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6. Standardization of Methodologies

6.1. Sequence Homology as Derived from Allergen Databases

The commonly used protein databases (PIR, SwissProt and TrEMBL) contain the amino acid sequences of most allergens for which this information is known. However, these databases are currently not fully up-to-date. A specialized allergen database is under construction.

Suggested procedure on how to determine the percent amino acid identity between the expressed protein and known allergens.

Step 1: obtain the amino acids sequences of all allergens in the protein databases (for SwissProt and TrEMBL: see; for PIR see in FASTA-format (using the amino acids from the mature proteins only, disregarding the leader sequences, if any). Let this be data set (1).

Step 2: prepare a complete set of 80-amino acid length sequences derived from the expressed protein (again disregarding the leader sequence, if any). Let this be data set (2).

Step 3: go to EMBL internet address: and compare each of the sequences of the data set (2) with all sequences of data set (1), using the FASTA program on the web site for alignment with the default settings for gap penalty and width.

Cross-reactivity between the expressed protein and a known allergen (as can be found in the protein databases) has to be considered when there is:

1) more than 35 % identity in the amino acid sequence of the expressed protein (i.e. without the leader sequence, if any), using a window of 80 amino acids and a suitable gap penalty (using Clustal-type alignment programs or equivalent alignment programs)


2) identity of 6 contiguous amino acids.

If any of the identity scores equals or exceeds 35 %, this is considered to indicate significant homology within the context of this assessment approach. The use of amino acid sequence homologies to identify prospective cross-reacting allergens in genetically modified foods has been discussed in more detail elsewhere (Gendel, 1998a; Gendel, 1998b).

Structural similarity with known allergens may still be important if significant amino acid identity is found, but it is below 35 %. In this case significant cross-reactivity is unlikely. However, some families of -structurally related proteins are known to contain several allergens. Some examples are:

If the expressed protein belongs to such a family, it may be considered to have a higher probability to be an allergenic protein.

Functional similarity without structural similarity is unlikely to result in significant cross-reactivity. For example, protease inhibitors that belong to distinct protein families are not known to be cross-reactive. Similarly, proteins belonging to structurally unrelated classes of pathogenesis-related proteins (PR-proteins) are not known to be cross-reactive.

Since identity of 6 contiguous amino acids has an appreciable risk of occurring by chance, verification of potential crossreactivity is warranted when criterion (1) is negative, but criterion (2) is positive. In this situation suitable antibodies (from human or animal source) have to be tested to substantiate the potential for crossreactivity.

Specific serum screening

In the evaluation of the reactivity of IgE antibodies in the sera of patients with known allergies to relevant source materials, an appropriate in vitro method should be applied. A variety of well validated immunoassays are available for this purpose. The Consultation agrees that any of these tests can be used.

In addition to the precautions cited earlier with respect to selection of suitable sera for such screening, the importance of glycosylation and glycan epitopes must also be considered. Proteins to be expressed in plant hosts may be posttranslationally modified, which may have an impact on their allergenic potential. The effects of glycosylation are particularly relevant to consider, because:

1. The degree of glycosylation may affect the susceptibility of the protein to processing and proteolysis;

2. Glycosylation may alter the epitope structure, either by shielding part of the protein surface (particularly if the glycosylation is extensive), or by introducing glycan epitopes. Glycan epitopes are known to be highly cross-reactive

Glycans may be attached either via an N-link or via an O-link. N-linked sites can be predicted with some accuracy, but the prediction of sites for O-glycosylation is still unreliable.

Cross-reactivity of IgE antibodies to glycan epitopes is important not so much because of their potential contribution to allergic symptomatology (which may be minimal in many cases), but because the structure of the protein part of these glycoproteins is in this situation largely irrelevant: all proteins with these glycan structures will be cross-reactive. When target glycoproteins are screened for cross-reactivity, it is important to make a clear distinction between IgE antibodies to the glycan part on the one hand and IgE antibodies to the protein part on the other hand. In general, it is advisable to select serum samples without IgE antibodies to glycans, absorb out such IgE antibodies with irrelevant glycoproteins obtained from the same host, or perform such tests with non-glycosylated variants, e.g. expressed in a bacterial host.

Information on glycan epitopes in relation to allergy is largely based on work with plant glycoproteins and invertebrate glycoproteins. Less is known about glycoproteins of eukaryotic microorganisms such as yeast. However, it is likely that similar precautions may need to be taken.

6.3. Targeted serum screening

When no sequence homology has been found between the expressed protein and an allergen, this does not mean that there is no such homologous allergen. It may be due to a lack of information on the relevant allergen. Random screening of serum samples from the allergic population is unlikely to be rewarding. However, some more targeted approach may, in some situations, be more appropriate.

The use of large serum pools (> 5 sera) is discouraged, because this will dilute any cross-reactive antibody present. For maximal sensitivity, individual sera should be tested.

Typically, a screen with 25 individual serum samples with high levels of IgE to the selected group of airborne allergens and (if applicable) 25 with IgE to the selected group of food allergens would be used.

6.4. Pepsin Resistance

Purified or enriched expressed protein (non-heated and non-processed) should be subjected to pepsin degradation conditions using Standard Operating Procedures and Good Laboratory Practices (SOP/GLP). In addition, the expressed protein should be assessed in its principal edible form under identical pepsin degradation conditions to those used to examine the expressed protein. Both known non-allergenic (soybean lipoxygenase, potato acid phosphatase or equivalent) and allergenic (milk beta lactoglobulin, soybean trypsin inhibitor or equivalent) food proteins should be included as comparators to determine the relative degree of the expressed proteins pepsin resistance. The protein concentrations should be assessed using a colorimetric assay (e.g., Bicinchoninic acid assay (BCA), Bradford Protein Assay, or equivalent protein assay) with bovine serum albumin (BSA) as a standard. Pepsin proteolytic activity should be assessed (Ryle). Enzyme/protein mixtures should be prepared using 500 mg of protein in 200 mL of 0.32% pepsin (w/v) in 30 mM/L NaCl, pH 2.0, and maintained in a shaking 37 C water bath for 60 minutes. Individual 500 microgram aliquots of pepsin/protein solution should be exposed for periods of 0, 15, 30 seconds and 1, 2, 4, 8, 15, and 60 minutes, at which time each aliquot should be neutralised with an appropriate buffer. Neutralised protein solutions should be mixed with SDS-PAGE sample loading buffer with and without reducing agent (DTT or 2-ME) and heated for 5 minutes at 90°C. Samples containing 5mg/cm gel of protein should be evaluated using 10-20% gradient Tricine SDS-PAGE gels or equivalent gel system under both non-reducing and reducing electrophoretic conditions. Protein in the gels should be visualised by silver or colloidal gold staining procedures. Evidence of intact expressed protein and/or intact fragments greater than 3.5 kDa would suggest a potential allergenic protein. Evidence of protein fragments less than 3.5 kDa would not necessarily raise issues of protein allergenicity and the data should be taken into consideration with other decision tree criteria. For detection of expressed protein in an edible food source, a polyclonal IgG immunoblot analysis should be performed according to the laboratory procedures. The immunoblot analysis should be compared to the silver or colloidal gold stained SDS-PAGE gel and reflect the stained pattern of the expressed protein run under identical conditions.

The investigator should be aware of and consider the following precautions. Edible food sources may contain protease inhibitors or other substances that may promote or reduce protein degradation. Resulting fragments may not be reactive with the polyclonal IgG antibody source. Finally, there is no absolute certainty that pepsin resistance or complete degradation of a protein will predict the allergenicity of novel proteins and must be taken into consideration with other decision tree criteria. Although the present pepsin resistance protocol is strongly recommended, it is recognized that other enzyme susceptibility protocols exist. Alternative protocols may be used for which adequate justification is provided. The producer is expected to take these results into consideration in combination with other decision tree criteria.

6.5. Animal Models

For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models in development. A number of animal models may be considered to assess on a relative scale the potential allergenicity using oral sensitisation routes with the Brown Norway rat model (Knippels et al., 1998) or intraperitoneal administration in murine models (Dearman et al 2000) or other relevant animal models. Results should be presented in characteristic Th1/Th2 antibody (isotype) profiles for assessing the potential immunogenic/allergenic activity. The different routes of administration in animal models (oral versus intraperitoneal) may not give the same results. Therefore, selection of one route of administration is not meant to exclude other routes of sensitisation. It is recommended to consider the results from two sensitisation routes in the same or different animal species.

It is recommended that the potential allergenicity of the expressed protein be ranked against well known strong and weak food allergens and non-allergenic proteins in the animal model. As additional information becomes available with respect to animal models, protocols may need to be modified to give optimal conditions for assessing protein allergenicity.

Although the present animal models provide additional information on potential allergenicity of novel proteins, they do not reflect all aspects of IgE-mediated food allergies in humans.

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