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Evaluation of indirect competitive and double antibody sandwich ELISAtests to determine β-lactoglobulin and ovomucoid in model processedfoodsRuth de Luis a; Luis Mata b; Gloria Estopañán c; María Lavilla a; Lourdes Sánchez a; María D. Pérez a

a Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain b

ZEU-Inmunotec, Zaragoza, Spain c Unidad de Calidad y Seguridad Alimentaria, Centro deInvestigación y Tecnología Agroalimentaria de Aragón, Zaragoza, Spain

To cite this Article de Luis, Ruth, Mata, Luis, Estopañán, Gloria, Lavilla, María, Sánchez, Lourdes and Pérez, MaríaD.'Evaluation of indirect competitive and double antibody sandwich ELISA tests to determine β-lactoglobulin andovomucoid in model processed foods', Food and Agricultural Immunology, 19: 4, 339 — 350To link to this Article: DOI: 10.1080/09540100802520755URL: http://dx.doi.org/10.1080/09540100802520755

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Evaluation of indirect competitive and double antibody sandwich ELISA teststo determine b-lactoglobulin and ovomucoid in model processed foods

Ruth de Luisa, Luis Matab, Gloria Estopananc, Marıa Lavillaa, Lourdes Sancheza andMarıa D. Pereza*

aTecnologıa de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain;bZEU-Inmunotec, Zaragoza, Spain; cUnidad de Calidad y Seguridad Alimentaria, Centro deInvestigacion y Tecnologıa Agroalimentaria de Aragon, Zaragoza, Spain

(Received 11 July 2008; final version received 30 September 2008)

Enzyme-linked immunosorbent assay (ELISA) kits (indirect competitive and sandwichformats) to determine either b-lactoglobulin or ovomucoid were evaluated in modelfoods. A cut-off value was established for each kit to consider food samples as positivefor milk or egg addition. Sausage and bread were positive at lower percentages of addedmilk using the sandwich format (0.005 and 0.05%) than the indirect competitive format(0.05 and 0.25%) and pate was positive at 0.25% milk addition for both formats. Sausagewas positive at 0.005%, and bread at 0.05% added egg for indirect competitive andsandwich formats, whereas pate was positive at 0.25% egg only by the indirectcompetitive assay. The concentration of added milk and egg to give a positive resultdepends on heat treatment, being higher for pate (sterilized), followed by bread (baked)and sausage (pasteurised). The particularities of each format and the heat processingapplied influenced the determination by ELISA of allergenic proteins in foods.

Keywords: b-lactoglobulin; ovomucoid; indirect competitive and sandwich ELISA; milkand egg allergy

Introduction

Food allergies represent an important health problem in industrialised countries. Although

accurate statistics are unavailable, data indicate that this health issue is on the rise (Yeung,

2006). Many studies have shown that egg and milk allergies are the most prevalent allergies

in children. In studies performed in children under 2 years of age, milk allergy represents

about 2�4% and egg allergy about 3�4% (Poms, Klein, & Anklam, 2004). Although milk

and egg allergies usually disappear during the first 3 years of age, some individuals

continue having severe reactions to those foods in adulthood.

The most allergenic proteins in eggs are present in the white, being ovomucoid (Gal d

1), the most important allergen followed by ovalbumin (Gal d 2), ovotransferrin (Gal d 3),

and lysozyme (Gal d 4). In the case of milk, the proteins that cause allergic reactions

include both caseins (Bos d 8) and whey proteins, mainly b-lactoglobulin (Bos d 5) and

a-lactalbumin (Bos d 4) (Bush & Hefle, 1996). Most of these proteins retain their

allergenicity after heating or other processing (Besler, Steinhart, & Paschke, 2001; Monaci,

Tregoat, van Hengel, & Anklam, 2006).

*Corresponding author. Email: dperez@unizar.es

Food and Agricultural Immunology

Vol. 19, No. 4, December 2008, 339�350

ISSN 0954-0105 print/ISSN 1465-3443 online

# 2008 Taylor & Francis

DOI: 10.1080/09540100802520755

http://www.informaworld.com

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The prevention of food allergies primarily involves strict avoidance of the offending

foods. This means that food processors should implement an allergen prevention plan and

inform consumers about the presence of allergens in their products (Deibel et al., 1997).

European Legislation, such as Directive 2003/89/EC and amendments (European

Commission, 2003) requires that all ingredients added to food products have to be

labelled and demands the obligatory declaration of those liable to cause allergies or

intolerances in which milk and egg and products thereof are included. Furthermore, the

inadvertent presence of allergens results from practices in the food industry such as the use

of contaminated raw materials, inadequate cleaning of shared equipment or other sources

of cross-contact (Hefle & Lambrecht, 2004). Therefore, reliable detection methods for food

allergens are necessary to ensure its compliance with the food labelling and to improve

consumer protection.

In the last few years, several immunochemical techniques have been developed to

determine milk and egg proteins in food products. These techniques are mainly based on

the interaction between the allergenic protein and the specific antibodies obtained against

it. At present, only the ELISA technique is used in the routine analysis due to its high

precision, simple handling and good potential for standardisation (de Luis, Perez, Sanchez,

Lavilla, & Calvo, 2007; Hefle & Lambrecht, 2004; Mariager, Solve, Eriksen, & Brogren,

1994; Poms et al., 2004).

According to studies performed by clinicians, it is considered that the detection limit of

techniques to detect allergenic sources should be in the range between 1 and 10 ppm, so

most commercial immunoassays have detection limits within this range (Poms et al., 2004;

van Hengel, 2007).

However, the determination of allergenic proteins using immunoassays has some

limitations related to the extraction of the target protein from the food and to interferences

by other components of the matrix. Food processing also poses a great challenge with

respect to allergen detection by immunoassays because it leads to partial denaturation of

proteins which can affect the ability of antibodies to recognise them (de Luis et al., 2007;

Julia et al., 2007). Simple spiking of foods with extracts of allergenic ingredients is not

considered appropriate in assessing the performance of allergen detection techniques.

Therefore, making model foods according to pilot plant or true industrial conditions will

be the key in the evaluation of allergen detection methods until well-characterised reference

materials are available (Poms, 2006).

On the other hand, the effectiveness of an immunoassay depends directly on the antigen

selected as a target and the quality of the antibodies used but in addition to this the

performance of the assay itself is also important (de Luis et al., 2007; Immer, 2006).

In this work, four ELISA tests based on the detection of b-lactoglobulin or ovomucoid

(an indirect competitive format and a double antibody sandwich format for each protein)

have been evaluated by three participating laboratories. Samples analysed were three model

processed foods elaborated at a pilot plant, which contained different amounts of skimmed

milk powder and egg powder.

Materials and methods

Materials

Low heat skimmed milk powder was supplied by Reny Picot (Anelo, Spain), egg powder by

Huevos Mayper S.A. (Valladolid, Spain), the mixture of spices and the soya protein isolate

by Anvisa (Arganda del Rey, Madrid, Spain), pea proteins by Cosucra (Fontenoy,

340 R. de Luis et al.

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Belgium) and soluble wheat proteins by Tate & Lyle (Zaragoza, Spain). Bovine serum

albumin was from Sigma Chemical (Poole, UK). The rest of the ingredients were

purchased from local retailers. ELISA kits (Proteon b-lactoglobulin indirect competitive

and sandwich kits and Proteon ovomucoid indirect competitive and sandwich kits) were

provided by ZEU-Inmunotec (Zaragoza, Spain).

Preparation of model processed foods

Three model processed foods (sausage, bread and pate) containing low heat skimmed

milk powder and egg powder as allergenic ingredients were prepared at the Pilot Plant of

Food Science and Technology (University of Zaragoza). They were prepared following

standard manufacturing processes. The allergenic ingredients were spiked at the

ingredient stage before processing to obtain final concentrations of 0.25%, 0.5% and

1% (w/w).

Sausage was made of 5 kg of pork leg meat, 1 kg of pork fat, 450 g of a mixture of

spices, 900 g of starch and 3 kg of ice water. The ingredients were mixed thoroughly and the

mixture ground using a cutter. Then, different amounts of skimmed milk powder and egg

powder were added and the mixture kneaded using a homogeniser. The mixture was

stuffed in cellulose casings (2.8 cm diameter), placed into the oven and cooked to 758C(internal temperature). Sausages were vacuum packed and heated at 908C (internal

temperature) for 1 min in a thermostatic water bath.

Bread was made using a bread and dough maker (Deluxe: Bread and Dough Maker,

Oster, USA). An amount of 480 g of wheat flour, 10 g of margarine, 20 g of sugar, 5 g of

salt, 8.8 g of yeast, skimmed milk powder and egg powder and 205 ml of water, were

kneaded for 30 min and left for 1 h at room temperature for dough formation. Then, the

dough was baked at 958C (internal temperature) for 40 min.

Pate was prepared with 1.6 kg of pork liver, 2.5 kg of pork fat, 100 g of salt, 11 g of

pepper, 2.8 g of sugar, 166 g of maize flour, 166 g of margarine and 840 ml of water. Pork

fat was grounded, heated in water at 858C for 30 min and then, drained to remove water.

The pork fat, the ground pork liver and the rest of ingredients were mixed thoroughly.

Then, different amounts of skimmed milk powder and egg powder were added and the

paste homogenised. A 250 g sample of the mixture was packaged in glass jars and heated at

1208C (internal temperature) for 1 h in an autoclave.

Food samples were ground and weighed into 50 ml plastic centrifugation tubes (3.009

0.01 g). To obtain model foods with final concentrations of 0.005%, 0.01%, 0.05% and

0.1%, of milk and egg powder, certain amounts of the finely ground 0.25% samples were

weighed directly into centrifugation tubes containing the corresponding blank sample to

give a total weight of 3.0090.01 g. All tubes were closed and stored at �208C before

dispatching to the participants.

Extraction of test samples

A 3.0090.01 g amount of ground sample was homogenised in 30 ml of extraction buffer

composed by 0.15M NaCl, 0.0015M potassium phosphate buffer, pH 7.4 (PBS) for 1 min

using a vortex mixer, and then heated at 608C for 15 min in a thermostatic water bath.

Extracts were clarified by centrifugation at 3000�g for 15 min, the supernatant was

filtered through paper and analysed.

Food and Agricultural Immunology 341

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ELISA test kits

ELISA kits contained coated plates, extraction and washing buffers, standard solutions,

antibody-enzyme conjugates, enzyme substrate and stopping solution. ELISA kits targeted

either bovine b-lactoglobulin or hen’s ovomucoid.

In the indirect competitive formats, antisera to b-lactoglobulin or ovomucoid raised in

rabbits were used. In the double antibody sandwich formats, specific antibodies purified

from their respective antisera using an immunosorbent of b-lactoglobulin or ovomucoid

were used. Protein standards contained purified proteins diluted in PBS and the

concentration was determined by spectrophotometry using an extinction coefficient at

280 nm of E1%�9.6 for b-lactoglobulin and E1%�4.6 for ovomucoid.For the sandwich format, a volume of 100 ml per well of standard or sample

solutions was added to a plate coated with antibodies against b-lactoglobulin or

ovomucoid, and incubated for 30 min at room temperature. The plate was washed five

times with 300 ml/well of washing buffer composed by PBS containing 0.05% Tween

(PBST) and then, 100 ml/ well of a solution of peroxidase labelled antibodies was added

and incubated for 30 min at room temperature. The plate was washed again and

100 ml/well of a solution of 3, 3?, 5, 5?-tetramethylbenzidine (TMB) was added and

incubated for 30 min at room temperature. The reaction was stopped by adding 50 ml/

well of stopping solution containing 1M sulphuric acid and the absorbance of wells was

measured at 450 nm.

For the indirect competitive formats, 100 ml of standard or sample solutions and

100 ml of rabbit antiserum against b-lactoglobulin or ovomucoid were added to each well

of a plate coated with b-lactoglobulin or ovomucoid, respectively, and incubated for

15 min at room temperature. The plate was washed five times with 300 ml/well of washing

buffer and then incubated with 100 ml/well of anti-rabbit IgG antibodies labelled with

peroxidase for 15 min at room temperature. After washing the plate, 100 ml/well of the

TMB substrate was added and incubated for 15 min at room temperature. The reaction

was stopped by adding 50 ml/well of stopping solution and the absorbance of wells was

measured at 450 nm.

Evaluation study

Three laboratories participated in this study coordinated by the University of Zaragoza.

Four trials, one for each protein (b-lactoglobulin and ovomucoid) and format (indi-

rect competitive and sandwich) were performed. The coordinator provided the 24 pre-

weighed test samples and ZEU-Inmunotec provided the four ELISA kits with

instructions.

The calibration standard solutions were assayed simultaneously with the food extracts.

Raw experimental absorbance data of calibration standards and test samples were sent to

the coordinator. The average absorbance of duplicate wells was used for the calculation.

Calibration curves were obtained using the relationship between the value of absorbance

and the logarithm of the concentration of standard solutions for b-lactoglobulin indi-

rect competitive test and ovomucoid indirect competitive and sandwich tests. For b-

lactoglobulin sandwich test, calibration curves obtained used the relationship between the

value of absorbance and the concentration of standard solutions (Figure 1a�d). The

concentration of b-lactoglobulin and ovomucoid in the test samples was calculated using

the correspondent calibration curves.

342 R. de Luis et al.

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Results and discussion

Single laboratory validation

A single laboratory validation of ELISA tests was performed to determine the limit of

detection (LOD), the homogeneity of the food samples and the specificity of the tests. The

LODs were calculated as the mean concentration value of eight replicates of the blank

standard plus three times the value of its standard deviation (Table 1). The homogeneity of

the final food samples was tested using the indirect competitive and sandwich formats tests

for b-lactoglobulin and ovomucoid. For this purpose, five samples of each concentration

and matrix were analysed in duplicate. The variation coefficient was less than 20%, which

was considered acceptable for the purpose of the study.

To verify the specificity of the ELISA tests, 15 basic ingredients were analysed. Most

ingredients showed a small decrease or increase of the background level compared to the

blank, for the indirect competitive and sandwich formats, respectively, indicating certain

interference (Figure 2a�d). In order to overcome this problem, a cut-off value was

established for each test. The cut-off value was calculated as the mean concentration value

of ingredients assayed plus three times the value of its standard deviation. Therefore, food

samples giving a concentration value equal or higher than the cut-off established for each

0.0

0.2

0.4

0.6

0.00 0.40 0.80 1.20 1.60Absorbance (450 nm)

ar2 = 0.9993

-0.5

0.0

0.5

1.0

1.5

2.0

0.00 0.50 1.00 1.50 2.00Absorbance (450 nm)

Log

β-l

acto

glob

ulin

(pp

m)

β-la

ctog

lobu

lin (

ppm

) br2 = 0.9899

-0.8

-0.4

0.0

0.4

0.8

0.00 0.30 0.60 0.90

Absorbance (450 nm)

Log

ovo

muc

oid

(ppm

)

cr2 = 0.9804

-0.5

0.0

0.5

1.0

1.5

2.0

0.00 0.20 0.40 0.60 0.80 1.00

Absorbance (450 nm) L

og o

vom

ucoi

d (p

pm) dr2 = 0.9967

Figure 1. Calibration curves obtained for the determination of b-lactoglobulin (a and b) and

ovomucoid (c and d) by double antibody sandwich (a and c) and indirect competitive (b and d)

ELISA tests.

Table 1. Limit of detection (LOD) and cut-off established for the ELISA tests to determine b-

lactoglobulin or ovomucoid in foods. Calibration points correspond to the protein concentration of

standards used in each ELISA test.

Test format Target protein LOD ppm Cut-off ppm Calibration points ppm

Sandwich b-lactoglobulin 0.04 0.05 0-0.05-0.1-0.25-0.50

Sandwich Ovomucoid 0.19 0.20 0-0.2-0.5-2.0-5.0

Competitive b-lactoglobulin 0.11 0.50 0-0.5-1.0-5.0-10.0-50.0

Competitive Ovomucoid 0.23 0.40 0-0.5-1.0-5.0-10.0-50.0

Food and Agricultural Immunology 343

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test were considered as positive for milk or egg addition. The cut-off values established for

each ELISA test and protein are shown in Table 1. The assumption of these cut-off values

assured that the effect of potential cross-reactivity of a food ingredient in the ELISA tests

is minimised. All ingredients used in the elaboration of model foods were assayed to know

cross-reactivity and concentrations determined were lower than the cut-off established for

each ELISA format (results were not shown).

The concentration of b-lactoglobulin in skimmed milk powder was found to be 28.5

and 114.1 mg/g and the concentration of ovomucoid in egg powder of 21.0 and 32.1 mg/g

for the sandwich and indirect competitive assays, respectively. These different results when

using the two formats for the same protein could be attributed to differences in protein

recognition by the antibodies used in each assay.

Evaluation of ELISA kits

Four trials, one for each protein and format, were performed by the three laboratories.

Data obtained from the four assays for the determination of b-lactoglobulin and

ovomucoid in food samples by the three laboratories were processed as described above.

Calibration curves were obtained for every ELISA plate using the protein standards

indicated in Table 1. When using those standards, a linear relationship was obtained

between absorbance values and protein concentration, with square coefficients of

correlation ]0.98 in all cases (Figure 1a�d). The concentration of b-lactoglobulin and

ovomucoid in test samples was calculated on the basis of the calibration curve for each

plate. Individual results obtained in the trials performed for the quantitation of b-

lactoglobulin and ovomucoid in model foods are presented in Tables 2 and 3, respectively,

and the mean values and coefficients of variation are summarised in Table 4.

0.00

0.01

0.02

0.03

0.04

0.05

1 2 3 4 5 6 7 8 9 10 11 12

β -la

ctog

lobu

lin (

ppm

)

a

0.00

0.10

0.20

0.30

0.40

0.50

1 2 3 4 5 6 7 8 9 10 11 12

β -la

ctog

lobu

lin (

ppm

)

b

0.00

0.10

0.20

0.30

3 4 5 6 7 8 9 10 11 12 13 14 15

Ovo

muc

oid

(ppm

)

d

0.00

0.05

0.10

0.15

0.20

3 4 5 6 7 8 9 10 11 12 13 14 15

Ovo

muc

oid

(ppm

)

c

Figure 2. Cross reactivity of basic ingredients in the double antibody sandwich (a and c) and

indirect competitive (b and d) ELISA test kits for b-lactoglobulin (a and b) and ovomucoid (b and d).

1: Ovalbumin, 2: lysozyme, 3: bovine serum albumin, 4: meat proteins, 5: fish gelatine, 6: soluble

wheat proteins, 7: wheat flour, 8: maize flour, 9: rice flour, 10: olive oil, 11: soya protein isolate, 12: pea

proteins, 13: milk, 14: whey, 15: casein. Data are the mean values of two sample extractions assayed

by duplicate and are expressed in ppm.

344 R. de Luis et al.

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Results obtained by the three participants for both indirect competitive and sandwich

tests showed that for the three model foods analysed, the blank food samples, without milk

and egg powder added, gave concentrations of b-lactoglobulin and ovomucoid below the

cut-off established for each ELISA test and, therefore, no false-positive samples were found

in this work.

For the food samples, it was found that the amount of added milk and egg powder

required to give a positive result was dependent on the intensity of heat treatment applied,

being that amount higher for pate (sterilised), followed by bread (baked) and sausage

(pasteurised). Furthermore, for the same percentage of milk and egg powder added to

samples considered as positive, b-lactoglobulin and ovomucoid estimated concentrations

were higher in sausage, followed by bread and pate when analysing by the two ELISA

formats. This fact is probably due in great part to the heat processing which causes protein

denaturation and aggregation and consequently a decrease in the degree of immunor-

eactivity depending on the treatment applied. This could also explain the low values of

b-lactoglobulin and ovomucoid concentration in processed foods, which were much lower

than those estimated from milk and egg powder added as ingredients.

Table 2. Results obtained by the three participating laboratories for the determination of b-

lactoglobulin (ppm) in model processed foods added with different percentages of skimmed milk

powder, using the double antibody sandwich and indirect competitive ELISA formats.

Sandwich Competitive

Milk powder (%) Lab 1 Lab 2 Lab 3 Lab 1 Lab 2 Lab 3

Sausage

0 0.00 0.04 0.00 0.35 0.30 0.32

0.005 0.05 0.05 0.07 0.37 0.33 0.12

0.01 0.10 0.12 0.13 0.21 0.22 0.43

0.05 0.34 0.39 0.44 0.91 0.69 0.89

0.1 0.54 0.60 0.70 1.53 0.88 1.12

0.25 0.67 0.68 0.77 3.88 1.94 2.06

0.5 0.72 0.65 0.79 3.34 3.08 2.81

1 0.75 0.67 0.72 8.28 3.45 11.69

Bread

0 0.01 0.04 0.02 0.10 0.23 0.32

0.005 0.02 0.02 0.02 0.33 0.27 0.12

0.01 0.02 0.03 0.04 0.14 0.16 0.29

0.05 0.15 0.18 0.20 0.24 0.24 0.41

0.1 0.20 0.30 0.29 0.37 0.39 0.50

0.25 0.34 0.54 0.43 1.16 0.74 1.90

0.5 0.42 0.58 0.60 2.20 0.94 2.15

1 0.55 0.72 0.62 6.75 4.49 7.97

Pate

0 0.00 0.00 0.01 0.27 0.14 0.14

0.005 0.00 0.00 0.01 0.37 0.23 0.30

0.01 0.00 0.00 0.02 0.10 0.22 0.33

0.05 0.03 0.00 0.03 0.15 0.31 0.36

0.1 0.00 0.01 0.03 0.14 0.32 0.45

0.25 0.09 0.18 0.23 1.44 1.06 0.55

0.5 0.10 0.17 0.23 1.67 1.82 1.29

1 0.17 0.40 0.24 4.42 2.91 4.70

Food and Agricultural Immunology 345

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Results obtained in this work indicate that the quantitative determination of b-

lactoglobulin and ovomucoid, in processed foods depends on the ELISA format used. For

the sandwich format, samples were found to be positive for b-lactoglobulin by the three

laboratories at percentages of added milk of 0.005 and 0.05%, for sausage and bread,

respectively, whereas for the indirect competitive format, samples were positive at

percentages of added milk of 0.05 and 0.25% for the same model foods. In the case of

the pate samples, both formats gave positive results at 0.25% of milk addition (Table 2).In the case of the ovomucoid assays, samples of sausage were positive for the three

laboratories at 0.005% of added egg and samples of bread at 0.05% of added egg for both

formats. For the pate samples, only the indirect competitive assay could detect ovomucoid

powder at 0.25% of egg addition (Table 3).

For the food samples with lower percentages of milk and egg addition than those

indicated above, the concentrations of b-lactoglobulin and ovomucoid were found to be

below the cut-off established for each ELISA format resulting in negatives, unless for bread

containing 0.1% of milk powder which was positive only for laboratory 3 by the indirect

competitive assay.

Table 3. Results obtained by the three participating laboratories for the determination of

ovomucoid (ppm) in model processed foods added with different percentages of egg powder, using

the double antibody sandwich and indirect competitive ELISA formats.

Sandwich Competitive

Egg powder (%) Lab 1 Lab 2 Lab 3 Lab 1 Lab 2 Lab 3

Sausage

0 0.13 0.14 0.15 0.20 0.09 0.22

0.005 0.36 0.34 0.34 0.72 0.41 0.53

0.01 0.52 0.68 0.56 2.05 1.36 0.49

0.05 0.57 0.75 1.03 1.62 9.88 4.93

0.1 1.42 0.95 1.32 17.18 19.36 15.27

0.25 1.99 1.11 1.21 30.73 27.97 34.32

0.5 1.47 1.38 1.55 35.00 35.63 43.38

1 1.95 1.06 1.52 42.81 45.68 42.16

Bread

0 0.16 0.19 0.27 0.21 0.15 0.21

0.005 0.14 0.14 0.15 0.12 0.10 0.13

0.01 0.14 0.14 0.16 0.17 0.11 0.22

0.05 0.21 0.25 0.26 0.48 0.64 0.56

0.1 0.20 0.18 0.30 0.53 0.56 0.62

0.25 0.95 0.80 0.91 2.91 5.09 2.47

0.5 1.24 1.72 1.29 2.71 4.80 8.26

1 1.52 1.81 1.61 12.52 31.57 10.12

Pate

0 0.14 0.14 0.15 0.07 0.20 0.04

0.005 0.14 0.14 0.16 0.03 0.17 0.09

0.01 0.13 0.14 0.15 0.10 0.11 0.11

0.05 0.14 0.14 0.16 0.21 0.14 0.07

0.1 0.15 0.14 0.14 0.07 0.36 0.08

0.25 0.14 0.15 0.15 0.66 1.51 0.42

0.5 0.16 0.15 0.16 2.07 1.77 1.11

1 0.14 0.15 0.15 1.09 5.27 0.98

346 R. de Luis et al.

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At higher percentages of milk and egg powder added than those indicated above,

samples were positive with the exception of the 0.1% bread sample analysed using sandwich

format for ovomucoid which was negative only for laboratory 2. The concentration of

immunoreactive b-lactoglobulin or ovomucoid in positive samples increased gradually with

the amount of added milk and egg powder. However, for sausage samples analysed by the

sandwich ELISA formats, the concentration of these proteins did not increase at

percentages of milk or egg powder higher than 0.25% that possibly due to a saturation

effect, in spite of the fact that the calibration curve of ovomucoid was linear up to a

standard concentration of 5 ppm.

The coefficients of variation for sausage and bread samples were in most cases higher

for the indirect competitive than for the sandwich formats (Table 4). This higher variability

may be attributed to the higher slope of the calibration curve obtained in the formats.

Table 4. Mean values (X) (mg/kg) and coefficients of variation (CV) obtained by the participating

laboratories for the determination of b-lactoglobulin and ovomucoid in model processed foods added

with different percentages of milk or egg powder, using the sandwich and competitive ELISA formats.

b-lactoglobulin Ovomucoid

Sandwich Competitive Sandwich Competitive

Milk and

egg powder (%)

X CV X CV X CV X CV

Sausage

0 0.01 173 0.32 8 0.14 7 0.17 41

0.005 0.05* 20 0.27 49 0.35* 3 0.55* 28

0.01 0.12* 13 0.29 43 0.59* 14 1.30* 60

0.05 0.39* 13 0.83* 15 0.78* 30 5.48* 76

0.1 0.61* 13 1.18* 28 1.23* 20 17.27* 12

0.25 0.71* 8 2.63* 41 1.44* 34 31.01* 10

0.5 0.72* 10 3.07* 9 1.47* 6 38.01* 12

1 0.72* 6 7.81* 53 1.51* 29 43.55* 4

Bread

0 0.02 65 0.22 51 0.16 28 0.19 18

0.005 0.02 0 0.30 45 0.14 4 0.12 13

0.01 0.03 33 0.20 41 0.15 8 0.17 33

0.05 0.17* 14 0.30 26 0.26* 11 0.56* 14

0.1 0.26* 21 0.42 17 0.23* 28 0.57* 8

0.25 0.44* 23 1.27* 46 0.88* 9 3.49* 40

0.5 0.53* 18 1.77* 40 1.42* 19 5.26* 53

1 0.63* 14 6.40* 28 1.65* 9 18.07* 65

Pate

0 0.00 173 0.18 41 0.14 4 0.10 82

0.005 0.00 173 0.30 23 0.14 8 0.10 73

0.01 0.01 173 0.22 53 0.14 7 0.10 5

0.05 0.02 87 0.27 40 0.15 8 0.14 50

0.1 0.02 115 0.30 51 0.14 4 0.17 97

0.25 0.17* 43 1.02* 44 0.15 4 0.85* 66

0.5 0.17* 39 1.59* 17 0.16 4 1.65* 30

1 0.27* 44 4.01* 24 0.15 4 2.45* 100

*Food samples with concentration values above the cut-off established for each ELISA format.

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Thus, for a certain change in the absorbance values, the variation of the concentration

values is higher in the indirect competitive than in the sandwich format. For the pate

samples, the variation coefficients were very high in most samples probably due to matrix

interferences produced by the high fat content of this food product.

In an interlaboratory study performed by Poms et al. (2005) to determine peanut

proteins added to biscuit samples at a level of 2.5, 5 and 10 mg/kg, reproducibility values

reported, expressed as the interlaboratory relative standard deviation (RSDR), were

127.0%, 73.6% and 58.2%, respectively, for the same ELISA test. Sanchez, Perez, Puyol,

Calvo, and Brett (2002) performed an interlaboratory study to validate an ELISA test to

determine soy proteins in milk samples and reported RSDR values between 14.0 and

74.5%. In the study carried out by Matsuda et al. (2006) to detect egg and milk proteins in

processed foods, RSDR values for the two ELISA test used were less than 17% in all food

samples analysed.

On the other hand, it is also remarkable from results obtained in our work that the

concentration of b-lactoglobulin and ovomucoid in positive food samples containing

the same amount of milk and egg powder was higher for the indirect competitive than for

the sandwich format. Because the food extracts and the calibration standards were shared

between the two tests for the same proteins, these discrepancies may be due in part to

differences in the reactivity between the denatured protein and the antibodies used in each

format.

Furthermore, there are some differences that could be attributed to the format used.

The sandwich format requires antibodies directed against two or more distinct epitopes

whereas the indirect competitive format is a technique that uses a one-epitope approach for

the antibody to recognise a protein in the sample. The type of immunoassay is also

important with respect to the way that a protein is presented to its specific antibodies. In

the sandwich format, the immobilised antibodies serve to specifically capture the soluble

antigen that is present in the sample. However, in the indirect competitive format, the

antigen in the sample will compete with the antigen coating on the wells for the binding of

a limited amount of antibodies and thus, the accessibility of determinants for an adsorbed

protein might differ from the protein in solution (de Luis et al., 2007; Yeung, 2006).

Several interlaboratory studies have shown that quantitative results obtained when

using different ELISA tests can vary significantly. In the work of Matsuda et al. (2006),

they compared two ELISA tests to determine egg proteins and two ELISA tests to

determine milk proteins in model foods and found differences in the average concentration

value for the same sample of up to 127 and 198%, respectively. In the interlaboratory

validation study of five commercial test kits for the determination of peanut proteins in

foods, the variation in the recoveries found between the different test kits had a spread of

44�191% across all concentrations (Poms et al., 2005).

Conclusions

This work was performed to compare indirect competitive and double antibody sandwich

ELISA kits to determine either b-lactoglobulin or ovomucoid in three model processed

foods containing milk and egg as ingredients.

The performance of each format was dependant on the target protein and the type

of food sample. The detection of milk addition in food products by ELISA using

b-lactoglobulin as the target protein was better when using the sandwich format than the

indirect competitive format, due to its higher sensitivity and specificity.

348 R. de Luis et al.

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However, in the case of the detection of egg addition in foods by ELISA using

ovomucoid as the target protein, the indirect competitive format resulted better than

the sandwich format because, although both formats had the same sensitivity for two of

the products analysed (sausage and bread), only the indirect competitive format could

detect ovomucoid in pate.

The intensity of heat processing also had a great influence on the detection of the

allergenic proteins, b-lactoglobulin and ovomucoid, in model foods. The amount of milk

and egg powder necessary to give a positive result was lower in pasteurised products,followed by baked and sterilised products.

These findings underline the fact that the determination of allergenic proteins in food

products is greatly influenced by the particularities of each ELISA format used as well as

by heat processing conditions applied to food products. These considerations should be

taken into account for a correct interpretation of results obtained when using different

immunoassays to detect allergens in foods.

Acknowledgements

This work has been supported by grant PLANICYT AGL2005-05494/ALI from the ComisionInterministerial de Ciencia y Tecnologıa and by PM035 (2006) from the Gobierno de Aragon. Ruthde Luis and Marıa Lavilla are recipients of a Fellowship from the Gobierno de Aragon.

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