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Enzyme-linked immunosorbent assay for the detection of bovine rennet whey powder in milk powder and buttermilk powder
ARTICLE IN PRESS
International Dairy Journal 18 (2008) 294–302 www.elsevier.com/locate/idairyj
Enzyme-linked immunosorbent assay for the detection of bovine rennet whey powder in milk powder and buttermilk powder Maria G.E.G. Bremer, Anna E.M. Kemmers-Voncken, Eduard A.M. Boers, Rob Frankhuizen, Willem Haasnoot RIKILT-Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands Received 24 April 2007; accepted 30 August 2007
Abstract An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of bovine rennet whey (BRW) solids in skim milk powders (SMP) and buttermilk powders is presented. The BRW content was determined in a neutralised trichloroacetic acid sample extract by binding of the dissolved caseinomacropeptide to an enzyme-labelled anti-bovine-k-casein monoclonal antibody. Calibration curves were constructed by analysing SMP standards with different known concentrations of BRW (0–5.8% (w/w)).The assay has a limit of detection of 0.1% (w/w) BRW powder in SMP and has high repeatability and reliability. The ELISA reached the sensitivity required for screening to conform with European Union (EU) legislation. It is easy to use, has a short assay time and is of low cost. The successful applicability of this new screening assay was demonstrated in comparison with chromatographic methods imposed by the EU, where 60 industrial samples taken by the Dutch General Inspection Service were analysed. r 2007 Elsevier Ltd. All rights reserved. Keywords: ELISA; Bovine rennet whey; Skim milk powder; Caseinomacropeptide
1. Introduction Bovine rennet whey (BRW) is a low-priced by-product obtained during cheese production from cows’ milk and it can be used to adulterate high-priced milk products. Adulteration of skim milk powders (SMP) is particularly attractive as the European Union (EU) subsidises the public storage of SMP and the processing of these powders intended for animal feed. To be eligible for EU subsidy, milk powders must be prepared exclusively from skim milk and should not contain solids from whey. Fraudulent addition of BRW solids to dairy products can be detected by determining the presence of caseinomacropeptide (CMP), a compound specific to BRW. In long-life liquid milk, CMP or products similar to CMP can be formed due to the action of psychotropic proteases (De Noni & Resmini, 2005; Recio, Garcı´ a-Risco, Lo´pez-Fandinˇo,
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E-mail address: [email protected] (M.G.E.G. Bremer). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.08.008
Olano, & Ramos, 2000). This might result in false positive results. However, the increase in CMP concentration will only occur under extreme conditions, e.g., very high bacterial counts and/or high storage temperature. Furthermore, milk powders, the target commodity for EU subsidy, are not prone to bacterial growth. Several methods have been developed for the detection of CMP in dairy products, e.g., colorimetric (De Koning, Eisses, & De Vries, 1966; Fukada, Roig, & Prata, 2004), chromatographic (Elgar et al., 2000; Kawakami, Kawasaki, Dosako, Tanimoto, & Nakajima, 1992; Le´onil & Molle´, 1991; Olieman & van den Bedem, 1983; Olieman & van Riel, 1989), immunological (Bitri, Rolland, & Besanc- on, 1993; Picard, Plard, Rongdaux-Gaida, & Collin, 1994) and, more recently, methods based on capillary zone electrophoresis (Cherkaoui, Doumenc, Tachon, Neeser, & Veuthey, 1997; Recio, Lo´pez-Fandinˇo, Olano, Olieman, & Ramos, 1996; Recio et al., 2000; Van Riel & Olieman, 1995), mass spectrometry (De Noni & Resmini, 2005; Molle´ & Le´onil, 2005) and biosensors (Haasnoot, 2005; Haasnoot, Marchesini, & Koopal, 2006). Most of the
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immunoassays were developed for the detection of CMP as a marker for proteolysis in raw milk or as a marker for bovine milk in sheep and goat milk products and not for the detection of rennet whey. The biosensor immunoassays are suitable for the detection of BRW powder in milk powder above 1% (w/w). However, specific, high-priced equipment (Haasnoot, 2005) or equipment not yet commercially available (Haasnoot et al., 2006) are required. For the detection of BRW solids in milk powders the colorimetric methods do not have sufficient specificity and detection limits. The chromatographic methods suffer from low resolution, and hence co-eluting compounds lead to false positive results, and are timeconsuming. Capillary electrophoresis is more specific but is also time-consuming. Mass spectrometry is even more specific but is labour intensive and requires expensive equipment and materials and is therefore not suitable for routine analysis. To combat subsidy fraud, the EU imposed a two-step procedure (Official Journal of the European Communities L 037, 2001) that consists of an initial screening step using gel permeation chromatography (GPC) for all samples, followed by a confirmation step using reversed-phase (RP) high-performance liquid chromatography (HPLC) for samples suspected to contain BRW according to the screening step. This screening method, however, is also lengthy and requires trained personnel and high-cost equipment. Hence, the need for an inexpensive easy-to-use specific screening method is evident. In this study, a rapid and low-cost alternative screening method for the detection of BRW powder in milk powder, based on an inhibition enzyme-linked immunosorbent assay (ELISA), was developed. The ELISA uses a monoclonal antibody (MAb) developed against bovine k-casein (Haasnoot, Smits, Kemmers-Voncken, & Bremer, 2004) recognising bovine CMP as a marker. The determination of BRW content by this ELISA depends on the removal of interfering proteins, in particular caseins, by precipitation with trichloroacetic acid (TCA). However, the solubility of CMP, consisting of glycosylated and non-glycosylated macropeptides, also depends on the TCA concentration. At higher TCA concentrations, non-glycosylated CMP becomes insoluble and only glycosylated CMP remains in solution. Consequently, the recovery of CMP decreases with increasing TCA concentration (Le´onil & Molle´, 1991; Kawakami et al., 1992; Li & Mine, 2004; Lieske & Konrad, 1996). Thus, for optimal detection of CMP in milk powder, a TCA concentration has to be selected at which both the removal of caseins and recovery of CMP are optimal. To assess the suitability of the ELISA, 60 industrial samples, taken by the Dutch General Inspection Service, were analysed by ELISA, GPC and RP-HPLC. For quantitative analysis, calibration curves were constructed by analysing SMP standards with different known concentrations of BRW (0–5.8% w/w). Additionally, samples were prepared with BRW contents close to the decision threshold.
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2. Materials and methods 2.1. MAbs Affinity-purified anti-bovine k-casein MAbs were prepared as described elsewhere (Haasnoot et al., 2004). MAb 4G10 and MAb 6A10 were conjugated to horseradish peroxidase (HRP) using the EZ-Link Plus Activated Peroxidase Kit (Pierce, Rockford, IL, USA). 2.2. Standards and samples SMP standards containing 0% (w/w), 1.0% (w/w) and 5.8% (w/w) BRW powder were bought from NIZO food research (Ede, The Netherlands). SMP containing 2.9% (w/w), 1.5% (w/w) and 0.75% (w/w) BRW powder were prepared by mixing the SMP standards containing 0% (w/w) and 5.8% (w/w) BRW powder in the proper ratio. Industrial samples, i.e., milk powders and buttermilk powders, were taken by the Dutch General Inspection Service (Kerkrade, The Netherlands). CMP (C-7278) and k-casein (C-0406) were bought from Sigma-Aldrich Chemie (Zwijndrecht, The Netherlands). According to the manufacturer, the purity of the k-casein was over 80% as determined by electrophoreses; the purity of CMP was not stated. 2.3. Buffers The following buffers were used: phosphate-buffered saline, pH 7.4 (PBS, 5.4 mM sodium phosphate, 1.3 mM potassium phosphate and 150 mM sodium chloride), PBST (PBS containing 0.05% Tween-20, Merck Schuchardt OHG, Hohenbrunn, Germany), washing buffer (PBS containing 0.1% Tween-20 and 0.004% antifoam A, Sigma-Aldrich Chemie), coating buffer (50 mM sodium carbonate, pH 9.6) and block buffer (coating buffer containing 0.1% ovalbumin, A-5503, Sigma-Aldrich Chemie). 2.4. ELISA 2.4.1. Sample preparation Eight samples were prepared simultaneously. To 0.5 g of sample (milk powder or buttermilk powder), 5 mL of TCA solution (12%, Merck KGaA, Darmstadt, Germany) was added and this mixture was vortexed for 30 s. After vortexing, the mixture was immediately filtered (No. 1, 90 mm qualitative circles, Whatman International, Maidstone, UK). To 100 mL of filtrate, 840 mL of PBS and 60 mL of 0.1 M NaOH were added. Prior to introduction in the ELISA, the pH of the diluted filtrate was tested using pH-indicator strips. If not approximately pH 7, the ratio of PBS and NaOH (final volume 900 mL) was adjusted before addition to 100 mL of fresh filtrate.
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2.4.2. ELISA procedure Because k-casein was commercially available at reasonable purity and price, compared with CMP, this was used for coating the microtitre plates. In this way, the coating could be maintained as reproducible and low cost as possible. High-binding 96-well microtitre plates (Greiner Bio One, Alphen a/d Rijn, The Netherlands) were coated with k-casein (100 mL per well of a 1 mg mL 1 solution in coating buffer) for 1 h at room temperature (RT). Subsequently, the wells were emptied and blocked with 200 mL of block buffer for 1 h at RT. The plates were emptied and sealed, and could be stored at 20 1C for a maximum of 1 month. Prior to use and after every step, as described in the following, the plate was washed three times with washing buffer. To each well, 50 mL of standard or sample extract, and 50 mL of MAb4G10-HRP in a 1:5000 dilution in PBST, were added. The plate was shaken for 30 s on a plate shaker and incubated for 1 h at RT. After washing the plate, the bound peroxidase was assessed by adding 100 mL of a freshly prepared mixture (1:1 v/v) of tetramethylbenzidine (TMB) peroxidase substrate and peroxide (Kirkegaard and Perry Labs, Gaithersburg, MD, USA) to each well. After incubation in the dark for 30 min at RT, the reaction was stopped by adding 100 mL of 1 M phosphoric acid to each well and the colored product was measured at 450 nm using an Argus 400 microplate reader (Canberra Packard, Downers Grove, IL, USA). A calibration curve was constructed by analysing SMP standards with known concentrations of BRW (0%, 0.75%, 1.0%, 1.45%, 2.9% and 5.8% all w/w). 2.5. GPC and RP-HPLC Sample preparations and analyses were performed as prescribed by EC No. 213/2001 annex XVIII and XIX (Official Journal of the European Communities L 037, 2001). 3. Results and discussion 3.1. Development and characterisation of MAbs After two fusions and sub-cloning, two MAb-containing cell culture supernatants were obtained that displayed competition with free k-casein in an indirect ELISA (Haasnoot et al., 2004). From the raw cell culture supernatants, MAbs were isolated by ammonium precipitation and affinity chromatography. The affinity-purified MAbs, called MAb 4G10 and MAb 6A10, were conjugated to HRP and tested in the inhibition ELISA for binding to k-casein and CMP (Fig. 1). Both MAbs could bind to the k-casein-coated plate and to k-casein and CMP in solution. With MAb 4G10, inhibition was obtained with k-casein concentrations in the range 0.1–1 mg mL 1 and CMP concentrations in the range 0.25–10 mg mL 1. With MAb 6A10, inhibition with k-casein and CMP was obtained in the range 0.1–10 mg mL 1 and the range 5–100 mg mL 1,
100 90 80 Absorbance %
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70 60 50 40 30 20 10 0 0.01
0.1
1
10
100
1000
Concentration (µg mL-1) Fig. 1. Inhibition curves obtained with k-casein and caseinomacropeptide (CMP) in the inhibition ELISA using monoclonal antibody (MAb) 4G10 and MAb 6A10. (&, 4G10 k-casein; ’, 6A10 k-casein; J, 4G10 CMP; K, 6A10 CMP).
respectively. These lower limits of the detection ranges for CMP are a factor of 10 lower than those observed for CMP by Mikkelsen et al. (2005). Picard et al. (1994) reported detection ranges comparable with ours. Lower limits of detection ranges were reached in an ELISA for the detection of bovine CMP in ovine and caprine dairy products (Bitri et al., 1993) and in a microparticle-enhanced nephelometric immunoassay for caseinmacropeptide detection in milk (Prin et al., 1996), however, the assay times were from 2 to almost 15 times longer than in the present assay. MAb 4G10 performed much better in the inhibition ELISA than Mab 6A10. This was also observed by Haasnoot et al. (2004, 2006), who applied these MAbs in fast biosensor immunoassays for the detection of cows’ milk in the milk of ewes and goats and for CMP detection. For both MAbs, the affinity for k-casein was higher than for CMP. In other words, the MAbs recognise their epitopes better in complete k-casein than in isolated CMP, which is in agreement with the findings of Mikkelsen et al. (2005). Both MAbs were also tested as capture and detection antibodies (in all possible combinations) in a sandwich ELISA format. In each combination, k-casein could be detected at low concentrations (o1 mg mL 1). However, CMP could only be detected using very high concentrations of a second antibody. The signals obtained were low and the difference in signal of milk powder with and without added BRW powder was small. This rendered this sandwich assay format unfit for the detection of BRW powder in milk powder. Therefore, in further experiments, MAb 4G10 was applied in the inhibition ELISA for detecting BRW solids in milk powder. 3.2. Optimisation of sample preparation The aim of this work was to develop a screening assay that can detect the presence of BRW solids in milk powder
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by using CMP as a marker. However, as shown above, the antibody to be applied in this assay not only binds to free CMP but also to k-casein molecules normally present in milk. Therefore, to be able to selectively detect free CMP in milk powder, interfering caseins need to be removed before analysis. Caseins and other interfering milk proteins can be removed from solution by ultrafiltration (Prin et al., 1996) or precipitation at low pH. TCA selectively precipitates interfering casein and whey proteins (Van Hooydonk & Olieman, 1982). A drawback of using TCA is the decrease in recovery of CMP with increasing TCA concentration (Le´onil & Molle´, 1991; Lieske & Konrad, 1996). For each detection method, the optimal TCA concentration can be different, e.g., 8% TCA is optimal for RP-HPLC measurements but 6% TCA is optimal for spectrophotometric analysis (Lieske & Konrad, 1996; Van Hooydonk & Olieman, 1982). For optimal detection of BRW solids in milk powder using our ELISA, a TCA concentration has to be selected at which, on the one hand, the removal of caseins and, on the other hand the recovery of CMP are
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both satisfactory. In particular, the difference in signal between background (milk powder without BRW) and the decision threshold set by the EU (milk powder with 1% (w/w) BRW) should be optimal. Furthermore, to keep the analysis as uncomplicated as possible, the sample preparation procedure should be fast and easy to perform. In the light of these requirements, milk powder was extracted without reconstitution to milk, in contrast to the sample preparation used for GPC, RP-HPLC and ultrafiltration. In Figs. 2(A) and (B) the effects of the TCA concentration on the solubility of caseins and recovery of CMP as detected with our ELISA are shown. As the developed assay is in the inhibition format, the signal (absorbance) inversely correlates with the concentration of TCA-soluble casein (fragments) and/or CMP. As shown in Fig. 2(A), the absorbance of blank SMP (0%, w/w, rennet whey) increased with increasing TCA concentration. This indicates a decrease in casein (fragment) concentration and hence background signal with increasing TCA concentration. The absorbance for milk powder containing 1%
2 1.8
Absorbance (AU)
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 19 20 TCA concentration (%)
0.4
Absorbance difference (AU)
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
5
10 TCA concentration (%)
15
20
Fig. 2. Effect of TCA concentration on (A) the ELISA signal of milk powder with 0% (w/w) ( ) and 1% (w/w) ( ) bovine rennet whey (BRW) and (B) the difference in signal (absorbance) between milk powder with 0% (w/w) and 1% (w/w) BRW.
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(w/w) rennet whey also increased with increasing TCA concentration. This increase is a result of both the decrease in casein (fragment) concentration (background) and, unfortunately, precipitation of CMP. As shown in Fig. 2(B), the difference in absorbance of SMP and SMP containing 1% (w/w) BRW is maximal between 11% and 14% TCA. In other words, the BRW concentration around the decision threshold of the EU (1%, w/w) can be determined most accurately within this TCA concentration range. For practical reasons concerning the ELISA, a sample extract should remain stable for some hours. At concentrations below 11% TCA, filtrates were cloudy and precipitation occurred. The appearance of additional precipitation after filtration was also observed by Olieman & van den Bedem (1983) using 8% TCA. They solved this problem by allowing 60 min settling before filtration. We observed, however, that clear and stable filtrates can be obtained with TCA concentrations of 11% or more. Using 12% TCA, we are assured of a stable sample extract and an accurate determination of the BRW concentration around the decision threshold, with a minimal use of chemical. As the applied sample preparation (extraction of milk powder with 12% TCA, without reconstitution to milk, and immediate filtration) differs significantly from the sample preparation procedure used in GPC and RP-HPLC methods in EU legislation (reconstituted milk at elevated temperature, slow addition of TCA up to 8% and allowing 60 min settling before filtration), the effect of both sample preparation methods on the ELISA signal was studied. For this, standards were prepared according to both methods. Before analysis, filtrates were diluted tenfold and adjusted to pH 7. As shown in Fig. 3, the absorbance of blank milk powder (0%, w/w, BRW) treated according to both protocols was lower than the absorbance of a PBS solution. This indicates the presence of complete caseins or casein fragments in both extracts and, hence, background signals.
Therefore, to quantify the concentration of BRW powder in SMP, a standard curve should be constructed from SMP with known concentrations of BRW powder instead of PBS solutions with known CMP concentrations. Moreover, the absorbance of blank SMP treated according to the GPC sample preparation was lower than when treated according to our sample preparation method. This indicates a higher concentration of caseins or fragments thereof in solution and hence a higher background signal. The difference in signal of blank milk powder and milk powder containing 1% (w/w) BRW is larger for powders treated according to our method than using the GPC sample preparation. Thus, the BRW concentration can be determined more accurately using our sample preparation method, which is much faster and easier and results in clear and stable filtrates. Furthermore, to construct an accurate calibration curve, standard milk powders containing known concentrations of BRW powder need to be used.
3.3. ELISA standard curve, limit of detection and decision level Standard curves were constructed by analysing milk powder standards containing known concentrations of BRW powder (0.0%, 0.75%, 1.0%, 1.45%, 2.9% and 5.8%, all w/w). A typical standard curve showing the means and standard deviations (SD) from nine curves is shown in Fig. 4. All standards were measured in triplicate on 3 consecutive days. The SDs of the BRW standards ranged between 1.2% and 4.7%. To determine the limit of detection and the decision threshold, a blank milk powder (as determined by RP-HPLC) and the 1% (w/w) BRW standard were analysed on 8 consecutive days. From the blank milk powder, a limit of detection (average background plus 3SD) of 0.1% (w/w) was determined for the ELISA.
2.5
100 90 80 Absorbance %
Absorbance (AU)
2 1.5 1 0.5
70 60 50 40 30 20 10
0
0 PBS
0
1.45 2.9 0.75 1 Concentration BRW (%, w/w)
5.8
Fig. 3. Effect of sample preparation, according to ELISA ( ) and GPC ( ) protocols, on the ELISA signal of PBS and skim milk powders with bovine rennet whey (BRW) powder contents between 0% (w/w) and 5.8% (w/w).
0
1
3 5 2 4 Concentration BRW (%, w/w)
6
7
Fig. 4. Typical ELISA standard curve constructed by analysing milk powder standards containing known concentrations of bovine rennet whey (BRW) powder. The average characteristics are derived from nine different curves. Error bars indicate the SD.
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To minimise the risk of false positive test results as a consequence of background levels of (pseudo) CMP formed due to the action of psychotropic proteases (De Noni & Resmini, 2005; Olieman & van Riel, 1989), the EU has set the decision level at a relatively high concentration, i.e., 1% (w/w) of BRW solids. We set the decision level of our assay at the 95% confidence interval of the milk powder standard containing 1% (w/w) BRW powder. From the 8 analyses of the 1% (w/w) BRW standard, the average concentration and SD were 1.04% and 0.08%, respectively. Therefore, the decision level (i.e., average minus 2SD) was set at 0.9% (w/w). Thus, all samples containing 0.9% (w/w) or more BRW powder according to our assay are potentially positive and need to be confirmed by RP-HPLC. 3.4. Determination of intra- and inter-assay precision To estimate the intra-assay variance, the BRW concentration of 3 buttermilk and 5 SMP were determined in triplicate, and the averages of measurements on 3 different days are shown in Table 1. In the case of the inter-assay variance, results of 3 different days were combined. The ELISA showed a high repeatability for all samples, with coefficients of variation ofo8% in intra-assay variance, ando14% for inter-assay variance.
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RP-HPLC false-positive test results will not lead to erroneous end results in any case. Using a decision threshold of 0.9% (w/w), no false negative results were observed in the ELISA for either the SMP samples or the buttermilk powders. Also the GPC method did not lead to any false-negative results. For the SMP (Table 2), five false positive results were observed with GPC and only one false positive with ELISA. For the buttermilk samples (Table 3), false-positive results were obtained for both the ELISA and the GPC method. The number of false positive results was comparable for the two methods (8 for ELISA and 6 for GPC). The higher number of false-positive results observed for buttermilk can be attributed to the formation of interfering degradation products, ‘‘pseudo CMP,’’ by proteolytic activity of the starters used (De Noni & Resmini, 2005; Olieman & van Riel, 1989). The RP-HPLC method developed by Olieman & van Riel (1989) is, however, capable of separating closely related ‘‘CMP species’’ and is suited for confirming positive results. All positive samples contained high BRW concentrations (between 2% and 18%, w/w) compared with the decision threshold of the EU (1%, w/w). To verify that the ELISA could be used for screening to conform to EU legislation and in particular that no false-negative results would be obtained, the ELISA was additionally tested for samples prepared with BRW contents slightly over the decision threshold, i.e., between 1% and 2% (w/w).
3.5. Analysis of industrial samples 3.6. Analysis of skim milk powders with added BRW To test the applicability of the ELISA, 60 industrial samples (40 SMP and 20 buttermilk powders) taken by the Dutch General Inspection Service were analysed for the presence of BRW by ELISA. For comparison and evaluation of the ELISA all samples were also measured by GPC which is the EU screening method. For final confirmation, all samples were analysed by RP-HPLC. The results are presented in Tables 2 and 3. For screening assays, like the ELISA and the GPC method, it is important that no false negative results are obtained as this would lead to erroneous end results. The number of false positive results should preferably be low. However, as all positive results will be confirmed by
Fifteen samples (5 buttermilk and 10 SMP) were prepared with BRW powder contents between 1% (w/w) and 2% (w/w). For this, milk and buttermilk powders with high and low concentrations of BRW powder (as determined in previous RP experiments) were mixed. The samples were analysed by ELISA, GPC and RP-HPLC and the difference between the expected concentration as determined by RP-HPLC (EU confirmation method) and the concentration measured by ELISA and GPC are added between brackets. Results are summarised in Table 4. For both the buttermilk and SMP samples, no false-negative results were obtained by
Table 1 Intra- and inter-assay variance data obtained in the enzyme-linked immunosorbent assay (ELISA) with buttermilk powders and skim milk powders containing bovine rennet whey (BRW) Sample
Concentration BRW (%, w/w)
Intra-assay CV (%)
Inter-assay CV (%)
Buttermilk powder 1 Buttermilk powder 2 Buttermilk powder 3 Skim milk powder 1 Skim milk powder 2 Skim milk powder 3 Skim milk powder 4 Skim milk powder 5
1.8 1.6 2.1 1.6 1.6 1.8 1.4 2.2
3.8 8.0 4.2 3.9 3.2 3.1 4.6 3.7
13.9 11.1 5.6 3.9 4.2 3.5 6.4 3.7
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Table 2 Bovine rennet whey (BRW) content of skim milk powders taken by Dutch General Inspection Service as analysed by ELISA, gel permeation chromatography (GPC) and reversed-phase (RP)-HPLC
Table 3 Bovine rennet whey (BRW) content of buttermilk powders taken by Dutch General Inspection Service as analysed by ELISA, gel permeation chromatography (GPC) and reversed phase (RP)-HPLC
Sample
Sample
Concentration BRW (%, w/w) GPC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
a
2.6 3.0b 13.9a 0.0 0.8 0.5 0.5 0.9 1.0a 0.4 0.3 0.3 0.5 0.2 0.6 0.6 0.2 0.2 0.5 0.4 20.7b 32.6b 33.5b 0.4 0.5 0.3 0.2 0.2 0.0 0.7 0.1 0.0 0.5 0.0 1.3a 0.1 0.1 0.0 4.4b 3.2a
RP
ELISA
0.1 2.2b 0.0 0.2 0.3 0.3 0.8 0.4 0.2 0.2 0.0 0.2 0.3 0.2 0.1 0.6 0.2 0.3 0.2 0.3 10.9b 17.0b 17.8b 0.2 0.2 0.3 0.4 0.4 0.2 0.9 0.2 0.2 0.3 0.1 0.4 0.3 0.2 0.1 1.1b 0.1
o0 2.6b 0.6 0.2 0.8 0.6 0.7 0.3 0.5 0.5 0.4 0.6 0.8 0.2 0.2 0.5 0.6 0.8 0.5 0.8 7.9b 11.8b 20.4b 0.7 0.7 0.4 0.1 o0 o0 1.0a 0.1 o0 0.8 o0 0.5 o0 o0 o0 2.8b o0 c
a
False-positive sample. Positive sample. c Samples having an absorbance higher, and hence a concentration of BRW lower, than the blank standard are indicated by o0. b
the ELISA or the GPC method. This means that the ELISA can be used for screening milk powders with BRW powder contents around the decision threshold. The average difference between the expected concentration and the measured concentration was much smaller for ELISA than for GPC, 0.2% (w/w) and 1.5% (w/w), respectively. In almost all our samples, the ELISA gave results closer to the conformation method than the GPC method did. These results indicate that the ELISA method offers a good alternative to the GPC screening method.
Concentration BRW (%, w/w) GPC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
a
1.3 1.6a 0.1 0.5 0.3 0.2 1.0a 6.7b 1.2a 0.6 0.7 1.8a 1.1a 6.7b 0.2 0.1 0.1 0.1 0.1 0.1
RP
ELISA
0.1 0.4 0.0 0.3 0.5 0.2 0.0 2.5b 0.5 0.3 0.3 0.3 0.3 2.4b 0.2 0.1 0.1 0.3 0.2 0.1
1.1a 0.9a 0.3 1.4a 0.5 0.2 1.1a 2.1b 1.5a 0.7 1.4a 2.7a 2.0a 1.9b 0.7 0.7 o0c o0 0.4 0.4
a
False-positive sample. Positive sample. c Samples having an absorbance higher, and hence a concentration of BRW lower, than the blank standard, are indicated by o0. b
Table 4 Results of ELISA, gel permeation chromatography (GPC) and reversed phase (RP)-HPLC analyses of prepared samples with bovine rennet whey (BRW) content between 1% and 2% (w/w) Sample
Measured concentration BRW (%, w/w) RP GPC ELISA
Buttermilk powder 1 Buttermilk powder 2 Buttermilk powder 3 Buttermilk powder 4 Buttermilk powder 5 Skim milk powder 1 Skim milk powder 2 Skim milk powder 3 Skim milk powder 4 Skim milk powder 5 Skim milk powder 6 Skim milk powder 7 Skim milk powder 8 Skim milk powder 9 Skim milk powder 10
1.1 1.3 1.3 1.5 1.5 1.4 1.4 1.5 1.9 1.1 1.4 1.9 1.2 1.5 1.7
3.0 3.8 4.1 4.3 4.2 1.6 1.7 1.8 1.8 3.0 3.5 2.0 2.6 3.2 2.9
(1.9)a (2.5) (2.8) (2.8) (2.7) (0.2) (0.3) (0.3) (0.1) (1.9) (2.1) (0.1) (1.4) (1.7) (1.2)
1.2 2.0 1.4 1.6 2.0 1.3 1.4 1.2 1.6 1.3 1.2 1.9 1.3 1.6 1.2
(0.1) (0.7) (0.1) (0.1) (0.5) (0.1) (0.0) (0.3) (0.3) (0.2) (0.2) (0.0) (0.1) (0.1) (0.5)
a
The difference between the expected concentration as determined by RP-HPLC (EU confirmation method) and the concentrations measured by ELISA and GPC are between brackets.
3.7. Evaluation of the ELISA and comparison with other (screening) methods As opposed to the colorimetric methods (De Koning et al., 1966; Fukada et al., 2004), the present immunoassay
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has sufficient specificity and detection limit for screening milk powders for the presence of BRW solids at a 1% (w/w) level. The specificity of the ELISA and the EU screening method (GPC) were comparable, as a lower or comparable number of false positive test results were obtained analysing industrial samples. Using (tandem) mass spectrometry, the amino acid sequence or the exact molecular weight of (pseudo) CMP can be determined (Molle´ & Le´onil, 2005) which would give ultimate specificity. However, these methods are labour intensive and require expensive equipment and materials, and might therefore be suitable for confirmation analysis but not for routine screening. The major advantages of the present ELISA, in comparison with other screening methods like capillary electrophoresis (Recio et al., 2000) and the GPC method, are its easy sample preparation and short assay time. As discussed before, the sample preparation for the ELISA method is much faster than the sample preparation of the GPC method. Moreover, using the ELISA, extracts of 40 samples and 8 standards for the calibration curve can be analysed in duplicate within 2 h, whereas with GPC, analysis time for the sample extracts alone takes at least 12 h. In addition, for each set of 4 samples, extracts of standards (0%, 1% and 5%, all w/w) need to be analysed in duplicate due to the reproducibility of the GPC method. This results in extra analysis time of about 18 h. Furthermore, before and after a series of analyses, columns need to be rinsed and conditioned, which takes a few hours. All in all, analysis of 40 samples with ELISA only takes a few hours whereas analysis with GPC takes over 2 days. 4. Conclusion An inhibition ELISA, based on a MAb recognising bovine rennet CMP, for the determination of BRW solids in SMP and buttermilk powders was developed. The method is easy to use, reliable and reaches the sensitivity required for screening conforming to EU legislation. Its major advantages are its simple and fast sample preparation and short assay time. Studies of industrial samples taken by the Dutch General Inspection Service demonstrated its satisfactory performance and ease of use compared to the GPC method imposed for screening by the EU. The ELISA did not result in any false negative test results and the number of false positive results was lower or comparable to GPC results. Therefore, this ELISA offers a good alternative to the GPC screening and may have wide application in the routine analysis of milk powders and buttermilk powders. References Bitri, L., Rolland, M. P., & Besanc- on, P. (1993). Immunological detection of bovine caseinomacropeptide in ovine and caprine dairy products. Milchwissenschaft, 48, 367–371. Cherkaoui, S., Doumenc, N., Tachon, P., Neeser, J. R., & Veuthey, J. L. (1997). Development of a capillary zone electrophoresis method for
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caseinoglycomacropeptide determination. Journal of Chromatography A, 790, 195–205. De Koning, P. J., Eisses, J., & De Vries, H. (1966). A method for the detection of small percentages of whey powder in milk powder. Netherlands Milk and Dairy Journal, 20, 204–212. De Noni, I., & Resmini, P. (2005). Identification of rennet-whey solids in ‘‘traditional butter’’ by means of HPLC/ESI-MS of non-glycosilated caseinomacropeptide A. Food Chemistry, 93, 65–72. Elgar, D. F., Norris, C. S., Ayers, J. S., Pritchard, M., Otter, D. E., & Palmano, K. P. (2000). Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene. Journal of Chromatography A, 878, 183–196. Fukada, S. P., Roig, S. M., & Prata, L. F. (2004). Correlation between acidic ninhydrin and HPLC methods to evaluate fraudulent addition of whey in milk. Lait, 84, 501–512. Haasnoot, W. (2005). Detection of adulterants in milk products. Biacore Journal, 5, 12–15. Haasnoot, W., Smits, N. G. E., Kemmers-Voncken, A. E. M., & Bremer, M. G. E. G. (2004). Fast biosensor immunoassays for the detection of cows’ milk in the milk of ewes and goats. Journal of Dairy Research, 71, 322–329. Haasnoot, W., Marchesini, G. R., & Koopal, K. (2006). Spreetabased biosensor immunoassays to detect fraudulent adulteration in milk and milk powder. Journal of the AOAC International, 89, 849–855. Kawakami, H., Kawasaki, Y., Dosako, S., Tanimoto, M., & Nakajima, I. (1992). Determination of k-casein glycomacropeptide by high performance liquid chromatography without trichloroacetic acid pretreatment. Milchwissenschaft, 47, 688–693. Le´onil, J., & Molle´, D. (1991). A method for determination of macropeptide by cation-exchange fast protein liquid chromatography and its use for following action of chymosin in milk. Journal of Dairy Research, 58, 321–328. Li, E. W. Y., & Mine, Y. (2004). Technical note: Comparison of chromatographic profile of glycomacropeptide from cheese whey isolated using different methods. Journal of Dairy Science, 87, 174–177. Lieske, B., & Konrad, G. (1996). A new method to estimate caseinomacropeptide and glycomacropeptide from trichloroacetic acid filtrates. Milchwissenschaft, 51, 431–435. Mikkelsen, T. L., Frøkiær, H., Topp, C., Bonomi, F., Iametti, S., Picariello, G., et al. (2005). Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in k-casein. Journal of Dairy Science, 88, 4228–4238. Molle´, D., & Le´onil, J. (2005). Quantitative determination of bovine k-casein macropeptide in dairy products by liquid chromatography/electrospray coupled to mass spectrometry (LC-ESI/MS) and liquid chromatography/electrospray coupled to tandem mass spectrometry (LC-ESI/MS/MS). International Dairy Journal, 15, 419–428. Official Journal of the European Communities L 037, 2001. Regulation No. 213/2001, 07/02/2001, pp. 1–99. Olieman, C., & van den Bedem, J. W. (1983). A sensitive HPLC method of detecting and estimating rennet whey total solids in skim milk powder. Netherlands Milk and Dairy Journal, 37, 27–36. Olieman, C., & van Riel, J. A. M. (1989). Detection of rennet whey solids in skim milk powder and buttermilk powder with reversed-phase HPLC. Netherlands Milk and Dairy Journal, 43, 171–184. Picard, C., Plard, I., Rongdaux-Gaida, D., & Collin, J.-C. (1994). Detection of proteolysis in raw milk stored at low temperature by an inhibition ELISA. Journal of Dairy Research, 61, 395–404. Prin, C., El Bari, N., Montagne, P., Cuilliere, M.-L., Bene, M.-C., Faure, G., et al. (1996). Microparticle-enhanced nephelometric immunoassay for caseinomacropeptide in milk. Journal of Dairy Research, 63, 73–81.
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Recio, I., Lo´pez-Fandinˇo, R., Olano, A., Olieman, C., & Ramos, M. (1996). Study of the formation of caseinomacropeptides in stored ultra-high-temperature-treated milk by capillary electrophoresis. Journal of Agricultural and Food Chemistry, 44, 3845–3848. Recio, I., Garcı´ a-Risco, M. R., Lo´pez-Fandinˇo, R., Olano, A., & Ramos, M. (2000). Detection of rennet whey solids in UHT milk by capillary electrophoresis. International Dairy Journal, 10, 333–338.
Van Hooydonk, A. C. M., & Olieman, C. (1982). A rapid and sensitive high-performance liquid chromatography method following the action of chymosin in milk. Netherlands Milk and Dairy Journal, 36, 153–158. Van Riel, J., & Olieman, C. (1995). Determination of caseinomacropeptide with capillary zone electrophoresis and its application to the detection and estimation of rennet whey solids in milk and buttermilk powder. Electrophoresis, 16, 529–533.
International Dairy Journal 18 (2008) 294–302 www.elsevier.com/locate/idairyj
Enzyme-linked immunosorbent assay for the detection of bovine rennet whey powder in milk powder and buttermilk powder Maria G.E.G. Bremer, Anna E.M. Kemmers-Voncken, Eduard A.M. Boers, Rob Frankhuizen, Willem Haasnoot RIKILT-Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands Received 24 April 2007; accepted 30 August 2007
Abstract An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of bovine rennet whey (BRW) solids in skim milk powders (SMP) and buttermilk powders is presented. The BRW content was determined in a neutralised trichloroacetic acid sample extract by binding of the dissolved caseinomacropeptide to an enzyme-labelled anti-bovine-k-casein monoclonal antibody. Calibration curves were constructed by analysing SMP standards with different known concentrations of BRW (0–5.8% (w/w)).The assay has a limit of detection of 0.1% (w/w) BRW powder in SMP and has high repeatability and reliability. The ELISA reached the sensitivity required for screening to conform with European Union (EU) legislation. It is easy to use, has a short assay time and is of low cost. The successful applicability of this new screening assay was demonstrated in comparison with chromatographic methods imposed by the EU, where 60 industrial samples taken by the Dutch General Inspection Service were analysed. r 2007 Elsevier Ltd. All rights reserved. Keywords: ELISA; Bovine rennet whey; Skim milk powder; Caseinomacropeptide
1. Introduction Bovine rennet whey (BRW) is a low-priced by-product obtained during cheese production from cows’ milk and it can be used to adulterate high-priced milk products. Adulteration of skim milk powders (SMP) is particularly attractive as the European Union (EU) subsidises the public storage of SMP and the processing of these powders intended for animal feed. To be eligible for EU subsidy, milk powders must be prepared exclusively from skim milk and should not contain solids from whey. Fraudulent addition of BRW solids to dairy products can be detected by determining the presence of caseinomacropeptide (CMP), a compound specific to BRW. In long-life liquid milk, CMP or products similar to CMP can be formed due to the action of psychotropic proteases (De Noni & Resmini, 2005; Recio, Garcı´ a-Risco, Lo´pez-Fandinˇo,
Corresponding author. Tel.: +31 317 475532; fax: +31 317 417717.
E-mail address: [email protected] (M.G.E.G. Bremer). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.08.008
Olano, & Ramos, 2000). This might result in false positive results. However, the increase in CMP concentration will only occur under extreme conditions, e.g., very high bacterial counts and/or high storage temperature. Furthermore, milk powders, the target commodity for EU subsidy, are not prone to bacterial growth. Several methods have been developed for the detection of CMP in dairy products, e.g., colorimetric (De Koning, Eisses, & De Vries, 1966; Fukada, Roig, & Prata, 2004), chromatographic (Elgar et al., 2000; Kawakami, Kawasaki, Dosako, Tanimoto, & Nakajima, 1992; Le´onil & Molle´, 1991; Olieman & van den Bedem, 1983; Olieman & van Riel, 1989), immunological (Bitri, Rolland, & Besanc- on, 1993; Picard, Plard, Rongdaux-Gaida, & Collin, 1994) and, more recently, methods based on capillary zone electrophoresis (Cherkaoui, Doumenc, Tachon, Neeser, & Veuthey, 1997; Recio, Lo´pez-Fandinˇo, Olano, Olieman, & Ramos, 1996; Recio et al., 2000; Van Riel & Olieman, 1995), mass spectrometry (De Noni & Resmini, 2005; Molle´ & Le´onil, 2005) and biosensors (Haasnoot, 2005; Haasnoot, Marchesini, & Koopal, 2006). Most of the
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immunoassays were developed for the detection of CMP as a marker for proteolysis in raw milk or as a marker for bovine milk in sheep and goat milk products and not for the detection of rennet whey. The biosensor immunoassays are suitable for the detection of BRW powder in milk powder above 1% (w/w). However, specific, high-priced equipment (Haasnoot, 2005) or equipment not yet commercially available (Haasnoot et al., 2006) are required. For the detection of BRW solids in milk powders the colorimetric methods do not have sufficient specificity and detection limits. The chromatographic methods suffer from low resolution, and hence co-eluting compounds lead to false positive results, and are timeconsuming. Capillary electrophoresis is more specific but is also time-consuming. Mass spectrometry is even more specific but is labour intensive and requires expensive equipment and materials and is therefore not suitable for routine analysis. To combat subsidy fraud, the EU imposed a two-step procedure (Official Journal of the European Communities L 037, 2001) that consists of an initial screening step using gel permeation chromatography (GPC) for all samples, followed by a confirmation step using reversed-phase (RP) high-performance liquid chromatography (HPLC) for samples suspected to contain BRW according to the screening step. This screening method, however, is also lengthy and requires trained personnel and high-cost equipment. Hence, the need for an inexpensive easy-to-use specific screening method is evident. In this study, a rapid and low-cost alternative screening method for the detection of BRW powder in milk powder, based on an inhibition enzyme-linked immunosorbent assay (ELISA), was developed. The ELISA uses a monoclonal antibody (MAb) developed against bovine k-casein (Haasnoot, Smits, Kemmers-Voncken, & Bremer, 2004) recognising bovine CMP as a marker. The determination of BRW content by this ELISA depends on the removal of interfering proteins, in particular caseins, by precipitation with trichloroacetic acid (TCA). However, the solubility of CMP, consisting of glycosylated and non-glycosylated macropeptides, also depends on the TCA concentration. At higher TCA concentrations, non-glycosylated CMP becomes insoluble and only glycosylated CMP remains in solution. Consequently, the recovery of CMP decreases with increasing TCA concentration (Le´onil & Molle´, 1991; Kawakami et al., 1992; Li & Mine, 2004; Lieske & Konrad, 1996). Thus, for optimal detection of CMP in milk powder, a TCA concentration has to be selected at which both the removal of caseins and recovery of CMP are optimal. To assess the suitability of the ELISA, 60 industrial samples, taken by the Dutch General Inspection Service, were analysed by ELISA, GPC and RP-HPLC. For quantitative analysis, calibration curves were constructed by analysing SMP standards with different known concentrations of BRW (0–5.8% w/w). Additionally, samples were prepared with BRW contents close to the decision threshold.
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2. Materials and methods 2.1. MAbs Affinity-purified anti-bovine k-casein MAbs were prepared as described elsewhere (Haasnoot et al., 2004). MAb 4G10 and MAb 6A10 were conjugated to horseradish peroxidase (HRP) using the EZ-Link Plus Activated Peroxidase Kit (Pierce, Rockford, IL, USA). 2.2. Standards and samples SMP standards containing 0% (w/w), 1.0% (w/w) and 5.8% (w/w) BRW powder were bought from NIZO food research (Ede, The Netherlands). SMP containing 2.9% (w/w), 1.5% (w/w) and 0.75% (w/w) BRW powder were prepared by mixing the SMP standards containing 0% (w/w) and 5.8% (w/w) BRW powder in the proper ratio. Industrial samples, i.e., milk powders and buttermilk powders, were taken by the Dutch General Inspection Service (Kerkrade, The Netherlands). CMP (C-7278) and k-casein (C-0406) were bought from Sigma-Aldrich Chemie (Zwijndrecht, The Netherlands). According to the manufacturer, the purity of the k-casein was over 80% as determined by electrophoreses; the purity of CMP was not stated. 2.3. Buffers The following buffers were used: phosphate-buffered saline, pH 7.4 (PBS, 5.4 mM sodium phosphate, 1.3 mM potassium phosphate and 150 mM sodium chloride), PBST (PBS containing 0.05% Tween-20, Merck Schuchardt OHG, Hohenbrunn, Germany), washing buffer (PBS containing 0.1% Tween-20 and 0.004% antifoam A, Sigma-Aldrich Chemie), coating buffer (50 mM sodium carbonate, pH 9.6) and block buffer (coating buffer containing 0.1% ovalbumin, A-5503, Sigma-Aldrich Chemie). 2.4. ELISA 2.4.1. Sample preparation Eight samples were prepared simultaneously. To 0.5 g of sample (milk powder or buttermilk powder), 5 mL of TCA solution (12%, Merck KGaA, Darmstadt, Germany) was added and this mixture was vortexed for 30 s. After vortexing, the mixture was immediately filtered (No. 1, 90 mm qualitative circles, Whatman International, Maidstone, UK). To 100 mL of filtrate, 840 mL of PBS and 60 mL of 0.1 M NaOH were added. Prior to introduction in the ELISA, the pH of the diluted filtrate was tested using pH-indicator strips. If not approximately pH 7, the ratio of PBS and NaOH (final volume 900 mL) was adjusted before addition to 100 mL of fresh filtrate.
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2.4.2. ELISA procedure Because k-casein was commercially available at reasonable purity and price, compared with CMP, this was used for coating the microtitre plates. In this way, the coating could be maintained as reproducible and low cost as possible. High-binding 96-well microtitre plates (Greiner Bio One, Alphen a/d Rijn, The Netherlands) were coated with k-casein (100 mL per well of a 1 mg mL 1 solution in coating buffer) for 1 h at room temperature (RT). Subsequently, the wells were emptied and blocked with 200 mL of block buffer for 1 h at RT. The plates were emptied and sealed, and could be stored at 20 1C for a maximum of 1 month. Prior to use and after every step, as described in the following, the plate was washed three times with washing buffer. To each well, 50 mL of standard or sample extract, and 50 mL of MAb4G10-HRP in a 1:5000 dilution in PBST, were added. The plate was shaken for 30 s on a plate shaker and incubated for 1 h at RT. After washing the plate, the bound peroxidase was assessed by adding 100 mL of a freshly prepared mixture (1:1 v/v) of tetramethylbenzidine (TMB) peroxidase substrate and peroxide (Kirkegaard and Perry Labs, Gaithersburg, MD, USA) to each well. After incubation in the dark for 30 min at RT, the reaction was stopped by adding 100 mL of 1 M phosphoric acid to each well and the colored product was measured at 450 nm using an Argus 400 microplate reader (Canberra Packard, Downers Grove, IL, USA). A calibration curve was constructed by analysing SMP standards with known concentrations of BRW (0%, 0.75%, 1.0%, 1.45%, 2.9% and 5.8% all w/w). 2.5. GPC and RP-HPLC Sample preparations and analyses were performed as prescribed by EC No. 213/2001 annex XVIII and XIX (Official Journal of the European Communities L 037, 2001). 3. Results and discussion 3.1. Development and characterisation of MAbs After two fusions and sub-cloning, two MAb-containing cell culture supernatants were obtained that displayed competition with free k-casein in an indirect ELISA (Haasnoot et al., 2004). From the raw cell culture supernatants, MAbs were isolated by ammonium precipitation and affinity chromatography. The affinity-purified MAbs, called MAb 4G10 and MAb 6A10, were conjugated to HRP and tested in the inhibition ELISA for binding to k-casein and CMP (Fig. 1). Both MAbs could bind to the k-casein-coated plate and to k-casein and CMP in solution. With MAb 4G10, inhibition was obtained with k-casein concentrations in the range 0.1–1 mg mL 1 and CMP concentrations in the range 0.25–10 mg mL 1. With MAb 6A10, inhibition with k-casein and CMP was obtained in the range 0.1–10 mg mL 1 and the range 5–100 mg mL 1,
100 90 80 Absorbance %
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70 60 50 40 30 20 10 0 0.01
0.1
1
10
100
1000
Concentration (µg mL-1) Fig. 1. Inhibition curves obtained with k-casein and caseinomacropeptide (CMP) in the inhibition ELISA using monoclonal antibody (MAb) 4G10 and MAb 6A10. (&, 4G10 k-casein; ’, 6A10 k-casein; J, 4G10 CMP; K, 6A10 CMP).
respectively. These lower limits of the detection ranges for CMP are a factor of 10 lower than those observed for CMP by Mikkelsen et al. (2005). Picard et al. (1994) reported detection ranges comparable with ours. Lower limits of detection ranges were reached in an ELISA for the detection of bovine CMP in ovine and caprine dairy products (Bitri et al., 1993) and in a microparticle-enhanced nephelometric immunoassay for caseinmacropeptide detection in milk (Prin et al., 1996), however, the assay times were from 2 to almost 15 times longer than in the present assay. MAb 4G10 performed much better in the inhibition ELISA than Mab 6A10. This was also observed by Haasnoot et al. (2004, 2006), who applied these MAbs in fast biosensor immunoassays for the detection of cows’ milk in the milk of ewes and goats and for CMP detection. For both MAbs, the affinity for k-casein was higher than for CMP. In other words, the MAbs recognise their epitopes better in complete k-casein than in isolated CMP, which is in agreement with the findings of Mikkelsen et al. (2005). Both MAbs were also tested as capture and detection antibodies (in all possible combinations) in a sandwich ELISA format. In each combination, k-casein could be detected at low concentrations (o1 mg mL 1). However, CMP could only be detected using very high concentrations of a second antibody. The signals obtained were low and the difference in signal of milk powder with and without added BRW powder was small. This rendered this sandwich assay format unfit for the detection of BRW powder in milk powder. Therefore, in further experiments, MAb 4G10 was applied in the inhibition ELISA for detecting BRW solids in milk powder. 3.2. Optimisation of sample preparation The aim of this work was to develop a screening assay that can detect the presence of BRW solids in milk powder
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by using CMP as a marker. However, as shown above, the antibody to be applied in this assay not only binds to free CMP but also to k-casein molecules normally present in milk. Therefore, to be able to selectively detect free CMP in milk powder, interfering caseins need to be removed before analysis. Caseins and other interfering milk proteins can be removed from solution by ultrafiltration (Prin et al., 1996) or precipitation at low pH. TCA selectively precipitates interfering casein and whey proteins (Van Hooydonk & Olieman, 1982). A drawback of using TCA is the decrease in recovery of CMP with increasing TCA concentration (Le´onil & Molle´, 1991; Lieske & Konrad, 1996). For each detection method, the optimal TCA concentration can be different, e.g., 8% TCA is optimal for RP-HPLC measurements but 6% TCA is optimal for spectrophotometric analysis (Lieske & Konrad, 1996; Van Hooydonk & Olieman, 1982). For optimal detection of BRW solids in milk powder using our ELISA, a TCA concentration has to be selected at which, on the one hand, the removal of caseins and, on the other hand the recovery of CMP are
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both satisfactory. In particular, the difference in signal between background (milk powder without BRW) and the decision threshold set by the EU (milk powder with 1% (w/w) BRW) should be optimal. Furthermore, to keep the analysis as uncomplicated as possible, the sample preparation procedure should be fast and easy to perform. In the light of these requirements, milk powder was extracted without reconstitution to milk, in contrast to the sample preparation used for GPC, RP-HPLC and ultrafiltration. In Figs. 2(A) and (B) the effects of the TCA concentration on the solubility of caseins and recovery of CMP as detected with our ELISA are shown. As the developed assay is in the inhibition format, the signal (absorbance) inversely correlates with the concentration of TCA-soluble casein (fragments) and/or CMP. As shown in Fig. 2(A), the absorbance of blank SMP (0%, w/w, rennet whey) increased with increasing TCA concentration. This indicates a decrease in casein (fragment) concentration and hence background signal with increasing TCA concentration. The absorbance for milk powder containing 1%
2 1.8
Absorbance (AU)
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 19 20 TCA concentration (%)
0.4
Absorbance difference (AU)
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
5
10 TCA concentration (%)
15
20
Fig. 2. Effect of TCA concentration on (A) the ELISA signal of milk powder with 0% (w/w) ( ) and 1% (w/w) ( ) bovine rennet whey (BRW) and (B) the difference in signal (absorbance) between milk powder with 0% (w/w) and 1% (w/w) BRW.
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(w/w) rennet whey also increased with increasing TCA concentration. This increase is a result of both the decrease in casein (fragment) concentration (background) and, unfortunately, precipitation of CMP. As shown in Fig. 2(B), the difference in absorbance of SMP and SMP containing 1% (w/w) BRW is maximal between 11% and 14% TCA. In other words, the BRW concentration around the decision threshold of the EU (1%, w/w) can be determined most accurately within this TCA concentration range. For practical reasons concerning the ELISA, a sample extract should remain stable for some hours. At concentrations below 11% TCA, filtrates were cloudy and precipitation occurred. The appearance of additional precipitation after filtration was also observed by Olieman & van den Bedem (1983) using 8% TCA. They solved this problem by allowing 60 min settling before filtration. We observed, however, that clear and stable filtrates can be obtained with TCA concentrations of 11% or more. Using 12% TCA, we are assured of a stable sample extract and an accurate determination of the BRW concentration around the decision threshold, with a minimal use of chemical. As the applied sample preparation (extraction of milk powder with 12% TCA, without reconstitution to milk, and immediate filtration) differs significantly from the sample preparation procedure used in GPC and RP-HPLC methods in EU legislation (reconstituted milk at elevated temperature, slow addition of TCA up to 8% and allowing 60 min settling before filtration), the effect of both sample preparation methods on the ELISA signal was studied. For this, standards were prepared according to both methods. Before analysis, filtrates were diluted tenfold and adjusted to pH 7. As shown in Fig. 3, the absorbance of blank milk powder (0%, w/w, BRW) treated according to both protocols was lower than the absorbance of a PBS solution. This indicates the presence of complete caseins or casein fragments in both extracts and, hence, background signals.
Therefore, to quantify the concentration of BRW powder in SMP, a standard curve should be constructed from SMP with known concentrations of BRW powder instead of PBS solutions with known CMP concentrations. Moreover, the absorbance of blank SMP treated according to the GPC sample preparation was lower than when treated according to our sample preparation method. This indicates a higher concentration of caseins or fragments thereof in solution and hence a higher background signal. The difference in signal of blank milk powder and milk powder containing 1% (w/w) BRW is larger for powders treated according to our method than using the GPC sample preparation. Thus, the BRW concentration can be determined more accurately using our sample preparation method, which is much faster and easier and results in clear and stable filtrates. Furthermore, to construct an accurate calibration curve, standard milk powders containing known concentrations of BRW powder need to be used.
3.3. ELISA standard curve, limit of detection and decision level Standard curves were constructed by analysing milk powder standards containing known concentrations of BRW powder (0.0%, 0.75%, 1.0%, 1.45%, 2.9% and 5.8%, all w/w). A typical standard curve showing the means and standard deviations (SD) from nine curves is shown in Fig. 4. All standards were measured in triplicate on 3 consecutive days. The SDs of the BRW standards ranged between 1.2% and 4.7%. To determine the limit of detection and the decision threshold, a blank milk powder (as determined by RP-HPLC) and the 1% (w/w) BRW standard were analysed on 8 consecutive days. From the blank milk powder, a limit of detection (average background plus 3SD) of 0.1% (w/w) was determined for the ELISA.
2.5
100 90 80 Absorbance %
Absorbance (AU)
2 1.5 1 0.5
70 60 50 40 30 20 10
0
0 PBS
0
1.45 2.9 0.75 1 Concentration BRW (%, w/w)
5.8
Fig. 3. Effect of sample preparation, according to ELISA ( ) and GPC ( ) protocols, on the ELISA signal of PBS and skim milk powders with bovine rennet whey (BRW) powder contents between 0% (w/w) and 5.8% (w/w).
0
1
3 5 2 4 Concentration BRW (%, w/w)
6
7
Fig. 4. Typical ELISA standard curve constructed by analysing milk powder standards containing known concentrations of bovine rennet whey (BRW) powder. The average characteristics are derived from nine different curves. Error bars indicate the SD.
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To minimise the risk of false positive test results as a consequence of background levels of (pseudo) CMP formed due to the action of psychotropic proteases (De Noni & Resmini, 2005; Olieman & van Riel, 1989), the EU has set the decision level at a relatively high concentration, i.e., 1% (w/w) of BRW solids. We set the decision level of our assay at the 95% confidence interval of the milk powder standard containing 1% (w/w) BRW powder. From the 8 analyses of the 1% (w/w) BRW standard, the average concentration and SD were 1.04% and 0.08%, respectively. Therefore, the decision level (i.e., average minus 2SD) was set at 0.9% (w/w). Thus, all samples containing 0.9% (w/w) or more BRW powder according to our assay are potentially positive and need to be confirmed by RP-HPLC. 3.4. Determination of intra- and inter-assay precision To estimate the intra-assay variance, the BRW concentration of 3 buttermilk and 5 SMP were determined in triplicate, and the averages of measurements on 3 different days are shown in Table 1. In the case of the inter-assay variance, results of 3 different days were combined. The ELISA showed a high repeatability for all samples, with coefficients of variation ofo8% in intra-assay variance, ando14% for inter-assay variance.
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RP-HPLC false-positive test results will not lead to erroneous end results in any case. Using a decision threshold of 0.9% (w/w), no false negative results were observed in the ELISA for either the SMP samples or the buttermilk powders. Also the GPC method did not lead to any false-negative results. For the SMP (Table 2), five false positive results were observed with GPC and only one false positive with ELISA. For the buttermilk samples (Table 3), false-positive results were obtained for both the ELISA and the GPC method. The number of false positive results was comparable for the two methods (8 for ELISA and 6 for GPC). The higher number of false-positive results observed for buttermilk can be attributed to the formation of interfering degradation products, ‘‘pseudo CMP,’’ by proteolytic activity of the starters used (De Noni & Resmini, 2005; Olieman & van Riel, 1989). The RP-HPLC method developed by Olieman & van Riel (1989) is, however, capable of separating closely related ‘‘CMP species’’ and is suited for confirming positive results. All positive samples contained high BRW concentrations (between 2% and 18%, w/w) compared with the decision threshold of the EU (1%, w/w). To verify that the ELISA could be used for screening to conform to EU legislation and in particular that no false-negative results would be obtained, the ELISA was additionally tested for samples prepared with BRW contents slightly over the decision threshold, i.e., between 1% and 2% (w/w).
3.5. Analysis of industrial samples 3.6. Analysis of skim milk powders with added BRW To test the applicability of the ELISA, 60 industrial samples (40 SMP and 20 buttermilk powders) taken by the Dutch General Inspection Service were analysed for the presence of BRW by ELISA. For comparison and evaluation of the ELISA all samples were also measured by GPC which is the EU screening method. For final confirmation, all samples were analysed by RP-HPLC. The results are presented in Tables 2 and 3. For screening assays, like the ELISA and the GPC method, it is important that no false negative results are obtained as this would lead to erroneous end results. The number of false positive results should preferably be low. However, as all positive results will be confirmed by
Fifteen samples (5 buttermilk and 10 SMP) were prepared with BRW powder contents between 1% (w/w) and 2% (w/w). For this, milk and buttermilk powders with high and low concentrations of BRW powder (as determined in previous RP experiments) were mixed. The samples were analysed by ELISA, GPC and RP-HPLC and the difference between the expected concentration as determined by RP-HPLC (EU confirmation method) and the concentration measured by ELISA and GPC are added between brackets. Results are summarised in Table 4. For both the buttermilk and SMP samples, no false-negative results were obtained by
Table 1 Intra- and inter-assay variance data obtained in the enzyme-linked immunosorbent assay (ELISA) with buttermilk powders and skim milk powders containing bovine rennet whey (BRW) Sample
Concentration BRW (%, w/w)
Intra-assay CV (%)
Inter-assay CV (%)
Buttermilk powder 1 Buttermilk powder 2 Buttermilk powder 3 Skim milk powder 1 Skim milk powder 2 Skim milk powder 3 Skim milk powder 4 Skim milk powder 5
1.8 1.6 2.1 1.6 1.6 1.8 1.4 2.2
3.8 8.0 4.2 3.9 3.2 3.1 4.6 3.7
13.9 11.1 5.6 3.9 4.2 3.5 6.4 3.7
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Table 2 Bovine rennet whey (BRW) content of skim milk powders taken by Dutch General Inspection Service as analysed by ELISA, gel permeation chromatography (GPC) and reversed-phase (RP)-HPLC
Table 3 Bovine rennet whey (BRW) content of buttermilk powders taken by Dutch General Inspection Service as analysed by ELISA, gel permeation chromatography (GPC) and reversed phase (RP)-HPLC
Sample
Sample
Concentration BRW (%, w/w) GPC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
a
2.6 3.0b 13.9a 0.0 0.8 0.5 0.5 0.9 1.0a 0.4 0.3 0.3 0.5 0.2 0.6 0.6 0.2 0.2 0.5 0.4 20.7b 32.6b 33.5b 0.4 0.5 0.3 0.2 0.2 0.0 0.7 0.1 0.0 0.5 0.0 1.3a 0.1 0.1 0.0 4.4b 3.2a
RP
ELISA
0.1 2.2b 0.0 0.2 0.3 0.3 0.8 0.4 0.2 0.2 0.0 0.2 0.3 0.2 0.1 0.6 0.2 0.3 0.2 0.3 10.9b 17.0b 17.8b 0.2 0.2 0.3 0.4 0.4 0.2 0.9 0.2 0.2 0.3 0.1 0.4 0.3 0.2 0.1 1.1b 0.1
o0 2.6b 0.6 0.2 0.8 0.6 0.7 0.3 0.5 0.5 0.4 0.6 0.8 0.2 0.2 0.5 0.6 0.8 0.5 0.8 7.9b 11.8b 20.4b 0.7 0.7 0.4 0.1 o0 o0 1.0a 0.1 o0 0.8 o0 0.5 o0 o0 o0 2.8b o0 c
a
False-positive sample. Positive sample. c Samples having an absorbance higher, and hence a concentration of BRW lower, than the blank standard are indicated by o0. b
the ELISA or the GPC method. This means that the ELISA can be used for screening milk powders with BRW powder contents around the decision threshold. The average difference between the expected concentration and the measured concentration was much smaller for ELISA than for GPC, 0.2% (w/w) and 1.5% (w/w), respectively. In almost all our samples, the ELISA gave results closer to the conformation method than the GPC method did. These results indicate that the ELISA method offers a good alternative to the GPC screening method.
Concentration BRW (%, w/w) GPC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
a
1.3 1.6a 0.1 0.5 0.3 0.2 1.0a 6.7b 1.2a 0.6 0.7 1.8a 1.1a 6.7b 0.2 0.1 0.1 0.1 0.1 0.1
RP
ELISA
0.1 0.4 0.0 0.3 0.5 0.2 0.0 2.5b 0.5 0.3 0.3 0.3 0.3 2.4b 0.2 0.1 0.1 0.3 0.2 0.1
1.1a 0.9a 0.3 1.4a 0.5 0.2 1.1a 2.1b 1.5a 0.7 1.4a 2.7a 2.0a 1.9b 0.7 0.7 o0c o0 0.4 0.4
a
False-positive sample. Positive sample. c Samples having an absorbance higher, and hence a concentration of BRW lower, than the blank standard, are indicated by o0. b
Table 4 Results of ELISA, gel permeation chromatography (GPC) and reversed phase (RP)-HPLC analyses of prepared samples with bovine rennet whey (BRW) content between 1% and 2% (w/w) Sample
Measured concentration BRW (%, w/w) RP GPC ELISA
Buttermilk powder 1 Buttermilk powder 2 Buttermilk powder 3 Buttermilk powder 4 Buttermilk powder 5 Skim milk powder 1 Skim milk powder 2 Skim milk powder 3 Skim milk powder 4 Skim milk powder 5 Skim milk powder 6 Skim milk powder 7 Skim milk powder 8 Skim milk powder 9 Skim milk powder 10
1.1 1.3 1.3 1.5 1.5 1.4 1.4 1.5 1.9 1.1 1.4 1.9 1.2 1.5 1.7
3.0 3.8 4.1 4.3 4.2 1.6 1.7 1.8 1.8 3.0 3.5 2.0 2.6 3.2 2.9
(1.9)a (2.5) (2.8) (2.8) (2.7) (0.2) (0.3) (0.3) (0.1) (1.9) (2.1) (0.1) (1.4) (1.7) (1.2)
1.2 2.0 1.4 1.6 2.0 1.3 1.4 1.2 1.6 1.3 1.2 1.9 1.3 1.6 1.2
(0.1) (0.7) (0.1) (0.1) (0.5) (0.1) (0.0) (0.3) (0.3) (0.2) (0.2) (0.0) (0.1) (0.1) (0.5)
a
The difference between the expected concentration as determined by RP-HPLC (EU confirmation method) and the concentrations measured by ELISA and GPC are between brackets.
3.7. Evaluation of the ELISA and comparison with other (screening) methods As opposed to the colorimetric methods (De Koning et al., 1966; Fukada et al., 2004), the present immunoassay
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has sufficient specificity and detection limit for screening milk powders for the presence of BRW solids at a 1% (w/w) level. The specificity of the ELISA and the EU screening method (GPC) were comparable, as a lower or comparable number of false positive test results were obtained analysing industrial samples. Using (tandem) mass spectrometry, the amino acid sequence or the exact molecular weight of (pseudo) CMP can be determined (Molle´ & Le´onil, 2005) which would give ultimate specificity. However, these methods are labour intensive and require expensive equipment and materials, and might therefore be suitable for confirmation analysis but not for routine screening. The major advantages of the present ELISA, in comparison with other screening methods like capillary electrophoresis (Recio et al., 2000) and the GPC method, are its easy sample preparation and short assay time. As discussed before, the sample preparation for the ELISA method is much faster than the sample preparation of the GPC method. Moreover, using the ELISA, extracts of 40 samples and 8 standards for the calibration curve can be analysed in duplicate within 2 h, whereas with GPC, analysis time for the sample extracts alone takes at least 12 h. In addition, for each set of 4 samples, extracts of standards (0%, 1% and 5%, all w/w) need to be analysed in duplicate due to the reproducibility of the GPC method. This results in extra analysis time of about 18 h. Furthermore, before and after a series of analyses, columns need to be rinsed and conditioned, which takes a few hours. All in all, analysis of 40 samples with ELISA only takes a few hours whereas analysis with GPC takes over 2 days. 4. Conclusion An inhibition ELISA, based on a MAb recognising bovine rennet CMP, for the determination of BRW solids in SMP and buttermilk powders was developed. The method is easy to use, reliable and reaches the sensitivity required for screening conforming to EU legislation. Its major advantages are its simple and fast sample preparation and short assay time. Studies of industrial samples taken by the Dutch General Inspection Service demonstrated its satisfactory performance and ease of use compared to the GPC method imposed for screening by the EU. The ELISA did not result in any false negative test results and the number of false positive results was lower or comparable to GPC results. Therefore, this ELISA offers a good alternative to the GPC screening and may have wide application in the routine analysis of milk powders and buttermilk powders. References Bitri, L., Rolland, M. P., & Besanc- on, P. (1993). Immunological detection of bovine caseinomacropeptide in ovine and caprine dairy products. Milchwissenschaft, 48, 367–371. Cherkaoui, S., Doumenc, N., Tachon, P., Neeser, J. R., & Veuthey, J. L. (1997). Development of a capillary zone electrophoresis method for
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