Biosensors & Bioelectronics 16 (2001) 971–977

Comparison of a conventional immunoassay (ELISA) with asurface plasmon resonance-based biosensor for IGF-1 detection in

cows’ milk

Alessandra Guidi a,*, Leopoldo Laricchia-Robbio b, Daniela Gianfaldoni a,Roberto Revoltella b, Giancarlo Del Bono a

a Department of Animal Pathology Prophylaxis and Food Hygiene, Uni�ersity of Pisa, Via delle Piaggie 2, 56100 Pisa, Italyb Institute of Mutagenesis and Differentiation, CNR Research Area, Via Moruzzi, Ghezzano, 56100 Pisa, Italy

Received 4 July 2000; received in revised form 17 April 2001; accepted 19 April 2001

Abstract

Recombinant bovine somatotropin (rBST) treatment is adopted in dairy cows to augment milk yield. Previous studies showedthat insulin-like growth factor-1 (IGF-1) is present in milk from cows treated with rBST. Since IGF-1 is a suspected carcinogen,its presence in milk for human consumption is potentially a health hazard. Therefore rBST use, still authorized in the UnitedStates, has been revoked in Canada and is under evaluation in the EU. The rising attention on IGF-1 presence in alimentary milkfocused the necessity to develop specific, sensitive and rapid IGF-1 detection systems. We have developed a solid phaseenzyme-linked immunoassay (ELISA) and also an automated surface plasmon resonance-based biosensor system (BIA-technol-ogy) for evaluating IGF-1 in fresh cow’s milk. Hyperimmune polyclonal anti-IGF-1 antibodies were characterized with respect totheir specific binding capacity to IGF-1. The results obtained with these two methods have been compared. This study shows thepotential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fullyautomated, measures in real time, sharpened yes/no response) offer several advantages compared to ELISA in the detection ofcompounds in fresh cows’ milk (MURST 40%; CNR P.F. MADESS 2). © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Immunosensors; BIA-technology; IGF-1; Sensors for food application

www.elsevier.com/locate/bios

1. Introduction

The use of recombinant bovine Somatotropin (rBST)in cows enhances milk yields. It has therefore becomeessential to inquire into its secondary effects. At presentthe US government authorizes the use of rBST whilethe EU is still waiting for the results of a study en-trusted to the Scientific Committee on Veterinary mea-sures relating to Public Health (SCVPH) and theScientific Committee on Animal Health and AnimalWelfare (SCAHAW). The WHO and FAO are stilluncertain about the health implications of possibleglobal scale applications of rBST treatment (Gianfal-

doni, 1989; Juskevih and Guyer, 1990; Macrı, 1999). Ithas been shown that rBST stimulates insulin-likegrowth factor-1 (IGF-1)-production (Prosser et al.,1989; Sharma et al., 1994), leading to increased valuesin the milk and blood of treated cows; this raisesquestions over the possible persistence of rBST residuesas well as the increased levels of IGF-1 in milk.

IGF-1 is a 70 amino acid-linked polypeptide pro-duced in the liver and other body tissues. It is capableof stimulating various cellular responses, including cellproliferation and differentiation (Manousos et al.,1999). It is also known to stimulate proliferation ofboth normal and malignant cells (Mantzoros et al.,1997; Zumkeller and Burdach, 1999); many recent stud-ies have evinced a link between cancer risk and highlevels of IGF-1 (Vadgama et al., 1999; Wolk et al.,1998; Hankinson and Willet, 1998).

* Corresponding author. Tel.: +390-50-541180; fax: +390-50-540464.

E-mail address: aguidi@vet.unipi.it (A. Guidi).

0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 9 5 6 -5663 (01 )00245 -7

A. Guidi et al. / Biosensors & Bioelectronics 16 (2001) 971–977972

Synthesis of IGF-1 is regulated by growth hormone(GH) and for this reason, as well as those cited above,treatment of cows with rBST should be strictly conrolled. Mammals produce structurally highly similarIGF-1 and the human and bovine molecules in particu-lar are completely identical (Juskevih and Guyer, 1990).

Current concerns over the potential health hazard ofIGF-1 as a cancer-causing agent is attracting increasingattention to develop rapid, sensitive and accurate IGF-1 detection assays in foodstuffs. This contribution re-views our laboratory experiments using an ELISA anda surface plasmon resonance (SPR)-based biosensorsystem (BIA-technology) that we have developed in anattempt to detect and quantitate IGF-1 in fresh cows’milk.

2. Materials and methods

2.1. Reagents

Recombinant human IGF-1, cytochrome-c, horsespleen ferritin, hyperimmune goat polyclonal anti hu-man-IGF-1 serum antibodies (Goat anti-IGF-1 Abs),alkaline phosphatase (AP)-labeled goat anti-rabbit IgG(AP-Goat anti Ra IgG) and bovine gelatine were allfrom Sigma (St. Louis, MU); recombinant human gran-ulocyte-macrophage colony-stimulating factor (GM-CSF), from Carlo Erba-Farmitalia (Milan, Italy); allfrom Sigma; hyperimmune rabbit polyclonal anti-IGF-1 serum antibodies (Ra-anti IGF-1) were from Pepro-Tech EC (London, UK) and p-nitro-phenyl-ortho-phosphate (PNPP) was from BDH (Europe, UK).

Sensor Chip CM5, surfactant P20, ethylene-di-aminetetracetic acid (EDTA), 4-(2 hydroxyethyl)-1-piperazine sulfonic acid (HEPES), N-ethyl-N �-(3-di-ethylaminpropyl) carbodiimide (EDC), N-hydrox-ysuccinamide (NHS), ethanolamine hydrochloride(ETH) were all from BIACORE AB, Uppsala, Sweden.

Milk samples obtained from 20 reliably untreatedcows were directly milk-machined at the milking shedand quick-frozen at −20° C. In some cases, individualmilk samples were defatted by centrifugation and auto-claved before use, to denature the native IGF-1, asdescribed (Burton and McBride, 1994).

2.2. Enzyme-linked immunosorbent assays (ELISA)

Immunoassays for IGF-1 have been previously de-scribed with alternative results (Zhao et al., 1991; Ep-pard et al., 1994). Preliminary evidence suggested thatthe use of highly specific antibodies is a critical factorfor a reliable immunoassay of this growth factor.

Direct and competition ELISA assays for IGF-1detection were developed as follows:

2.2.1. Direct assayIGF-1 (0.05–0.1 �g/50 �l carbonate buffer, pH 9.0)

was added to each well in a 96-well Falcon plate(Becton-Dickinson, Oxnard, CA) and allowed to ad-sorb for 6 h at room temperature under continuousshaking. After washing, unreacted binding sites on theplate were blocked with 2% bovine gelatin diluted inphosphate buffered saline, pH 7.0 (PBS). After repeatedwashings in PBS-0.05% Tween (T-PBS), dilutions of Raanti-IGF-1 Abs (or a normal rabbit serum as negativecontrol) were incubated for 4 h at room temperature,under continuous shaking. Bound immunocomplexeswere revealed using AP-Goat anti-Ra IgG second anti-body. The developed reactions were read at OD 405 nmafter 15 min from the addition of PNPP as chromogen.

2.2.2. Competition assayAliquots containing a constant amount of anti-IGF-1

serum antibodies (diluted to approx. 70% maximumbinding capacity to IGF-1 adsorbed on the plate) werepre-incubated in solution for 2 h at 37° C with varyingconcentrations of recombinant IGF-1. The tubes con-taining samples were then centrifuged (8000×g for 10min) and the free residual antibody-binding activity inthe mixture supernatant was assayed as above, usingAP-Goat anti Ra IgG second Abs. The developedreactions were evaluated at OD 405 nm, reading after15 min from the addition of PNPP as chromogen.

2.3. BIA-technology

Measurements were performed using the BIA-tech-nology (Liedberg et al., 1983; Lundstrom, 1994; Lofasand Jonsson, 1992; Liedberg et al., 1993, 1996; Jonssonand Malmqvist, 1992; Fagerstam et al., 1992; Fager-stam, 1991), surface plasmon resonance (SPR)-basedbiosensor system (BIACORE AB, Uppsala, Sweden),as described (Laricchia Robbio et al., 1998; Revoltellaet al., 1998).

The BIA-technology allows real-time measurementsof ligand binding to a specific antigen coupled to anextended carboxymethylated hydrogel matrix layeredon a gold surface (Sensor Chip CM5). Covalent bindingof ligands was performed using conventional carbodi-imide coupling chemistry (Sjolander and Urbaniczky,1991). The SPR detects and measures changes in refrac-tive index due to the binding and dissociation of inter-acting molecules at or in proximity to the gold surface.The change in refractive index (proportional to theconcentration of the interacting molecules) causes ashift in the angle of incidence at which the SPR phe-nomenon occurs. Such shifts are monitored continu-ously over time and are shown as sensorgrams. Thedifferential in signals at the beginning and at the end ofthe injection cycle reflects the amount of immobilizedligand bound to an acceptor molecule within the sens-

A. Guidi et al. / Biosensors & Bioelectronics 16 (2001) 971–977 973

ing layer. Values are expressed as resonance signal units(RU) and reflect the relative amount of ligandimmobilized.

2.3.1. Direct assayRecombinant IGF-1 was immobilized to the NHS/

EDC pre-activated carboxymethylated dextran-matrixby covalent coupling (Jonsson et al., 1991). Immobiliza-tion was performed by flowing 25 �l of a stock solutionof IGF-1 (40 �g/ml) solubilized in 10 mM Na-acetatebuffer, pH 4, at a constant flow rate of 5 �l/min at25° C.

The residual binding sites were then saturated withETH, pH 8.5. The HEPES-buffered saline (HBS), pH7.4, with 0.15 M NaCl, 3.4 mM EDTA and 0.05%surfactant P20, was used as running buffer. The anti-IGF-1 antibody diluted in 10 mM HBS was theninjected (20 �l) and run at a constant flow rate of 5�l/min. Regeneration of the IGF-1 dextran matrix wasperformed by subsequent pulses of HCl 10 mM or SDS0.05%; this procedure allows a total regeneration of thesensing surface. Analysis of direct antibody interactionwith the captured antigen was then performed withoutsecondary tracer molecules and antibody binding wasmeasured in real time.

2.3.2. Competition assayThe competition assay was set up, using a fixed

anti-IGF-1 antibody concentration, corresponding toapproximately 60% maximum antibody binding capac-ity against the recombinant IGF-1 immobilized on thesensor surface. HBS was used as diluent buffer. Theantibody was pre-incubated in microfuge tubes, in theabsence or presence of varying concentrations of theinhibitory molecule (recombinant IGF-1 or an unre-lated protein such as GM-CSF, ferritin or cytochrome-c), for 2 h at 37° C. The test tubes were thencentrifuged (8000×g for 10 min) and the antibodybinding capacity in the supernatant was evaluated.Intermediate washing with HCl 10 mM or SDS 0.05%allowed full detachment of antibodies from the sensingsurface, before beginning any subsequent analysis.

Two procedures were used to evaluate the possibilityof IGF-1 antibody cross-reactivity with endogenousIGF-1 present in milk samples:1. In a preliminary series of experiments, recombinant

IGF-1 diluted in HBS, was first immobilized ontothe chip surface. Decreasing concentrations of re-combinant IGF-1, diluted either in HBS or in auto-claved milk, were first pre-incubated with the abovefixed concentration of anti-IGF-1 Abs, and then themixture was injected for antibody measurement.

2. In a second series of experiments, recombinant IGF-1, diluted in HBS, was first linked to the chipsensing surface which was then flowed with auto-claved milk. Subsequently, anti-IGF-1 antibodies

pre-incubated with recombinant IGF-1 at varyingconcentrations in autoclaved milk, were flowed.

3. Results

3.1. ELISA

3.1.1. Direct assayAnti-IGF-1 serum antibodies and a normal rabbit

serum were assayed in a direct ELISA to evaluate theantibody binding capacity against recombinant IGF-1coated on plastic. Fig. 1a shows the results of a typicalexperiment, where the polyclonal anti-IGF-1 antibodiesbound specifically to IGF-1 in a concentration-depen-dent manner, while the normal serum did not exert anysignificant binding capacity to this protein, unless athigh concentration.

3.1.2. Competition assayIn a competition assay, anti-IGF-1 antibodies were

first incubated in solution with varying amounts ofrecombinant IGF-1 or other unrelated proteins as nega-tive controls (e.g. GM-CSF, ferritin or cytochrome-c),and the immunoreactivity of these mixtures was tested.In a first approach, all the samples were diluted in PBS.As shown in the insert of Fig. 1b, anti-IGF-1 antibodybinding was specifically inhibited by recombinant IGF-1 in a dose-dependent manner, while no inhibition wasfound with the other control proteins (at any testedconcentrations). Using the same reagents, the competi-tion assay was repeated, by diluting both the antigensand the anti-IGF-1 antibodies in autoclaved milk. Fig.2 compares the results of a representative ELISA com-petition assay, with PBS and milk used as ‘diluents’. Asshown in the figure, two very similar curves of inhibi-tion by free recombinant human IGF-1 were obtained,suggesting that antibody binding to the immobilizedIGF-1 was not influenced by unrelated milk con-stituents. The limiting low sensitivity of this assay wasof approximately 0.5–1 ng IGF-1/ml. The aim of thispreliminary experiment was to detect the presence ofendogenous IGF-1 or other potential milk constituentsthat could non-specifically influence the antibody bind-ing to recombinant IGF-1.

When antibodies were pre-incubated with varyingconcentrations of fresh milk, some inhibition wasshown with undiluted milk or with poorly diluted milk.Using the reference dilution scale obtained with recom-binant IGF-1 as specific inhibitor, we performed ascreening of 20 different fresh milk samples in anattempt to evaluate the normal level of IGF-1 in milk(data not shown); the content of natural IGF-1 wasnever higher than 2 ng of IGF-1/ml of undiluted milk.

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3.2. BIA-technology

Fig. 3 shows the results of a representative competi-tion binding assay performed in triplicate, with poly-clonal Ra. anti-IGF-1 antibodies binding to immo-bilized recombinant IGF-1 in the presence or absenceof inhibitors, using the BIAcore. In these experiments,

recombinant IGF-1 was first immobilized on the chipsurface; polyclonal anti-IGF-1 antibodies, diluted at aconcentration of 60% maximum binding (as previouslydetermined in a direct binding assay), were preincu-bated for 2 h at 37° C with varying concentrations ofrecombinant IGF-1 diluted in HBS. These samples werethen flowed over the sensing layer. In competition

Fig. 1. Direct ELISA (a). Dilution curves of Ra-anti-IGF-1 polyclonal antibodies (�) binding to IGF-1 coated on plastic and a rabbit normalserum (�) used as negative control. Experiments were performed in duplicate wells. In the insert, the results of a Competition ELISA (b)experiment are reported. Antibodies at a fixed dilution (1:1300) giving approximately 70% of maximal binding to immobilized IGF-1, werepre-incubated for 2 h at 37° C with increasing concentration of IGF-1 (�), ferritin, cytochrome-c or GM-CSF (�) as inhibitor. Aftercentrifugation, the residual antibody binding activity was evaluated.

Fig. 2. Competition ELISA. The assay was performed with Ra anti-IGF-1 polyclonal antibodies on microtiter plates coated with IGF-1 (0.05�g/well). Antibodies at a fixed dilution (giving approx. 70% of maximal binding in direct ELISA) were pre-incubated for 2 h at 37° C withincreasing IGF-1 concentrations (0.2–125 ng/ml) and the residual antibody binding was then evaluated in the mixture supernatant. The figureshows the comparison between two representative competition assays performed with antibodies and competitors, respectively diluted in PBS (�)or in autoclaved milk (�). The results of the experiments performed in duplicate wells are reported (mean values�S.E.).

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Fig. 3. BIA technology. The results of two representative experiments run in triplicate are reported as mean RU values. Ra anti-IGF-1 polyclonalantibodies diluted in HBS to a concentration giving 60% of maximal binding to recombinant IGF-1 pre-immobilized on the sensor surface, wereincubated for 2 h at 37° C with varying IGF-1 concentrations. The residual antibody binding capacity in the mixture supernatant was thenevaluated.

assays both recombinant IGF-1 and anti-IGF-1 antibod-ies were diluted either in HBS or in autoclaved milk.After incubation, the samples were injected and the freeantibody binding was evaluated.

The results obtained using autoclaved milk were com-parable with those obtained with HBS. Nevertheless,using milk as buffer the values are constantlyhigher due to the presence of milk compounds than canstick to the surface; however, this raised baseline canbe tolerated because its value is constant in all theinjections.

Table 1 summarizes the results of three different

representative competition experiments, run in dupli-cates. Using the reference dilution scale obtained withrecombinant IGF-1 as inhibitor, the optimum sensitivityof this assay using the biosensor was obtained in therange of 4–100 ng IGF-1/ml. When 20 different fresh rawmilk samples were tested for the content of endogenousIGF-1 by this assay, none of them appeared to containmore than 2–3 ng IGF-1 equivalent/ml (data not shown).

As shown in the table, there were no significantdifferences by comparing inhibition obtained from sam-ples diluted in HBS or autoclaved milk, confirming theresults in ELISA.

Table 1Binding to pre-immobilized IGF-1 by polyclonal Rb. anti-IGF-1 serum antibodies after pre-incubation with or without IGF-1 or unrelatedGM-CSF in HBS or in autoclaved milk (see Sections 2 and 3)

Inhibitors Samples diluted in milkSamples diluted in HBS

Antibody binding Percentage inhibitionconcentration Antibody binding Percentage inhibition(R.U.)(ng/ml)

IGF-172% 234 (�8)125 122 (�3) 69%

42%496 (�5)38%302 (�5)2515%669 (�4)5 396 (�6) 12%

4% 702 (�5)1 412 (�3) 7%0%763 (�1)0%435 (�2)0

GM-CSF:2%740 (�2)20 427 (�2) 1%

Antibodies (diluted to approx. 60% of maximal binding to the immobilized IGF-1, as pre-determined in a direct assay) were incubated withvarying IGF-1 concentrations (from 125 to 1 ng/ml) for 2 h at 37° C The mixture was then injected over the sensing surface to evaluate theresidual antibody binding activity. All the reagents were diluted either in HBS or in autoclaved milk. RU values are the means (�S.E.) oftriplicates.

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4. Discussion

In this paper we describe two sensitive and accurateimmunoassays for measuring IGF-1 in fresh untreatedcow’s raw milk, with no requirement of previous purifi-cation. We compared measurements made on the samereagents by a solid phase ELISA with those obtainedby the BIA-technology using an optical biosensor. Arequirement in both assays is to use highly specificanti-IGF-1 polyclonal antibodies capable of reactingagainst epitopes on the protein which are not modifiedby the unknown gross structural modifications of theantigen occurring during its immobilization onto thesensing surface. In preliminary competition binding ex-periments, a fixed antibody dose (corresponding toapprox. 70% maximal antibody binding capacityagainst the immobilized IGF-1) was pre-incubated withvarying IGF-1 amounts and the residual antibody bind-ing activity was measured in order to construct acalibration curve of values. Using recombinant IGF-1as inhibitor, the range of optimal measurements ineither fresh or autoclaved cow’s milk for this proteinwas consistently found within 1–50 ng/ml. Evaluationof native IGF-1 content in normal fresh raw milk wasnever found to be higher than 4 ng/ml, in line withsimilar findings reported previously in the literaturewith other methods of measurement (Collier et al.,1991). However, higher levels of IGF-1 (�30 ng/ml)have been consistently measured in the milk of cowstreated with BST (Barton et al., 1994).These findingsstrongly suggest a possible role of IGF-1 as a probeindicator, i.e. increases in IGF-1 levels in milk could beused for evaluating animal pre-treatments with BST.

The results of our present study have shown thatboth ELISA and the BIAcore are suitable for detectingIGF-1 at these concentration levels in cow’s milk. How-ever, the BIA-technology offers several advantages overELISA: biosensors require very low amounts ofreagents without any special preparation; do not re-quire antibody labeling or a second reagent for im-munocomplexes detection; allow measurements in realtime with high precision and repeatability of the assay,sharpening yes/no results. Furthermore, since milk is ahighly perishable product, the length of traditionallaboratory methods makes real-time monitoring ofsamples of great interest.

In conclusion, biosensors appear to be good candi-dates for routine IGF-1 monitoring in fresh cow’ smilk, opening the way for other applications in labora-tory investigations for the dairy industry.

Acknowledgements

This work was supported in part by the NationalResearch Council of Italy grants: P.F. MADESS II and

MURST 40%. The authors thank Dr Lorenzo Cas-tigliego for his excellent assistance and Dr GiorgioIannone for his technical support. The work was madein equal parts by the authors.

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