1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationFoodstuffs Detection of food allergens by immunological methodsPart 2: Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and bicinchon
2、inic acid-protein detectionPD CEN/TS 15633-2:2013National forewordThis Published Document is the UK implementation of CEN/TS 15633-2:2013. The UK participation in its preparation was entrusted to Technical CommitteeAW/275, Food analysis - Horizontal methods.A list of organizations represented on thi
3、s committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions of acontract. Users are responsible for its correct application. The British Standards Institution 2013. Published by BSI Standards Limited 2013 ISBN 978 0 580 75409 8 ICS
4、 67.050Compliance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30 April 2013.Amendments issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 15633-2:2
5、013TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15633-2 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 an
6、d bicinchoninic acid-protein detection Produits alimentaires - Dtection des allergnes alimentaires par des mthodes danalyse immunologiques -Partie 2: Dtermination quantitative de la prsence de noisette par un immuno-essai enzymatique laide danticorps monoclonaux et dtection des protines avec lacide
7、bicinchoninique Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren - Teil 2: Quantitative Bestimmung von Haselnuss mit einem Enzym-Immunoassayverfahren unter Verwendung von monoklonalen Antikrpern und Proteindetektion mit Bicinchoninsure This Technical Specification (CE
8、N/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 E
9、uropean 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
10、 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
11、, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: A
12、venue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 15633-2:2013: EPD CEN/TS 15633-2:2013CEN/TS 15633-2:2013 (E) 2 Contents Page Foreword . 3 Introduction 4 1 Scope 5 2 Principles 5 3 Reagents
13、. 6 4 Apparatus and equipment . 6 5 Procedure . 7 6 Evaluation . 11 Annex A (informative) Internal validation (manufacturers 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 Robu
14、stness of the method (Ruggedness) 24 A.6 Calibration curve 26 A.7 Stability testing/data 27 Annex B (informative) Collaborative trial . 30 Bibliography 32 PD CEN/TS 15633-2:2013CEN/TS 15633-2:2013 (E) 3 Foreword This document (CEN/TS 15633-2:2013) has been prepared by Technical Committee CEN/TC 275
15、“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 do
16、cument 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 immunoassa
17、y 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
18、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
19、, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 15633-2:2013CEN/TS 15633-2:2013 (E) 4 Introduction Hazelnuts (Corylus avellana) have a wi
20、de 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 a
21、re 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
22、 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 hazeln
23、uts 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 birc
24、h 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. PD CEN/TS 15633-2:2013CEN/TS 15633-
25、2:2013 (E) 5 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) bec
26、ause 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
27、, 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 hazeln
28、ut 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
29、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
30、 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 an
31、tibodies 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
32、 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. Bou
33、nd 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 concentr
34、ation 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 Rmer, Barcelona Giants). These six varieties, raw and roa
35、sted, 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 ac
36、cording to BCA (Pierce). 1) RIDASCREENFAST 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. Equival
37、ent products may be used if they can be shown to lead to the same results. PD CEN/TS 15633-2:2013CEN/TS 15633-2:2013 (E) 6 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 t
38、he 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/k
39、g 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 dete
40、ctor anti-Cor a9 monoclonal antibody. The amount, which is necessary, has been determined by titration. The conjugate buffer consists of 10 mmol/l phospate buffer one plus one mixed with Stabilzyme2)containing finally 150 mmol/l saline, 5 % sorbitol, 2 % BSA (bovine serum albumin) 0,1 % Kathon2), Ta
41、rtrazin/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 concent
42、rated, 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 buffe
43、r, containing 0,9 % saline, 0,01 % Synperonic2), 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 powd
44、er (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 maintainin
45、g (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, Synperonicand Vortexare examples of suitable products available commercially. This information is given for the convenience of the users of this standard and d
46、oes not constitute an endorsement by CEN-CENELEC of these products. PD CEN/TS 15633-2:2013CEN/TS 15633-2:2013 (E) 7 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 Prec
47、ision micropipettes, capable of delivering 20 l to 200 l and 200 l to 1 000 l. 4.7 Mixer, e.g. Vortex2). 4.8 Multi-channel pipette or a repetitive pipette (25 l to 1 000 l) (optional). 4.9 Reagent reservoirs for multi-channel dispensing (optional). 4.10 Automated plate washer (optional). 5 Procedure
48、 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 sk
49、in 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 s
