Food Chemistry 158 (2014) 445–448
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
A rapid immunomagnetic beads-based immunoassay for the detection of b-casein in bovine milk F. Song a, Y. Zhou a,⇑, Y.S. Li a, X.M. Meng b, X.Y. Meng a, J.Q. Liu c, S.Y. Lu a, H.L. Ren a, P. Hu a, Z.S. Liu a, Y.Y. Zhang a, J.H. Zhang a a b c
Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, PR China Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun 130062, PR China Production Quality Test Institute of Jilin Province, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 19 October 2012 Received in revised form 22 September 2013 Accepted 26 February 2014 Available online 12 March 2014 Keywords: Immunomagnetic beads Sandwich structure b-Casein Enzyme-linked immunosorbent assay Detection
a b s t r a c t An immunomagnetic beads-based enzyme-linked immunosorbent assay (IMBs-ELISA) was developed for the detection of b-casein in bovine milk. Immunomagnetic beads (IMBs) were employed as the solid phase. The anti-b-casein monoclonal antibody (McAb) bound to IMBs was used as capture probe and an anti-b-casein polyclonal antibody (PcAb), labelled with horseradish peroxidase (HRP), was employed as detector probe. Three reaction and two washing steps were needed. Each reaction needed 10 min or less, which significantly shortened detection compared with classic sandwich ELISA. b-Casein in bovine milk was detected across a linear range (2–128 lg mL 1). Application results were in accordance with the Kjejdahl method, which suggests the IMBs-ELISA is rapid and reliable for the detection of b-casein in bovine milk. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bovine milk is a dairy product with high nutritional value that is popular amongst consumer of all ages. It contains protein, fat, carbohydrate, minerals, vitamin and water. In recent years, the consumption of dairy products has rapidly increased (Bashir, 2011). However, milk and milk product have become the target of adulteration (fraudulent incorporation of less costly ingredients), which is a significant problem for the dairy industry in many countries in the world and consumers, for example, the pet-food contamination by melamine adulterant in America in 2007 (Dobson et al., 2008) and the melamine-tainted-milk powder event in China in September 2008 (Chen, 2009). The common adulterants also include urea, nitrates, alum, and soya-bean meal, besides melamine (Attia, Bakir, Abdel-aziz, & Abdel-mottaleb, 2011), which are all nitrogen-containing materials. There is no distinction between protein nitrogen and non-protein nitrogen when the adulterate milk is tested using the standard Kjeldahl method, which only measures total nitrogen not nitrogen source/types, and gives a false impression of levels of milk protein content. It may be harmful to human health and is unethical. There is a need to develop a rapid and reliable method not only for such safety ⇑ Corresponding author. Tel.: +86 0431 87835734; fax: +86 13634318992. E-mail address: [email protected] (Y. Zhou). http://dx.doi.org/10.1016/j.foodchem.2014.02.150 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
problems but also for quality in cases of adulteration with other milks (Chen, 2009). Milk quality is generally evaluated by determining total proteins, and it contains whey proteins (20%) and caseins (80%) (Muller-Renaud, Dupont, & Dulieu, 2004). There are four kinds of caseins in bovine milk, namely aS1- (37%), aS2- (10%), b- (37%), and j-caseins (10%), respectively (Johansson et al., 2009). Among these, b-casein is the major indigenous. It consistently makes up 35–45% total casein content (Colin, Laurent, & Vignon, 1992; Remeuf, Lenoir, & Duby, 1989; Song, Xue, & Han, 2011). Thus, the quantity of b-casein in bovine milk could be used as an index to evaluate its quality and detect dairy adulteration. Various analytical techniques have been proposed for milk authentication including optical immunosensor (Muller-Renaud et al., 2004), single frequency electrical conductance measurements (Mabrook & Petty, 2003), isoelectric focusing (Kim & Jimenez-Flores, 1994; Rodríguez, Ortiz, Sarabia, & Gredilla, 2010), capillary electrophoresis (Miralles, Ramos, & Amigo, 2000; Recio, Amigo, & Lopez-Fandino, 1997), hydrophobic interaction chromatography (Bramanti, Sortino, Onor, Beni, & Raspi, 2003) and enzyme-linked immunosorbent assay (ELISA) (Hurley, Coleman, Ireland, & Williams, 2006). However, most of these methods require sophisticated, technical expertise. Immunomagnetic beads (IMBs) have more surface area than a flat solid phase, permitting more ‘active molecules’ to be immobilised on the surface and enhancing sensitivity (Soh et al.,
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2004; Teste et al., 2010). IMBs also can be easily separated from the reaction mixtures with a magnet and re-dispersed immediately following removal of the magnet (Wei et al., 2012). IMBs allow for a nearly ‘in solution’ reaction (Kim et al., 2009). These characteristics lead to increased sensitivity and shorter times. Therefore, a range of assays based on IMBs have been used in a variety of research fields, such as food safety (Xu et al., 2012), environment monitoring (Schreier et al., 2012; Tudorache, Tencaliec, & Bala, 2008), and clinical diagnosis (Eguílaz et al., 2010; Wei et al., 2012; Yang, Lien, Huang, Lei, & Lee, 2008). ELISA is the most widely used bio-chemical techniques in food analysis because no expensive instrumentation or complicated pre-treatment are required (Giovannacci et al., 2004). However, classic ELISAs are tedious and time-consuming, requiring of several washes and long reaction times. In order to conquer the drawbacks of the classic ELISA, we used mono- (McAb) and polyclonal (PcAb) antibodies specific to b-casein to develop a rapid IMBs-based enzyme-linked immunosorbent assay (IMBs-ELISA) for b-casein in bovine milk. (Fig. 1). 2. Experimental 2.1. Materials and reagents b-Casein, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and biphenyl phenylenediamine were purchased from Sigma Chemicals Co (St. Louis, MO, USA). Activated HRP (type B) kids were obtained from Tai Tianhe (Beijing, China). Carboxylated immunomagnetic beads (350 nm in diameter) (IMBs) were obtained from Wa Wasaina (Wu Han, China). McAb and PcAb specific for b-casein were produced in our previous study (Zhou et al., 2013). Phosphate-buffered saline (PBS, 0.01 mol L 1, pH 7.4) was prepared with 8 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, and 0.2 g potassium dihydrogen phosphate dissolving in 1000 mL distilled water. PBST (0.01 mol L 1, pH 7.4) was prepared by dropping 500 lL of Tween 20 into 1000 mL 0.01 mol L 1 PBS (pH 7.4). Borate buffer (0.1 mol L 1 pH 9.5) was prepared with 6.18 g H3BO3 dissolved in 1 L distilled water, and adjusted pH to 9.5 using 10 mol L 1 NaOH. TMB solution was prepared by using 0.01% (w/v) TMB, 0.005% (v/v) H2O2 and 50 mmol L 1 sodium citrate buffer (pH 5.0). All other reagents were of analytical grade.
Fig. 2. Analysis of HRP-PcAb conjugation by polyacrylamide gel electrophoresis. Lane 1. HRP-PcAb, Lane 2. HRP, Lane 3. Maker, Lane 4. PcAb. Each sample was 10 lL.
temperature with slow rotation. Next, the IMBs were washed three times with 2 mL PBST solution, and then 200 lg McAb was added and incubated for 16–18 h at 37 °C with slight stirring. Thirdly, the IMBs were washed twice with PBST to remove the excess McAbs by magnetic separation process. The non-specific sites on IMBs were blocked by incubating with PBS buffer (containing 2% BSA) at room temperature for 30 min with slight stirring. Finally, the IMB probes were obtained and stored at 4 °C for further use.
2.2. Preparation of IMB probes
2.3. HRP molecules labelled with PcAb
The IMBs (200 lL, 10 mg mL 1) were firstly activated by incubating with EDC (150 lL, 50 mg mL 1) and NHS (150 lL, 50 mg mL 1) solution in 500 lL PBST for 30 min at room
Labelling was performed according to the manufacturer’s instruction as follows: Aliquots of 100 lL b-casein specific PcAbs (1 mg mL 1) were added into a tube containing 1 mg HRP. Then,
Fig. 1. Schematic illustration of the detector probe preparation [A (a)], capture probe preparation [A (b)] and IMBs-based immunoassay procedure (B).
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Fig. 3. Standard curves for the detection of b-casein in bovine milk by IMBs-based immunoassay (A) and correlation of b-casein detection between IMBs-based immunoassay and Kjejdahl method in bovine milk (B).
10 lL of priming agent was dropped into the above solution and incubated at 37 °C for 0.5–1 h or 4 °C for overnight. Afterward, 30 lL of stop buffer was added and the pH of HRP-PcAbs solution was adjusted to 7.0. After addition of 140 lL aseptic glycerol, the conjugation was stored at 20 °C for further use. 2.4. Process of immunoassay The IMBs-based ELISA was carried out as follows: Aliquots of 20 lL IMB probes (1 mg mL 1) were added into EP tubes and mixed with different concentrations of b-casein at 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 lg mL 1. After 10 min incubation at 37 °C, the EP tubes were positioned on the magnet for 5 s. The conjugations of IMB probes and b-casein were precipitated on the bottom of the EP tubes and the supernatant was discarded. The IMBs were then re-dispersed with 200 lL of washing buffer and collected to remove the uncombined antigen. After the IMBs were washed 3 times with washing buffer, 100 lL of detector probe (diluted with PBS in the ratio of 1:1000) was dropped in and incubated for 10 min at 37 °C. The sandwich structure (IMBs probe-target antigen-detector probe) was formed through the reaction of b-casein, McAb and PcAb specific for b-casein. After washing 4 times, 100 lL TMB solution was added and incubated for 5 min at 37 °C avoiding of light. Finally, the absorbance at 450 nm wavelength was measured after the blocking reaction with 50 lL of 11% H2SO4. The calibration curve was obtained using the relationship between the values of positive/negative (P/N) and logarithm of the concentration of b-casein. Data were the means of triplicates. The detection limits (LOD) of the assay was calculated as the mean value of 10 blank samples plus 3 times standard deviations of the mean (Peng et al., 2008). The Kjeldahl method (Lynch, Barbano, & Fleming, 1998) was used in this study for standardization of the developed assay.
than that of HRP and PcAb. Therefore, the migration velocity of HRP-PcAb became slower. 3.2. The protocol of the assay The basic principle of the IMBs-based ELISA was illustrated in Fig. 1. The anti-b-casein PcAb, labelled with HRP was employed as detector probe. The anti-b-casein McAb, bond with IMBs which served as solid phase, was employed as capture probe. In the presence of the antigen, the sandwich structure (capture probeb-casein-detector probe) was formed. HRP, labelled with PcAb can catalyse the oxidation of TMB solution into colourful products to indicate the presence of b-casein antigen. And the absorbance value of the colourful products was proportional to the concentration of antigen. The negative control was treated as the same as the positive group, only without addition of antigen. Three reaction steps and two washing steps, which demand only 30 min was needed to fulfill the procedure of the IMBs-based immunoassay. However, to fulfill the procedure of the traditional sandwich ELISA, four reaction steps and there washing steps were needed, which demand more than 2 h (Hurley et al., 2006; Song et al., 2011). Furthermore, compared with the flat solid phase, the easily separated and re-dispersed nature of IMBs allowed for a ‘‘in solution’’ reaction (Kim et al., 2009; Wei et al., 2012), which also significantly shortened the reaction time of the assay. 3.3. Standard curve As shown in Fig. 3A, a calibration curve was obtained using the relationship between the different concentrations of b-casein (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 lg mL 1) and the positive/negative (P/N) values. The linear range of this method for the detection of b-casein was 2–128 lg mL 1 with a linear regression equation: y = 4.3782x 0.7154 (R2 = 0.9706). The detection limits (LOD) of b-casein in this assay was 0.4 lg mL 1.
3. Results and discussion 3.1. Characteristics of PcAb labelled HRP
3.4. Correlation studies between IMBs-based assay and Kjejdahl method analysis
The activated HRP was directly immobilised on the detector antibody (PcAb). The HRP molecules on PcAb here were used for signal amplification, superseding a secondary antibody labelled with the HRP molecules (Jia et al., 2009), which reduced the procedure of this assay. The result of the conjugation was shown in Fig. 2 by denaturing polyacrylamide gel electrophoresis. The band migration of HRP-PcAb was different from those of activated HRP and purified PcAb. The molecular weight of HRP-PcAb became greater
To validate the performance of the IMBs-based assay, bovine milk samples of five different brands were purchased from local supermarket. The concentration of b-casein was measured by Kjejdahl method and the developed assay simultaneously and the results were compared. The milk samples just need 100 times dilution with distilled water without trivial pretreatment. Fig. 3B showed the detection results of b-casein in bovine milk samples of five different brands by using IMBs-based assay and Kjejdahl
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method. Linear regression analysis showed a good correlation between the two methods, with R2 values 0.9502. The results indicated that the IMBs-based assay is a credible immunoassay for detection of b-casein in bovine milk. 4. Conclusion In conclusion, we presented an IMBs-based immunoassay for the detection of b-casein in bovine milk. The linear detection range was 2–128 lg mL 1 with the detection limits of 0.4 lg mL 1, which fits the concentration of b-casein in bovine milk samples after 100 times dilution. The procedure of the assay can be fulfilled within 30 min without complicated handling procedures. The application results showed a good correlation between the developed assay and Kjejdahl method, demonstrating this IMBs-ELISA would be a reliable tool for rapid detection of b-casein in bovine milk samples. With respect to its overall rapidity and simplicity, the IMBs-based immunoassay is superior to traditional ELISA. Acknowledgements The authors are thankful to the financial support of the National Nature Science Foundation of China (NSFC, Nos. 61171022, 60971011 and 30771657). Talented man support project of Jilin University (No. 4305050102J9). Science and Technology development project of Jilin Province (No. 201205054). References Attia, M. S., Bakir, E., Abdel-aziz, A. A., & Abdel-mottaleb, M. S. A. (2011). Determination of melamine in different milk batches using a novel chemosensor based on the luminescence quenching of Ru (II) carbonyl complex. Talanta, 84, 27–33. Bashir, K. A. (2011). Consumption of dairy products in the UAE: A comparison of nationals and expatriates. Journal of the Saudi Society of Agricultural Sciences, 10, 121–125. Bramanti, E., Sortino, C., Onor, M., Beni, F., & Raspi, G. (2003). Separation and determination of denatured as1-, as2-, b- and j-caseins by hydrophobic interaction chromatography in cows’ ewes’ and goats’ milk, milk mixtures and cheeses. Journal of Chromatography A, 994, 59–74. Chen, J. S. (2009). What can we learn from the 2008 melamine crisis in China? Biomedical and Environmental Sciences, 22, 109–111. Colin, O., Laurent, F., & Vignon, B. (1992). Relationship with milk composition and coagulation parameters. Lait, 72, 307–319. Dobson, R. L., Motlagh, S., Quijano, M., Cambron, R. T., Baker, T. R., Pullen, A. M., et al. (2008). Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicological Sciences, 106, 251–262. ˇ ezEguílaz, M., Moreno-Guzmán, M., Campuzano, S., González-Cortés, A., Yán ˇ , P., & Pingarrón, J. M. (2010). An electrochemical immunosensor for Sedeon testosterone using functionalized magnetic beads and screen-printed carbon electrodes. Biosensors and Bioelectronics, 26, 517–522. Giovannacci, I., Guizard, C., Carlier, M., Duval, V., Martin, J. L., & Demeulemester, C. (2004). Species identification of meat products by ELISA. International Journal of Food Science and Technology, 39, 863–867. Hurley, I. P., Coleman, R. C., Ireland, H. E., & Williams, J. H. H. (2006). Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese. International Dairy Journal, 16, 805–812.
Jia, C. P., Zhong, X. Q., Hua, B., Liu, M. Y., Jing, F. X., Lou, X. H., et al. (2009). NanoELISA for highly sensitive protein detection. Biosensors and Bioelectronics, 24, 2836–2841. Johansson, A., Lugand, D., Rolet-Répécaud, O., Mollé, D., Delage, M. M., Peltre, G., et al. (2009). Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: The case of casein micelle. Molecular Immunology, 46, 1058–1066. Kim, H. J., Ahn, K. C., González-Techera, A., González-Sapienza, G. G., Gee, S. J., & Hammock, B. D. (2009). Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides. Analytical Biochemistry, 386, 45–52. Kim, H. H. Y., & Jimenez-Flores, R. (1994). Comparison of milk proteins using preparative isoelectric focusing followed by polyacrylamide gel electrophoresis. Journal of Dairy Science, 77, 2177–2190. Lynch, J. M., Barbano, D. M., & Fleming, J. R. (1998). Indirect and direct determination of the casein content of milk by Kjeldahl nitrogen analysis: Collaborative study. Journal of AOAC International, 81, 763–774. Mabrook, M. F., & Petty, M. C. (2003). A novel technique for the detection of added water to full fat milk using single frequency admittance measurements. Sensors and Actuators B, 96, 215–218. Miralles, B., Ramos, M., & Amigo, L. (2000). Application of capillary electrophoresis to the characterization of processed cheeses. Journal of Dairy Research, 67, 91–100. Muller-Renaud, S., Dupont, D., & Dulieu, P. (2004). Quantification of b-casein in milk and cheese using an optical immunosensor. Food Chemistry, 52, 659–664. Peng, C. F., Chen, Y. W., Chen, W., Xu, C. L., Kim, J. M., & Jin, Z. Y. (2008). Development of a sensitive heterologous ELISA method for analysis of acetylgestagen residues in animal fat. Food Chemistry, 109, 647–653. Recio, I., Amigo, L., & Lopez-Fandino, R. (1997). Assessment of the quality of dairy products by capillary electrophoresis of milk proteins. Journal of Chromatography B, 697, 231–242. Remeuf, F., Lenoir, J., & Duby, C. (1989). A study of the relations between, physicochemical characteristics of goat milks and their renneting properties. Lait, 69, 499–518. Rodríguez, N., Ortiz, M. C., Sarabia, L., & Gredilla, E. (2010). Analysis of protein chromatographic profiles joint to partial least squares to detect adulterations in milk mixtures and cheeses. Talanta, 81, 255–264. Schreier, S., Doungchawee, G., Triampo, D., Wangroongsarb, P., Hartskeerl, R. A., & Triampoa, W. (2012). Development of a magnetic bead fluorescence microscopy immunoassay to detect and quantify Leptospira in environmental water samples. Acta Tropica, 122, 119–125. Soh, N., Nishiyama, H., Asano, Y., Imato, T., Masadome, T., & Kurokawa, Y. (2004). Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads. Talanta, 64, 1160–1168. Song, H. X., Xue, H. Y., & Han, Y. (2011). Detection of cow’s milk in Shaanxi goat’s milk with an ELISA assay. Food Control, 22, 883–887. Teste, B., Vial, J., Descroix, S., Georgelin, T., Siaugue, J. M., Petr, J., et al. (2010). A chemometric approach for optimizing protein covalent immobilization on magnetic core-shell nanoparticles in view of an alternative immunoassay. Talanta, 81, 1703–1710. Tudorache, M., Tencaliec, A., & Bala, C. (2008). Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis. Talanta, 77, 839–843. Wei, B., Li, F., Yang, H. C., Yu, L., Zhao, K. H., Zhou, R., et al. (2012). Magnetic beadsbased enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae. Biosensors and Bioelectronics, 35, 390–393. Xu, J., Yin, W. W., Zhang, Y. Y., Yi, J., Meng, M., Wang, Y. B., et al. (2012). Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk. Food Chemistry, 134, 2526–2531. Yang, S. Y., Lien, K. Y., Huang, K. J., Lei, H. Y., & Lee, G. B. (2008). Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection. Biosensors and Bioelectronics, 24, 855–862. Zhou, Y., Song, F., Li, Y. S., Liu, J. Q., Lu, S. Y., Ren, H. L., et al. (2013). Double-antibody based immunoassay for the detection of b-casein in bovine milk samples. Food Chemistry, 141, 167–173.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
A rapid immunomagnetic beads-based immunoassay for the detection of b-casein in bovine milk F. Song a, Y. Zhou a,⇑, Y.S. Li a, X.M. Meng b, X.Y. Meng a, J.Q. Liu c, S.Y. Lu a, H.L. Ren a, P. Hu a, Z.S. Liu a, Y.Y. Zhang a, J.H. Zhang a a b c
Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, PR China Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun 130062, PR China Production Quality Test Institute of Jilin Province, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 19 October 2012 Received in revised form 22 September 2013 Accepted 26 February 2014 Available online 12 March 2014 Keywords: Immunomagnetic beads Sandwich structure b-Casein Enzyme-linked immunosorbent assay Detection
a b s t r a c t An immunomagnetic beads-based enzyme-linked immunosorbent assay (IMBs-ELISA) was developed for the detection of b-casein in bovine milk. Immunomagnetic beads (IMBs) were employed as the solid phase. The anti-b-casein monoclonal antibody (McAb) bound to IMBs was used as capture probe and an anti-b-casein polyclonal antibody (PcAb), labelled with horseradish peroxidase (HRP), was employed as detector probe. Three reaction and two washing steps were needed. Each reaction needed 10 min or less, which significantly shortened detection compared with classic sandwich ELISA. b-Casein in bovine milk was detected across a linear range (2–128 lg mL 1). Application results were in accordance with the Kjejdahl method, which suggests the IMBs-ELISA is rapid and reliable for the detection of b-casein in bovine milk. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bovine milk is a dairy product with high nutritional value that is popular amongst consumer of all ages. It contains protein, fat, carbohydrate, minerals, vitamin and water. In recent years, the consumption of dairy products has rapidly increased (Bashir, 2011). However, milk and milk product have become the target of adulteration (fraudulent incorporation of less costly ingredients), which is a significant problem for the dairy industry in many countries in the world and consumers, for example, the pet-food contamination by melamine adulterant in America in 2007 (Dobson et al., 2008) and the melamine-tainted-milk powder event in China in September 2008 (Chen, 2009). The common adulterants also include urea, nitrates, alum, and soya-bean meal, besides melamine (Attia, Bakir, Abdel-aziz, & Abdel-mottaleb, 2011), which are all nitrogen-containing materials. There is no distinction between protein nitrogen and non-protein nitrogen when the adulterate milk is tested using the standard Kjeldahl method, which only measures total nitrogen not nitrogen source/types, and gives a false impression of levels of milk protein content. It may be harmful to human health and is unethical. There is a need to develop a rapid and reliable method not only for such safety ⇑ Corresponding author. Tel.: +86 0431 87835734; fax: +86 13634318992. E-mail address: [email protected] (Y. Zhou). http://dx.doi.org/10.1016/j.foodchem.2014.02.150 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
problems but also for quality in cases of adulteration with other milks (Chen, 2009). Milk quality is generally evaluated by determining total proteins, and it contains whey proteins (20%) and caseins (80%) (Muller-Renaud, Dupont, & Dulieu, 2004). There are four kinds of caseins in bovine milk, namely aS1- (37%), aS2- (10%), b- (37%), and j-caseins (10%), respectively (Johansson et al., 2009). Among these, b-casein is the major indigenous. It consistently makes up 35–45% total casein content (Colin, Laurent, & Vignon, 1992; Remeuf, Lenoir, & Duby, 1989; Song, Xue, & Han, 2011). Thus, the quantity of b-casein in bovine milk could be used as an index to evaluate its quality and detect dairy adulteration. Various analytical techniques have been proposed for milk authentication including optical immunosensor (Muller-Renaud et al., 2004), single frequency electrical conductance measurements (Mabrook & Petty, 2003), isoelectric focusing (Kim & Jimenez-Flores, 1994; Rodríguez, Ortiz, Sarabia, & Gredilla, 2010), capillary electrophoresis (Miralles, Ramos, & Amigo, 2000; Recio, Amigo, & Lopez-Fandino, 1997), hydrophobic interaction chromatography (Bramanti, Sortino, Onor, Beni, & Raspi, 2003) and enzyme-linked immunosorbent assay (ELISA) (Hurley, Coleman, Ireland, & Williams, 2006). However, most of these methods require sophisticated, technical expertise. Immunomagnetic beads (IMBs) have more surface area than a flat solid phase, permitting more ‘active molecules’ to be immobilised on the surface and enhancing sensitivity (Soh et al.,
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2004; Teste et al., 2010). IMBs also can be easily separated from the reaction mixtures with a magnet and re-dispersed immediately following removal of the magnet (Wei et al., 2012). IMBs allow for a nearly ‘in solution’ reaction (Kim et al., 2009). These characteristics lead to increased sensitivity and shorter times. Therefore, a range of assays based on IMBs have been used in a variety of research fields, such as food safety (Xu et al., 2012), environment monitoring (Schreier et al., 2012; Tudorache, Tencaliec, & Bala, 2008), and clinical diagnosis (Eguílaz et al., 2010; Wei et al., 2012; Yang, Lien, Huang, Lei, & Lee, 2008). ELISA is the most widely used bio-chemical techniques in food analysis because no expensive instrumentation or complicated pre-treatment are required (Giovannacci et al., 2004). However, classic ELISAs are tedious and time-consuming, requiring of several washes and long reaction times. In order to conquer the drawbacks of the classic ELISA, we used mono- (McAb) and polyclonal (PcAb) antibodies specific to b-casein to develop a rapid IMBs-based enzyme-linked immunosorbent assay (IMBs-ELISA) for b-casein in bovine milk. (Fig. 1). 2. Experimental 2.1. Materials and reagents b-Casein, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and biphenyl phenylenediamine were purchased from Sigma Chemicals Co (St. Louis, MO, USA). Activated HRP (type B) kids were obtained from Tai Tianhe (Beijing, China). Carboxylated immunomagnetic beads (350 nm in diameter) (IMBs) were obtained from Wa Wasaina (Wu Han, China). McAb and PcAb specific for b-casein were produced in our previous study (Zhou et al., 2013). Phosphate-buffered saline (PBS, 0.01 mol L 1, pH 7.4) was prepared with 8 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, and 0.2 g potassium dihydrogen phosphate dissolving in 1000 mL distilled water. PBST (0.01 mol L 1, pH 7.4) was prepared by dropping 500 lL of Tween 20 into 1000 mL 0.01 mol L 1 PBS (pH 7.4). Borate buffer (0.1 mol L 1 pH 9.5) was prepared with 6.18 g H3BO3 dissolved in 1 L distilled water, and adjusted pH to 9.5 using 10 mol L 1 NaOH. TMB solution was prepared by using 0.01% (w/v) TMB, 0.005% (v/v) H2O2 and 50 mmol L 1 sodium citrate buffer (pH 5.0). All other reagents were of analytical grade.
Fig. 2. Analysis of HRP-PcAb conjugation by polyacrylamide gel electrophoresis. Lane 1. HRP-PcAb, Lane 2. HRP, Lane 3. Maker, Lane 4. PcAb. Each sample was 10 lL.
temperature with slow rotation. Next, the IMBs were washed three times with 2 mL PBST solution, and then 200 lg McAb was added and incubated for 16–18 h at 37 °C with slight stirring. Thirdly, the IMBs were washed twice with PBST to remove the excess McAbs by magnetic separation process. The non-specific sites on IMBs were blocked by incubating with PBS buffer (containing 2% BSA) at room temperature for 30 min with slight stirring. Finally, the IMB probes were obtained and stored at 4 °C for further use.
2.2. Preparation of IMB probes
2.3. HRP molecules labelled with PcAb
The IMBs (200 lL, 10 mg mL 1) were firstly activated by incubating with EDC (150 lL, 50 mg mL 1) and NHS (150 lL, 50 mg mL 1) solution in 500 lL PBST for 30 min at room
Labelling was performed according to the manufacturer’s instruction as follows: Aliquots of 100 lL b-casein specific PcAbs (1 mg mL 1) were added into a tube containing 1 mg HRP. Then,
Fig. 1. Schematic illustration of the detector probe preparation [A (a)], capture probe preparation [A (b)] and IMBs-based immunoassay procedure (B).
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Fig. 3. Standard curves for the detection of b-casein in bovine milk by IMBs-based immunoassay (A) and correlation of b-casein detection between IMBs-based immunoassay and Kjejdahl method in bovine milk (B).
10 lL of priming agent was dropped into the above solution and incubated at 37 °C for 0.5–1 h or 4 °C for overnight. Afterward, 30 lL of stop buffer was added and the pH of HRP-PcAbs solution was adjusted to 7.0. After addition of 140 lL aseptic glycerol, the conjugation was stored at 20 °C for further use. 2.4. Process of immunoassay The IMBs-based ELISA was carried out as follows: Aliquots of 20 lL IMB probes (1 mg mL 1) were added into EP tubes and mixed with different concentrations of b-casein at 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 lg mL 1. After 10 min incubation at 37 °C, the EP tubes were positioned on the magnet for 5 s. The conjugations of IMB probes and b-casein were precipitated on the bottom of the EP tubes and the supernatant was discarded. The IMBs were then re-dispersed with 200 lL of washing buffer and collected to remove the uncombined antigen. After the IMBs were washed 3 times with washing buffer, 100 lL of detector probe (diluted with PBS in the ratio of 1:1000) was dropped in and incubated for 10 min at 37 °C. The sandwich structure (IMBs probe-target antigen-detector probe) was formed through the reaction of b-casein, McAb and PcAb specific for b-casein. After washing 4 times, 100 lL TMB solution was added and incubated for 5 min at 37 °C avoiding of light. Finally, the absorbance at 450 nm wavelength was measured after the blocking reaction with 50 lL of 11% H2SO4. The calibration curve was obtained using the relationship between the values of positive/negative (P/N) and logarithm of the concentration of b-casein. Data were the means of triplicates. The detection limits (LOD) of the assay was calculated as the mean value of 10 blank samples plus 3 times standard deviations of the mean (Peng et al., 2008). The Kjeldahl method (Lynch, Barbano, & Fleming, 1998) was used in this study for standardization of the developed assay.
than that of HRP and PcAb. Therefore, the migration velocity of HRP-PcAb became slower. 3.2. The protocol of the assay The basic principle of the IMBs-based ELISA was illustrated in Fig. 1. The anti-b-casein PcAb, labelled with HRP was employed as detector probe. The anti-b-casein McAb, bond with IMBs which served as solid phase, was employed as capture probe. In the presence of the antigen, the sandwich structure (capture probeb-casein-detector probe) was formed. HRP, labelled with PcAb can catalyse the oxidation of TMB solution into colourful products to indicate the presence of b-casein antigen. And the absorbance value of the colourful products was proportional to the concentration of antigen. The negative control was treated as the same as the positive group, only without addition of antigen. Three reaction steps and two washing steps, which demand only 30 min was needed to fulfill the procedure of the IMBs-based immunoassay. However, to fulfill the procedure of the traditional sandwich ELISA, four reaction steps and there washing steps were needed, which demand more than 2 h (Hurley et al., 2006; Song et al., 2011). Furthermore, compared with the flat solid phase, the easily separated and re-dispersed nature of IMBs allowed for a ‘‘in solution’’ reaction (Kim et al., 2009; Wei et al., 2012), which also significantly shortened the reaction time of the assay. 3.3. Standard curve As shown in Fig. 3A, a calibration curve was obtained using the relationship between the different concentrations of b-casein (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 lg mL 1) and the positive/negative (P/N) values. The linear range of this method for the detection of b-casein was 2–128 lg mL 1 with a linear regression equation: y = 4.3782x 0.7154 (R2 = 0.9706). The detection limits (LOD) of b-casein in this assay was 0.4 lg mL 1.
3. Results and discussion 3.1. Characteristics of PcAb labelled HRP
3.4. Correlation studies between IMBs-based assay and Kjejdahl method analysis
The activated HRP was directly immobilised on the detector antibody (PcAb). The HRP molecules on PcAb here were used for signal amplification, superseding a secondary antibody labelled with the HRP molecules (Jia et al., 2009), which reduced the procedure of this assay. The result of the conjugation was shown in Fig. 2 by denaturing polyacrylamide gel electrophoresis. The band migration of HRP-PcAb was different from those of activated HRP and purified PcAb. The molecular weight of HRP-PcAb became greater
To validate the performance of the IMBs-based assay, bovine milk samples of five different brands were purchased from local supermarket. The concentration of b-casein was measured by Kjejdahl method and the developed assay simultaneously and the results were compared. The milk samples just need 100 times dilution with distilled water without trivial pretreatment. Fig. 3B showed the detection results of b-casein in bovine milk samples of five different brands by using IMBs-based assay and Kjejdahl
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method. Linear regression analysis showed a good correlation between the two methods, with R2 values 0.9502. The results indicated that the IMBs-based assay is a credible immunoassay for detection of b-casein in bovine milk. 4. Conclusion In conclusion, we presented an IMBs-based immunoassay for the detection of b-casein in bovine milk. The linear detection range was 2–128 lg mL 1 with the detection limits of 0.4 lg mL 1, which fits the concentration of b-casein in bovine milk samples after 100 times dilution. The procedure of the assay can be fulfilled within 30 min without complicated handling procedures. The application results showed a good correlation between the developed assay and Kjejdahl method, demonstrating this IMBs-ELISA would be a reliable tool for rapid detection of b-casein in bovine milk samples. With respect to its overall rapidity and simplicity, the IMBs-based immunoassay is superior to traditional ELISA. Acknowledgements The authors are thankful to the financial support of the National Nature Science Foundation of China (NSFC, Nos. 61171022, 60971011 and 30771657). Talented man support project of Jilin University (No. 4305050102J9). Science and Technology development project of Jilin Province (No. 201205054). References Attia, M. S., Bakir, E., Abdel-aziz, A. A., & Abdel-mottaleb, M. S. A. (2011). Determination of melamine in different milk batches using a novel chemosensor based on the luminescence quenching of Ru (II) carbonyl complex. Talanta, 84, 27–33. Bashir, K. A. (2011). Consumption of dairy products in the UAE: A comparison of nationals and expatriates. Journal of the Saudi Society of Agricultural Sciences, 10, 121–125. Bramanti, E., Sortino, C., Onor, M., Beni, F., & Raspi, G. (2003). Separation and determination of denatured as1-, as2-, b- and j-caseins by hydrophobic interaction chromatography in cows’ ewes’ and goats’ milk, milk mixtures and cheeses. Journal of Chromatography A, 994, 59–74. Chen, J. S. (2009). What can we learn from the 2008 melamine crisis in China? Biomedical and Environmental Sciences, 22, 109–111. Colin, O., Laurent, F., & Vignon, B. (1992). Relationship with milk composition and coagulation parameters. Lait, 72, 307–319. Dobson, R. L., Motlagh, S., Quijano, M., Cambron, R. T., Baker, T. R., Pullen, A. M., et al. (2008). Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicological Sciences, 106, 251–262. ˇ ezEguílaz, M., Moreno-Guzmán, M., Campuzano, S., González-Cortés, A., Yán ˇ , P., & Pingarrón, J. M. (2010). An electrochemical immunosensor for Sedeon testosterone using functionalized magnetic beads and screen-printed carbon electrodes. Biosensors and Bioelectronics, 26, 517–522. Giovannacci, I., Guizard, C., Carlier, M., Duval, V., Martin, J. L., & Demeulemester, C. (2004). Species identification of meat products by ELISA. International Journal of Food Science and Technology, 39, 863–867. Hurley, I. P., Coleman, R. C., Ireland, H. E., & Williams, J. H. H. (2006). Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese. International Dairy Journal, 16, 805–812.
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