Hall, J. Agric. Food Chem. 50 (2002) 933. [21] ISO, Milk and milk products, in: Guidelines for a Standardized Description of Competitive Enzyme Immunoassays-Determination of AFM1 Content. Standard 14675, International Standards Organisation, Geneva, Switzerland, 2002. [22] M.J. Warwick, Immunoassay, in: A Practical Guide, Taylor & Francis Ltd., London, UK, 1996, p. 160. [23] A. Dankwardt, Encyclopedia of Analytical Chemistry, Wiley, Chichester, 2001, pp. 1–25.
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Talanta journal homepage: www.elsevier.com/locate/talanta
Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection A. Radoi ∗ , M. Targa, B. Prieto-Simon, J.-L. Marty IMAGES, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France
a r t i c l e
i n f o
Article history: Received 4 March 2008 Received in revised form 22 May 2008 Accepted 29 May 2008 Available online 5 June 2008 Keywords: Superparamagnetic nanoparticles ELISA Aflatoxin M1 Milk
a b s t r a c t Five different clones of antibodies developed against the aflatoxin M1 were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM1 in milk, in a wide linear working range (4–250 ng/L). The IC50 value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM1 ), showing an excellent percentage of recovery, close to 100%. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxins are highly toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus on a variety of agricultural commodities [1] and [2]. These fungi grow under particular conditions of temperature and humidity on a great variety of food commodities and animal feed materials. Contamination, either before or after harvest, of corn, peanuts, cereal crops, figs, etc., is a common occurrence [3–5]. Aflatoxin M1 (AFM1 ) is the hydroxylated metabolite of aflatoxin B1 (AFB1 ). Mammals that ingest aflatoxin B1 contaminated diets excrete amounts of the principal 4-hydroxylated metabolite known as aflatoxin M1 into milk, and subsequently it can be found in a large variety of dairy products. The toxic and carcinogenic effects of AFM1 have been convincingly demonstrated in laboratory investigations [6]. The demonstrated toxic and carcinogenic effects of AFM1 recently lead WHO-IARC to change its classification from group 2 to group 1 [7]. AFM1 is relatively stable during pasteurisations, storage, and preparation of various dairy products [6] and [8] and therefore AFM1 contamination poses a significant threat to human health, especially to children, who are the major consumers of milk. European Community legislation limits the concentration of aflatoxin M1 , in milk and dried or processed milk products intended for adults, at 0.050 ppb (g/kg) [9] and at 0.025 ppb (g/kg) for milk
intended for infants or for baby-food production [10]. The official methods of sampling and analysis are regulated by the European Commission Directives [11]. A high performance liquid chromatography analysis with fluorimetric detection (HPLC-FD) coupled with a clean-up treatment by immunoaffinity columns (IC) is the reference method used for the determination of aflatoxins in milk [12]. This procedure is long and laborious and requires expensive equipment and well-trained personnel. Other methods for AFM1 determination have also been proposed. Of significant importance are thin-layer chromatography [13], fluorescence detection after immunoaffinity clean-up [14], liquid chromatography coupled to mass spectrometry [15], and immunoenzymatic assays. Immunochemical assays are rapid, simple, specific, sensitive and even in portable format, have become the most common quick methods for the routine analysis of mycotoxins in food and feed materials [16–20]. ELISA (Enzyme-Linked Immunosorbent Assay) is wellestablished as a high throughput assay with low sample volume requirements, and often has less sample clean-up procedures compared to conventional HPLC methods, and its standardisation for the application to milk sample analysis has been reported in International Standards Organisation guidelines [21]. 2. Experimental 2.1. Safety note
∗ Corresponding author. E-mail address: [email protected] (A. Radoi). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.05.048
Aflatoxins are highly carcinogenic and should be handled with extreme care. Aflatoxin contaminated labware should be
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decontaminated with an aqueous solution of sodium hypochlorite (5%). 2.2. Materials and apparatus Polystyrene microtitre plates, MaxiSorpTM and PolySorpTM , were purchased from NUNCTM (Roskilde, Denmark). The ERM (European Reference Material) BD282 (zero level of AFM1 ) was purchased from the Institute for Reference Materials and Measurements (IRMM, Belgium). I’screen AFLA M1 MILK test kit was from Tecna S.r.l. (Trieste, Italy). Milk samples were obtained from local supermarkets. Aflatoxin M1 (AFM1 , A6428), aflatoxin M1 linked to bovine seric albumin (BSA-AFM1 , A6412), anti-rat goat IgG linked to alkaline phosphatase (rat AbII -AP, A8438), anti-mouse IgG linked to alkaline phosphatase (AbII -AP, A3562) or to horseradish peroxidase (AbII HRP, A4416), 3,3 ,5,5 -tetramethylbenzidine (TMB, T0440) liquid substrate for ELISA, bovine seric albumin (A9647) were purchased from Sigma (St. Louis, MO, USA). Alkaline phosphatase substrate (p-nitrophenol) was from Fluka Chemie (Buchs, Switzerland). Superparamagnetic nanoparticles (d = 300 nm) coated with affinity purified goat anti-mouse IgG (Bio-Adembeads Antibodies Goat anti-Mouse IgG) and Bio-Adembeads Protein G (uniform sized superparamagnetic nanoparticles conjugated with protein G) were from Ademtech S.A. (Pessac, France). Five different types of monoclonal antibodies against aflatoxin M1 were tested: clone 1C6 (Acris Antibodies GmbH, Hiddenhausen, Germany, 0.5 mg/mL), clones 3G11 and 6G4 (Soft Flow Biotech˝ Hungary, 1 mg/mL) and clones (confidential nology Ltd., Gödöllo, source) ATX9 (1.78 mg/mL) and ATX2 (0.93 mg/mL). All other reagents were from Sigma (St. Louis, MO, USA). A Multiskan EX (Thermo Life Sciences, Cergy-Pontoise, France) microplate photometer was utilised for colorimetric measurements. Adem-Mag 96 (adapted for 96-well microtitre plates) and Adem-Mag SV (single magnet position adapted for both 1.5 mL or 2 mL microtubes) were from Ademtech S.A. (Pessac, France). A horizontal shaker (IKA, Vibrax-VXR) was also utilised. 2.3. Samples preparation Milk samples (available on local markets) were centrifuged (10 min, 3000 × g, 10 ◦ C, Beckman centrifuge-model J2-21), and the skimmed milk was assayed. All the samples were also assayed using the I’screen AFLA M1 MILK test kit, following the recommendations indicated by the supplier. The ERM-BD282 milk powder was handled as recommended in the instructions for use, data provided with the certified material. For recovery studies, it was spiked before centrifugation (10 min, 3000 × g, 10 ◦ C) and recovery percentage was calculated. 2.4. Spectrophotometric ELISA 2.4.1. Indirect competitive ELISA (ic-ELISA) protocol In the indirect competitive ELISA (ic-ELISA) format, the BSAAFM1 was adsorbed onto the wells of the microtitre plate (MaxiSorpTM ) during the coating step, performed in 50 mM carbonate buffer (CB), pH 9.80. The coating volume was 100 L/well and the plate was incubated at 4 ◦ C, over night (ON). Then the incubated ELISA plate was covered with 150 L/well of 1% (w/v) BSA solution (blocking step) prepared in 15 mM phosphate buffer saline (PBS), pH 7.40, for 45 min, at room temperature (RT, 22 ◦ C). The competition was allowed to proceed, by adding inside the wells non-labelled aflatoxin M1 (90 L) and primary (AbI ) antiAFM1 antibody (10 L); solutions were prepared in PBS and the
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competition time was 1 h, at room temperature. Then a solution (100 L) prepared in PBS of secondary (AbII ) anti-IgG antibody was added and allowed to react (1 h), at RT. Finally, 100 L of substrate solution was added and the absorbance was read. Washing (3 × 200 L) was performed after each step, by using a solution of 0.05% (v/v) of Tween prepared in PBS (2 × 200 L) followed by only PBS (1 × 200 L). All the five antibodies (clones 1C6, 3G11, 6G4, ATX9 and ATX2) developed against AFM1 were assayed in the ic-ELISA format. The optimised conditions (coating concentrations and dilutions of primary antibody), for the three different clones, when using an alkaline phosphatase labelled secondary antibody (AbII -AP, 1/1000, v/v), were: 3G11 (1/1280, v/v) and BSA-AFM1 (25 ng/mL); 6G4 (1/1280, v/v) and BSA-AFM1 (25 ng/mL); ATX2 (1/1600, v/v) and BSA-AFM1 (12.5 ng/mL). A solution of para-nitrophenyl phosphate (p-NPP, 2 mg/mL) solubilised in 10% diethanolamine buffer (DEA, pH 9.80) was used as substrate. The absorbance was read after 30 min, using the 405 nm filter. When using as secondary antibody an anti-mouse IgG labelled with horseradish peroxidase (AbII -HRP, 1/2500, v/v) the dilutions of primary antibodies were kept constant as before (i.e. for clones 3G11 and 6G4 the dilution was 1/1280, v/v and for ATX2 clone it was 1/1600, v/v), only the coating concentration varied (25 ng/mL of BSA-AFM1 ). The AbII -HRP was allowed to bind the anti-AFM1 primary antibody during an incubation time of 30 min, and the absorbance was read also after 30 min. TMB liquid substrate for ELISA was used as chromogen, the absorbance being measured at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). 2.4.2. Direct competitive ELISA (dc-ELISA) protocol In the direct competitive ELISA (dc-ELISA) assay, the 96-well ELISA plate (MaxiSorpTM ) was coated with anti-AFM1 antibodies (clones G11, 6G4, and ATX2). The coating step (100 L/well, 4 ◦ C, ON) was performed in 50 mM carbonate buffer, pH 9.80. The blocking step (45 min) was performed at 22 ◦ C, using 150 L/well of 1% (w/v) BSA solution (prepared in 15 mM PBS, pH 7.40). The competition was allowed to proceed for 45 min and at RT, by adding inside the wells non-labelled aflatoxin M1 (90 L) and horseradish peroxidase labelled aflatoxin M1 (AFM1 -HRP, 10 L). Finally, 100 L of substrate solution was added and the absorbance was read 15 min after the TMB solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). Washing (3 × 200 L) was performed after each step, by using a solution of 0.05% (v/v) of Tween prepared in PBS (2 × 200 L) followed by only PBS (1 × 200 L). The optimised conditions (coating and dilution of labelled AFM1 ) for the three different clones were: 3G11 (0.4 g/mL) and AFM1 -HRP (1/180, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/180, v/v); ATX2 (0.5 g/mL) and AFM1 -HRP (1/120, v/v). 2.5. Direct competitive ELISA based on Bio-Adembeads Protein G This simplified version of dc-ELISA format relies on the use of uniform sized superparamagnetic nanoparticles conjugated with protein G as support for immobilizing the anti-AFM1 antibodies. No blocking step was necessary and as working buffer a solution at 0.05% (v/v) of Tween prepared in PBS was employed for all the steps:
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coating, competition and washing. Prior to use the nanoparticles were washed twice with working buffer (1400 L) for removing the Proclin 300. Briefly the optimised procedure was the following:
3G11 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); ATX2 (1.5 g/mL) and AFM1 -HRP (1/80, v/v). 2.7. Calibration curves for spectrophotometric ELISA
coating: 14 L of superparamagnetic nanoparticles conjugated with protein G were dispersed into 1400 L of antibody solution prepared in working buffer and allowed to react for 20 min; then the particles were collected using the Adem-Mag SV and washed twice with working buffer (1400 L) and finally resuspended in 1400 L; 30 L of this dispersion were added inside the wells and the buffer was removed, meanwhile the nanoparticles were collected onto the inner wall of the well by using the Adem-Mag 96; competition: 90 L of AFM1 and 10 L of AFM1 -HRP were allowed to compete (PolySorpTM microtitre plates were utilised) for the antibody binding sites, during 20 min; during coating and competition, an horizontal shaker (200 rpm) was employed. Washing steps were performed (300 L/well, 1 min) and when the different solutions were discarded from the wells, the superparamagnetic nanoparticles were collected by using the magnets available on the Adem-Mag 96. The TMB liquid substrate for ELISA was utilised (150 L/well) and the absorbance was read 15 min after the substrate solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). The optimised amount of antibody against AFM1 and the dilution of AFM1 -HRP used, for each of the three tested clones, is: 3G11 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); 6G4 (0.2 g/mL) and AFM1 -HRP (1/150, v/v); ATX2 (0.5 g/mL) and AFM1 -HRP (1/120, v/v). 2.6. Direct competitive ELISA based on Bio-Adembeads anti-Mouse IgG This simplified version of dc-ELISA format relies on the use of superparamagnetic nanoparticles coated with affinity purified goat anti-mouse IgG as support for antibody binding. The procedure is similar with the one previously described, when the Bio-Adembeads Protein G superparamagnetic particles were utilised. Prior to use the nanoparticles were washed twice with working buffer (1400 L) for removing the Proclin 300. The optimised procedure was the following: coating: 13 L of superparamagnetic nanoparticles coated with affinity purified goat anti-mouse IgG were dispersed into 1400 L of antibody solution prepared in working buffer and allowed to react for 20 min; then the particles were collected using the AdemMag SV and washed twice with working buffer (1400 L) and finally resuspended in 1400 L; 100 L of this dispersion were added inside the wells and the buffer was removed; competition: 90 L of AFM1 and 10 L of AFM1 -HRP were allowed to compete (PolySorpTM microtitre plates were utilised) for the antibody binding sites, during 20 min; during coating and competition, an horizontal shaker (200 rpm) was employed. Washing steps were performed as described above. The TMB liquid substrate for ELISA was utilised (150 L/well) and the absorbance was read 15 min after the substrate solution was added inside the wells, at both 650 and 450 nm (after quenching with 100 L/well of 1N H2 SO4 ). The optimised amount of antibody against AFM1 and the dilution of AFM1 -HRP used, for each of the three tested clones, is:
Spectrophotometric ELISA standard curves were obtained using AFM1 standard solutions prepared in PBS or in BD282 reference material. Each experiment was performed in triplicate and the mean of each value was used for curve fitting. The calibration curves (absorbance at 650 or 405 nm vs. antigen concentration) were fitted using “non-linear four parameter logistic calibration plots” [22]. To allow the direct comparison of different calibration curves, absorbance values were converted into their corresponding test inhibition values (A/A0 , %) as follows: %
A A − Asat = × 100 A0 A0 − Asat
(1)
where A is the absorbance value of competitors, Asat and A0 are the absorbance values corresponding to the saturating and the non-competition antigen, respectively (as evaluated by the four parameters logistic function). The matrix effect, the recovery percentage and the limit of detection were assayed using blank samples prepared using the ERM-BD282 (zero level of AFM1 ) powder milk. Recovery was assessed by spiking blank milk samples with a known amount of AFM1 standard solution. The detection limit (LOD) was calculated as the concentration corresponding to 90% of A/A0 [23]. The midpoint value (IC50 ) was evaluated as the concentration of AFM1 at 50% A/A0 . The working range was evaluated as the toxin concentration that gives test inhibition values of 80 and 20% of A/A0 . The data obtained for each curve were plotted and fitted using a SigmaPlot software (SPSS), and a regression analysis on the linear portion of the sigmoidal curves was also performed. The slopes obtained from the regression analysis were used to evaluate the matrix effect and the recovery of the assay. 3. Results and discussion 3.1. Spectrophotometric ELISA Indirect and direct competitive ELISA formats were assayed to characterise analytically 5 clones of antibodies developed against AFM1 . This study was aimed also towards the possibility to reduce the time of analysis with respect to the classical ELISA competitive techniques. The indirect competitive format of ELISA was utilised to ascertain how many of the five antibodies (clones 1C6, 3G11, 6G4, ATX9 and ATX2) available in our laboratory were able to bind the aflatoxin M1 . Three of them (clones 3G11, 6G4 and ATX2) were able to recognise and compete for the free AFM1 during the competition step, meanwhile the other two clones were not able to recognise AFM1 (data not shown). Two types of secondary antibodies, labelled with alkaline phosphatase (Fig. 1) or horseradish peroxidase (Fig. 2), were used to trace the AFM1 , but no significant difference, with respect to linear working range or midpoint value, was achieved (Table 1), since the determining factor for this format is the coating step. In fact, maintaining constant the amount of one primary antibody (for example ATX2, 1/1600, v/v) and varying the coating concentrations (50, 25 and 12.5 ng/mL of BSA-AFM1 ), the midpoint value shifted from 430 to 120 and from 120 to 47 ng/L, respectively, when an AbII -AP was used. When using an AbII -HRP only the time analysis was shortened by 30 min, since it was possible to reduce the binding time with the primary antibody,
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Fig. 1. Indirect competitive ELISA; secondary antibody labelled with alkaline phosphatase; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 60 min competition time.
Fig. 2. Indirect competitive ELISA; secondary antibody labelled with horseradish peroxidase; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 60 min competition time. Table 1 Analytical parameters for spectrophotometric ELISA Clone
LWR (ng/L)
IC50 (ng/L)
LOD (ng/L)
ic-ELISA (AbII -AP)-165 min 3G11 15–250 6G4 15–500 ATX2 15–125
75 84 47
15 15 15
ic-ELISA (AbII -HRP)-135 min 3G11 15–250 6G4 8–500 ATX2 8–125
76 72 35
15 8 8
dc-ELISA-90 min 3G11 6G4 ATX2
36 40 36
12.5 12.5 12.5
12.5–80 12.5–80 12.5–100
dc-ELISA Bio-Adembeads Protein G-20 min 3G11 4–250 6G4 12–500 ATX2 30–500
15 50 150
4 12 30
dc-ELISA Bio-Adembeads anti-Mouse IgG-20 min 3G11 8–125 30 6G4 10–500 50 ATX2 10–250 50
8 10 10
LWR: linear working range; IC50 : midpoint value; LOD: limit of detection.
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Fig. 3. Direct competitive ELISA; over night coating, at 4 ◦ C; blocking step (45 min) performed at room temperature; 45 min competition time.
presumably due to a better affinity of AbII -HRP towards the primary antibody. When the three clones (3G11, 6G4 and ATX2) were assayed in the direct competitive format (Fig. 3), all of them showed, more or less, an identical midpoint value (around 40 ng/L). The linear working range (LWR) was narrower then the one obtained in the ic-ELISA procedure but the limits of detection were comparable with the ones obtained for the ic-ELISA. However, the dc-ELISA format is more rapid then the ic-ELISA format (90 min against 165 or 135 min) therefore it was further optimised, by transferring the entire procedure (coating, competition, etc.) onto the superparamagnetic nanoparticles coated with protein G or anti-IgG. One of the main advantage of using nanoparticles resides in the short interaction time between the biological components and the protein G or anti-IgG coated nanoparticles. The whole process could be defined as “homogeneous”, since the interaction between the Ag and the Ab takes place at “nano” level. Moreover, being superparamagnetic, these nanoparticles can be easily separated from bulk solution, allowing also a versatile manipulation. Confronted with the ic- and dc-ELISA, the coating time was reduced to 20 min, instead of an over night incubation time (12–14 h). Moreover, the already incubated nanoparticles could be stored separately as ready to use nanobeads for performing just the competition step in a dc-ELISA. One time consuming factor was a priori overcame, since no blocking step was necessary. This was achieved by adding a non-ionic surfactant into the working buffer, as described before. Then, the competition time was even further reduced, when compared with an ic- or dc-ELISA, till 20 min (Figs. 4 and 5). When the competition curves performed by the aid of superparamagnetic nanoparticles were compared with the ones obtained from ordinary ic- and dc-ELISA, a different shape of these curves was evident, since the affinity of the tested clones towards the reactive coating biomaterial of the nanoparticles, protein G and anti-IgG, respectively, hardly depends on this factor. When a dcELISA based on Bio-Adembeads anti-Mouse IgG was performed, the behaviour of the clones 3G11, 6G4 and ATX2 was more or less identical, and similar with the one observed in the classical icand dc-ELISA. A better discrimination was further achieved when a dc-ELISA based on Bio-Adembeads Protein G was assayed. The clone 3G11 showed to be the best antibody, with the lowest IC50 (15 ng/L) and it was further used to assess the matrix effect, the recovery percentage, when ERM-BD282 powder milk was fortified with known amounts of AFM1 , and to test locally available milk samples.
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Fig. 4. Direct competitive ELISA based on Bio-Adembeads Protein G; coating (20 min) performed at room temperature; no blocking step; 20 min competition time.
Fig. 6. Competition curves performed in buffer and in certified reference material (BD282, zero level of AFM1 ); clone 3G11 immobilised on Bio-Adembeads Protein G was utilised to assess the matrix effect.
3.2. Matrix effect, recovery and real sample analysis Certified reference material (BD282, zero level of AFM1 ) was reconstituted as indicated in the certification report supplied by the IRMM, Belgium. After reconstitution and centrifugation, a calibration curve in matrix was performed and it was observed that the calibration curve in milk was influenced by the new environment (Fig. 6). The IC50 shifted almost 3 times (41 ng/L) then the midpoint value obtained in buffer (15 ng/L). The linear working range (4–250 ng/L) was slightly modified, being essentially the same as for the standard competition curve performed in PBS. This is very important since the maximum accepted level of AFM1 (50 ng/L) is well fitted into this linear working range. Recovery was assessed (Table 2) by spiking with aflatoxin M1 the BD282 reconstituted material. The fortified (30, 60 120 ng/L of AFM1 ) blank milk samples were interpolated from the calibration curve performed using reconstituted certified reference material. The precision was determined by calculating the relative standard deviation (%R.S.D.) for the replicate measurements and the accuracy (%R.E.) was calculated by assessing the agreement between measured and nominal concentration of the fortified samples.
Table 2 BD282, zero level of AFM1 certified reference material was employed; clone 3G11 immobilised on Bio-Adembeads Protein G was utilised to asses the recovery efficiency AFM1 added (ng/L)
AFM1 found (ng/L)
R.S.D. (%)
R.E. (%)
Recovery (%)
30 60 120
28 58 118
7 5 3
−7 −3 −2
93 97 98
%R.E. (relative error) = [(measured value − true value)/true value] × 100; %R.S.D. (relative standard deviation) = standard deviation/mean × 100; n = 6.
Six different brands of commercially available milk samples, three declared to be produced following the rules of biological agriculture and three as fresh milk, were assayed and for all of them the response was negative, since the absorbance values were at the same level as for the “no competition” point of the standard calibration curve. The obtained results were confronted with the response obtained by using a commercial available kit for aflatoxin M1 detection (AFLA M1 MILK), and again a negative response was obtained. 4. Conclusions
Fig. 5. Direct competitive ELISA based on Bio-Adembeads anti-Mouse IgG; coating (20 min) performed at room temperature; no blocking step; 20 min competition time.
In this work, five different antibodies developed against the aflatoxin M1 were investigated by using different ELISA schemes based on classical indirect and direct competitive formats, and on the use of superparamagnetic nanoparticles. From all the five investigated antibodies, the most powerful, i.e. showing the best IC50 value and limit of detection, was the clone 3G11. Superparamagnetic nanoparticles conjugated with protein G and anti-mouse IgG were utilised to further reduce the coating (till 20 min, instead of 12–14 h necessary for the over night coating procedure) and the competition time. Also, one time consuming factor was a priori minimised, since no blocking step was necessary. Being superparamagnetic, these nanoparticles are easily separated from the bulk solution, allowing also a versatile manipulation. The use of superparamagnetic nanoparticles allowed us to demonstrate that the classical ELISA procedures, which sometimes are time consuming, could be further improved by decreasing the coating and competition time and by eliminating steps that cannot be neglected, like the blocking step. The feasibility of this dc-ELISA based on the use of pre-coated (protein G or anti-IgG) nanoparticles was confirmed by performing
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competition curves in milk reference material, showing that the matrix effect is not greatly affecting neither the linear working range, neither the recovery rate nor the midpoint value. In conclusion, we have demonstrated that a competitive immunoassay for AFM1 based on the use of superparamagnetic nanoparticles is reliable, easy to perform and time efficient. Acknowledgement This work was supported by a financial contribution from the programme EraSME Food for Better Human Health (project FASTDETECT). References [1] W.F. Busby Jr., G.N. Wogan, Food-Borne Infections and Intoxications, second ed., Academic Press, New York, 1979, pp. 519–610. [2] W.H. Butler, Mycotoxins, Elsevier Scientific Publishing, New York, 1974, pp 1–28. [3] J.W.O. Ellis, P. Smith, B.K. Simpson, Food Sci. Nutr. 30 (1991) 403. [4] J.P. Jouany, Anim. Res. 51 (2002) 81. [5] D. Miller, J. Stor. Prod. Res. 30 (1994) 1. [6] L. Stoloff, J. Food Prot. 43 (1989) 226. [7] IARC, International Agency for Research on Cancer, Monograph on the Evaluation of Carcinogenic Risk to Humans, vol. 82, World Health Organisation, Lyon, France, 2002, p. 171. [8] R.D. Stubblefield, G.M. Shannon, J. Assoc. Off. Anal. Chem. 57 (1974) 847. [9] European Commission Regulation, No. 466/2001/EC of 8 March 2001, Setting Maximum Levels for Certain Contaminants in Foodstuffs, Official Journal of European Communities L077, 2001, pp. 1–13.
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[10] European Commission Regulation, No. 683/2004/EC of 13 April 2004, Amending Regulation (EC) No. 466/2001 as Regards Aflatoxins and Ochratoxin A in Foods for Infants and Young Children, Official Journal of European Communities L106, 2004, pp. 3–5. [11] European Commission Directive, 2002/26/EC of 13 March 2002 Laying Down the Sampling Methods and the Methods of Analysis For The Official Levels of Ochratoxin A in Foodstuffs, Official Journal of European Communities L075, 2002, pp. 38–43. [12] ISO, Milk and milk powder. Determination of aflatoxin M1 content, in: Clean-up by Immunoaffinity Chromatography and Determination by High-Performance Liquid Chromatography. Standard 14501, International Standards Organisation, Geneva, Switzerland, 1998. [13] F. Grosso, J.M. Fremy, S. Bevis, S. Dragacci, Food Addit. Contam. 21 (2004) 348. [14] E. Chiavaro, C. Cavicchioli, E. Berni, E. Spotti, Food Addit. Contam. 22 (2005) 1154. [15] C. Cavaliere, P. Foglia, E. Pastorini, R. Samperi, A. Lagana, J. Chromatogr. A 1101 (2006) 69. [16] M. Magliulo, M. Mirasoli, P. Simoni, R. Lelli, O. Portanti, A. Roda, J. Agric. Food Chem. 53 (2005) 3300. [17] L. Micheli, R. Greco, M. Badea, D. Moscone, G. Palleschi, Biosens. Bioelectron. 21 (2005) 588. [18] M.L. Rodriguez Velasco, M.M. Calonge Delso, D. Ordonez Escudero, Food Addit. Contam. 20 (2003) 276. [19] J. Stroka, E. Anklam, Trends Anal. Chem. 21 (2002) 90. [20] K. Thirumala-Devi, M.A. Mayo, A.J. Hall, J. Agric. Food Chem. 50 (2002) 933. [21] ISO, Milk and milk products, in: Guidelines for a Standardized Description of Competitive Enzyme Immunoassays-Determination of AFM1 Content. Standard 14675, International Standards Organisation, Geneva, Switzerland, 2002. [22] M.J. Warwick, Immunoassay, in: A Practical Guide, Taylor & Francis Ltd., London, UK, 1996, p. 160. [23] A. Dankwardt, Encyclopedia of Analytical Chemistry, Wiley, Chichester, 2001, pp. 1–25.