Human IgE-binding epitopes of the latex allergen Hev b 5

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Presentation transcript:

Human IgE-binding epitopes of the latex allergen Hev b 5 Donald H. Beezhold, PhDa, Vicky L. Hickey, BSa, Jay E. Slater, MDb, Gordon L. Sussman, MDc Journal of Allergy and Clinical Immunology Volume 103, Issue 6, Pages 1166-1172 (June 1999) DOI: 10.1016/S0091-6749(99)70194-6 Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 1 Latex-specific antibody recognition of recombinant allergens. Western blot of IgE reactivity of patients allergic to latex or rabbit IgG with NAL (lanes 1, 3, and 5 ) and recombinant Hev b 5 (lanes 2, 4, and 6 ). IgE reactivity was identified by using pooled sera from health care workers with latex allergy. No reactivity was observed with the MBP construct without an insert, which served as a control (data not shown). rHev b 5/MBP shows a fusion protein of ~80 kd (lane 2) . Little reactivity was observed with the recombinant protein by using rabbit sera from AL- or NAL-immunized rabbits (lanes 4 and 6 ). Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 2 Western blot inhibition of latex-specific immunoglobulin recognition of NAL protein. IgE-reactive proteins were identified by using pooled sera from health care workers allergic to latex or IgG from rabbits immunized with NAL proteins (lane 1) . Inhibitions were performed with recombinant Hev b 5 (lane 2) , and inhibition with MBP (lane 3) served as a negative control. Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 3 Octapeptide sequences of Hev b 5. Overlapping synthetic peptides of the Hev b 5 molecule were synthesized on the SPOTs membrane according to the manufacturer’s instructions. Peptides corresponding to the N- and C-terminal regions (spots 1 to 6 and 51 to 95) were synthesized with overlaps of 2 AAs. To more narrowly define potential epitopes, the peptides corresponding to AA 19-56 (Spots 7 to 50) were overlapped by a single AA. Underlining represents spots that were positive with the pooled human anti-latex IgE (see Fig 4). Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 4 Reactivity of latex-specific antibodies with synthetic peptides of Hev b 5. Each spot represents a separate octapeptide as described in Fig 3. BSA (negative control protein), rHev b 5/MBP, and NAL were spotted (a, b, and c from left to right) in the upper right hand corner of the membrane as negative and positive controls for antibody recognition. The membrane was reacted with high IgE control sera (A ) or with sera pooled from 10 health care workers allergic to latex (B ), pooled sera from AL-immunized rabbits (C ), and pooled sera from NAL-immunized rabbits (D ). Regions of antibody binding were identified by AP-labeled secondary antibodies followed by reaction with the development solution (Vistra ECF) to produce chemifluorescence. Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 5 Densitometry scan of the SPOTs membrane as recognized by human IgE. Chemifluorescence was recorded by using STORM 840 and quantified by using Imagequant software. Data is expressed as arbitrary units minus background. Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 6 Alignment of Hev b 5 to kiwi protein: location of lgE epitopes. Sequence alignment was performed by using the computer program BLASTP 2.0.5 with the 4blosum90 matrix (National Center for Biotechnology Information). Human (H) and rabbit (R) epitope regions are numbered and indicated by underlining. Dashes represent gaps introduced to maximize alignment, vertical lines indicate identical sequence, and periods indicate similar sequences. Journal of Allergy and Clinical Immunology 1999 103, 1166-1172DOI: (10.1016/S0091-6749(99)70194-6) Copyright © 1999 Mosby, Inc. Terms and Conditions