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ANALYSIS OF THE ALLERGENIC COMPONENTS IN TRADITIONAL APPLE CULTIVARS BY SINGLE POINT HIGHEST INHIBITION ACHIEVABLE ASSAY (SPHIAa) FOR IgE
Palazzo Paola, Pasquariello Maria Silvia, Tuppo Lisa, Liso Marina, Petriccione Milena, Rega Pietro,Tartaglia Alfonso, Tamburrini Maurizio, Alessandri Claudia, Mari Adriano, Ciardiello Maria Antonietta.
Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, Italy. Institute of Protein Biochemistry, CNR, Naples, Italy. CRA-FRC Unità di Ricerca per la Frutticoltura , Caserta, Italy. STAPA -Ce.PI.C.A., Centro direzionale Collina Liguorini, Avellino, Italy.
BACKGROUND
APPLE (Malus domestica):
is one of the most commonly eaten fruit worldwide
some individuals have to avoid apple consumption because this fruit may cause allergic reactions
literature reports have described a significant variation of the allergen composition in different apple cultivars
their characterization may allow the selection of apple cultivars the consumption of which could be suggested to subjects having mild symptoms following apple ingestion.
AIMS
The objective of this study was the characterization of apple cultivars traditionally cultivated in Southern Italy
RESULTS
Protein extracts obtained from the pulp of apple samples were analyzed by estimating the protein concentration (Figure 1) and the protein profile on SDS-PAGE (Figure 2).
The allergen composition of each apple extract was investigated by the Single Point Highest Inhibition Achievable assay (SPHIAa) performed by detecting IgE on the ISAC 103 microarray (Phadia Multiplexing Diagnostics, Vienna, Austria) and using a pool of sera from allergic subjects recognizing profilins, LTPs, and Bet v 1-like (Figure 3).
Only 6 cultivars have a IgE inhibition higher than 50% on Mal d allergen, and three cultivars have a IgE inhibition higher than 50% on all the other Bet v 1-like allergens present on the microarray.
Six cultivars have a inhibition higher than 50% on two LTPs (Art v 3 and Cor a ), three on Pru p 3, no one on Par j
The same range (30-90%) of IgE inhibition was obtained on all the five profilins available on the ISAC 103 microarray for all the 12 cultivars.
The results highlight a significant variation of the protein concentration in the protein extracts without a correlation with the potential allergenicity.
ANALYSIS OF THE PROTEIN CONTENT IN THE APPLE EXTRACTS
Relative quantity (%)
Apple cultivars -
ANALYSIS OF ALLERGEN PROFILES IN APPLE EXTRACTS BY SPHIAa
Figure 1. Relative abundance of total proteins in the protein extracts.
LTP
BET V 1-LIKE
PROFILIN
Figure 2. SDS-PAGE of the protein extracts.
APPLE CULTIVARS
1. Chianella di Volturara (AV) Renetta di Serino (AV)
2. Cassanese di Volturara (AV) Fierro di Bucciano (BN)
3. Bianca di Grottolella (AV) Mela Rosa di S. Nazzaro (BN)
4. Annurca Rossa del Sud di Pignataro (CE) Sergente di Banoli Irpino (AV)
5. Red Chief di Pignataro (CE) Golden B di Pignataro (CE)
6. Suricillo di Volturara (AV) Limoncella di Paternopoli (AV)
CONCLUSIONS
IgE SPHIAa is a simple method for a fast identification of allergenic molecules in allergenic foods.
The assay can give a preliminary idea about the presence of the allergens in the source, though the results largely depend on the concentration, the affinity, and the avidity of the molecules in the extract compared to the homologous ones spotted on the microarray.
In case of no inhibition for some risky allergens further biochemical studies should be carried out to grant the use of some cultivars by allergic patients. Improving the assay for allergen detecting purposes would require the availability of all the known species-related allergens on the microarray (i.e. Mal d 2, 3, 4….).
Figure 3. Single Point Highest Inhibition Achievable assay (SPHIAa)