Tatiana Cucu

Céline Platteau

Isabel Taverniers

Bart Devreese

Marc De

Marc De Loose

Bruno De Meulenaer
email: bruno.demeulenaer@ugent.be

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Keywords: Hazelnut allergy, ELISA, detectability, Maillard reaction, reactive lysine, protein bound carbonyls, aggregation 18

come

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Introduction

Food allergy is an abnormal immunological response due to sensitization to a food 20 component. Food allergies have become an important health problem, especially in industrialized countries, where they have been estimated to affect about 2% of the adult 22 population and up to 8% of children [START_REF] Monaci | Milk allergens, their 526 characteristics and their detection in food: A review[END_REF]. Together with the hen's egg, cow's milk, fish, crustacean, peanut, soybean and wheat, tree nuts are typically associated with 24 severe allergic reactions. Among the latter, hazelnut (Corylus avellana) allergy is becoming more important because of its increased incidence, which is related to the homology between 26 the major birch pollen allergen Bet v 1 and the allergens from hazelnut [START_REF] Hansen | Roasted hazelnuts -allergenic activity 520 evaluated by double-blind, placebo-controlled food challenge[END_REF].

As a consequence, hazelnut allergies are predominantly secondary allergies. Hazelnut allergy 28 can cause severe reactions and occurs mostly throughout the European population with a prevalence between 0.1 and 0.5% [START_REF] Birmingham | Hazelnut allergy: 484 Evidence that hazelnut can directly elicit specific IgE antibody response via activating type 2 cytokines in mice[END_REF][START_REF] Degroot | Birch pollinosis and atopy 496 caused by apple, peach, and hazelnut comparison of three extraction procedures with two apple strains[END_REF]. Moreover, 30 numerous cases of hazelnut contamination of foods have been reported [START_REF] Kiening | Sandwich immunoassays for the determination of peanut and hazelnut traces in foods[END_REF] Pele [START_REF] Maria | Peanut and 538 hazelnut traces in cookies and chocolates: Relationship between analytical results and declaration of food allergens on product labels[END_REF]. 32

Given that the only effective treatment for hazelnut or other food allergies is the complete avoidance of the food containing the allergen, this represents an important food 34 safety problem. Directive 2007/68/EC specifies a list of 14 allergic raw materials which must be labeled when present in the food as an ingredient (European Parliament andCouncil 36 Directive 2007/68/EC, 2007). However, due to the growing complexity of food formulation and food processing, food may be unintentionally contaminated via allergen-containing 38 ingredients or carry-over during processing. For this reason, accurate screening of the raw materials and the end products is essential. However, no "gold standard methods" exists for 40 detection of food allergens. At present, the enzyme-linked immunosorbent assay (ELISA) is most frequently used in routine food analysis because it is relatively cheap, easy to perform 42 and highly sensitive. ELISA methods are based on specific molecular interactions between the antibodies used in the assay and the analyte, in this case proteins. During food processing, 44 however, proteins undergo changes such as aggregation, polymerization, degradation and random unfolding. Moreover, food proteins often interact with other food components during 46 processing and can lead to additional modifications at the protein level. Therefore, the antibody-protein interaction during the analytical procedure might be affected and potentially 48 lead to erroneous assay results [START_REF] De Meulenaer | Development of an enzyme-linked immunosorbent assay for peanut proteins using 494 chicken immunoglobulins[END_REF].

Hazelnuts are frequently used in food products containing significant amounts of sugar 50 (e.g. cookies, chocolate, breakfast cereals and, nut spreads). During thermal treatment and storage, hazelnut proteins might be modified by the Maillard reaction, which occurs between 52 a reducing sugar and amino groups in proteins. The products formed as a result of Maillard reaction are significant in foods because they are responsible for flavor and color (Brands and 54 van Boekel, 2001). Abundant evidence exists for how thermal processing together with Maillard reaction can greatly influence the IgE binding activity of food allergens (Gruber et 56 al., 2004;[START_REF] Gruber | Influence of the Maillard reaction on the allergenicity of rAra h 2, a recombinant major allergen from peanut (Arachis 512 hypogaea), its major epitopes, and peanut agglutinin[END_REF][START_REF] Nakamura | Effect of Maillard 532 reaction on allergenicity of scallop tropomyosin[END_REF][START_REF] Nakamura | Reduction of in vitro allergenicity of buckwheat Fag e 1 through the Maillard-type 536 glycosylation with polysaccharides[END_REF]. In contrast, little information is available on how thermal processing and the Maillard reaction influence the 58 performance of commercially available ELISA kits for allergen detection. Such kits are often used in the food industry and official food agency laboratories to detect and quantify hidden 60 allergens in food. Recently, Scaravelli et al. demonstrated that thermal treatment of cookies spiked with peanut caused a decline in the detection of Ara h1 and Ara h2 by two commercial 62 ELISA kits [START_REF] Scaravelli | The effect of heat treatment on the detection of peanut allergens as determined by ELISA and real-time 546 PCR[END_REF].

The aim of this study was to investigate whether the Maillard reaction in the presence or 64 absence of soluble wheat proteins affects the detectability of hazelnut proteins in solution.

This was done in 1% buffered protein solutions incubated with 6% glucose at 70°C for up to 66 48 h. A model system with soluble wheat proteins was chosen, given that wheat is one of the bulk ingredients in cookies and breakfast cereals. First, the impact of the Maillard reaction on 68

Materials and methods

Reagents and standards 76

Soluble wheat proteins "Meripro 711" were kindly provided by Syral Belgium N.V.

(Aalst, Belgium). Chemicals and standards of analytical grade were purchased from Sigma-78 Aldrich (Bornem, Belgium), VWR (Leuven, Belgium) and Acros Organics (Geel, Belgium).

Analytical grade solvents and technical grade hexane were obtained from Chem-Lab 80 (Zedelgem, Belgium). Gel and standards for SDS-PAGE came from Bio-Rad (Nazareth, Belgium) while the Krypton protein stain was provided by Thermo Scientific (Aalst,82 Belgium). The commercial ELISA kits used were Veratox® for hazelnut (Neogen, Lansing, USA), Ridascreen® FAST Hazelnut (R-Biopharm, Darmstadt, Germany), BioKits Hazelnut 84 Assay (Tepnel, Flintshire, UK) and Hazelnut Residue (ELISA Systems, Queensland,

Australia). 86

Hazelnut protein extraction

Hazelnuts from nine different brands were purchased in Belgian local supermarkets; 88 eight brands were virgin and one was roasted. A mixture of the hazelnuts was made by taking equal amounts of each kind. Hazelnuts were frozen with liquid nitrogen and ground in a two-90 step process, first with a blender (Moulinex, France) then with an Ultra Turrax t25 (IKA,

Wilmington, US). 92

The in-house prepared hazelnut protein extracts were obtained following Yeung and Collin's protocol [START_REF] Yeung | Enzyme immunoassay for determination of peanut 550 proteins in food products[END_REF]. In summary, 100 g of ground nuts were defatted 94 four times using 1 L technical hexane each time. After allowing the solvent to evaporate overnight at ambient temperature, 10 g of the defatted powder was mixed with 100 mL of 96 extraction buffer and mixed at 4°C over night. Protein extraction buffer consisted of 1 M NaCl, 1.47 mM KH 2 PO 4 , 2.7 mM KCl and 10 mM Na 2 HPO 4 . Sodium azide 0.2 g L -1 was 98 [START_REF] Bjorksten | Extraction and properties of 488 apple allergens[END_REF]. The protein mixture was further centrifuged for 1h at 4°C and 20000 g to separate the lees. 104 Subsequently, the clear supernatant was dialyzed (Spectra/Por 3 membrane tubing, MWCO 3.5 kDa) using the extraction buffer with the protease inhibitors containing only 0.137 M 106 NaCl, for 48h at 4°C and then filtered through a 0.45 µm membrane filter (Puradisc FP 30,Novolab,Belgium). Finally, the protein solution was lyophilized and stored at -20°C until 108 further use. This extract was used as the reference extract.

Maillard reaction in the absence of soluble wheat proteins 110

Hazelnut protein extracts were dialyzed in 0.1 M phosphate buffer pH 7.4 (Spectra/Por 3 membrane tubing, MWCO 3.5 kDa) before treatment to remove the nitrogen-containing 112 protease inhibitors. All reaction systems were prepared in the same phosphate buffer (pH 7.4) by mixing 1% (w/v) hazelnut proteins with 6% (w/v) glucose. The reaction systems in closed 114 falcon tubes were incubated in a water bath at 70°C for up to 48 h. A glucose-free reaction system was used as a control. Samples were retrieved from the water bath and placed in an ice 116 bath to stop the reaction at specific times. Further, the modified protein solutions were immediately used for the determination of reactive lysine residues, protein bound carbonyls, 118 free thiol and free amino groups. Aliquots for SDS-PAGE were frozen at -20°C until further use. 120

Maillard reaction in the presence of soluble wheat proteins

Two types of glucose-containing model systems were prepared. One model system was 122 obtained by mixing 0.25% (w/v) of hazelnut proteins with 0.75% (w/v) of wheat proteins and 6% (w/v) of glucose. Another model system was obtained by mixing 0.5% (w/v) hazelnut 124 proteins with 0.5% (w/v) wheat proteins and 6% (w/v) glucose. Respective model systems without glucose were prepared as control. These systems were then treated similarly as the 126 model systems containing no soluble wheat proteins.

Assessment of the Maillard reaction 128

Determination of the protein bound carbonyls

Protein bound carbonyls were measured after converting them to the corresponding 2, 130 4-dinitrophenylhydrazones. Briefly, 300 µL protein solution was incubated with 400 µL 2.4dinitrophenylhydrazine (DNPH) 10 mM in 2 M HCl. Further, the proteins were precipitated 132 with 10% trichloroacetic acid (final concentration) and thoroughly washed with 1 mL ethanol/ethyl acetate (1:1) after derivatization; hence any unreacted DNPH or DNPH bound to 134 any other components than protein, was removed. Finally, the pellet was redissolved in 6 M urea and absorbance was measured at 370 nm using a Bio-Rad Benchmark Plus microplate 136 spectrophotometer (Nazareth, Belgium). The carbonyl content was calculated using a molar absorption coefficient of 22000 M -1 cm -1 and the results were expressed in nmol bound 138 carbonyls/mg protein.

Loss of reactive lysine residues 140

The loss of reactive lysine residues was monitored using derivatization with orthophthaldialdehyde (OPA) in the presence of 2-mercaptoethanol yielding a fluorescent product 142 with a maximum excitation wavelength of 340 nm and emission of 450 nm [START_REF] Ferrer | Evolution of 504 available lysine and furosine contents in milk-based infant formulas throughout the shelf-life storage period[END_REF] and measured by a Spectramax Gemini XPS fluorimeter from Molecular Devices 144 (Brussels, Belgium). A standard of β-casein was used to prepare a calibration curve and samples were pre-treated and analyzed using a fluorimeter as described previously (Morales 146 et al., 1996).

Free amino groups determination 148

The modification of free amino groups was determined using derivatization with 2, 4, 6trinitrobenzene sulfonic acid (TNBS) as previously described [START_REF] Fields | The Rapid Determination of Amino Groups with TNBS[END_REF]. Briefly, 0.5 mL 150 of protein solution (containing between 0.05 -0.25 mg proteins) was incubated with 0.5 mL of 0.1 M tetraborate buffer prepared in 0.1 M NaOH. Next, 20 µL of 0.5% TNBS solution 152 was added to the proteins and the reaction mixture was incubated for exactly 10 minutes at room temperature. To stop the reaction, 2 mL of stop solution (1.5 mL of 0,1 M Na 2 S 2 O 3 with 154 98.5 mL of 0.1 M NaH 2 PO 4 ) were added. The absorbance of the resulting mixture was read at 420 nm using a Cary 50 UV-Vis Spectrophotometer from Varian (Sint-Katelijne-Waver, 156 Belgium). The amount of free amino groups was calculated using a molar absorption coefficient of 22000 M -1 cm -1 and results were expressed in nmol free amino groups/mg 158 protein.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 160

Protein cross-linking or fragmentation was analyzed by SDS-PAGE under reducing conditions. A 4% acrylamide stacking gel and a 10% acrylamide resolving gel were used for 162 protein separation. An aliquot of 10 µL sample containing 6 µg of protein was added to 10 µL Laemmli sample buffer containing β-mercaptoethanol and heated at 95°C for 5 min. The 164 samples were further loaded on the gel along with a Precision plus protein unstained molecular weight marker (Bio Rad, Nazareth, Belgium). Krypton fluorescence staining was 166 used to visualize the bands. The gels were read using the Molecular Imager PharosFX Plus

Protein determination 170

Protein determination was performed by determining the nitrogen content according to the Kjeldahl procedure (AOAC Official Method 981.10, 1981). A factor of 5.41 was used to 172 convert nitrogen to total hazelnut protein, and 6.25 to convert to total protein content for the hazelnut wheat protein mixtures. The non-protein nitrogen (NPN) fraction was determined in 174 the supernatant after a previous protein precipitation with 15% TCA (final concentration).

ELISA assays 176

Samples of the above-described model systems were analyzed using four different commercial ELISA kits: Veratox® for hazelnut (Neogen), Ridascreen® FAST Hazelnut (R-178 Biopharm), BioKits Hazelnut Assay (Tepnel) and Hazelnut Residue (ELISA Systems). The four kits were applied to the different model systems. These model systems included (1) 1% 180 hazelnut proteins, (2) 1% hazelnut proteins with 6% glucose, (3) 0.5% hazelnut + 0.5% soluble wheat proteins, (4) 0.25% hazelnut + 0.75% soluble wheat proteins, (5) 0.5% 182 hazelnut + 0.5% soluble wheat proteins and 6% glucose, (6) 0.25% hazelnut + 0.75% soluble wheat proteins and 6% glucose. Of the different model systems, samples retrieved after 0, 24 184 and 48 h incubation at 70°C were analyzed. The untreated sample (= 0 h of thermal treatment) of the glucose-free model system (1) containing only hazelnut proteins is referred to as the 186 reference sample. Before the analysis, dilution series were made of the different samples in the extraction buffer or dilution solution of the respective kits. The actual concentrations are 188 the concentrations of hazelnut proteins based on the absolute protein determination using the Kjeldahl procedure and do not include the wheat proteins that were added in some of the 190 model systems. The dilution series of the samples were analyzed in duplicate in the different [START_REF] Englebienne | Data Analysis, Processing and Presentation. in Immuno and Receptor Assays in Theory and Practice. 500[END_REF]. 200

( )

a d c conc a b Absorbance +                   + - = 1 (2.1)
For the Ridascreen® FAST Hazelnut data were also fitted according to a cubic spline function 202 as described in the manufacturer's instructions. In case of the Veratox for Hazelnut the kit's

Progress of the Maillard reaction

The interaction of reducing sugars or dicarbonyl compounds derived from sugars may 212 lead to the formation of protein-bound carbonyls [START_REF] Stadtman | Free radical-mediated oxidation of free amino acids 548 and amino acid residues in proteins[END_REF]. Therefore, the amount of protein bound carbonyls was used as an indicator of the Maillard reaction. A time-214 dependent increase of protein bound carbonyls was observed in all model systems used (Figure 1). In the pure hazelnut protein samples, an almost seven-fold increase was observed 216 after incubation for 48 h in the presence of glucose while incubation in the absence of glucose led to only a slight increase in protein carbonyls (about a three-fold increase). Incubation of 218 hazelnut proteins in the presence of wheat proteins and glucose similarly led to an increase in the measured protein bound carbonyls, but less than the similar model systems in which no 220 wheat proteins were present.

When incubating hazelnut proteins with glucose a gradual decrease of the reactive 222 lysine residues was observed as incubation time increased. This led to a 64% loss after 48 h of incubation. (Figure 2). In contrast, incubation in the absence of glucose did not affect the 224 reactive lysine content of the hazelnut protein, even after 48 h of incubation. The reaction systems containing 0.5% and 0.75% wheat proteins contained slightly lower amounts of 226 reactive lysine residues as compared with the model system containing no wheat proteins, which is in line with the lysine deficiency of wheat proteins. Upon incubation, 60 to 70% 228 losses of reactive lysine residues during the first 24 h were observed, while further incubation did not further affect the reactive lysine content. 230 Upon incubation of hazelnut proteins with glucose, high molecular weight protein 242 clusters appear as a smear in the SDS-PAGE pattern. Moreover, prominent aggregate formation occurred, which is manifested by their presence at the boundary of the resolving 244 and stacking gel, as well as in the stacking gel. The high-molecular mass aggregates on top of the gel could not migrate into the stacking gel. They probably have molecular masses larger 246 than 300 kDa. Upon addition of the soluble wheat proteins, it was obvious that the formation of these high molecular weight complexes was partially inhibited in a wheat protein 248 concentration dependent manner. Nevertheless, after 48 h of incubation, smearing and formation of high molecular weight aggregates were still visible. 250

Detection with commercial ELISA kits

The concentration of the standards included in three of the four kits tested (Veratox® 252 for hazelnut, Ridascreen® FAST Hazelnut and BioKits Hazelnut Assay) is expressed as a particular amount of hazelnut. However, the samples of the different prepared model systems 254 have a known or actual hazelnut protein concentration. To be able to compare the actual concentration of the samples that were analyzed with the measured concentration derived 256 from the calibration curve obtained with the kit's standards, the concentration of the standards 14 was converted to the same dimension of that from the model system samples. This was done 258 according to the formula below (3.1), taking into consideration the protein content of the hazelnut standards as indicated in the manual of the kit or, if not indicated, a protein content 260 of 12% was used (Belser et al., 1999).

(3.1) 264

The hazelnut or hazelnut protein concentration of the kit's standards is expressed in parts per million (ppm) which equals ng analyte/mg food product. However, as the 266 concentration of the hazelnut proteins in the model systems is expressed in ng mL -1 , the concentrations of the kit's standards were converted according to formula 3.2, considering the 268 weight of the sample (mg) extracted by a particular volume of extraction buffer (mL) according to the respective manufacturers' instructions.

(3.2) 272

For the evaluation of the kits' performance, a dilution series of the hazelnut protein 274 solutions obtained from the six different model systems were applied in the appropriate concentration range of each kit. The obtained absorbance values were converted to a 276 measured hazelnut protein concentration, which was compared with the actual protein concentration presents in each diluted solution (Figure 4). 278

Veratox for hazelnut

For the "Veratox® for hazelnut" kit, calibration curves established with the standard 280 hazelnut solutions provided in the kit, were fitted to the four-parameter-logistic function. The log-logit fit, according to the manufacturer's instructions, is a linearization of the sigmoid 282 shape of the four-parameter logistic function. Consequently, both fittings return the same concentration for a given absorbance value. After conversion of the concentrations according 284 to formulas 3.1 and 3.2, the standards covered a range from zero to 120 ng mL -1 hazelnut protein. The actual hazelnut protein concentration present in the reference sample and its 286 dilutions was dramatically underestimated -on average by 94% (Figure 4A). Upon heating the reference sample for 24 h at 70°C without glucose, the discrepancy increased between the 288 actual hazelnut protein concentration present and the protein concentration measured by the kit. Only up to 4% of the actual hazelnut protein present could be detected (data not shown). 290

When the heat treatment up to 48 h the detectability decreased down to about 3.3% on average. 292

Once soluble wheat proteins (0.75%) were added to the reference hazelnut protein sample (0.25%), an underestimation was again observed. Only 12.6% of the actual hazelnut 294 protein concentration by the kit. This was only marginally less dramatic compared to the reference protein samples to which no wheat proteins were added. Remarkably, if a lower 296 amount of wheat proteins was present (0.5% hazelnut + 0.5% soluble wheat proteins) even less of the actual hazelnut protein concentration was observed (9.6% detected, not shown). 298

Upon heating the mixture of hazelnut and soluble wheat protein solutions a further decrease in the measured hazelnut protein concentration was observed, although the decrease was less 300 pronounced compared to the similarly treated samples in the absence of soluble wheat proteins (17% compared to 45% loss in signal compared to the native reference sample in the 302 absence and presence of soluble wheat proteins, respectively).

Upon addition of glucose during heating, the discrepancy between the actual and 304 measured hazelnut protein concentration increased dramatically, both in the absence and

Ridascreen® FAST Hazelnut 308

The values obtained for the standards using the "Ridascreen® FAST Hazelnut" kit were fitted to the four-parameter-logistic and a cubic spline function. After conversion according to 310 the formulas given above, the standards ranged from zero up to 90 ng mL -1 hazelnut protein.

After analysis of a dilution series of the reference hazelnut protein sample with the 312

Ridascreen® FAST Hazelnut only the data for the samples actually containing less than 50 ng mL -1 hazelnut protein could be considered, as the samples with a higher concentration 314 returned absorbance values outside the range of the kits standard curve.

The measured concentrations of the reference sample were underestimated by about 316 17% using the logistic standard curve, except for the lowest concentration (5 ng mL -1 ), which was underestimated by 49% (Figure 4B). With the cubic spline fit the calculated 318 concentrations were about 10% higher than with the four-parameter logistic function.

However, using the former fitting, no calculations could be made for samples with an 320 absorbance value between zero and that of the first standard. Heat-treatment of the reference sample at 70°C led to a limited loss in detectability after 48 h (about 13%). 322

When soluble wheat proteins were added (0.75%) to the reference hazelnut protein sample (0.25%) the protein concentration was either underestimated by 12% (40 ng mL -1 ) or 324 overestimated by 15% (20 ng mL -1 ). For the lowest concentration (4 ng mL -1 ) the obtained signal resulted in a calculated concentration equal to zero. In the presence of a lower amount 326 of soluble wheat proteins (0.5% hazelnut protein + 0.5% soluble wheat proteins) the samples were underestimated by 80% on average (data not shown). Heating alone of the mixture of 328 hazelnut and soluble wheat proteins, led to a loss in detectability (signal reduced on average by 30% after 24 h with an additional loss of 15% after 48 h incubation as compared to the 330 native samples). This loss in detectability was more pronounced as compared with the similarly treated reference hazelnut protein solution in the absence of wheat proteins (13% 332 reduction). Addition of glucose during heating led to a greater discrepancy between the actual and measured concentrations. In the presence of wheat proteins the signal further decreased 334 further by about 30%. Interestingly, in the absence of wheat proteins an increased detectability was observed after heating for 48 h. 336

Hazelnut Residue

The calibration curves obtained with the standards of the "Hazelnut Residue" kit were 338 fitted to the four-parameter logistic function. The converted concentrations of the standards ranged from zero to 500 ng mL -1 . The actual hazelnut protein concentration of a dilution 340 series of the reference sample was underestimated on average by 45% (Figure 4C). Overall, small fluctuations in detection were observed upon heating the samples at 70°C up to 48h in 342 the absence of glucose as compared with the reference sample. Addition of soluble wheat proteins (0.25% hazelnut + 0.75% soluble wheat proteins) resulted in increased detection, 344 leading to an overestimation by an average of 34% for the samples considered. It should be noted that the overestimation occurred especially in the more diluted sample solutions. In 346 addition, due to the overestimation in the presence of soluble wheat proteins, some samples were out of range (800 and 400 ng mL -1 ). When a lower amount of wheat proteins was added 348 (0.5% hazelnut + 0.5% wheat proteins) the considered samples were now underestimated by 24% on average (data not shown). Upon heating the samples containing hazelnut and soluble 350 wheat proteins alone, the measured hazelnut protein concentration decreased by 45% on average. Upon addition of glucose to the reference sample during heating, the discrepancy 352 between the actual and measured hazelnut protein concentration increased further (44% loss in signal compared to the sample heated in the absence of glucose), which was even more 354 pronounced in the presence of soluble wheat proteins (63% loss in signal).

Biokits Hazelnut Assay 356

After conversion according to formula 3.1 and 3.2, the standards of the "Biokits Hazelnut Assay" covered a range from zero up to 192 ng mL -1 hazelnut protein and the corresponding 358 absorbencies were fitted according to the four-parameter-logistic function. The actual concentrations of the considered dilutions of the reference sample were on average 360 overestimated by 27% (Figure 4D). Upon heating the reference sample for 24 h at 70°C without glucose, the measured concentration of the samples decreased by 7% (data not 362 shown) with an additional loss in signal of 20% when the heating was extended to 48 h. When adding soluble wheat proteins (0.75%) to the reference sample (0.25% hazelnut proteins) the 364 actual concentration was overestimated by about 50% on average, especially at the lower concentration range, while this overestimation became only 20% when a lower amount of 366 wheat proteins (0.5% hazelnut and 0.5% wheat proteins) was added (not shown). Upon heating in the absence of glucose the overestimation became an underestimation (14% on 368 average) When glucose was included during the heat-treatment of the samples, the discrepancy between the actual and the measured hazelnut protein concentrations further 370 increased, both in the presence and the absence of wheat proteins. This observation was also more pronounced in the presence of soluble wheat proteins (48% loss in signal compared to 372 the sample heat in the absence of glucose). A comparison of the kits manuals revealed interesting differences that affected the interpretation of the analytical results. Some of the kits express the results as amount of 376 hazelnut present, while others express the results as amount of hazelnut protein present. Given that the allergen in question is hazelnut proteins and since the protein content of hazelnut 378

shows some variability, it seems more appropriate to express the results as the content of hazelnut protein. Another difference observed between Ridascreen Fast Hazelnut and the 380 other kits concerned the fitting of the calibration curve to a particular equation. The latter kit suggested using the cubic spline function. However, the equilibrium reactions involved in a 382 immunoassay make it more appropriate to use the four logistic function, or its linearized loglogit equivalent. Moreover, the use of the alternative cubic spline function does not allow 384 quantification of signals between zero and the first point of the calibration curve.

Use of the reference extract (obtained from a mixture of various raw and one roasted 386 hazelnut lots) in the various kits yielded remarkable differences in their performance. The Ridascreen Fast Hazelnut kit and the Biokits Hazelnut assay proved to be the most accurate, 388 while the Veratox for Hazelnut kit showed results that underestimated by one order of magnitude the actual hazelnut protein present in the reference extract. Interestingly, the 390 accuracy of some kits depended somewhat upon the actual hazelnut protein concentration present in the solutions applied. Two possible causes for the differences observed between the 392 actual hazelnut protein concentration present in the applied reference solutions and the concentration obtained by the respective kits could be suggested. One, the antibodies used in 394 the respective kits could have been raised towards pooled hazelnut proteins extracted from different hazelnut varieties than those used for our reference extract. Two, the ratio's of the 396 different varieties in our reference mixture could also have differed. extracted from Runner peanuts and those extracted from various other peanut varieties. The kits' manufacturers could also have used a protein extract from roasted hazelnuts instead. Our 400 reference protein extract was based upon a mixture of 9 different hazelnut samples, of which only one was roasted. Indeed, as can be observed from various evaluated chemical parameters 402 linked to the Maillard reaction, it was obvious that our reference extract did not contain severely modified proteins. 404

Heating the reference hazelnut protein solution in the absence of glucose, which restricted the Maillard reaction as confirmed from the Maillard reaction parameters monitored 406 (Figures 1234), further decreased the accuracy for the Veratox for Hazelnut kit and the Biokits Hazelnut assay. In the case of the former assay, the loss in accuracy became quite 408 dramatic, with less than 10% of the hazelnut protein present being detected. Some of the hazelnut proteins seem to be heat labile and denature upon incubation at 70°C. Because of 410 this, the kits were able to detect less as compared with the native proteins. For the other kits evaluated only minor losses in accuracy could be noted, or in the case of the Hazelnut 412

Residue kit, even a slightly increased accuracy.

Upon heating the reference protein solution in the presence of glucose typical chemical 414 indicators used to assess the impact of the reaction showed a progressive increase as a function of incubation time (Figure 1 and2). These changes also clearly indicated that severe 416 modifications on the molecular level of the hazelnut proteins occurred. Moreover, these modifications gave rise to a dramatic loss in the accuracy in three of the four kits evaluated 418 (only the Ridascreen Fast Hazelnut assay remained accurate). For the latter assay, even a better binding occurred between the antibodies and the Maillard modified proteins compared 420 to the proteins in the non-heated reference extract, most probably indicating that the antibodies used in that assay alone were developed towards modified hazelnut proteins. 422 proteins are linked to each other resulting in the formation of large molecular complexes. It is thus important to note that allergen quantification is typically performed in foods in which the 430 considered allergen is present as a minor compound next to major ingredients, including bulk proteins. As a consequence, the traces of hazelnut proteins will primarily cross-link with these 432 bulk proteins instead of cross-linking with the traces of other hazelnut proteins present.

Therefore, the hazelnut proteins surface may become covered with bulk proteins, making the 434 receptor-based detection using antibodies even more vulnerable to inaccuracy. In order to evaluate this hypothesis, mixtures of the reference protein with soluble wheat proteins were 436 heated in the presence of glucose. Soluble wheat proteins were taken as a case example in order to avoid solubility problems of the modified proteins as a result of progressive Maillard 438 reaction. Since the soluble wheat protein consisted of a partially hydrolyzed mixture of wheat proteins, considerably less protein aggregation occurred. This can be explained by the lower 440 chance of retrieving multiple reactive nucleophillic groups in smaller protein fragments than in intact proteins. Nevertheless, cross-linking between hazelnut proteins and soluble wheat 442 proteins did occur. Cross-linking proved to have dramatic consequences on the accuracy of all kits evaluated, which could be easily explained by the molecular modification induced on 444 proteins and consecutively on the receptor-based principle of immune assays.

In view of the results obtained, it is obvious that extreme care should be taken by using 446 receptor-based assays, such as ELISA kits for allergen detection. None of the evaluated ELISA kits produced accurate analytical results, even when non -treated reference protein 448 solution was used. Even if the observed underestimation with the reference proteins solution used in our study is due to the use of a hazelnut pool which is not similar to the ones used to 450 raise antibodies in these kits, it should be noted that these kits are used worldwide to asses contamination levels of hazelnuts of different origins, and modified to different degrees. As a 452 consequence, care should be taken when using these kits for quantitative analysis, especially in view of enforcement of for instance EC Directive 2007/68/EU. Special attention should be 454 also drown when qualitatively interpreting the analytical data obtained. It was striking to observe that the accuracy of the kits evaluated not only depended upon the chemical 456 modifications induced in the reference protein extract, but also depended upon the actual protein concentration present in the evaluated solutions. For some of the kits evaluated, a 458 considerable lack of accuracy was observed, especially at low hazelnut protein concentrations.

Given that kits for allergen detection are used for trace analysis, one would expect that the 460 concentration of the target proteins in the food extract to be low. The above results clearly show that both false positive or false negative results from the commercial ELISA kits are 462 likely to occur. False negative results present a potentially fatal risk for allergic consumers, while false positive results may harm the food industry through unnecessary and costly 464 product withdrawal. Finally, it should be realized that the final outcome of any analytical method is influenced by the extractability of the analyte from the food matrix. In this 466 perspective it is important to note that protein solubility is typically affected by progressive Maillard reactions. Considering the results obtained in this study, it can be concluded that, 468 apart from issue related to the extractability of the proteins, also chemical modifications occurring on protein level as a result of the Maillard reaction may seriously affect the 470 outcome of receptor based analytical methods, such as ELISA's. soluble wheat proteins due to incubation with 6% glucose. The data points represent mean values ±SD of four independent determinations. ◊ 1% hazelnut proteins with 6% glucose; □ 556 0.5% hazelnut with 0.5% soluble wheat proteins and 6% glucose; ∆ 0.25% hazelnut and 0.75% soluble wheat proteins and 6% glucose; ○ 1% hazelnut proteins 558

Figure 2 Loss of reactive lysine due to incubation of hazelnut proteins in the presence or in the absence of soluble wheat proteins with 6% glucose, data points represent mean values 560 ±SD of four independent determinations. ◊ 1% hazelnut proteins with 6% glucose; □ 0.5% hazelnut with 0.5% soluble wheat proteins and 6% glucose; ∆ 0.25% hazelnut and 0.75% 562 soluble wheat proteins and 6% glucose; ○ 1% hazelnut proteins Figure 3 SDS-PAGE pattern of the hazelnut proteins incubated in the presence or absence of 564 wheat proteins with glucose: 1% hazelnut proteins and 6% glucose non treated (lane 1), incubated for 24 h (lane 2) and 48 h (lane 3). 0,5% hazelnut proteins with 0,5% wheat 566 proteins and 6% glucose non treated (lane 4), incubated for 24 h (lane 5) and for 48 h (lane 6). 0,25% hazelnut proteins with 0,75% wheat proteins and 6% glucose non treated (lane 7), 568 incubated for 24 h (lane 8) and 48 h (lane 9). Molecular weight marker (lane 10) Figure 3 SDS-PAGE pattern of the hazelnut proteins incubated in the presence or absence of wheat proteins with glucose: 1% hazelnut proteins and 6% glucose non treated (lane 1), incubated for 24 h (lane 2) and 48 h (lane 3). 0,5% hazelnut proteins with 0,5% wheat proteins and 6% glucose non treated (lane 4), incubated for 24 h (lane 5) and for 48 h (lane 6). 0,25% hazelnut proteins with 0,75% wheat proteins and 6% glucose non treated (lane 7), incubated for 24 h (lane 8) and 48 h (lane 9). Molecular weight marker (lane 10)

  was assessed by evaluating the losses of reactive lysine residues and of free amino groups, generation of protein bound carbonyls and changes in the SDS-PAGE 70 pattern. Additionally, the influence on the hazelnut protein detectability was assessed by analyzing the modified protein solutions by four commercially available ELISA kits: 72 Veratox® for Hazelnut, Ridascreen® FAST Hazelnut, Hazelnut Residue and BioKits Hazelnut Assay were used for this purpose. 74

  extraction buffer to avoid microbial growth. Further, a mixture of protease inhibitors (2 mM ethylenediaminetetraacetic acid (EDTA), 5 mM sodium diethyl-100 dithiocarbamate (DIECA), 0.5 mM benzamidine hydrochloride (BAHC) and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)) was added to the extraction buffer to increase the 102 stability of the protein isolate during extraction and storage

  (Nazareth, Belgium) and the respective molecular weights of the hazelnut 168 proteins were recorded.

  the manufacturers' instructions. Absorbance measurements were made in a 192 microtiter plate reader (Multiskan MCC/340, Titertek, Huntsville, Alabama). Each kit was calibrated using the respective standard protein solutions included in each kit. For fitting the 194 absorbance values to the protein concentration of the standard solutions, a four-parameterlogistic dose-response curve was used with equation 2.1 below. This equation represents a 196 standard way to express immunoassay data, where a is the maximum dose (protein concentration) of the curve, b is the minimum concentration, c is the E 50 -value (concentration 198 at half-maximal saturation), d is the slope of the curve and conc is the concentration of the standard or sample

  procedure recommended fitting the data to log-logit function, which is a linearization of the 204 four-parameter logistic function. For the Hazelnut Residue and the BioKits Hazelnut Assay no suggestion was made with respect to curve fitting. Only the data obtained within the range 206 of the respective standard curves were considered in the results. Outlying data are indicated

Figure 3

 3 Figure3shows the SDS-PAGE pattern of the hazelnut protein incubated with glucose

  wheat proteins (2% or less of the actual hazelnut protein concentration 306 was measured).

  Indeed, De Meulenaer et al. (2005) observed a difference in the response of antibodies raised towards the protein 398

  the gel electrophoresis experiments (Figure3), upon heating the reference hazelnut protein solution in the presence of glucose, severe protein cross-linking 424 occurred resulting in formation of high molecular weight aggregate formation. Protein crosslinking in the Maillard reaction typically originates because reactive nucleophilic groups such 426 as lysine (Figure2) react with the reactive carbonyl intermediates originating from the degraded reducing sugars. Since these reactive groups are present in several proteins, multiple 428

  472

Figure 1

 1 Figure captions

Figure 4 Figure 1 Figure 2

 412 Figure 4 Ratio of measured over actual hazelnut protein concentration (Y-axis) in different 570

Figure 4 Figure 4 Figure 4 Figure 4

 4444 Figure 4 Ratio of measured over actual hazelnut protein concentration (Y-axis) in different ELISA kits after in duplo analysis of a dilution series (X-axis) of a reference hazelnut protein sample (darkest) and after heating in the presence or absence of glucose and in the absence (left concentration on X-axis) or presence (right concentration on X-axis) of soluble wheat proteins. (* =absorbance value of sample outside the range of the standard curve) -reference sample, -reference heated for 48 h, -reference heated for 48 h in the presence of glucose, -0.25% hazelnut proteins and 0.75% soluble wheat proteins, -0.25% hazelnut proteins and 0.75% soluble wheat proteins heated for 48 h, -0.25% hazelnut proteins and 0.75% soluble wheat proteins heated for 48 h in the presence of glucose

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Acknowledgements

We greatly appreciate the Belgian Science Policy (SD/AF/03A) for funding this research. 474

The authors would like to thank the Neogen Corporation for kindly providing us the Veratox for Hazelnut ELISA kits for this study. 476