To assure equal loading and minimize variability among gels, samples were normalized to GAPDH

food allergens. Meanwhile, hypoallergens of the major shellfish allergen tropomyosin that could be translated into specific immunotherapy are unavailable. Although several shrimp allergens including arginine kinase, sacroplasmic calcium-binding protein, myosin light chain and troponin C have been identified and registered by the IUIS-allergen database, tropomyosin is reactive to.80% patients allergic to shrimp and is regarded as the major shrimp and shellfish cross-reactive allergen. Herein, we have defined the IgE-binding epitopes of the shrimp tropomyosin Met e 1 by ELISA, dot-immunoblotting and three online 1268798 web models as prediction tool represents an emerging strategy in epitope mapping studies among food and drug allergies. Using this combination, we aimed to achieve higher accuracy, including a lower chance of missing important epitopes, more complete recovery and a higher PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19691102/ resolution of epitopes. Using this approach, nine major IgE-binding Met e 1 epitopes were identified. These epitopes range from five to twenty one amino acid residues in length, with some of these allergenic regions longer than the IgEbinding epitopes of other allergens. This variation may be due to the relatively simple coiled-coiled secondary structure of tropomyosin and/or the high flexibility of this molecule, possibly resulting in the higher proportion of surface-exposing IgEbinding sequences. The discovery that six IgE-binding epitopes no. of mice reacted MEM49 IgE OD Discussion 0 1.778 60.037 no. of mice reacted IgG 6 0.40560.056 no. of mice reacted Met e 1 IgE 6 Immunized with rMet e 1 Immunized with MED171 Group 0 0.092 60.003 OD 6 1.089 60.085 OD 0 0.069 60.009 6 0.857 60.073 OD 0 0.089 60.005 Hypoallergen-immunized mice produced Met e 1specific IgG antibodies and inhibited IgE binding to Met e1 no. of mice reacted importantly, none of the MEM49- or MED171-immunized mice produced Met e 1-recognizing IgE antibodies and hypoallergen-specific IgE antibodies, comparing to an IgE level of OD 0.40560.056 upon Met e 1 immunization. These clearly PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692133 demonstrated that both MEM49 and MED171 had marked reduction in their in vivo allergenicity. IgG 0.754 60.087 0.283 60.015 6 6 OD 6 1.121 60.098 OD Hypoallergens of Shrimp Tropomyosin Met e 1 identified in our work overlap with those previously reported for Pen a 1 is not surprising because the two proteins only have one amino acid difference at residue 69. The three Met e 1 IgE epitopes newly identified in this study may partly account for the limited success of a Pen a 1 hypoallergen in reducing allergenicity to shrimp tropomyosin. Incidentally, serum samples from adults were used in the Pen a 1 study while serum samples from children and adolescents were used in determining the IgE-binding epitopes of Met e 1. The presumed greater epitope diversity in children with shrimp allergy than adults may account for the additional epitopes revealed in the present study. Interestingly, some of the Met e 1 epitopes predicted by Bepipred Antibody Epitope Prediction are only one to five amino acid residues apart. Although this model was designed for continuous B cell epitope prediction, a recent study suggests that the results are similar to the predicted discontinuous B cell epitopes. Hence, the epitopes predicted by this model may possibly represent the discontinuous epitopes of Met e 1, although more sophisticated experiments such as crystal structure resolution of allergen/IgE complex could be conducted to confirm the identity