SIST-TS CEN/TS 15633-2:2013
(Main)Foodstuffs - Detection of food allergens by immunological methods - Part 2: Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and bicinchoninic acid-protein detection
Foodstuffs - Detection of food allergens by immunological methods - Part 2: Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and bicinchoninic acid-protein detection
This Technical Specification specifies an enzyme linked immunsorbent assay (ELISA) method for the determination of hazelnut from food samples. In the ELISA the antibodies bind to hazelnut proteins from the food sample. The result of the ELISA is given in mg hazelnut/kg (ppm) because the calibrators consist of an extract of whole hazelnut. Matrices like cereals, ice cream, cookies, chocolate, sausage, cottage cheese, yogurt and salad dressing were validated by spiking experiments with a carboxymethylcellulose-suspension containing hazelnut paste [2]. The monoclonal antibodies, raised against the whole aqueous extract of hazelnut, detect proteins with approximate molecular weights of 14 kDa, 18 kDa, and 42 kDa. The antibodies detect the major thermostable allergen Cor a9 (11S storage protein). Both antibodies were evaluated by western blots with partially purified hazelnut extracts and purified allergenic proteins.
The ELISA test method is commercially available1). The performance has been validated by an in house validation performed by the manufacturer. All parameters of interest are indicated. In addition, the ELISA was successfully validated by a collaborative study in order to determine the interlaboratory reproducibility. This ring trial was organised by the working group established by the Federal Office of Consumer Protection and Food Safety (BVL) for the execution of § 64 of the German Food and Feed Code (LFGB) for the determination of hazelnut content in dark chocolate. Fourteen German laboratories participated in this collaborative study.
Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren - Teil 2: Quantitative Bestimmung von Haselnuss mit einem Enzym-Immunoassayverfahren unter Verwendung von monoklonalen Antikörpern und Proteindetektion mit Bicinchoninsäure
Dieser Teil von EN 15633 legt ein ELISA-Verfahren (enzymgebundener Immunosorbent-Test, en: Enzyme-linked Immunosorbent Assay) für den Nachweis von Haselnuss aus Lebensmittelproben fest.
Dieses Verfahren wurde zum Nachweis von Haselnuss in Lebensmitteln entwickelt. Matrices wie Zerealien, Eiscreme, Kekse, Schokolade, Würstchen, Hüttenkäse, Joghurt und Salatdressing wurden durch Aufstock-versuche mit einer Haselnusspaste enthaltenden Carboxymethylcellulose-Suspension [2] validiert.
Die monoklonalen Antikörper, die gegen den gesamten wässrigen Haselnussextrakt gerichtet sind, weisen sowohl Proteine mit Molekulargewichten von 14 kDa und 18 kDa als auch ein Protein mit einem scheinbaren Molekulargewicht von 42 kDa sowie das thermostabile Major Allergen Cor a9 (11S-Speicherprotein) nach. Das wurde für beide Antikörper durch Western-Blotting mit teilweise gereinigten Haselnussextrakten und gereinigten allergenen Proteinen bewertet.
Das Immunoassay System ist kommerziell erhältlich ). Die Leistungsfähigkeit wurde in einer vom Hersteller durchgeführten betriebsinternen Validierung bestätigt. Alle interessierenden Parameter sind angegeben.
Zusätzlich wurde das ELISA Verfahren durch einen Ringversuch erfolgreich validiert, um die Vergleich-präzision zwischen den Laboratorien zu bestimmen. Dieser Ringversuch, an dem 14 deutsche Laboratorien beteiligt waren, wurde durch die beim Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) bestehende Arbeitsgruppe zur Durchführung von § 64 des Lebensmittel-, Bedarfsgegenstände- und Futter-mittelgesetzbuches (LFGB) für die Bestimmung des Haselnussgehalts in feinherber Schokolade organisiert.
Produits alimentaires - Détection des allergènes alimentaires par méthodes immunologiques - Partie 2: Détermination quantitative de la présence de noisette par un immuno-essai enzymatique à l'aide d'anticorps monoclonaux et détection des protéines avec l'acide bicinchoninique
La présente Norme européenne spécifie une méthode ELISA pour le dosage des noisettes dans des échantillons de produits alimentaires.
La méthode a été développée pour déterminer la quantité de noisettes dans les produits alimentaires. Les matrices telles que les céréales, les crèmes glacées, les biscuits, le chocolat, les saucisses, le fromage blanc, le yaourt et les sauces salade ont été validées par des expériences de dopage (additions volontaires de noisettes) réalisé avec une suspension de carboxyméthylcellulose contenant de la pâte de noisettes [2].
Les anticorps monoclonaux de cette méthode ont été obtenus contre un extrait aqueux de noisettes : ils détectent des protéines ayant des poids moléculaires apparents de 14 kDa et 18 kDa, une protéine ayant un poids moléculaire apparent de 42 kDa ainsi que l’allergène majeur thermostable Cor a9 (protéine de stockage 11S). Cette spécificité des 2 anticorps a été évaluée par technique de Western blot avec des extraits de noisettes partiellement purifiés et des protéines allergéniques purifiées.
La méthode est commercialisée ). Les performances ont été validées lors d’une validation interne réalisée par le fabricant. Tous les paramètres étudiés sont indiqués.
Cette méthode ELISA a également été validée lors d’un essai interlaboratoires afin de déterminer la reproductibilité interlaboratoires. Cet essai interlaboratoires a été organisé par le groupe de travail constitué par l’Office fédéral allemand pour la protection des consommateurs et la sécurité alimentaire (BVL) dans le cadre de la mise en application du paragraphe 64 du règlement allemand sur les denrées alimentaires et les produits de consommation (LFGB), en vue de doser les noisettes dans le chocolat noir. Cette étude regroupait 14 laboratoires allemands.
Živila - Odkrivanje prisotnosti alergenov v živilih z imunološkimi metodami - 2. del: Kvantitativno določanje lešnika z encimsko-imunološko metodo z uporabo monoklonskih protiteles in detekcijo beljakovin z bicinhoninsko kislino
Ta tehnična specifikacija določa encimsko-imunološko metodo ELISA za določanje lešnika v vzorcih živil. Pri metodi ELISA se protitelesa vežejo na beljakovine lešnika iz vzorca živila. Rezultat metode ELISA je naveden v mg lešnika/kg (ppm), ker kalibratorji vsebujejo ekstrakt celega lešnika. Matrice, kot so žita, sladoled, piškoti, čokolada, klobasa, skuta, jogurt in solatni preliv, so bile potrjene s preskusi z dodatkom razredčine karboksimetilceluloze z lešnikovo pasto [2]. Monoklonska protitelesa v celotnem vodnem ekstraktu lešnika odkrijejo prisotnost beljakovin s približno molekularno težo 14 kDa, 18 kDa in 42 kDa. Protitelesa zaznajo glavni termostabilni alergen Cor a9 (skladiščni protein 11S). Obe protitelesi so ocenili z metodo western blot z delno prečiščenimi ekstrakti lešnika in prečiščenimi alergenskimi beljakovinami.
Preskusna metoda ELISA je na voljo na tržišču (1). Učinkovitost je bila potrjena z internim potrjevanjem, ki ga je izvedel proizvajalec. Vsi obravnavani parametri so navedeni. Poleg tega je bila metoda ELISA uspešno potrjena z medlaboratorijsko študijo za določitev medlaboratorijske ponovljivosti. Ta primerjalni preskus je organizirala delovna skupina, ki jo je ustanovil Zvezni urad za zaščito potrošnikov in varnost živil (BVL) za izvedbo člena 64 nemškega zakonika za živila in krmo (LFGB) za določitev vsebnosti lešnikov v temni čokoladi. V tej medlaboratorijski študiji je sodelovalo štirinajst nemških laboratorijev.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 15633-2:2013
01-junij-2013
äLYLOD2GNULYDQMHSULVRWQRVWLDOHUJHQRYYåLYLOLK]LPXQRORãNLPLPHWRGDPLGHO
.YDQWLWDWLYQRGRORþDQMHOHãQLND]HQFLPVNRLPXQRORãNRPHWRGR]XSRUDER
PRQRNORQVNLKSURWLWHOHVLQGHWHNFLMREHOMDNRYLQ]ELFLQKRQLQVNRNLVOLQR
Foodstuffs - Detection of food allergens by immunological methods - Part 2: Quantitative
determination of hazelnut with an enzyme immunoassay using monoclonal antibodies
and bicinchoninic acid-protein detection
Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren -
Teil 2: Quantitative Bestimmung von Haselnuss mit einem Enzym-
Immunoassayverfahren unter Verwendung von monoklonalen Antikörpern und
Proteindetektion mit Bicinchoninsäure
Produits alimentaires - Détection des allergènes alimentaires par méthodes
immunologiques - Partie 2: Détermination quantitative de la présence de noisette par un
immuno-essai enzymatique à l'aide d'anticorps monoclonaux et détection des protéines
avec l'acide bicinchoninique
Ta slovenski standard je istoveten z: CEN/TS 15633-2:2013
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 15633-2:2013 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 15633-2:2013
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SIST-TS CEN/TS 15633-2:2013
TECHNICAL SPECIFICATION
CEN/TS 15633-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
April 2013
ICS 67.050
English Version
Foodstuffs - Detection of food allergens by immunological
methods - Part 2: Quantitative determination of hazelnut with an
enzyme immunoassay using monoclonal antibodies and
bicinchoninic acid-protein detection
Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen mit
alimentaires par des méthodes d'analyse immunologiques - immunologischen Verfahren - Teil 2: Quantitative
Partie 2: Détermination quantitative de la présence de Bestimmung von Haselnuss mit einem Enzym-
noisette par un immuno-essai enzymatique à l'aide Immunoassayverfahren unter Verwendung von
d'anticorps monoclonaux et détection des protéines avec monoklonalen Antikörpern und Proteindetektion mit
l'acide bicinchoninique Bicinchoninsäure
This Technical Specification (CEN/TS) was approved by CEN on 7 January 2013 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15633-2:2013: E
worldwide for CEN national Members.
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Contents Page
Foreword . 3
Introduction . 4
1 Scope . 5
2 Principles . 5
3 Reagents . 6
4 Apparatus and equipment . 6
5 Procedure . 7
6 Evaluation . 11
Annex A (informative) Internal validation (manufacturer's in house study) . 14
A.1 Precision (intra- and inter-assay variance) . 14
A.2 Sensitivity . 15
A.3 Accuracy/Trueness . 19
A.4 Specificity/Selectivity (Interferences) . 22
A.5 Robustness of the method (Ruggedness) . 24
A.6 Calibration curve . 26
A.7 Stability testing/data . 27
Annex B (informative) Collaborative trial . 30
Bibliography. 32
2
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Foreword
This document (CEN/TS 15633-2:2013) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document consists of the following parts:
EN 15633-1, Foodstuffs — Detection of food allergens by immunological methods — Part 1: General
considerations;
CEN/TS 15633-2, Foodstuffs — Detection of food allergens by immunological methods — Part 2:
Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and
bicinchoninic acid-protein detection;
CEN/TS 15633-3, Foodstuffs — Detection of food allergens by immunological methods — Part 3:
Quantitative determination of hazelnut with an enzyme immunoassay using polyclonal antibodies and
Lowry protein detection.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
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Introduction
Hazelnuts (Corylus avellana) have a wide distribution in food industry, especially in chocolate and nougat
production. In these cases, the content of hazelnut determines the quality of a product. Hazelnuts are also
frequently used in confectionaries, bakery products, biscuits, breakfast cereals and ice-creams.
Unfortunately, hazelnuts are one of the major causes of food allergy. The amount of hazelnut which causes an
allergic reaction depends on the sensitivity of the individuals. Even consumption of a few milligrams of
hazelnut can induce allergic reactions in highly sensitive allergic consumers. Amounts ranging from 0,7 mg/kg
to 100 mg/kg can induce reactions in sensitised individuals [1]. Symptoms of an allergic reaction include local
itching of the mouth and throat to severe life-threatening anaphylaxis. Thus deliberately added non-declared
hazelnuts in food products are particularly dangerous. Also trace amounts of hazelnuts or nougat, as a result
of cross contamination, pose a health risk.
The allergy is caused among other proteins by glycoproteins like corylin, an 18 kDa storage protein contained
in the hazelnut, which is similar to the Cor a1-antigen of hazelnut pollen and homologous to the Bet v1 antigen
of birch pollen. Corylin is one of the main allergenic proteins beside Cor a8, Cor a9 and Cor a11 as
representatives of seed storage and lipid transfer proteins (LTP-proteins). Corylin is differentiated between
pollen associated allergy and non-pollen associated allergy.
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1 Scope
This Technical Specification specifies an enzyme linked immunsorbent assay (ELISA) method for the
determination of hazelnut from food samples. In the ELISA the antibodies bind to hazelnut proteins from the
food sample. The result of the ELISA is given in mg hazelnut/kg (ppm) because the calibrators consist of an
extract of whole hazelnut.
Matrices like cereals, ice cream, cookies, chocolate, sausage, cottage cheese, yogurt and salad dressing
were validated by spiking experiments with a carboxymethylcellulose-suspension containing hazelnut paste
[2].
The monoclonal antibodies, raised against the whole aqueous extract of hazelnut, detect proteins with
approximate molecular weights of 14 kDa, 18 kDa, and 42 kDa. The antibodies detect the major thermostable
allergen Cor a9 (11S storage protein). Both antibodies were evaluated by western blots with partially purified
hazelnut extracts and purified allergenic proteins.
1)
The ELISA test method is commercially available . The performance has been validated by an in house
validation performed by the manufacturer. All parameters of interest are indicated.
In addition, the ELISA was successfully validated by a collaborative study in order to determine the
interlaboratory reproducibility. This ring trial was organised by the working group established by the Federal
Office of Consumer Protection and Food Safety (BVL) for the execution of § 64 of the German Food and Feed
Code (LFGB) for the determination of hazelnut content in dark chocolate. Fourteen German laboratories
participated in this collaborative study.
2 Principles
A direct sandwich ELISA is used for detection of hazelnut. The basis of the test is an antigen-antibody
reaction. Two hazelnut specific monoclonal antibodies are used to detect the analyte. The antibodies
recognise the hazelnut specific protein Cor a9. A microtiter plate is coated with the capture monoclonal
antibody mouse anti Cor a9 antibody. Hazelnut standards provided with the kit or sample extracts are
incubated for 10 min. After washing, a detector monoclonal antibody mouse anti Cor a9 antibody, labelled with
peroxidase, is added as the enzyme conjugate for further 10 min. The conjugate binds to the hazelnut protein
antibody complex on the plate. Any unbound enzyme conjugate is then removed by a washing step.
Chromogen/substrate is added to the wells and incubated for 10 min. Bound enzyme converts the chromogen
into a blue coloured product. The addition of stop reagent inhibits the enzymatic process and causes a shift of
the coloured product to yellow. Absorbance measurement is performed at 450 nm against air. The resulting
absorbance values are proportional to the concentration of hazelnut of a sample. The result is expressed as
hazelnut in mg/kg. The standard stock solution used is an aqueous hazelnut extract of six different varieties of
hazelnut (Hallesche Riesen, Levantiner, Kerassunder, Piemonteser, round Römer, Barcelona Giants). These
six varieties, raw and roasted, are representative for the hazelnuts used in food products world-wide by food
industry. The standard stock solution is further diluted (see 3.1.2). The extract from the different hazelnuts has
a protein content of approx. 9 % protein, measured by the photometric protein determination method
according to BCA (Pierce).
®
1) RIDASCREEN FAST Hazelnut is the trade name of a product supplied by R-Biopharm AG, Darmstadt, Germany.
This information is given for the convenience of users of this Technical Specification and does not constitute an
endorsement by CEN-CENELEC of the product named. Equivalent products may be used if they can be shown to lead to
the same results.
5
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3 Reagents
The assay can be performed in a standard laboratory environment. The test kit shall be stored under cool
conditions (4 °C to 8 °C).
3.1 Reagents usually provided with the test kit.
All reagents used for preparing the components and buffers shall be of analytical grade.
3.1.1 Microtiter plate, 48 wells (6 strips with 8 wells each) coated with anti-Cor a9 monoclonal antibody.
3.1.2 Standards, 1,3 ml each, namely 0 mg/kg (zero standard), 2,5 mg/kg , 5 mg/kg , 10 mg/kg and
20 mg/kg hazelnut in aqueous solution; ready to use. (These concentrations correspond to the actual hazelnut
amounts of 0 ng/ml, 125 ng/ml, 250 ng/ml, 500 ng/ml and 1000 ng/ml hazelnut in the vials).
3.1.3 Conjugate, 11-fold concentrated aqueous solution of horseradish peroxidase labelled detector anti-
Cor a9 monoclonal antibody. The amount, which is necessary, has been determined by titration. The
®2)
conjugate buffer consists of 10 mmol/l phospate buffer one plus one mixed with Stabilzyme containing
®2)
finally 150 mmol/l saline, 5 % sorbitol, 2 % BSA (bovine serum albumin) 0,1 % Kathon , Tartrazin/Patent
blue as color.
3.1.4 Chromogen/Subtrate, coloured, ready to use Tetramethylbenzidine (TMB)/urea peroxide solution
(commercial product provided by e. g. KemEnTec, Denmark).
3.1.5 Stop reagent, 1 mol/l sulphuric acid ready to use solution.
3.1.6 Sample extraction buffer, 20-fold concentrated, PBS-Tween buffer, diluted to 0,01 mol/l phosphate
buffer containing 0,9 % saline, 0,05% Tween 20 Ph (8,0 ± 0,2), store at 4 °C to 8 °C over the shelf life of the
component (at least 36 months).
3.1.7 Washing buffer, 10-fold concentrate, PBS buffer, finally consisting of 0,01 mol/l phosphate buffer,
®2)
containing 0,9 % saline, 0,01 % Synperonic , 0,01 % Thimerosal Ph (7,2 ± 0,2), store at 4 °C to 8 °C over
the shelf life of the component (at least 18 months).
3.2 Chemicals not supplied with the test kit
3.2.1 Distilled water
Mono-distilled water or purified by reverse osmosis.
3.2.2 Skim milk powder (food grade like offered in a normal supermarket).
It is necessary to make sure that the skim milk powder is hazelnut free.
4 Apparatus and equipment
Usual laboratory equipment should be used and in particular as listed in 4.1 to 4.10:
4.1 Temperature controlled water bath, capable for maintaining (37 ± 4) °C and (60 ± 5) °C.
4.2 Centrifuge, capable for producing a centrifugal acceleration of at least 2 500 g at 4 °C.
® ® ® ®
2) Stabilzyme , Kathon , Synperonic and Vortex are examples of suitable products available commercially. This
information is given for the convenience of the users of this standard and does not constitute an endorsement by CEN-
CENELEC of these products.
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4.3 Microtiter plate reader, capable of reading absorbance at 450 nm.
4.4 Analytical Balance, capable of weighing gram amounts (max. (150 ± 0,01) g).
4.5 Laboratory mill/grinder
4.6 Precision micropipettes, capable of delivering 20 µl to 200 µl and 200 µl to 1 000 µl.
®2)
4.7 Mixer, e.g. Vortex .
4.8 Multi-channel pipette or a repetitive pipette (25 µl to 1 000 µl) (optional).
4.9 Reagent reservoirs for multi-channel dispensing (optional).
4.10 Automated plate washer (optional).
5 Procedure
5.1 Warnings or precautions
WARNING — Wash buffer (thiomersal), harmful by inhalation in contact with skin and if swallowed.
Chromogen/substrate (tetramethylbenzidine/urea peroxide), avoid skin and eye contact. Stop solution
contains diluted sulfuric acid (1 mol/l), irritating and corrosive, avoid skin and eye contact. Chemicals
should be treated with care. Waste shall be disposed according to good laboratory practice.
5.2 Sample collection, transport, preservation and storage
From the whole food aggregated samples shall be collected (according to a sampling protocol). The
aggregated sample (combination of different parts of the food) is mixed and the laboratory samples are taken.
The sample material is mixed very well to ensure homogeneity of the sample prior to weighing. The food stuff
should be transported in sealed bags or closed vials to prevent cross contamination. The samples should be
stored in a cool room until use, unless the sample requires freezing (e.g. ice cream) or storage at room
temperature (e.g. muesli).
5.3 Sample preparation
Sample preparation shall be carried out according appropriate instructions. An example for an appropriate
sample preparation for the test procedure described in this Technical Specification the following:
collect 5 g of the food sample and ground as fine as possible by high speed for 3 min to 5 min (the
temperature of the sample should not be higher than 40 °C); chocolate should be melted at 30 °C to
40 °C and mixed well before weighing;
weigh 1 g of the milled or melted sample and add 1 g of skim milk powder (3.2.2);
add 20 ml of pre-heated diluted extraction buffer; mix intensively;
extract 10 min at 60 °C in a water bath by shaking casually (4.1);
− cool down to room temperature (if possible on ice), centrifuge (10 min at 2 500 g at 4 °C (if possible))
and/or filter the extract on a folded paper filter (3 hw grade or similar grade) to avoid particles in the
extract;
− use 100 µl of the extract per well.
Sample extracts can be stored at –20 °C for three months.
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5.4 Method Performance
5.4.1 General
All reagents shall be brought to room temperature (20 °C to 25 °C) prior to use. All reagents shall be returned
to 2 °C to 8 °C immediately after use. Microwells shall not dry between working steps. Reproducibility in any
ELISA is largely dependent upon the consistency with which the microwells are washed. Carefully follow the
recommended washing sequence as outlined in the ELISA test procedure. Direct exposure to sunlight during
all incubations shall be avoided. Covering the microtiter plates is recommended. Chromogen/Substrate
reaction should be carried out in the dark.
5.4.2 Physical/environmental conditions
No special laboratory conditions are required. Equipment for standard biochemical working condition is
needed.
5.4.3 Instrument calibration
The pipettes shall be calibrated and the laboratory balance shall be checked regularly as written in the
laboratories’ quality management documents. The microtiter plate reader should be calibrated according to
the laboratories’ quality management documents.
5.4.4 Cleanliness of work area
The sample preparation should be performed in a preparation room, separate from the ELISA room to avoid
cross contamination to the kit components. Laboratory equipment shall be clean and free from hazelnut
residues. After each sample is weighed, the equipment (e. g. spatula, mills) shall be cleaned to avoid cross
contamination.
5.5 Preparation of reagents
5.5.1 Antibody Enzyme Conjugate
The conjugate is a 11fold concentrate solution. Since the conjugate has a limited stability only the amount that
is required should be diluted. Before diluting the concentrated conjugate should be shaken carefully. The
conjugate shall be diluted 1 + 10 with distilled water (3.2.1).
5.5.2 Washing Buffer
The washing buffer is a 10-fold concentrate solution. Before use the buffer shall be diluted 1:10 (1 + 9) with
water (i.e. 100 ml buffer concentrate solution + 900 ml distilled water). The diluted ready-to-use buffer is stable
at 2 °C to 8 °C for four weeks. Before dilution, dissolve any crystals of the concentrated buffer in a water bath
at 37 °C completely.
5.5.3 Sample Extraction Buffer
The sample extraction buffer is a 20-fold concentrated solution. Before dilution of the buffer concentrate
solution, dissolve any crystals in a water bath at 37 °C completely and mix well. Then dilute the heated buffer
concentrate 1:20 (1 + 19) with dist. water (3.2.1) before use (i. e. 100 ml buffer concentrate + 1 900 ml water)
or alternatively follow the kit manufacturer’s instruction. The diluted buffer is stable at 2 °C to 8 °C for max.
four weeks.
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5.6 Test performance
Insert a sufficient number of wells into the microwell holder. Not more than 3 strips shall be done per run for all
standards and samples. Standard and sample positions should be recorded. Standards and samples should
be run at least in duplicates. Incubation steps shall occur without shaking unless otherwise stated.
Add 100 µl of each standard solution or prepared sample to separate wells and incubate for 10 min at room
temperature (20 °C to 25 °C).
Pour the liquid out of the wells and tap the microwell holder upside down vigorously (consecutively three
times) against absorbent paper to ensure complete removal of liquid from the wells. Fill all wells with
approximately 250 µl washing buffer (3.1.7) and pour out the liquid again. Repeat four more times.
Add 100 µl of the diluted enzyme conjugate to each well. Mix gently by rocking the plate. Incubate for 10 min.
Pour the liquid out of the wells and tap the microwell holder upside down vigorously (consecutively three
times) against absorbent paper to ensure complete removal of liquid from the wells. Fill all wells with approx.
250 µl washing buffer and repeat four more times as mentioned above.
Add 100 µl of red colored chromogen/substrate solution to each well.
Mix gently by rocking the plate manually and incubate for 10 min at room temperature (20 °C to 25 °C) in the
dark.
Add 100 µl stop solution to each well. Mix gently by rocking the plate manually and measure the absorbance
at 450 nm against an air blank. Read within 30 min after addition of stop solution.
5.7 Reading/interpretation and test result report
Sample concentrations are calculated on the basis of the standard curve. The curve is generated with an
aqueous hazelnut extract corresponding to 0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, and 20 mg/kg hazelnut
(real concentrations see 3.1.2), whereas the extraction dilution factor is already included. Absorbances
obtained for hazelnut extract are plotted into a system of coordinates onto semi-logarithmic graph paper
versus hazelnut concentration (mg/kg) manually or by suitable software applying cubic spline fitting or 4-
parameter curve logistic. Sample results are expressed in mg/kg hazelnut as indicated on the standard vials.
The dilution factor of 20 is already included in the standard concentration. If a dilution factor other than 20 is
used, this shall be taken into account when calculating the result. All results of the internal study were
®
obtained with commercial available software (RIDA SOFT Win, Version 1.34, R-Biopharm AG).
A positive internal control sample or a reference material is recommended with each test run. The result shall
be expressed as mg/kg hazelnut. The hazelnut content can be converted into approximately hazelnut protein
content by multiplying by the factor 0,09. (The protein content of the standard is approximately 9 %. This was
measured by the BCA method.) For those who will prepare their own standards, the protein content shall be
determined e.g. using BCA assay or equivalent.
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5.8 Flowcharts
Figure 1 — Extraction flowchart
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Figure 2 — ELISA procedure flowchart
6 Evaluation
6.1 Summary of validated performance characteristics
The performance characteristics of the presented Hazelnut ELISA meet the following specification:
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Time required for completion of the sample preparation was below 20 min and less than 40 min for the test
implementation.
Specificity of the test with hazelnut-free blank samples was 100 % (no false positive samples were detected).
Analytical Sensitivity was found at LOD < 1,5 mg/kg hazelnut as measured by 10-fold determination of various
hazelnut-free food matrices. LOQ was set at 2,5 mg/kg hazelnut, which was confirmed by multiple
measurement of a sample contaminated close to that value. Accuracy was investigated with spiked samples
of various matrices at different defined levels between 2,5 mg/kg and 20 mg/kg hazelnut. Mean recovery was
found at 98,2 %.
The ELISA is not sensitive to temperature changes between 18 °C and 37 °C.
The ELISA is not sensitive to variation of incubation time between 3 × 9 min and 3 × 11 min.
The ELISA is not sensitive to small changes of reagent volumes between 90 µl and 110 µl.
The test kit components are stable as indicated on the test kit labels (shelf life is usually more than
12 months).
This Hazelnut ELISA was successful evaluated by a collaborative study performed by the working group
“Lebensmittel-Allergene“ (Food Allergens) of the Bundesamt für Verbraucherschutz und Lebensmittel-
sicherheit (Federal Office of Consumer Protection and Food Safety, BVL), Germany, for implementing LFGB,
using dark chocolate as matrix. Four spiked samples at different levels (in the range of 2,5 mg/kg to 20 mg/kg)
have been investigated; a mean recovery of 106,3 % has been calculated. The RSD was calculated at 9,5 %
r
and the RSD at 17,3 %.
R
6.2 Internal validation (manufacturer’s in house study)
To determine the precision three samples (one negative and two hazelnut containing samples) have been
included into the experiments. The mean, the standard deviation (SD) and the recovery have been calculated
concerning the intra-assay and the inter-assay variance. Over all a mean recovery was found at 99,3 % with a
CV (%) of 8,7 %.
The limit of detection (LOD) of the Hazelnut ELISA, evaluated with four clear negative kinds of samples
(cookies, cereals, ice cream and chocolate), was measured close to standard 1 (zero mg/kg) at 0,99 mg/kg
under consideration of the three times standard deviation and was set at 1,5 mg/kg hazelnut.
The limit of quantitation (LOQ) was carried out with a sample, which had a hazelnut content of about
2,5 mg/kg, near standard two. The mean value and the standard deviation of the sample and of standard 2
have been calculated and compared with standard 1 under consideration of the three times standard
deviation. It could be shown that the test is able to determine samples near standard point 2 sure, significant
different from zero.
Zero samples and spiked samples on a range of 2,5 mg/kg to 20 mg/kg hazelnut were analysed with the test
for determination of accuracy. Mean value and recovery have been calculated. A recovery over all of 97,8 %
was found. Hazelnut free samples have been validated clearly negative, no false positive results occur.
Cross reactivity of the test was checked with 43 different compounds of interest (grains, vegetables, seeds,
nuts and miscellaneous samples). All samples were extracted with the extraction buffer included in the kit. As
a result most of the compounds were measured close to standard one without any measurable cross reaction.
Some of the substances showed reactivities at a 100 % level. These compounds have been repeated at a
level of 100 mg/kg (0,01 %) related to the AOAC Research Institute validation requirements for performance
tested methods [3]. All of the tested compounds have been measured well below standard 2 at the level of
100 mg/kg (0,01 %). Matrices which have been checked were:
cereals like corn, rice, wheat, rye, barley, oats, buckwheat;
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