SIST-TS CEN/TS 15633-3:2012
(Main)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
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
This Technical Specification specifies an ELISA-method for the determination of hazelnut concentration in food samples.Spiking experiments using diluted ground hazelnut have been used to validate the method’s use on food matrices such as mixed grain cereals, dark chocolate (45 % cocoa) and ice cream. The range of the method is 0,5 mg to 5,0 mg hazelnut protein per kg of food sample. As hazelnut kernels typically contain between 12 % to 15 % protein [2], [3], this equates to approximately 3,7 mg to 37 mg hazelnut kernel per kg of food sample. The upper limit of the range of quantitation can be extended, if required, by further dilution of sample extracts.The method is commercially available ) and has been validated in-house by the manufacturer. The data is included in A.2.The method has been successfully validated by a collaborative study. The study was organized 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. Thirteen German laboratories participated. The data are included in A.3.
Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren - Teil 3: Quantitative Bestimmung von Haselnuss mit einem Enzym-Immunoassayverfahren unter Verwendung von polyklonalen Antikörpern und Proteindetektion nach Lowry
Diese Technische Spezifikation legt ein ELISA-Verfahren zur Bestimmung der Haselnusskonzentration in Lebensmittelproben fest. Zur Validierung der Anwendung des Verfahrens auf Lebensmittelmatrices, wie z. B. Mehrkorn-Zerealien, feinherbe Schokolade (45 % Kakao) und Eiscreme, wurden Aufstockversuche unter Verwendung von verdünnter gemahlener Haselnuss durchgeführt. Der Bereich des Verfahrens reicht von 0,5 mg bis 5,0 mg Haselnussprotein je kg Lebensmittelprobe. Da Haselnusskerne üblicherweise zwischen 12 % bis 15 % Protein enthalten [2], [3], entspricht das etwa 3,7 mg bis 37 mg Haselnusskernen je kg Lebensmittelprobe. Die obere Grenze des Quantifizierungsbereichs kann bei Bedarf durch weitere Verdünnung von Probenextrakten erweitert werden. Das Verfahren ist im Handel erhältlich1) und wurde vom Hersteller intern validiert. Die Daten sind in A.2 enthalten. Das Verfahren wurde in einem Ringversuch erfolgreich validiert. Der Versuch wurde von der Arbeitsgruppe organisiert, die vom Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) zur Durchführung von § 64 des Lebensmittel-, Bedarfsgegenstände- und Futtermittelgesetzbuches (LFGB) für die Bestimmung des Haselnussgehaltes in feinherber Schokolade eingerichtet wurde. Es nahmen 13 deutsche Laboratorien teil. Die Daten sind in A.3 enthalten.
Produits alimentaires - Détection des allergènes par des méthodes immunologiques - Partie3 : Détermination quantitative de la présence de noisette par un immuno-essai enzymatique à l'aide d'anticorps polyclonaux et détection des protéines par la méthode de Lowry
Cette Spécification Technique décrit une méthode ELISA utilisée pour la détermination de la concentration de noisette dans des échantillons de produits alimentaires.
Des expériences d’addition réalisées avec des noisettes moulues et diluées ont permis de valider l’utilisation de la méthode avec des matrices alimentaires telles que des mélanges de céréales, du chocolat noir (45 % de cacao) et de la crème glacée. L’étendue de la méthode se situe entre 0,5 mg et 5,0 mg de protéine de noisette par kg d’échantillon de produit alimentaire. Comme les cerneaux de noisettes contiennent habituellement entre 12 % et 15 % de protéine [2], [3], ceci équivaut à environ 3,7 mg à 37 mg de cerneau de noisette par kg d’échantillon de produit alimentaire. Si nécessaire, la limite supérieure de la gamme de quantification peut être agrandie, en diluant davantage les extraits d’échantillons.
Cette méthode est disponible sur le marché ) et a été validée en interne par le fabricant. Les données correspondantes figurent en Annexe au point A.2.
La méthode a été validée avec succès via une étude collaborative. L’étude a été organisée par le Groupe de Travail établi par le Bureau Fédéral de la Protection des Consommateurs et de la Sécurité alimentaire (BVL) en vue de la mise en application du § 64 du Code Allemand sur les produits alimentaires et la nourriture (LFGB) pour la détermination de la teneur en noisette dans le chocolat noir. Treize laboratoires allemands y ont participé. Les données correspondantes figurent en Annexe au point A.3.
Živila - Odkrivanje prisotnosti alergenov v živilih z imunološkimi metodami - 3. del: Kvantitativno določanje lešnika z encimsko-imunološko metodo z uporabo poliklonalnih protiteles in detekcijo beljakovin po Lowryju
Ta tehnična specifikacija določa metodo ELISA za določitev koncentracije lešnikov v vzorcih hrane. Preizkusi z dodatkom lešnikov z razredčenim semenom so bili uporabljeni za potrditev uporabe metode pri matricah, kot so mešane žita v zrnju, temna čokolada (45 % kakava) in sladoled. Razpon te metode je od 0,5 mg do 5,0 mg beljakovin lešnikov na kg vzorca hrane. Ker jedrca lešnikov običajno vsebujejo od 12 % do 15 % beljakovin, to ustreza približno od 3,7 mg do 37 mg lešnikovih jedrc na kg vzorca hrane. Zgornja meja količinskega razpona se po potrebi lahko zviša, in sicer z dodatnim redčenjem ekstrakta vzorca. Ta metoda je na voljo na tržišču in jo proizvajalec interno potrdi. Ti podatki so vključeni v Dodatek A.2. Ta metoda je uspešno potrjena z medlaboratorijsko študijo. Študijo je organizirala delovna skupina, ki jo je ustanovil Zvezni urad za zaščito potrošnikov in varnost živil za izvedbo ? 64 nemškega zakonika za hrano in krmo (LFGB) za določitev vsebnosti lešnikov v temni čokoladi. V medlaboratorijski študiji je sodelovalo trinajst nemških laboratorijev. Ti podatki so vključeni v Dodatek A.3.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 15633-3:2012
01-december-2012
äLYLOD2GNULYDQMHSULVRWQRVWLDOHUJHQRYYåLYLOLK]LPXQRORãNLPLPHWRGDPLGHO
.YDQWLWDWLYQRGRORþDQMHOHãQLND]HQFLPVNRLPXQRORãNRPHWRGR]XSRUDER
SROLNORQDOQLKSURWLWHOHVLQGHWHNFLMREHOMDNRYLQSR/RZU\MX
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
Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren -
Teil 3: Quantitative Bestimmung von Haselnuss mit einem Enzym-
Immunoassayverfahren unter Verwendung von polyklonalen Antikörpern und
Proteindetektion nach Lowry
Produits alimentaires - Détection des allergènes par des méthodes immunologiques -
Partie3 : Détermination quantitative de la présence de noisette par un immuno-essai
enzymatique à l'aide d'anticorps polyclonaux et détection des protéines par la méthode
de Lowry
Ta slovenski standard je istoveten z: CEN/TS 15633-3:2012
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 15633-3:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 15633-3:2012
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SIST-TS CEN/TS 15633-3:2012
TECHNICAL SPECIFICATION
CEN/TS 15633-3
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2012
ICS 67.050
English Version
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
Produits alimentaires - Détection des allergènes par des
Lebensmittel - Nachweis von Lebensmittelallergenen mit
méthodes immunologiques - Partie3 : Détermination immunologischen Verfahren - Teil 3: Quantitative
quantitative de la présence de noisette par un immuno- Bestimmung von Haselnuss mit einem Enzym-
essai enzymatique à l'aide d'anticorps polyclonaux et Immunoassayverfahren unter Verwendung von
détection des protéines par la méthode de Lowry polyklonalen Antikörpern und Proteindetektion nach Lowry
This Technical Specification (CEN/TS) was approved by CEN on 4 March 2012 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
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15633-3:2012: E
worldwide for CEN national Members.
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Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Principle .5
5 Reagents .6
5.1 Reagents usually supplied with the kit, as standard .6
5.2 Preparation of Working Strength Solutions from Concentrates .7
5.3 Reagents not supplied with the test kit .7
6 Apparatus and equipment .8
7 General instructions/recommendations/preparation .8
7.1 General .8
7.2 Preparation of sample .9
7.3 Immunoassay procedure/Operational Scheme . 10
7.4 Reading/Interpretation and test result report (Calculations, Reporting) . 11
8 Estimation of measurement uncertainty . 13
Annex A (informative) Validation status and performance criteria/method performance . 14
A.1 General . 14
A.2 Internal validation (manufacturer´s in house study). . 14
A.3 Collaborative study. 31
Bibliography . 33
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CEN/TS 15633-3:2012 (E)
Foreword
This document (CEN/TS 15633-3:2012) 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 Technical Specification consists of the following parts:
CEN/TS 15633-1, Foodstuffs — Detection of food allergens by immunological methods — Part 1: General
considerations
FprCEN/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 organizations 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
The hazelnut (Corylus avellana) belongs to a group of foods commonly referred to as tree nuts. Allergic
reactions to hazelnut ranging from oral allergy syndrome to severe anaphylaxis have been widely reported.
Food labelling legislation requires the presence of hazelnut in food to be declared in numerous nations of the
European Union, North America, Asia and Australasia.
IgE binding studies from the sera of sensitized patients have revealed a number of allergens, including both
pollen related and non-pollen related allergens [1]. Threshold dose studies have reported provocative doses
as low as 1 mg of hazelnut protein [1].
Hazelnut material may occur unintentionally in foods for several reasons. It may be present in contaminated
ingredients or cross contamination may occur during food manufacture. Consequently there is a need for
sensitive and reliable tests for the detection of hazelnut in food samples.
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1 Scope
This Technical Specification specifies an enzyme-linked immunosorbent assay (ELISA)-method for the
determination of hazelnut concentration in food samples.
Spiking experiments with diluted ground hazelnut have been used to validate the method’s use on food
matrices such as mixed grain cereals, dark chocolate (45 % cocoa) and ice cream. The range of the method is
0,5 mg to 5,0 mg hazelnut protein per kg of food sample. As hazelnut kernels typically contain between 12 %
to 15 % protein [2], [3], this equates to approximately 3,7 mg to 37 mg hazelnut kernel per kg of food sample.
The upper limit of the range of quantitation can be extended, if required, by further dilution of sample extracts.
1)
The method is commercially available and has been validated in-house by the manufacturer. These data are
included in Annex A.2.
The method has been successfully validated by a collaborative study. The study was organized 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. Thirteen German laboratories participated in the collaborative study. These data are included
in Annex A.3.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN 15633-1:2008, Foodstuffs – Detection of food allergens by immunological methods – Part 1: General
considerations
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 15633-1 apply.
4 Principle
A direct sandwich ELISA is used for detection of hazelnut protein.
A 13 kDa to 14 kDa protein band common to both raw and roasted hazelnuts was identified using polyacryl-
amide gel electrophoresis. This protein marker was purified by high performance liquid chromatography and
used to raise rabbit anti-hazelnut polyclonal anti-sera. The IgG fraction of this antiserum was purified by
affinity chromatography then used to develop a direct sandwich ELISA as outlined below:
1) Soluble hazelnut protein is extracted from a food sample.
2) The extract is then added to micro-titre wells coated with the anti-hazelnut capture antibody. The sample
is allowed to react before thorough washing.
1) ELISA Systems Hazelnut Residue ELISA is the trade name of a product supplied by ELISA Systems Pty Ltd, Brisbane, Australia.
This information is given for the convenience of users of this Technical Specification and does not constitute an endorsement by CEN of
the product named. Equivalent products may be used if they can be shown to lead to the same results.
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3) The conjugate (horseradish peroxidase labelled anti-hazelnut antibodies) is then added and allowed to
react before thorough washing. Hazelnut protein captured in step 2 will bind the conjugate to form a sandwich.
4) Tetramethylbenzidine (TMB), the chromogenic substrate, is added and allowed to react. Hazelnut protein-
antibody sandwiches will cause development of a blue colour.
5) The reaction is stopped by the addition of 1 mol/l phosphoric acid which causes a colour change to
yellow.
A spectrophotometer (450 nm primary wavelength and 620 nm to 650 nm reference wavelength) is used to
measure the optical density (OD) of each well.
The OD produced is determined by the hazelnut protein concentration. The hazelnut concentration of a
sample can be calculated by comparison of the OD it generates with the OD generated by standards. The
standards are calibrated in hazelnut protein mg/kg. They were prepared from an aqueous extract of
commercial hazelnuts (Corylus avellana). The protein concentration was determined by the Lowry method.
5 Reagents
5.1 Reagents usually supplied with the kit, as standard
5.1.1 Extraction Solution
The routine extraction solution is phosphate buffered saline with Tween 20 (PBST).The solution is supplied as
a 20-fold concentrate and requires dilution to working strength prior to use (see 5.2.1).
Table 1 — Composition of ELISA Systems Working Strength PBST
Compound Working
Concentration
KH PO 0,2 g/l
2 4
NaH PO 1,2 g/l
2 4
Tween 20 2 ml/l
NaCl 8 g/l
KCl 0,2 g/l
Ethyl(2-mercaptobenzoato-(2-)-O,S)mercurate(1-) sodium 0,2 g/l
(Thiomersal (INN))
5.1.2 ESADDSOL (Enhanced Extraction Solution for Samples containing Polyphenols)
A specialized extraction solution should be used for foods containing polyphenols. The solution is supplied as
a concentrate and requires dilution prior to use (see 5.2.2). The working strength solution contains PBST (see
Table 1) plus 1,5 % Polyvinylpyrrolidone (PVP10 average molecular weight 10 000).
5.1.3 Antibody Coated Strips
48 Micro-titre wells coated with anti-hazelnut protein capture antibodies, with one strip holder.
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5.1.4 Standards
Each vial contains 1,7 ml of hazelnut protein in aqueous solution to provide standards of the following values:
0 mg/kg (negative control); 0,5 mg/kg; 1,0 mg/kg; 2,5 mg/kg and 5,0 mg/kg. Coloured pale purple with food
dye and ready to use.
NOTE The true concentration of each vial is one tenth of that listed on the label. This compensates for the dilution
effect of the extraction procedure.
5.1.5 Enzyme Conjugate, 7 ml rabbit anti-hazelnut polyclonal antibody conjugated to horseradish
peroxidase (HRP) coloured with green food dye, ready to use.
2),
5.1.6 Substrate Enhanced KBlue 7 ml of chromogenic substrate, ready to use.
Contains 3,3’,5,5’-tetramethylbenzidine (TMB) and hydrogen peroxide.
5.1.7 Stop Solution, 7 ml of 1 mol/l phosphoric acid, ready to use.
5.1.8 Wash Buffer 20××Concentrate
××
This PBST solution is supplied as a 20-fold concentrate and requires dilution to working strength prior to use
(see 5.2.3). The components are listed in Table 1.
5.2 Preparation of Working Strength Solutions from Concentrates
5.2.1 Extraction Solution
Use distilled or deionised water to dilute 25 ml of the extraction solution concentrate supplied to a final volume
of 500 ml. The working strength extraction solution can be stored at room temperature (20 °C to 25 °C) for up
to 12 months.
5.2.2 ESADDSOL (Enhanced Extraction Solution for Samples containing Polyphenols)
A specialized extraction solution should be used for foods containing polyphenols. Use distilled or deionised
water to dilute 30 ml of the enhanced extraction solution concentrate supplied to a final volume of 500 ml. The
working strength enhanced extraction solution can be stored at room temperature (20 °C to 25 °C) for up to
12 months.
5.2.3 Wash Buffer
Use distilled or deionised water to dilute 25 ml of the wash buffer concentrate supplied to a final volume of
500 ml. The working strength wash buffer can be stored at room temperature (20 °C to 25 °C) for up to
12 months.
5.3 Reagents not supplied with the test kit
5.3.1 pH indicator strips
5.3.2 Distilled or deionised water
5.3.3 Reagents for pH adjustment (optional)
2) Enhanced KBlue Substrat is the trade name of a product supplied by Neogen, Michigan, U.S.A. This information is given for the
convenience of users of this Technical Specification and does not constitute an endorsement by CEN of the product named. Equivalent
products may be used if they can be shown to lead to the same results.
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5.3.3.1 General
The reagents described in 5.3.3.2 and 5.3.3.3 may be required for pH adjustment of sample-extraction
solution mixtures (see 7.2.3). These solutions should be prepared using analytical grade reagents and distilled
or deionised water.
5.3.3.2 Sodium hydroxide solution, c = 1 mol/l.
5.3.3.3 Hydrochloric acid solution, c = 1 mol/l.
6 Apparatus and equipment
Standard laboratory equipment and in particular the following:
6.1 Temperature-controlled water bath, capable of maintaining temperatures of 60 °C.
6.2 Laboratory balance, capable of weighing to the nearest 0,01 g.
6.3 Plastic containers, of suitable size for the extraction procedure, low protein binding disposable
containers are recommended.
6.4 Device to allow sample homogenization, such as blender, grinder, stomacher or tissue homogenizer.
)
®3
6.5 Laboratory mixer, as a Vortex style mixer.
6.6 Precision pipette, capable of measuring 100 µl.
6.7 Laboratory wash bottle
6.8 Spectrophotometer, plate reader or strip reader capable of reading at 450 nm (primary) and 620 nm to
650 nm (reference).
6.9 pH meter, (optional).
6.10 Incubator, if room temperatures falls outside the range of 20 °C to 25 °C.
6.11 Centrifuge or micro-centrifuge, (optional).
6.12 Equipment required for larger batches (> 16 wells)
6.12.1 Multichannel pipette, capable of measuring 100 µl.
6.12.2 Automated plate washer, (recommended).
7 General instructions/recommendations/preparation
7.1 General
Kits should be stored at 2 °C to 8 °C.
Do not substitute reagents and wells from kits of different lot numbers.
3) Vortex is an example of suitable products available commercially. This information is given for the convenience of the
users of this Technical Specification and does not constitute an endorsement by CEN of these products.
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If fewer than 48 wells are required then mix the reagents thoroughly and remove the required wells and
aliquots of reagents. Reseal the remaining reagents and wells and return the remainder of the kit to
refrigeration.
Reagents should be allowed to reach assay temperature (20 °C to 25 °C) before the assay is commenced.
The incubation times listed in 7.3.2 should be adhered to.
7.2 Preparation of sample
7.2.1 Sample type and amounts, including sample identification
Solid and liquid samples can be analysed. Samples should be clearly and unambiguously labelled.
7.2.2 Sample collection, transport, preservation and storage
Representative samples should be collected in accordance with a HACCP (Hazard Analysis and Critical
Control Points) plan and sampling standards.
Samples should be transported in sealed bags or containers to prevent leakage and contamination.
Ideally samples should be extracted and analysed promptly. Proteases from microbes and other sources can
digest food allergens present in samples and decrease the concentrations detected. If a delay is expected
samples should be refrigerated or frozen.
7.2.3 Test sample preparation
A minimum of two portions of each submitted sample should be extracted. Samples should be allowed to
reach assay temperature (20 °C to 25 °C) before processing.
ESADDSOL (5.2.2) is required for samples containing polyphenols such as: dark chocolate, berries, coffee,
tea etc. Other samples can be extracted with the routine Extraction Solution.
After the sample is added to the extraction solution the pH of the mixture should be in the range of 6,8 to 7,4.
If it is below this range then add 1 mol/l NaOH drop by drop until the correct pH is achieved. If it is above the
6,8 to 7,4 range then add 1 mol/l HCl drop by drop until the pH is in range.
Solid Samples: The sample should be ground to a fine consistency to provide a homogenous mixture.
Routinely 5 g of sample is extracted in 50 ml of the extraction solution. If necessary this can be altered as long
as the ratio of 1 g of sample to 10 ml of the extraction solution is maintained. The sample/extraction solution
mixture should be blended or mixed until homogenous and only minimal clumps are present.
Liquid Samples: Liquid samples should be thoroughly mixed. Routinely 5 ml of sample is added to 45 ml of
the extraction solution. If necessary this can be altered as long as the ratio of 1 ml of sample added to 9 ml of
the extraction solution is maintained. The sample/extraction solution mixture should be blended or mixed until
homogenous.
Extraction: All samples are extracted at 60 °C. Incubate for 5 min then shake/mix for 1 min. Repeat two more
times (total incubation time is 15 min). Allow the extract to cool to room temperature (20 °C to 25 °C) and
settle. Samples may be centrifuged speeds up to 500 g for 5 min. The aqueous phase is used for the ELISA
procedure.
Sample extracts should be analysed promptly, or they may be stored refrigerated for up to 24 h. If longer
delays before analysis are expected, then aliquots of the aqueous phase should be stored frozen at – 20 °C.
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7.3 Immunoassay procedure/Operational Scheme
7.3.1 General
WARNING — Avoid skin and eye contact with all reagents. Dispose of reagents in accordance with
local best practice. Stop Solution contains 1 mol/l phosphoric acid. TMB Substrate contains
tetramethylbenzidine and hydrogen peroxide. Concentrates of the extraction solutions and wash
buffer contain 0,4 % Thiomersal as a preservative. Standards and negative control contain 0,2 %
Thiomersal as a preservative. Natural ventilation is sufficient.
The assay should be performed in the temperature range of 20 °C to 25 °C. Reagents should be allowed to
reach this temperature range before the assay is commenced. If room temperature falls outside this range
then an incubator should be used.
Avoid exposure to direct sunlight.
For quantitative results the full range of standards should be included with each run. Each standard and
sample extract should be run in duplicate. It is also recommended to include a positive internal control sample
or reference material if available.
Each wash step comprises of:
Invert the plate and flick the contents out into a sink or discard container.
Using a wash bottle fill each well to the brim with the working strength wash solution, then invert the plate
and flick out the contents.
Repeat four times (a total of 5 washes).
Then tap thoroughly onto absorbent paper towels to remove excess wash solution.
An automated plate washer with a 5 wash cycle may be used as an alternative. Washing with multi-channel
pipettes is not recommended. Do not allow the plate to totally dry out during the assay.
7.3.2 Assay Steps
Insert sufficient antibody-coated wells for each standard and sample into a test strip holder. If more than
2 strips are required then the use of a multichannel pipette is recommended to minimize timing errors.
Record the position of each standard, control and sample.
Add 100 µl of each standard solution, control and sample extract to their allocated well. Mix by moving
the strip holder sideways for 10 s.
Incubate at room temperature for 10 min then wash.
Add 100 µl of the enzyme conjugate to each well. Mix by moving the strip holder sideways for 10 s.
Incubate at room temperature for 10 min then repeat the wash step.
Add 100 µl of substrate to each well. Mix by moving the strip holder sideways for 10 s.
Incubate at room temperature for 10 min. Do not wash.
Add 100 µl of Stop Solution to each well. Mix thoroughly by moving the strip holder sideways for 10 s.
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SIST-TS CEN/TS 15633-3:2012
CEN/TS 15633-3:2012 (E)
Within 30 min, use a spectrophotometer to record the OD measurement of each well. A bi-chromatic
reading at 450 nm (primary) and 620 nm to 650 nm (secondary/reference) is recommended.
7.3.3 Physical/ Environmental conditions
The analysis should be performed between 20 °C and 25 °C. Temperature control (incubator or air
conditioning) is necessary if room temperatures stray outside this range.
7.3.4 Instrument calibration
Instruments (e. g. microplate readers, pipettes and balances) should be calibrated as per
EN ISO/IEC 17025 [5].
7.3.5 Pollution prevention/ waste disposal
Appropriate local best practices shall be adhered to.
7.4 Reading/Interpretation and test result report (Calculations, Reporting)
7.4.1 General
Using the OD results of the standards, generate a standard curve of “OD vs. Hazelnut Protein Concentration”.
The hazelnut protein concentration measured by the ELISA can then be read from the standard curve.
Mathematical models are discussed in 7.4.2.
The positive standards are in a range of 0,5 mg/kg to 5,0 mg/kg. A negative control (0 mg/kg) is also included.
The kit is calibrated to give results as mg/kg hazelnut protein.
NOTE The true concentration of each vial is one tenth of that listed on the label. This compensates for the dilution
effect of the extraction procedure.
Quantitative results should not be reported if calculated by extrapolation below the 0,5 mg/kg standard nor
above the 5 mg/kg standard. If quantitative results are required for samples with OD values above the range
of the standards, then the extract will need to be further diluted using the extraction solution and the
immunoassay will need to be repeated.
Sample Concentration = Concentration Measured by the ELISA × Dilution Factor.
If the extraction procedure is not modified and the extract is not further diluted the Dilution Factor = 1.
If the extraction procedure is modified, or if further dilution of the sample extract is required (for
results > 5 mg/kg) then the effect of these additional steps will need to be taken into consideration and the
dilution factor determined.
7.4.2 Mathematical models
7.4.2.1 General
These formulae refer to concentrations measured by the ELISA only.
Measured concentrations can be calculated by manually plotting “OD vs. Concentration” of the standards on
graph paper and then reading the concentration of the samples off the graph. The standard curve will not be a
straight line.
A number of mathematical models can be used to approximate the standard curve in the range of 0,5 mg/kg
®
to 5,0 mg/kg. Spreadsheet programs (such as Microsoft Excel ) or software loaded onto a microplate reader
2
can be used to perform the calculations. In each case the R value of the standard curve should be > 0,98.
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SIST-TS CEN/TS 15633-3:2012
CEN/TS 15633-3:2012 (E)
7.4.2.2 Quadratic or polynominal order 2 model
A quadratic or polynomial order 2 model fits the standard curve well at low concentrations. It has been used
for the validation data calculations in Annex A.2.6. However at high concentrations (> 2,5 mg/kg) results may
become less accurate, see Formula 1:
2
OD = ax + bx+ c (1)
where
x is the concentration;
a, b and c are constants for the run.
An example standard curve with a quadratic model is included in Figure 1.
The concentration of a sample can be calculated using Formula 2:
2
− b+ b − 4a(c− OD)
x= (2)
2a
2
y = -0,09x + 0,925 8x + 0,068 8
2
R = 0,997 9
Key
X Hazelnut Protein content, in mg/kg
Y OD at 450 nm
Figure 1 — Standard Curve – Quadratic model example
7.4.2.3 Logarithmic model
A logarithmic model is recommended for routine analyses. Results can only be quantified in the range of
0,5 mg/kg to 5 g/kg, see Formula 3:
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SIST-TS CEN/TS 15633-3:2012
CEN/TS 15633-3:2012 (E)
OD= aln(x)+ c (3)
where
x is the concentration;
a and c are constants for the run.
Figure 2 illustrates a
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