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 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.
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
Diese Technische Spezifikation legt ein ELISA-Verfahren (enzymgebundenes Immunosorbent-Verfahren, en: enzyme linked immunosorbent assay) für den Nachweis von Haselnuss aus Lebensmittelproben fest. Im ELISA-Verfahren binden die Antikörper an die Haselnussproteine aus den Lebensmittelproben. Das Ergebnis aus dem ELISA-Verfahren wird in mg Haselnuss/kg (ppm) angegeben, da die Kalibratoren aus einem Extrakt der ganzen Haselnuss bestehen.
Matrices wie Zerealien, Eiscreme, Kekse, Schokolade, Würstchen, Hüttenkäse, Joghurt und Salatdressing wurden durch Aufstockversuche mit einer Haselnusspaste enthaltenden Carboxymethylcellulose-Suspension [2] validiert.
Die monoklonalen Antikörper, die gegen den gesamten wässrigen Haselnussextrakt gerichtet sind, weisen sowohl Proteine mit ungefähren Molekulargewichten von 14 kDa, 18 kDa und 42 kDa als auch das thermostabile Major-Allergen Cor a9 (11S-Speicherprotein) nach. Beide Antikörper wurden durch den Western Blot, mit teilweise gereinigten Haselnussextrakten und gereinigten allergenen Proteinen, bewertet.
Das ELISA-Prüfverfahren ist kommerziell erhältlich1). 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 des méthodes d'analyse 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
01-junij-2013
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.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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
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
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.
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.
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.
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 m
...
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