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The latex allergen Hev b 5 is an antigen with repetitive murine B-cell epitopes.

Authors
Beezhold-DH; Hickey-VL; Sutherland-MF; O'Hehir-RE
Source
Int Arch Allergy Immunol 2004 Aug; 134(4):334-340
NIOSHTIC No.
20025318
Abstract
Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63-68), PAEGEK (120-125), and PAEEEK (126-131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K131 as critical aa in each epitope to change by site-directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that epitope 126-131 was the dominant epitope, but mutation of epitope 120-125 was also required to eliminate mAb reactivity to Hev b 5. The antibodies did not appear to recognize the epitope 63-68 in the recombinant fusion protein. We identified an immunodominant B-cell epitope in Hev b 5 that is repeated 3 times within the sequence, making Hev b 5 multivalent. Well-characterized monoclonals recognizing repeated epitopes would be a good choice for immunodetection of Hev b 5 in glove extracts where individual epitopes could get altered by the manufacturing process.
Keywords
Allergens; Antigens; Mutagenesis; Amino-acids; Proteins; Bioassays; Antibody-response
Contact
Dr. Donald Beezhold, NIOSH, 1095 Willowdale Road, Morgantown, WV 26505
CODEN
IAAIEG
Publication Date
20040801
Document Type
Journal Article
Email Address
zec1@cdc.gov
Fiscal Year
2004
NTIS Accession No.
NTIS Price
Issue of Publication
4
ISSN
1018-2438
NIOSH Division
HELD
Priority Area
Disease and Injury: Asthma and Chronic Obstructive Pulmonary Disease
Source Name
International Archives of Allergy and Immunology
State
WV; PA
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