Determination of lactoferrin in bovine milk, colostrum ...

ARTICLE IN PRESS

International Dairy Journal 15 (2005) 429–438 www.elsevier.com/locate/idairyj

Determination of lactoferrin in bovine milk, colostrum and infant formulas by optical biosensor analysis Harvey E. Indyka,, Enrı´ co L. Filonzib a

Fonterra, P.O. Box 7, Waitoa, New Zealand Biacore International Limited, Box Hill, Melbourne, Victoria 3128, Australia

b

Received 20 May 2004; accepted 14 September 2004

Abstract An automated, rapid, sensitive and label-free biosensor-based immunoassay for lactoferrin in bovine milk utilising surface plasmon resonance (SPR) optical detection is described. Lactoferrin content is estimated from its specific interaction with an antibovine lactoferrin antibody immobilised on the sensor surface in a direct- binding assay format. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilisation, flow-rate, contact time and regeneration have been defined and non-specific binding considerations evaluated. Performance parameters include a working range of 0–1000 ng mL 1, a method-detection limit of 19.9 mg mL 1 in undiluted milk, overall instrument response RSDR of 3.50%, a mean inter-assay RSDR of 10.8% for milk and surface stability over ca 500 samples. The technique was applied to the measurement of lactoferrin content of consumer milks, colostrum and infant formulas, and temporal change during early bovine lactation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Lactoferrin; Optical biosensor; Surface plasmon resonance; Immunoassay; Bovine milk; Colostrum

1. Introduction Milk contains numerous minor proteins with physiological properties targeted at providing immunoprotective, growth and antimicrobial factors to the neonate, as distinct from the nutritionally more significant major proteins. Many of these minor bioactive proteins are found in the serum fraction of mammalian milks, and are generally present at elevated levels in colostrum, reflecting their importance to early neonatal health. Lactoferrin is a ca 80 kDa basic (pI: ca. 9.0) ironbinding, bi-lobal secretory transport sialylated glycoprotein of known amino acid sequence, and is a member of the transferrin family characterised by carbonate anion dependent, high affinity (KD10 20 M, Baker & Baker, 2004) and reversible binding of two Fe3+ per molecule yielding a pink complex (lmax: 470 nm) Corresponding author. Tel.: +64 7 889 3989; fax: +64 7 887 1502.

E-mail address: [email protected] (H.E. Indyk). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.09.003

(Anderson, Baker, Norris, Rumball, & Baker, 1990; Lonnerdal & Atkinson, 1995; Wong, Camirand, & Pavlath, 1996; Steijns & van Hooijdonk, 2000; Weinberg, 2001). Although present in milk as a result of in situ synthesis within the mammary gland, it is also present in several other exocrine fluids. The fact that milk shares several antimicrobial components, including lactoferrin, with other glandular secretions may be evidence of functions that predate the nutritional role of lactogenesis (Blackburn, 1993). In addition to its antimicrobial activity, lactoferrin may function in intestinal iron uptake and regulation, immune response, growth factor activity and antioxidant activity (Wong et al., 1996; Pakkanen & Aalto, 1997; Steijns & van Hooijdonk, 2000; Fox & Kelly, 2003; Stevenson & Knowles, 2003). Lactoferrin content is species-dependent, with significantly higher levels in human milk and colostrum compared to the bovine equivalent (Fox & McSweeney, 1998). This has stimulated an increasing trend of supplementation of bovine milk-based infant

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H.E. Indyk, E.L. Filonzi / International Dairy Journal 15 (2005) 429–438

formulas with fractionated bovine lactoferrin (Boland, MacGibbon, & Hill, 2001; Stevenson & Knowles, 2003), despite a 69% primary sequence homology with human lactoferrin (Wong et al., 1996; Pakkanen & Aalto, 1997) and a poorly characterised understanding of its absorption in the infant gastrointestinal tract (Kuwata et al., 2001). The increasing commercial interest in exploiting the therapeutic value of lactoferrin has stimulated the need for reliable concentration assays for its determination at endogenous levels in milk and colostrum, at supplemental levels in infant formulas and at pharmaceutical levels in milk protein isolates. Such techniques should, however, be distinguished from methods intended for the determination of biological activity. Ion-exchange, gel permeation, adsorption and immunoaffinity chromatographic techniques have been utilised for the isolation of lactoferrin isolates from milk or whey (Ekstrand & Bjo¨rck, 1986; Hahn, Schulz, Schaupp, & Jungbauer, 1998; Tu, Chen, Chang, & Chang, 2002; Zhang, Nakano, & Ozimek, 2002; Zhang, Nakano, & Ozimek, 2003). Analytical chromatographic techniques for bovine whey proteins have been reviewed (Gonzalez–Llano, Polo & Ramos, 1990; Strange, Malin, Van Hekken, & Basch, 1992; Elgar et al., 2000), while several chromatographic techniques for the quantitation of lactoferrin in milk and whey have also been reported (Monti, Fumeaux, Barrios–Larouze, & Jolles, 1984; Law, Leaver, Banks, & Horne, 1993; Francis, Regester, Webb, & Ballard, 1995; Palmano & Elgar, 2002). A recent review of the different immunological techniques available to quantify lactoferrin concentration concludes that immunodiffusion techniques have inherently low sensitivity and have generally been superseded by the more sensitive ELISA techniques (Desmazeaud, 1993). More recent enzyme- and nephelometric-immunoassays for lactoferrin in milk have also been reported (Lindmark–Ma˚nsson et al., 2000; Montagne, Tregoat, Cuilliere, Bene, & Faure, 2000; Turner, Williamson, Thomson, Roche & Kolver, 2003; Chen & Mao, 2004; Yamauchi et al., 2004). Recent developments in real-time, label-free optical biosensor techniques based on surface plasmon resonance (SPR) as applied to milk proteins have included the quantitative detection of plant protein adulterants, IgG, folate-binding protein, insulin-like growth factor (IGF-1), b-casein and the kinetic study of milk casein interactions (Haasnoot, Olieman, Cazemier, & Verheijen, 2001; Guidi, Laricchia-Robbio, Gianfaldoni, Revoltella, & Del Bono, 2001; Marchesseau et al., 2002; Indyk & Filonzi, 2003; Nygren, Sternesjo¨, & Bjo¨rck, 2003; Indyk & Filonzi, 2004; Muller-Renaud, Dupont, & Dulieu, 2004). SPR-based techniques have also determined kinetic parameters for the interactions of lactoferrin with oocyte receptor (Hiesberger et al., 1995), the lipopolysaccharide receptor CD14 (Baveye et al.,

2000) and salivary agglutinin (Mitoma, Oho, Shimazaki, & Koga, 2001). Although ferritin, a cellular iron-binding storage protein, has recently been quantitated in serum by this technique (Cui, Yang, Sha, & Yang, 2003; Chou, Hsu, Hwang, & Chen, 2004), the biosensor-based analysis of lactoferrin content in milk has not yet been reported. SPR-based biosensor immunoassays exploit the specific and reversible interaction between antibody and antigen, and are versatile, robust and capable of producing rapid and reliable data for the quantitative analysis of components in complex food matrices with minimal sample preparation (Homola, 2003). The present study describes an automated SPR-biosensor assay for the quantitation of lactoferrin in protein isolates, milk, colostrum and lactoferrin-supplemented infant formula.

2. Materials and methods 2.1. Instrumentation The Biacores Q optical biosensor was from Biacore AB (Uppsala, Sweden). Instrument operation and data processing was performed with Biacore Q control software 3.0.1. 2.2. Reagents Amine coupling reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC, 0.4 M), N-hydroxysuccinimide (NHS, 0.1 M) and ethanolamine-HCl (1 M, pH 8.5), sodium acetate buffer (10 mM, pH 4.5–5.5), Sensor Chip CM5 and HBS-EP running buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) were all obtained from Biacore. Glycine (AR grade) was obtained from BDH (Poole, UK). Commercially available bovine lactoferrin was obtained from Sigma-Aldrich (L9507, MO, USA), Bethyl Laboratories (RC10-126, TX, USA) and BioX Diagnostics (BIO K187, Marche-en-Famenne, Belgium). Bovine lactoferrin (provided by D. Elgar and K. Palmano, Fonterra Research Centre, Palmerston North, New Zealand) was isolated from milk by cation exchange liquid chromatography and gel filtration. Briefly, a lactoferrin fraction from fresh skim milk was eluted from Sepharose S (25 mM phosphate buffer, pH 6.5 at 4 1C) with 1 M NaCl, dialysed, freeze-dried, and reprocessed under the same conditions. Final purification was achieved by gel filtration through Sephacryl S 300 (50 mM phosphate buffer, 0.15 M NaCl, pH 6.5) and the recovered lactoferrin was dialysed and lyophilised. Identity was confirmed by SDS-PAGE and purity estimated as 496% by reversed phase HPLC (Palmano

ARTICLE IN PRESS H.E. Indyk, E.L. Filonzi / International Dairy Journal 15 (2005) 429–438

& Elgar, 2002). Iron-saturation (ca. 15%) and ironbinding status (estimated with ferric nitrilotriacetate) indicated that virtually all the lactoferrin was able to bind iron stoichiometrically (Bates, Billups, & Saltman, 1967; Brock & Arzabe, 1976). Circular dichroism analysis gave near- and far-UV spectra typical of both native and iron-saturated lactoferrin (Parry & Brown, 1974). The final concentration of the isolated lactoferrin was determined by quantitative amino acid analysis. Affinity purified goat polyclonal anti-bovine lactoferrin antibody (1 mg mL 1) was obtained from Bethyl Laboratories (A10-126A) and an equivalent rabbit polyclonal (2 mg mL 1) from BioX Diagnostics (BIO 187). 2.3. Biosensor surface preparation Goat and rabbit anti-bovine lactoferrin antibody were separately immobilised on sensor chip CM5 at 25 1C via amine coupling under instrument control. Briefly, the designated flow cell was activated with a mixture of EDC and NHS (1:1 v/v) (10 mL min 1, 7 min) followed by antibody (50 mg mL 1 in 10 mM sodium acetate, pH 5.0) (10 mL min 1, 7 min). Finally, unreacted ester functionalities were deactivated with ethanolamine (1 M, pH 8.5, 10 mL min 1, 7 min). Following immobilisation, the chip was stored between analyses over dessicant at 4 1C in a sealed container. A reference flow cell was prepared similarly, but without the antilactoferrin ligand. 2.4. Lactoferrin standards Stock standards were initially prepared at 1.0 mg mL 1 in HBS-EP. Intermediate standards were then prepared at 100 mg mL 1 in HBS-EP, sub-aliquoted and stored at –181C. Working lactoferrin calibration standards (1000, 500, 250, 125, 62.5 ng mL 1) were prepared daily in HBS-EP containing elevated levels of NaCl to a final concentration of 0.5 M. 2.5. Samples Raw bovine milk was collected from a single 4-yearold Jersey cow (2nd calving) between days –1 prepartum and +15 post-partum. Samples were diluted 1:50 in HBS-EP buffer, divided into 1.0 mL aliquots and frozen (–18 1C) until analysed by SPR-biosensor assay. In preparation for analysis, aliquots were thawed at 37 1C and further diluted to a final 1:10,000 (oday 1 post-partum), 1:2000 (day 1) or 1:1000 (4day 2) with HBS-EP buffer containing a final concentration of 0.5 M NaCl. A range of retail liquid consumer milks, commercial milk powders, colostrum powders and infant formulas

431

were obtained from both New Zealand milk processing facilities and commercial sources, and all were prepared for analysis by dilution to 1:2000 in HBS-EP (0.5 M NaCl). 2.6. SPR-biosensor assay A calibration curve was established by serial dilution of a 1000 ng mL 1 bovine lactoferrin standard in HBSEP (0.5 M NaCl). Calibrants and sample extracts (in duplicate) were dispensed (200 mL) into 96-well microtitre plates and were injected for 5 min at 10 mL min 1 with HBS-EP (0.15 M NaCl) running buffer at a temperature of 25 1C. Binding responses acquired 30 s after the end of the injection were measured relative to the initial baseline and used for generation of calibration curve and interpolation of unknown samples. Regression analysis was routinely performed under software control by a 4-parameter fit. The surface was regenerated by injection of 27 mL of 10 mM glycine-HCl, pH 1.75 at 50 mL min 1.

3. Results and discussion 3.1. Selection of ligand Two commercially available, affinity purified, polyclonal anti-bovine lactoferrin antibodies were evaluated for their binding characteristics to bovine lactoferrin. Both goat and rabbit anti-lactoferrin were successfully immobilised at high ligand density under optimum buffer conditions (10 mM sodium actetate, pH 5.0) yielding ca. 15,000 RU (ca. 75 fmole) surface immobilisation levels, based on the relationship 1 RU=ca. 1 pg mm 2 for most proteins on a CM5 surface, as illustrated in Fig. 1. High ligand capacity is generally beneficial in concentration assays, since the relative binding response is directly related to the level of immobilised ligand, and a typical goat anti-lactoferrin immobilised surface yielded a maximum binding capacity (Rmax) of 5600 RU. Based on its significantly higher binding response (10 ) to bovine lactoferrin compared to rabbit antibody, the goat polyclonal was selected as the preferred ligand for an SPR-biosensor assay. 3.2. Selection of calibrant The identity of the isolated high-purity bovine lactoferrin was established by SDS-PAGE that yielded a single band at ca. 80 kDa and N-terminal amino acid sequencing. For calibration purposes, protein content was determined by quantitative amino acid analysis. Compared to commercial sources, equivalent concentrations of the purified bovine lactoferrin isolate yielded


432 RU 50000

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Fig. 1. Immobilisation of goat anti-bovine lactoferrin on CM5 sensor surface at 10 mL min-1. Captions indicate surface activation with 70 mL EDC:NHS (1), injection of 70 mL antibody at 50 mg mL-1, pH 5.0 (2), and blocking with 70 mL 1M ethanolamine (3). Ligand immobilisation level is indicated by WRU.

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Fig. 2. Overlay sensorgrams following injection of 50 mL duplicate bovine lactoferrin calibrants (0–1000 ng mL-1) over anti-lactoferrin immobilised surface at flowrate of 10 mL min-1. Conditions described in text. Captions indicate (1) baseline , (2) lactoferrin association phase, (3) lactoferrin binding level, (4) regeneration with 27 mL of 10 mM glycine, pH 1.75 at 50 mL min-1 and (5) baseline equilibration.

optimum binding responses, as determined by both SPR-biosensor and ELISA analysis (E10-126, Bethyl Laboratories) and was therefore selected as the primary calibrant in the SPR-biosensor assay.

3.3. Optical biosensor analysis Fig. 2 illustrates overlaid sensorgrams obtained from the analysis of duplicate multi-level working calibrants


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Fig. 3. Dose–response curve established from relative responses (duplicates) vs. lactoferrin concentration (ng mL 1) acquired in Fig. 2. Duplicate RSDr at each level o0.9%.

under the conditions described, while Fig. 3 displays the derived calibration curve obtained as relative responses (RU) against lactoferrin concentration.

3.4. Method validation 3.4.1. Specificity It is imperative for any immunoassay format that non-specific interferences be evaluated (Brynda, Houska, Brandenburg, & Wikerstal, 2002). The extent of potential non-specific binding from both target analyte and sample matrix components to the dextran support was therefore assessed over the reference ligand-free surface. Binding responses were measured for lactoferrin standards (o1 RU), individual milk proteins (o2 RU) and a representative range of consumer milks, colostrum and infant formulas (o5 RU). Suppression of the predominantly electrostatic interactions of the basic lactoferrin and other milk proteins with the hydrophilic and negatively charged carboxymethyl dextran surface to such analytically negligible levels was achieved by increasing diluent ionic strength to 0.5 M sodium chloride.

Further, binding specificity of the immobilised ligand was formally evaluated through measuring its crossreactivity for individual milk proteins (a-casein, bcasein, k-casein, a-lactalbumin, b-lactoblobulin, IgG, bovine serum albumin and lactoperoxidase) at levels expected in milk. Only k-casein was found to bind to the ligand with a cross-reactivity of ca. 0.5% relative to bovine lactoferrin. In the absence of lactoferrin-free milk, the potential for binding interferences originating from non-analyte sample components was also evaluated by analysis of serial dilutions of milk and infant formula (1:1000–1:4000) over the active immobilised surface. Measured lactoferrin estimates were found to be independent of dilution level (milk: 2.53% RSD; infant formula: 2.14% RSD) and confirmed the absence of any significant matrix interference. This data provides additional evidence that the analytical signal is uncompromised by non-specific interactions of sample components with immobilised ligand. Competitive inhibition experiments were performed to further evaluate specificity of the immobilised antibody for bovine lactoferrin based on inhibition of binding by free antibody in solution. When either authentic lactoferrin or diluted milk (ca. 1.25 pmol mL 1) was


initially equilibrated with excess antibody (270 pmol mL 1), the binding inhibition to surface ligand was estimated at 498%, thereby confirming both ligand specificity and minimal non-specific binding. A sandwich-immunoassay format was also investigated, primarily as an additional confirmation of assay specificity, but also for potential enhancement of analytical response. Although such assays generally utilise capture and enhancement antibodies of different epitope specificity, use of the same polyclonal antibovine lactoferrin antibody (pAb) as secondary reagent also yielded signal enhancement due exclusively to its interaction with surface-bound lactoferrin (confirmed by a negligible response following direct injection over immobilised pAb). The sandwich-assay protocol was, despite its enhancement of analytical signal and confirmation of specificity, not adopted for routine analysis of lactoferrin due to inherent requirements of increased analysis time and complexity.

fluid milk sample measured 3.64%, while inter-assay reproducibility RSDR over several immobilised surfaces for independent fluid low-fat milk samples and a control infant formula powder measured 10.8% (n: 10) and 12.3% (n: 13), respectively. Performance over sequential analyses illustrates the stability of the surface ligand and its analyte binding capacity, as well as the effectiveness of the regeneration protocol, as shown in Fig. 4 for replicate analysis of a typical infant formula (n: 30). Intra-run repeatability of the relative binding response was 1.0% RSD, while baseline drift was acceptably low (ca. 5RU cycle 1). Furthermore, it was found that the anti-bovine lactoferrin immobilised surface was durable over at least 500 injections with minimal baseline inter-run drift under the described assay conditions. In the absence of a certified reference milk or colostrum for lactoferrin content, analytical accuracy may be assessed based on recovery and method comparison studies. Recovery was determined following duplicate spiking of fluid low-fat milk with lactoferrin at 1 and 2 endogenous levels and measured 101.7–106.8%. The described biosensor-based method was evaluated against HPLC and ELISA techniques for samples representative of type, including a lactoferrin isolate, fluid skim milk, colostrum, spray dried colostrum, infant formulas, milks and yoghurt and the comparative data summarised in Table 1. Equivalence of data from independent analytical methods is generally considered indicative of an unbiased estimate of analyte content. Despite the fundamentally different analytical principles and the non-reference status of the alternative methods utilised, analysis of variance and Tukey’s HSD post-hoc comparison revealed that there were no significant

Relative response

3.4.2. Performance Over the optimised working calibration range (0–1000 ng mL 1), a 4-parameter curve fit adequately described the dose–response relationship. The instrumental limit of detection (response+3sd of blank calibrant) for bovine lactoferrin over several independent runs was estimated as 1.11 ng mL 1. The method detection limit (sd xtn 1,0.01) was estimated from replicate analysis of a fluid milk sample containing endogenous lactoferrin and measured 19.9 mg mL 1 (n: 7). Instrument precision was estimated from replicate analysis of the measured lactoferrin concentration of a single-level control calibrant (500 ng mL 1) over several runs and measured 3.50% (n: 23) relative standard deviation (RSD). Intra-assay repeatability RSDr for a RU

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Fig. 4. Absolute (RSD: 0.24%) baseline (m) and relative (RSD: 1.00%) binding response (’) of lactoferrin in an infant formula extract (1:1000) over goat anti-bovine lactoferrin immobilised surface for 30 sequential analyses. Assay conditions as described in Fig. 2.

ARTICLE IN PRESS H.E. Indyk, E.L. Filonzi / International Dairy Journal 15 (2005) 429–438 Table 1 Comparison of (mg 100 g 1)a

Lactoferrin isolate (g 100 g 1) Colostrum Colostrum Colostrum Colostrum Colostrum Infant Formulad Infant Formula Skim Milk Skim Milk Whole Milke Whole Milke Whole Milke Whole Milke Yoghurtd

analytical

methods

for

lactoferrin

content

Sepharose LCb

Heparin LCb

ELISAc

SPRbiosensor

90.5 214.7 214.0 93.0 170.0 240.0 18.9 4.5 2.3 16.3 6.1 118.2 49.1 13.1 173

87.0 235.2 226.8 nd nd nd 18.9 15.1 9.8 18.7 12.9 119.8 51.3 12.5 nd

98.4 224.0 219.5 nd nd nd 14.8 0.4 4.6 16.4 0.1 101.6 58.4 11.4 225

85.6 265.0 274.6 76.8 159.0 227.0 38.6 3.1 16.9 20.7 5.9 108.0 50.6 14.8 207

a

All samples (except liquid skim milk) are powders containing endogenous lactoferrin (except d). b LC methods are modifications of Francis et al., (1995). c Bethyl ELISA kit E10-126 (Bethyl Labs, Montgomery, TX, USA). d Lactoferrin-supplemented. e Raw milk from individual cows.

overall method differences with 95% confidence intervals of the average differences containing zero. However, only the SPR-biosensor technique recovered the expected content in supplemented infant formula based on the sum of known added and endogenous lactoferrin. 3.5. Method applications The described biosensor-based immunoassay, utilising an immobilised anti-bovine lactoferrin antibody, has been applied to a range of cows’ milk products following a sample preparation scheme involving simple dissolution in buffer to high dilution levels, a protocol facilitated by the inherent sensitivity of SPR detection. Such minimal manipulation avoids the potential for recovery losses of lactoferrin associated with casein removal, filtration and centrifugation protocols utilised with alternative analytical techniques. The mean lactoferrin content in consumer cows’ milk has been estimated by this technique as 170 mg mL 1 (range: 150–210 g mL 1; n: 10), a level consistent with the 20–750 mg mL 1 range reported using other techniques (Jensen, 1995; Pakkanen & Aalto, 1997; Fox & McSweeney, 1998; Steijns & van Hooijdonk, 2000; Tu, et al., 2002; Palmano, & Elgar, 2002; Farr et al., 2002; Fox & Kelly, 2003; Lindmark-Ma˚nsson, Fonden, Pettersson, 2003; Turner et al., 2003; Chen & Mao, 2004). The described SPR-immunoassay also reveals that lactoferrin levels expressed in raw milk from

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individual animals are more variable, probably dependent on genotype, diet, somatic cell status and collection time (Turner et al., 2003; Chen & Mao, 2004). In mammals, physiological expression of milk components is highly influenced by the stage of lactation, and the SPR-biosensor technique was therefore applied to investigate the variation in lactoferrin content during the progression from early bovine colostrum to mature milk. Fig. 5 illustrates the temporal trend for an individual animal over the first 15 days following parturition. As shown, the content of lactoferrin in early colostrum was found to be ca. 100 greater (ca. 5000 mg mL 1) than in mature milk (ca. 50m g mL 1), illustrating a significant physiological response to lactogenesis. These biosensor-SPR based results obtained from a single lactating cow are consistent with data based on alternative techniques, which have variously reported a dramatic reduction ranging from 1500–5000 mg mL 1 in colostrum to 20–350 mg mL 1 in milk (Senft & Klobasa, 1973; Pakkanen & Aalto, 1997; Fox & McSweeney, 1998; Tu et al., 2002; Turner et al., 2003). The higher content of lactoferrin in human compared to bovine milk by ca. 10 is suggestive of its greater essentiality to human infants, and in view of this, supplementation of infant formulas with isolated bovine lactoferrin is becoming increasingly prevalent (Steijns & van Hooijdonk, 2000; Kuwata et al., 2001; Tu et al., 2002; Fox & Kelly, 2003). Although recombinant human lactoferrin is available, bovine lactoferrin is currently preferred for reasons of availability, cost, consumer acceptability and functional similarity with its human equivalent. The potential for the described SPRbased biosensor immunoassay has been evaluated through the estimation of lactoferrin content in a limited group of infant formulas, and the data shown in Table 2. Measured lactoferrin content was reasonably consistent with label claims based on levels added during manufacture, although one heterogenous product produced by a dry-blending protocol demonstrated a significantly lower than expected level. Although only a limited number of consumer infant formula products were available for evaluation, the described technique is robust, rapid and reliable, and combined with minimal sample preparation requirements and automated operation, is suitable for the routine compliance control of infant formula production. Bioactive minor proteins in bovine milk will be increasingly exploited in the expanding international trade of milk products (Boland et al., 2001), and such trade will be increasingly reliant on the traceability of internationally validated analytical methods. In the absence of any currently accepted reference method, the described SPR-biosensor binding assay may fulfil


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Fig. 5. Lactoferrin content of early lactation milk acquired from the 2nd calving of a 4-year Jersey cow. Each lactoferrin level represents the mean of 3 independent analyses over 2 independent immobilised CM5 surfaces with 1x standard deviation error bars (mean RSD: 11.5%).

Table 2 Lactoferrin content (mg g 1) of supplemented infant formulas by SPR biosensor immunoassay against label claim Sample

Measured

Claim

1 2 3 4 5b

386 381 375 174 31

200 500 400 500a nd

a

heterogenous product due to dry-blending lactoferrin protocol. non lactoferrin-supplemented infant formula.

b

this need for lactoferrin in both colostrum and infant formula products. While discrepancies between analytical methods will generally occur due to differences in detection principles, it is also apparent that there are factors that critically influence the analysis of lactoferrin irrespective of the end-point measurement technique. It will therefore be expedient to identify an internationally accepted primary reference material, in order to facil-

itate a more realistic assessment of the equivalence of available analytical methods.

4. Conclusions The sensitivity, specificity and quantitation range of biospecific methods are generally determined by ligand:analyte affinity, and an optimised SPR-immunoassay, therefore, relies predominantly on appropriate ligand selection and immobilisation chemistry, as well as buffer conditions, contact time and regeneration protocol. Goat anti-bovine lactoferrin was found to provide excellent performance parameters, stable surfaces under multiple regeneration conditions, low detection limits and minimal non-specific binding at the high sample dilution levels facilitated by the inherent sensitivity of SPR detection. The described label-free, real-time and automated biosensor-immunoassay for the quantitation of lactoferrin in milk, colostrum and infant formula is rapid, sensitive, precise and accurate, and provides


analytical information comparable to alternative methods available.

Acknowledgements We thank Drs. David Elgar, Don Otter and Kate Palmano (Fonterra Research Centre, Palmerston North, New Zealand) for both the supply of fully characterised bovine lactoferrin purified from milk and for comparative HPLC and ELISA analyses. We thank Gaylene Watene and Lauryn Wati for their expert and enthusiastic technical assistance, and the Nutritional TCE Fonterra for support of the project. References Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V., & Baker, E. N. (1990). Apolactoferrin structure demonstrates ligandinduced conformational change in transferrins. Nature, 344, 784–787. Baker, H. M., & Baker, E. N. (2004). Lactoferrin and iron: structural and dynamic aspects of binding and release. BioMetals, 17, 209–216. Bates, G. W., Billups, C., & Saltman, P. (1967). The kinetics and mechanism of iron (III) exchange between chelates and transferrin. Journal of Biological Chemistry, 242, 2810–2815. Baveye, S., Elass, E., Fernig, D. G., Blanquart, C., Mazurier, J., & Legrand, D. (2000). Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, Eselectin and ICAM-1, induced by the CD14-lipolysaccharide complex. Infection and Immunity, 68, 6519–6525. Blackburn, D. G. (1993). Lactation: Historical patterns and potential for manipulation. Journal of Dairy Science, 76, 3195–3212. Boland, M., MacGibbon, A., & Hill, J. (2001). Designer milks for the new millennium. Livestock Production Science, 72, 99–109. Brock, J. H., & Arzabe, F. R. (1976). Cleavage of differic bovine transferrin into two monoferric fragments. FEBS Letters, 69, 63–66. Brynda, E., Houska, M., Brandenburg, A., & Wikerstal, A. (2002). Optical biosensor for real-time measurement of analytes in blood plasma. Biosensors & Bioelectronics, 17, 665–675. Chen, P.-W., & Mao, F. C. (2004). Detection of lactoferrin in bovine and goat milk by enzyme-linked immunosorbent assay. Journal of Food and Drug Analysis, 12, 133–139. Chou, S.-F., Hsu, W.-L., Hwang, J.-M., & Chen, C.-Y. (2004). Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance. Biosensors & Bioelectronics, 19, 999–1005. Cui, X., Yang, F., Sha, Y., & Yang, X. (2003). Real-time immunoassay of ferritin using surface plasmon resonance biosensor. Talanta, 60, 53–61. Desmazeaud, M. (1993). Determination of indigenous antimicrobial proteins of milk.In L. Bjo¨rck (Ed.), Bulletin of the international dairy federation (vol. 284). Lactoferrin (pp 29–42) (Chapter 3). Ekstrand, B., & Bjo¨rck, L. (1986). Fast protein liquid chromatography of antibacterial components in milk. Lactoperoxidase, lactoferrin and lysozyme. Journal of Chromatography, 358, 429–433. 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 HPLC on polystyrenedivinylbenzene. Journal of Chromatogaphy. A., 878, 183–196.

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Farr, V. C., Prosser, C. G., Clark, D. A., Tong, M., Cooper, C. V., Willix-Payne, D., & Davis, S. R. (2002). Lactoferrin concentration is increased in milk from cows milked once-daily. Proceedings of the New Zealand Society of Animal Production, 62, 225–236. Fox, P. F., & Kelly, A. L. (2003). Developments in the chemistry and technology of milk proteins. 2. Minor milk proteins. Food Australia, 55, 231–234. Fox, P. F., & McSweeney, P. L. H. (1998). Milk proteins. In P. F. Fox, & P. L. H. McSweeney (Eds.), Dairy chemistry and biochemistry (pp. 146–238). London: Blackie Academic (chapter 4). Francis, G. L., Regester, G. O., Webb, H. A., & Ballard, F. J. (1995). Extraction from cheese whey by cation-exchange chromatography of factors that stimulate the growth of mammalian cells. Journal of Dairy Science, 78, 1209–1218. Gonzalez-Llano, D., Polo, C., & Ramos, M. (1990). Update on HPLC and FPLC analysis of nitrogen compounds in dairy products. Lait, 70, 255–277. Guidi, A., Laricchia-Robbio, L., Gianfaldoni, D., Revoltella, R., & Del Bono, G. (2001). Comparison of a conventional immunoassay (ELISA) with a surface plasmon resonance-based biosensor for IGF-1 detection in cows’milk. Biosensors & Bioelectronics, 16, 971–977. Haasnoot, W., Olieman, K., Cazemier, G., & Verheijen, R. (2001). Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder. Journal of Agricultural and Food Chemistry, 49, 5201–5206. Hahn, R., Schulz, P. M., Schaupp, C., & Jungbauer, A. (1998). Bovine whey fractionation based on cation-exchange chromatography. Journal of Chromatography A, 795, 277–287. Hiesberger, T., Hermann, M., Jacobsen, L., Novak, S., Hodits, R. A., Bujo, H., Meilinger, M., Httinger, M., Schneider, W. J., & Nimpf, J. (1995). The chicken oocyte receptor for yolk precursors as a model for studying the action of receptor-associated protein and lactoferrin. Journal of Biological Chemistry, 270, 18219–18226. Homola, J. (2003). Present and future of surface plasmon resonance biosensors. Analytical and Bioanalytical Chemistry, 377, 528–539. Indyk, H., & Filonzi, E. (2003). The determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPRimmunoassay. Journal of AOAC International, 86(2), 386–393. Indyk, H., & Filonzi, E. (2004). Direct optical biosensor analysis of folate-binding protein in milk. Journal of Agricultural and Food Chemistry, 52, 3253–3258. Jensen, R. G. (1995). Defence agents in bovine milk. In R. G. Jensen (Ed.), Handbook of milk composition (pp. 746–748). New York: Academic Press (Chapter 9B). Kuwata, H., Yamauchi, K., Teraguchi, S., Ushida, Y., Shimokawa, Y., Toida, T., & Hayasawa, H. (2001). Functional fragments of injested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats. Journal of Nutrition, 131, 2121–2127. Law, A. J. R., Leaver, J., Banks, J. M., & Horne, D. S. (1993). Quantitative fractionation of whey proteins by gel permeation FPLC. Milchwissenschaft, 48, 663–666. Lindmark-Ma˚nsson, H., Fonden, R., & Pettersson, H.-E. (2003). Composition of Swedish dairy milk. International Dairy Journal, 13, 409–425. Lindmark-Ma˚nsson, H., Svensson, U., Paulsson, M., Alden, G., Frank, B., & Johnsson, G. (2000). Influence of milk components, somatic cells and supplemental zinc on milk processability. International Dairy Journal, 10, 423–433. Lonnerdal, B., & Atkinson, S. (1995). Nitrogenous components of milk. In R. G. Jensen (Ed.), Handbook of Milk Composition (pp. 351–368). New York: Academic Press (Chapter 5). Marchesseau, S., Mani, J.-C., Martineau, P., Roquet, F., Cuq, J.-L., & Pugniere, M. (2002). Casein interactions studied by the surface

ARTICLE IN PRESS 438

H.E. Indyk, E.L. Filonzi / International Dairy Journal 15 (2005) 429–438

plasmon resonance technique. Journal of Dairy Science, 85, 2711–2721. Mitoma, M., Oho, T., Shimzaki, Y., & Koga, T. (2001). Inhibitory effect of bovine lactoferrin on the interaction between a streptococcal surface protein antigen and human salivary agglutinin. Journal of Biological Chemistry, 276, 18060–18065. Montagne, P. M., Tregoat, V. S., Cuilliere, M. L., Bene, M., & Faure, G. C. (2000). Measurement of nine human milk proteins by nephelometric immunoassays: application to the determination of mature milk protein profile. Clinical Biochemistry, 33, 181–186. Monti, J. C., Fumeaux, D., Barrios-Larouze, V., & Jolles, P. (1984). Relative distribution of human milk whey proteins in normal and abnormal samples: an approach by HPLC. Milchwissenschaft, 39, 219–221. Muller-Renaud, S., Dupont, D., & Dulieu, P. (2004). Quantification of b-casein in milk and cheese using an optical immunosensor. Journal of Agricultural and Food Chemistry, 52, 659–664. Nygren, L., Sternesjo¨, A., & Bjo¨rck, L. (2003). Determination of folate-binding proteins from milk by optical biosensor analysis. International Dairy Journal, 13, 283–290. Pakkanen, R., & Aalto, J. (1997). Growth factors and antimicrobial factors of bovine colostrum. International Dairy Journal, 7, 285–297. Palmano, K. P., & Elgar, D. F. (2002). Detection and quantitation of lactoferrin in bovine whey samples by reversed-phase HPLC on polystyrene-divinylbenzene. Journal of Chromatography A, 947, 307–311. Parry, R.M., & Brown, E.M. (1974). Lactoferrin conformation and metal binding properties. In: M. Friedman (Ed.), Advances in experimental medicine and Biology. (vol 48) (pp. 141–160). Senft, B., & Klobasa, F. (1973). Studies on the concentration of lactoferrin in colostral and normal milk of cows. Milchwissenschaft, 28, 750–752.

Steijns, J. M., & van Hooijdonk, A. C. M. (2000). Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. British Journal of Nutrition, 84(Suppl 1), S11–S17. Stevenson, L., & Knowles, G. (2003). The role of dairy ingredients in enhancing immunity. Australin Journal of Dairy Technology, 58, 135–139. Strange, E. D., Malin, E. L., Van Hekken, D. L., & Basch, J. J. (1992). Chromatographic and electrophoretic methods used for analysis of milk proteins. Journal of Chromatography, 624, 81–102. Tu, Y. Y., Chen, C. C., Chang, J. H., & Chang, H. M. (2002). Characterization of lactoferrin (Lf) from colostral whey using antiLf antibody immunoaffinity chromatography. Journal of Food Science, 67, 996–1001. Turner, S-A., Williamson, J. H., Thomson, N. A., Roche, J. R., & Kolver, E. S. (2003). Diet and genotype affect milk lactoferrin concentrations in late lactation. Proceedings of the New Zealand Society of Animal Production, 63, 87–90. Weinberg, E. D. (2001). Human lactoferrin: a novel therapeutic with broad spectrum potential. Journal of Pharmacy and Pharmacology, 53, 1303–1310. Wong, D. W., Camirand, W. M., & Pavlath, A. E. (1996). Structures and functionalities of milk proteins. Critical Reviews in Food Science and Nutrition, 36, 807–844. Yamauchi, K., Soejima, T., Ohara, Y., Kuga, M., Nagao, E., Kagi, K., Tamura, Y., Kanbara, K., Fujisawa, M., & Namba, S. (2004). Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab’)2 fragments. BioMetals, 17, 349–352. Zhang, N. T., Nakano, T., & Ozimek, L. (2002). Isolation of lactoferrin from bovine colostrum by SP-sepharose cationexchange chromatography. Milschwissenschaft, 57, 614–617. Zhang, N. T., Nakano, T., & Ozimek, L. (2003). Purification of lactoferrin from bovine milk by chromatography on DNA-agarose. Milschwissenschaft, 58, 249–252.