BSI PD CEN/TS 15633-2:2013

$167.15

Foodstuffs. Detection of food allergens by immunological methods – Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and bicinchoninic acid-protein detection

Published By	Publication Date	Number of Pages
BSI	2013	36

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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.

PDF Catalog

PDF Pages	PDF Title
7	1 Scope 2 Principles
8	3 Reagents 4 Apparatus and equipment
9	5 Procedure 5.1 Warnings or precautions 5.2 Sample collection, transport, preservation and storage 5.3 Sample preparation
10	5.4 Method Performance 5.4.1 General 5.4.2 Physical/environmental conditions 5.4.3 Instrument calibration 5.4.4 Cleanliness of work area 5.5 Preparation of reagents 5.5.1 Antibody Enzyme Conjugate 5.5.2 Washing Buffer 5.5.3 Sample Extraction Buffer
11	5.6 Test performance 5.7 Reading/interpretation and test result report
12	5.8 Flowcharts
13	6 Evaluation 6.1 Summary of validated performance characteristics
14	6.2 Internal validation (manufacturer’s in house study)
15	6.3 Collaborative trial
16	Annex A (informative) Internal validation (manufacturer’s in house study) A.1 Precision (intra- and inter-assay variance) A.1.1 Intra-assay variance A.1.2 Inter-assay variance
17	A.2 Sensitivity A.2.1 Limit of detection (LOD)
20	A.2.2 Limit of quantitation (LOQ)
21	A.3 Accuracy/Trueness
24	A.4 Specificity/Selectivity (Interferences)
26	A.5 Robustness of the method (Ruggedness)
28	A.6 Calibration curve
29	A.7 Stability testing/data
32	Annex B (informative) Collaborative trial