Food Chemistry 158 (2014) 445–448

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Food Chemistry

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

Analytical Methods

A rapid immunomagnetic beads-based immunoassay for the detectionof b-casein in bovine milk

http://dx.doi.org/10.1016/j.foodchem.2014.02.1500308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +86 0431 87835734; fax: +86 13634318992.E-mail address: zhouyu69@sina.com (Y. Zhou).

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 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, PR Chinab Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun 130062, PR Chinac 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 2012Received in revised form 22 September 2013Accepted 26 February 2014Available online 12 March 2014

Keywords:Immunomagnetic beadsSandwich structureb-CaseinEnzyme-linked immunosorbent assayDetection

a b s t r a c t

An immunomagnetic beads-based enzyme-linked immunosorbent assay (IMBs-ELISA) was developed forthe detection of b-casein in bovine milk. Immunomagnetic beads (IMBs) were employed as the solidphase. The anti-b-casein monoclonal antibody (McAb) bound to IMBs was used as capture probe andan anti-b-casein polyclonal antibody (PcAb), labelled with horseradish peroxidase (HRP), was employedas detector probe. Three reaction and two washing steps were needed. Each reaction needed 10 min orless, which significantly shortened detection compared with classic sandwich ELISA. b-Casein in bovinemilk was detected across a linear range (2–128 lg mL�1). Application results were in accordance withthe Kjejdahl method, which suggests the IMBs-ELISA is rapid and reliable for the detection of b-caseinin bovine milk.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Bovine milk is a dairy product with high nutritional value that ispopular amongst consumer of all ages. It contains protein, fat, car-bohydrate, minerals, vitamin and water. In recent years, the con-sumption of dairy products has rapidly increased (Bashir, 2011).However, milk and milk product have become the target of adulte-ration (fraudulent incorporation of less costly ingredients), whichis a significant problem for the dairy industry in many countriesin the world and consumers, for example, the pet-food contamina-tion by melamine adulterant in America in 2007 (Dobson et al.,2008) and the melamine-tainted-milk powder event in China inSeptember 2008 (Chen, 2009). The common adulterants alsoinclude urea, nitrates, alum, and soya-bean meal, besidesmelamine (Attia, Bakir, Abdel-aziz, & Abdel-mottaleb, 2011), whichare all nitrogen-containing materials. There is no distinctionbetween protein nitrogen and non-protein nitrogen when theadulterate 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. Itmay be harmful to human health and is unethical. There is a needto develop a rapid and reliable method not only for such safety

problems but also for quality in cases of adulteration with othermilks (Chen, 2009). Milk quality is generally evaluated bydetermining total proteins, and it contains whey proteins (20%)and caseins (80%) (Muller-Renaud, Dupont, & Dulieu, 2004). Thereare four kinds of caseins in bovine milk, namely aS1- (37%),aS2- (10%), b- (37%), and j-caseins (10%), respectively (Johanssonet al., 2009). Among these, b-casein is the major indigenous. It con-sistently 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 usedas an index to evaluate its quality and detect dairy adulteration.Various analytical techniques have been proposed for milk authen-tication including optical immunosensor (Muller-Renaud et al.,2004), single frequency electrical conductance measurements(Mabrook & Petty, 2003), isoelectric focusing (Kim & Jimenez-Flo-res, 1994; Rodríguez, Ortiz, Sarabia, & Gredilla, 2010), capillaryelectrophoresis (Miralles, Ramos, & Amigo, 2000; Recio, Amigo, &Lopez-Fandino, 1997), hydrophobic interaction chromatography(Bramanti, Sortino, Onor, Beni, & Raspi, 2003) and enzyme-linkedimmunosorbent assay (ELISA) (Hurley, Coleman, Ireland, &Williams, 2006). However, most of these methods require sophis-ticated, technical expertise.

Immunomagnetic beads (IMBs) have more surface area than aflat solid phase, permitting more ‘active molecules’ to beimmobilised on the surface and enhancing sensitivity (Soh et al.,

446 F. Song et al. / Food Chemistry 158 (2014) 445–448

2004; Teste et al., 2010). IMBs also can be easily separated from thereaction mixtures with a magnet and re-dispersed immediatelyfollowing removal of the magnet (Wei et al., 2012). IMBs allowfor a nearly ‘in solution’ reaction (Kim et al., 2009). These charac-teristics lead to increased sensitivity and shorter times. Therefore,a range of assays based on IMBs have been used in a variety of re-search fields, such as food safety (Xu et al., 2012), environmentmonitoring (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 foodanalysis because no expensive instrumentation or complicatedpre-treatment are required (Giovannacci et al., 2004). However,classic ELISAs are tedious and time-consuming, requiring of severalwashes and long reaction times. In order to conquer the drawbacksof the classic ELISA, we used mono- (McAb) and polyclonal (PcAb)antibodies specific to b-casein to develop a rapid IMBs-based en-zyme-linked immunosorbent assay (IMBs-ELISA) for b-casein inbovine milk. (Fig. 1).

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.

2. Experimental

2.1. Materials and reagents

b-Casein, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS) and biphenylphenylenediamine were purchased from Sigma Chemicals Co (St.Louis, MO, USA). Activated HRP (type B) kids were obtained fromTai 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 pro-duced in our previous study (Zhou et al., 2013).

Phosphate-buffered saline (PBS, 0.01 mol L�1, pH 7.4) wasprepared with 8 g sodium chloride, 0.2 g potassium chloride,1.15 g disodium hydrogen phosphate, and 0.2 g potassium dihy-drogen phosphate dissolving in 1000 mL distilled water. PBST(0.01 mol L�1, pH 7.4) was prepared by dropping 500 lL of Tween20 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 in1 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 re-agents were of analytical grade.

2.2. Preparation of IMB probes

The IMBs (200 lL, 10 mg mL�1) were firstly activated byincubating 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

Fig. 1. Schematic illustration of the detector probe preparation [A (a)], captur

temperature with slow rotation. Next, the IMBs were washed threetimes with 2 mL PBST solution, and then 200 lg McAb was addedand incubated for 16–18 h at 37 �C with slight stirring. Thirdly, theIMBs were washed twice with PBST to remove the excess McAbs bymagnetic separation process. The non-specific sites on IMBs wereblocked by incubating with PBS buffer (containing 2% BSA) at roomtemperature for 30 min with slight stirring. Finally, the IMB probeswere obtained and stored at 4 �C for further use.

2.3. HRP molecules labelled with PcAb

Labelling was performed according to the manufacturer’sinstruction as follows: Aliquots of 100 lL b-casein specific PcAbs(1 mg mL�1) were added into a tube containing 1 mg HRP. Then,

e probe preparation [A (b)] and IMBs-based immunoassay procedure (B).

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 immunoassayand Kjejdahl method in bovine milk (B).

F. Song et al. / Food Chemistry 158 (2014) 445–448 447

10 lL of priming agent was dropped into the above solution andincubated 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 solutionwas adjusted to 7.0. After addition of 140 lL aseptic glycerol, theconjugation was stored at �20 �C for further use.

2.4. Process of immunoassay

The IMBs-based ELISA was carried out as follows: Aliquots of20 lL IMB probes (1 mg mL�1) were added into EP tubes andmixed 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 incuba-tion at 37 �C, the EP tubes were positioned on the magnet for 5 s.The conjugations of IMB probes and b-casein were precipitatedon the bottom of the EP tubes and the supernatant was discarded.The IMBs were then re-dispersed with 200 lL of washing bufferand collected to remove the uncombined antigen. After the IMBswere washed 3 times with washing buffer, 100 lL of detectorprobe (diluted with PBS in the ratio of 1:1000) was dropped inand incubated for 10 min at 37 �C. The sandwich structure (IMBsprobe-target antigen-detector probe) was formed through thereaction of b-casein, McAb and PcAb specific for b-casein. Afterwashing 4 times, 100 lL TMB solution was added and incubatedfor 5 min at 37 �C avoiding of light. Finally, the absorbance at450 nm wavelength was measured after the blocking reaction with50 lL of 11% H2SO4.

The calibration curve was obtained using the relationship be-tween the values of positive/negative (P/N) and logarithm of theconcentration of b-casein. Data were the means of triplicates. Thedetection limits (LOD) of the assay was calculated as the mean va-lue of 10 blank samples plus 3 times standard deviations of themean (Peng et al., 2008). The Kjeldahl method (Lynch, Barbano, &Fleming, 1998) was used in this study for standardization of thedeveloped assay.

3. Results and discussion

3.1. Characteristics of PcAb labelled HRP

The activated HRP was directly immobilised on the detectorantibody (PcAb). The HRP molecules on PcAb here were used forsignal amplification, superseding a secondary antibody labelledwith the HRP molecules (Jia et al., 2009), which reduced the proce-dure of this assay. The result of the conjugation was shown in Fig. 2by denaturing polyacrylamide gel electrophoresis. The band migra-tion of HRP-PcAb was different from those of activated HRP andpurified PcAb. The molecular weight of HRP-PcAb became greater

than that of HRP and PcAb. Therefore, the migration velocity ofHRP-PcAb became slower.

3.2. The protocol of the assay

The basic principle of the IMBs-based ELISA was illustrated inFig. 1. The anti-b-casein PcAb, labelled with HRP was employedas detector probe. The anti-b-casein McAb, bond with IMBs whichserved as solid phase, was employed as capture probe. In thepresence of the antigen, the sandwich structure (capture probe-b-casein-detector probe) was formed. HRP, labelled with PcAbcan catalyse the oxidation of TMB solution into colourful productsto indicate the presence of b-casein antigen. And the absorbancevalue of the colourful products was proportional to the concentra-tion of antigen. The negative control was treated as the same as thepositive group, only without addition of antigen. Three reactionsteps and two washing steps, which demand only 30 min wasneeded 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, whichdemand more than 2 h (Hurley et al., 2006; Song et al., 2011). Fur-thermore, compared with the flat solid phase, the easily separatedand re-dispersed nature of IMBs allowed for a ‘‘in solution’’ reac-tion (Kim et al., 2009; Wei et al., 2012), which also significantlyshortened the reaction time of the assay.

3.3. Standard curve

As shown in Fig. 3A, a calibration curve was obtained using therelationship 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) andthe positive/negative (P/N) values. The linear range of this methodfor the detection of b-casein was 2–128 lg mL�1 with a linearregression equation: y = 4.3782x �0.7154 (R2 = 0.9706). The detec-tion limits (LOD) of b-casein in this assay was 0.4 lg mL�1.

3.4. Correlation studies between IMBs-based assay and Kjejdahlmethod analysis

To validate the performance of the IMBs-based assay, bovinemilk samples of five different brands were purchased from localsupermarket. The concentration of b-casein was measured byKjejdahl method and the developed assay simultaneously and theresults were compared. The milk samples just need 100 times dilu-tion with distilled water without trivial pretreatment. Fig. 3Bshowed the detection results of b-casein in bovine milk samplesof five different brands by using IMBs-based assay and Kjejdahl

448 F. Song et al. / Food Chemistry 158 (2014) 445–448

method. Linear regression analysis showed a good correlationbetween the two methods, with R2 values 0.9502. The results indi-cated that the IMBs-based assay is a credible immunoassay fordetection of b-casein in bovine milk.

4. Conclusion

In conclusion, we presented an IMBs-based immunoassay forthe detection of b-casein in bovine milk. The linear detection rangewas 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 samplesafter 100 times dilution. The procedure of the assay can be fulfilledwithin 30 min without complicated handling procedures. Theapplication results showed a good correlation between the devel-oped assay and Kjejdahl method, demonstrating this IMBs-ELISAwould be a reliable tool for rapid detection of b-casein in bovinemilk 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 NationalNature Science Foundation of China (NSFC, Nos. 61171022,60971011 and 30771657). Talented man support project of JilinUniversity (No. 4305050102J9). Science and Technology develop-ment project of Jilin Province (No. 201205054).

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