Mutational analysis of the IgE epitopes in the latex allergen Hev b 5

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Presentation transcript:

Mutational analysis of the IgE epitopes in the latex allergen Hev b 5 Donald H. Beezhold, PhDa, Vicky L. Hickey, BSa, Gordon L. Sussman, MDb Journal of Allergy and Clinical Immunology Volume 107, Issue 6, Pages 1069-1076 (June 2001) DOI: 10.1067/mai.2001.115482 Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 1 A , SPOTs analysis of Hev b 5 epitopes with sera from highly sensitized patients. Each spot represents a sequential decapeptide from the Hev b 5 sequence overlapping by 2 AAs. Membrane was reacted with a 1:10 dilution of pooled sera from 7 individuals allergic to Hev b 5. Epitopes are numbered in the order in which they appear. Epitope 10 was previously identified with sera from patients with latex allergy. Control sera from patients without latex allergy failed to react to the membrane. B , Antigenic index for Hev b 5 determined by using the MacVector computer model. Theoretical epitopes are indicated by a positive antigenic index. Actual epitopes determined by SPOTs analysis above are indicated at the top of the figure. Journal of Allergy and Clinical Immunology 2001 107, 1069-1076DOI: (10.1067/mai.2001.115482) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 2 Heterogeneity in individual recognition of Hev b 5 epitopes. Six epitopes (5.1, 5.2, 5.4, 5.6, 5.7, and 5.8) were synthesized as 12 AA peptides on a derivatized cellulose membrane strip. The strip was then reacted with individual patient sera (lanes 1-7) and processed for IgE reactivity. A serum pool from 3 individuals (lane 8) with high IgE but without latex allergy served as the negative control. Journal of Allergy and Clinical Immunology 2001 107, 1069-1076DOI: (10.1067/mai.2001.115482) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 3 Alanine substitution analysis of Hev b 5 immunodominant epitopes. Alanine substitution SPOTs membrane reacted with pooled sera from 7 highly sensitized patients. Epitope designation is indicated at the left. Column N represents the native sequence, and in subsequent columns the AA that was changed to alanine is indicated to the right of each spot. Journal of Allergy and Clinical Immunology 2001 107, 1069-1076DOI: (10.1067/mai.2001.115482) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 4 Plot of the inhibition ELISA curves for selected mutant (Mut) proteins. The 50% inhibition level used for comparison of the clones is indicated. Journal of Allergy and Clinical Immunology 2001 107, 1069-1076DOI: (10.1067/mai.2001.115482) Copyright © 2001 Mosby, Inc. Terms and Conditions