Molecular and immunological analysis of hen’s egg yolk allergens with a focus on YGP42 (Gal d 6)
Introduction
Allergy to hen’s egg is one of the most common forms of food allergy in childhood, second only to cow’s milk allergy (Caubet et al., 2011). In developed countries, the prevalence of egg allergy is estimated to range from 0.5 to 2.5% among infants and young children (Rona et al., 2007). Albumen of the chicken egg contains the well-known dominant allergens namely, ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3) and lysozyme (Gal d 4). The egg yolk, on the other hand, also contains two minor allergens known as α-livetin (Gal d 5) and YGP42 (Gal d 6). The allergenic properties of the hen’s egg white proteome have been extensively studied. By contrast, egg yolk proteins and its involvement in food allergy have not come under the limelight of many studies over the years.
The knowledge regarding the association between allergy to egg white and allergy to egg yolk is very limited in the literature. Few studies in the literature support evidence about a significant positive correlation between the allergenicities of egg white and yolk. It has been speculated that this may be due to the common allergenic epitopes that some egg white and yolk proteins share (Anet et al., 1985, Walsh et al., 1987). Furthermore, Walsh et al. reported two egg yolk proteins namely; apovitellenin I and apovitellenin V to be IgE reactive indicating that there could be potentially undiscovered allergens in the egg yolk. Also, the same authors demonstrated a good correlation between IgE binding to egg yolk proteins and the clinical incidence in most egg allergy patients (Walsh et al., 1988).
The objective of this study was threefold. The first objective was to investigate if patients sensitised to hen’s egg white also would be allergic to egg yolk through immunological methods. We found that majority of egg white allergy patients to be seropositive to various egg yolk proteins. The second aim was to establish the identity of IgE reactive proteins as shown from the results of the first objective. The third objective involved production of recombinant version of Gal d 6 (rGal d 6) since it was found to be IgE reactive in a significant number of patients with egg white allergy indicating it is an important allergen. Here, the rGal d 6 was produced and purified as a soluble fraction and showed that it is allergenic using inhibition ELISA immunoassay.
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Human patients’ sera
Sera from 25 patients allergic to hen’s egg white were obtained from the Royal Children’s Hospital (Melbourne, VIC, Australia). The egg white specific IgE levels of patients are shown in Table 1. Experimentation involving patients’ sera was conducted in compliance with the National Statement on Ethical Conduct in Human Research (2007) with approval from Deakin University Faculty of Science, Engineering and Built Environment Human Ethics Advisory Group (HEAG), with a project approval number of
Immunological analysis of CEY and mass spectrometry analysis of IgE reactive hen’s egg yolk proteins
The complexity of the CEY is evident from the protein profile generated from SDS-PAGE (Fig. 1). The resulting protein profile of the CEY shows a large number of protein bands. Although there are some overlapping bands, around 13 different proteins ranging from 10 to 250 kDa could be clearly distinguished (Fig. 1). Furthermore, already known egg yolk allergens Gal d 5 and Gal d 6 were also clearly detectable on the SDS-PAGE of CEY approximately at 65 kDa and 42 kDa, respectively.
Western immunoassay
Discussion
Hen’s egg is one of the most well-known sources of allergens with several allergens present within the egg white and the yolk. The allergens of the egg white have much stronger allergenic potential than the yolk proteins. The role of egg white allergens in regard to food allergy has been well investigated compared to egg yolk allergy. Also, majority of studies reported in literature have investigated allergy to egg white and allergy to egg yolk separately and therefore, overlooking the aspect
Conflict of interest
There is no conflict of interest.
Author contributions
CS conceived the idea, contributed to the study design and supervised the study. CDS and PD performed experiments, collected data and prepared manuscript. TD provided chicken egg and tissue samples and MLKT provided reagents essential for the immunological analysis. All authors reviewed and edited the manuscript.
Acknowledgements
We would like to thank Poultry Cooperative Research Centre (CRC) (established and supported under the Australian Government’s Cooperative Research Centres Program) and Deakin University’s Centre for Molecular and Medical Research (MMR) for providing this study with the required research funding, Australian Animal Health Laboratory (AAHL) of Commonwealth Scientific and Industrial Research Organisation (CSIRO) for supplying animal tissues required for the study, and the Murdoch Children’s
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