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Keywords:

  • allergy;
  • atopy patch test;
  • egg allergens;
  • milk allergens;
  • SDS-PAGE

Abstract

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

Abstract:  The patch test with food antigens (atopy patch test, APT) has been reported as a more specific method than prick or RAST for the early detection of cow’s milk and/or egg sensitizations in children. Standardization of APT extracts is a major issue on the road towards full clinical exploitation of this assay. Here, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to characterize sensitivity and specificity of commercial preparations of APT for milk and egg allergies, which are expected to improve the reliability of this test, when compared with fresh food allergen sources. We found that: (i) SDS-PAGE is an appropriate technique for quality control of APT and (ii) commercial milk and egg APT are equivalent to fresh food preparations in terms of allergen content. Clinical trials aimed at characterizing sensitivity and specificity of APT in the diagnosis of food allergy in children will benefit from this technique.


Abbreviation:
APT

atopy patch test

Background

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

The atopy patch test (APT) is from many aspects an innovative approach in the diagnosis of food allergies in children. In principle, ATP allows the study of the presence of delayed, T-cell-dependent reactivity to allergens, with clinical expression in the skin (1–4). However, this technique, which has been in clinical practice for more than a decade (5) and represents in principle a useful in vivo diagnostic tool, has been criticized for its poor reproducibility (6, 7).

The working hypothesis underlying APT usage is that this test unveils delayed reactions to food allergens, similarly to specific IgE determination in the case of immediate type allergic reactions. However, there is controversy about this assumption, and a lack of association between delayed reactions to oral food challenge and positive APT was reported (1, 8). Another open issue is the lack of standardization of this technique, both in food preparation and in the concentration of the material used before application on the skin. In particular, the usage of fresh foods brings about the issue of possible contamination by heterologous proteins. Recently, with the aim to improve standardization of this test and to reduce side effects observed with fresh food preparations, commercial kits for APT have been made available, which were prepared with freeze-dried proteins from the specific allergen sources (9). No data are available on the purity of these preparations, particularly on allergen content and on the presence of possible contaminants.

Questions addressed and experimental design

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

Here, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate whether commercial preparations of APT (Allergon AB, Angelholm, Sweden and ALK Abellò, Milan, Italy) for milk and egg allergens could be proficiently compared with fresh food allergen sources in terms of relative protein distribution and purity. Details of the allergen extracts we used for SDS-PAGE, of the electrophoretic analysis and of the statistical evaluation of results are reported in the supplementary file.

Results

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

We found that SDS-PAGE profiles of milk APT and fresh milk showed four main bands, which were identified, based on their electrophoretic run, as α-casein, β-casein, β-lactoglobulin and α-lactalbumin (Fig. 1). The detection of κ-casein was not possible, due to its relatively limited quantity (10). Equal electrophoretic mobility of corresponding bands was observed in compared samples. Moreover, analogous minor protein bands were also visible, such as bovine serum albumin (BSA).

image

Figure 1.  SDS-PAGE of milk APT products (left lane of each pair) compared with fresh cow's milk (right lane of each pair). Electrophoretic runs were from three independent analyses. α-Cas: alpha-Casein, β-Cas: beta-Casein, β-Lg: beta-Lactoglobulin, α-La: alpha-Lactalbumin, BSA: Bovine serum albumin. Pre-stained MW standards are displayed in the left most lane.

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The distributions of signals in the four main co-migrating bands from milk APT and from fresh milk were visually equivalent. This was confirmed using quantitative densitometric image analysis (supplementary Fig. S1). In the case of egg protein analysis, due to the more complex protein content, we made preliminary gels to co-identify bands corresponding to the most relevant major allergens. To this aim, commercially available purified ovalbumin, ovomucoid and lysozime were run in parallel with fresh egg fractions, permitting a comparison between the electrophoretic runs and/or the molecular weight of any single band. Four major egg allergens (supplementary Table S1) were identified in fresh egg white and in whole egg preparation (supplementary Fig. S2). Subsequently, SDS-PAGE of egg APT, run in parallel with fresh egg white, allowed detecting four main bands, which were identified as ovotransferrin, ovomucoid, ovalbumin and lysozime (Fig. 2). The electrophoretic runs of bands corresponding to these major allergens were then analysed using densitometry in the compared samples. In both samples, other minor protein bands were also visible, but they were equally represented in the compared samples. No extra bands were visible in the egg APT sample, when compared with the fresh egg preparation. The distributions of the bands corresponding to the main allergens from egg APT and from fresh egg white were visually equivalent. This was confirmed using quantitative densitometric image analysis (supplementary Fig. S3).

image

Figure 2.  SDS-PAGE of the egg APT product (left lane of each pair) compared with fresh whole egg (right lane of each pair). Electrophoretic runs were from three independent analyses. OVA: ovalbumin, OVM: ovomucoid, OVT: ovotransferrin, LYS: lysozime. Pre-stained MW standards are displayed in the left most lane.

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Conclusions

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

Thus, commercial, freeze-dried preparations of APT for milk and egg allergies can be critically analysed qualitatively and quantitatively using the SDS-PAGE method we describe. We suggest that manufacturers of APT use this approach as a standard quality control procedure. Our study also demonstrates that the freeze-dried preparations we considered were fully satisfactory in terms of their antigenic content.

Currently, EAACI/GA2LEN recommendations still suggest the usage of fresh food as preferred reagents (11), whereas recombinant allergens may have a role in the future. However, difficulties in standardization of preparatory procedures are well known (12). In this context, presently available commercial extracts may represent useful tools for the physician, as they warrant higher stability, better reproducibility of results and a batch-to-batch consistency (9). Moreover, as products of pharmaceutical grade, they are expected to reduce the risk of bacterial contamination, which is quite frequent in the preparation of fresh food for APT. This is particularly relevant taking into consideration a recent report showing that colonization with exotoxin-producing bacteria may influence the outcome of patch tests in patients with atopic dermatitis (13). The procedure we used sets the stage for obtaining reproducible clinical results when using atopy patch test.

References

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed and experimental design
  5. Results
  6. Conclusions
  7. References
  8. Supporting Information

Figure S1. Relative distribution of four main milk allergens in APT product versus fresh milk.

Figure S2. SDS-PAGE of fresh whole egg (WE) compared with fresh egg white (EW).

Figure S3. Relative distribution of four main egg allergens in APT product versus fresh whole egg.

Table S1. Major egg allergens (2).

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
EXD_848_sm_FigS1.tif2032KSupporting info item
EXD_848_sm_FigS2.tif320KSupporting info item
EXD_848_sm_FigS3.tif2032KSupporting info item
EXD_848_sm_TableS1.doc43KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.