A4

A A4

(c) Related proteins, inhibition of IgE binding

(d) Heterogeneous mixture with a shared protein

Fig. 7.3 Schematic representation of ELISA inhibition to determine the similarity of two allergenic food sources.

• Concentration and/or extraction into suitable buffer

• Removal of particulate matter.

The efficiency of the extraction procedure will vary for individual allergens and for different food matrixes. The expected recovery should be estimated by experiment in all circumstances.

Dry powders and cereals

Many food sources will be dry or semi-dry. In order to achieve adequate extraction the matrix must be broken down. This can usually be achieved by grinding, using a warring blender. Matrixes such as chocolate may be most easily treated by liquefying by heating and then extracted as a liquid using a warm buffer. Most allergens are water soluble and so can be extracted directly into the assay buffer. Common methods employ mixing (using a rotary shaker) the ground food with phosphate-buffered saline overnight at 4°C. Once extracted, particulate material is removed by sedimentation with or without centrifugation and filtration where necessary.

Liquids

Liquids must of course be homogenised by mixing. It may be necessary to concentrate the sample. Common techniques involve dialysis to exchange buffers and/or remove low molecular weight contamination, followed by freeze-drying to concentrate. Proteins may also be concentrated by virtue of size using an Amicon filtration unit, or a Sephadex G25 column.

Oils

As the majority of allergens investigated are water soluble, the oil can simply be shaken overnight with an equal volume of aqueous assay buffer. The oil/aqueous layers are then separated by cold centrifugation and the aqueous layer decanted. Concentration can then be employed as above. Alternatively detergents may be used to extract the proteins, but these may interfere with the subsequent assays.

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